Compositions and methods of treating infections and tumours
SUBSTANCE: invention refers to medicine, namely to oncology and immunology, and can be used in treating an individual with a stable pathogenic infection or a tumour. That is ensured by administering a therapeutically effective amount of a programmed death (PD-1) peptide antagonist and a therapeutically effective amount of a pathogen or tumour antigen molecule (vaccine).
EFFECT: invention provides the synergetic action of the antigen and PD-1 antagonist (anti-PD-L1 antibody) combination by intensifying the T-cell immune response to the pathogen or tumour.
14 cl, 29 dwg, 5 tbl, 25 ex
This application claims the priority of provisional application U.S. No. 60/877518, filed December 27, 2006, which is incorporated into this description by reference.
Disclosed subject matter also relates to provisional application U.S. No. 60/688872, filed June 8, 2005, application No. 11/449919, filed June 8, 2006, and PCT application no PCT/US2006/22423. These prior applications are also included in the present description by reference in full.
The application for state support
This invention was supported by U.S. government grants NIH AI39671 and CA84500. The government has certain rights in the invention.
The technical field to which the invention relates
This invention relates to the use of antagonists, specifically to the use of antagonists PD-1, for the treatment of resistant infections and tumors.
Immunosuppression of the host immune response plays a role in resistant infections and immunosuppression tumors. Resistant infections are infections in which the virus does not appear, but remains in specific cells of infected individuals. Resistant infections often include stage as latent and productive infection without rapid destruction or even with the occurrence of the excessive damage to the host cells. There are three types of stable interaction of virus-host: latent, chronic and slow infection. Latent infection is characterized by the absence of detectable infectious virus between episodes of recurrent disease. Chronic infection is characterized by a prolonged presence of infectious virus after primary infection and may include chronic or relapsing disease. Slow infection is characterized by a long incubation period with subsequent disease progression. Unlike latent and chronic infections slow the infection may not begin with the acute period of the propagation of the virus. In the process of sustainable infections viral genome can either stably integrated into the cell's DNA, or be supported episomal. Persistent infection occurs during infection with these viruses, as viruses T-cell leukemia, Epstein-Barr, cytomegalovirus, herpes viruses, varicella zoster virus, measles virus, papovavirus, prions, hepatitis viruses, adenoviruses, parvoviruses and papilloma viruses.
The mechanisms by which supported resistant infections may include modulation of gene expression of the virus and cells and modification of the host immune response. Reactivation of latent infection can be triggered by various stimuli, including the I changes in cellular physiology, the superinfection with other viral and physical stress or trauma. Immunosuppression of the host is often associated with reactivation of a number of persistent viral infections.
Many studies have demonstrated defective immune responses in patients diagnosed with cancer. Identified a number of tumor antigens that are associated with specific types of cancer. Many tumor antigens defined as a characteristic of solid tumors: MAGE 1, 2 and 3, certain immunological; MART-1/Melan-A, gp100, removeability antigen (CEA), HER-2, mucines (i.e., MUC-1), prostatespecific antigen (PSA) and acid phosphatase prostate (PAP). In addition, viral proteins, such as proteins of hepatitis B virus (HBV), Epstein-Barr (EBV) and human papillomavirus (HPV), as shown, are important for the development of hepatocellular carcinoma, lymphoma and cervical cancer, respectively. However, due to immunosuppression in patients diagnosed with cancer, the innate immune system of these patients are often not able to respond to tumor antigens.
Both passive and active immunotherapy, as it is assumed, can be used in the treatment of tumors. Passive immunity provides component of the immune response, such as antibodies or cytotoxic T cells of interest of the individual. Active immunotherapy uses therapeutic agent, such as itoken, the antibody or chemical compound to activate endogenous immune response when the immune system premirovan to recognize the tumor as foreign. Induction of both passive and active immunity is successful in treating specific types of cancer.
In General, there is a need to provide safe and effective therapeutic methods of treatment of diseases, such as autoimmune diseases, inflammatory disorders, allergies, transplant rejection, cancer, immune deficiency and other disorders associated with the immune system.
Summary of the invention
In the present description revealed that antigen-specific CD8+ T cells become functionally tolerant ("depleted") in respect of an infectious agent or tumor antigen after induction of polypeptide-1 programmed death (PD-1). Accordingly, by reducing the expression or activity of PD-1 immune response, specific infectious agent or tumor cells can be increased. Individuals with infections, such as persistent infection can be treated with the use of antagonists PD-1. Individual tumors can also be treated using antagonists PD-1. Additionally, individuals can be treated with transplantation therapeutically effective if the ESCWA activated T-cells, they learn the antigen of interest, in combination with a therapeutically effective amount of the antagonist PD-1.
In some embodiments, implementation of the disclosed methods of induction of an immune response to any antigen in an individual, which is a mammal. The method includes the administration to an individual a therapeutically effective amount of activated T-cells, where the T cells specifically recognize the antigen of interest, and a therapeutically effective amount of an antagonist of the polypeptide-1 programmed death (PD)-1. An individual may represent any interest of the individual, including the individual with a viral infection, such as persistent viral infection, or a person with a tumor.
In additional embodiments, implementation of the disclosed methods of induction of an immune response to any antigen in the recipient mammal. The methods include introducing into contact population of donor cells from the same species of mammals, including T-cells, antigen presenting cells (APC), and a preliminary selection of the antigen of interest, where the pre-selected antigen is represented by the APC to T cells with obtaining donor population of activated T cells in the presence of antagonist PD-1. A therapeutically effective amount of a population of activated donor T cells Tran is planiruetsja recipient. The recipient also administered a therapeutically effective amount of the antagonist PD-1.
In some embodiments, implementation of the disclosed methods of treatment of an individual infected with a pathogen, such as the treatment of resistant infections. The methods include the introduction to the individual a therapeutically effective amount of an antagonist of the polypeptide-1 programmed death (PD-1) and a therapeutically effective amount of the antigenic molecule from a pathogen. Examples of pathogens include viral and fungal pathogens.
In additional embodiments, implementation of the disclosed methods of treatment of an individual with cancer. The methods include the introduction to the individual a therapeutically effective amount of an antagonist of the polypeptide-1 programmed death (PD-1) and a therapeutically effective amount of a tumor antigen or a nucleic acid that encodes a tumor antigen.
Below and other features and advantages will become more apparent from the subsequent detailed description of certain embodiments, which are presented with reference to the accompanying drawings.
Brief description of drawings
Fig.1A is a histogram showing the mRNA levels of PD-1 is specific for DbGP33-41 and/or DbGP276-286 T-cells from uninfected transgenic mice, mice immunized with limp citarum virus choriomeningitis (LCMV) Armstrong (approximately 30 days after infection), or mice with deficiency of CD-4 infected with LCMV-C1-13 (approximately 30 days after infection), the results of measurement using the method of genetic matrices. Fig.1B is a series of images of the experiment with flow cytometry, showing surface expression of PD-1 on containing CD8+tetramer+ T-cells in mice immunized with LCMV Armstrong, and mice with deficiency of CD-4 infected with LCMV-C1-13, approximately 60 days after infection. CD8+ T cells from anergy Express high levels of the polypeptide of PD-1 on the cell surface approximately 60 days after chronic virus infection LCMV-C1-13 (labeled "chronic"), but specific virus CD8+ T cells do not Express the polypeptide PD-1 after release from acute infection with LCMV Armstrong (labeled "immune"). Fig.1C is a series of images of the experiment with flow cytometry, demonstrating the presence of PD-L1 on splenocytes from chronically infected and uninfected mice. This shows that the expression of PD-L1 is highest on the splenocytes infected with the virus.
Fig.2A is a series of scatterplots showing that when mice infected with C1-13, heal from 23 to 27 days after infection, there is approximately 3-fold the expansion of the number of specific DbNP396-404 and specific in DbGP33-41 CD8+ T cells compared with the control, not being treated. In order to determine any changes in the function, we measured the production of IFN-γ and TNF-α in response to 8 different epitopes of LCMV. Fig.2B is a scatterplot showing that when all known specific markers CD8+ T cells measured, observed 2, 3-fold increase in the total number specific for LCMV SW+ T cells. Fig.2C is a series of diagrams flow cytometry, demonstrating the production of IFN-γ and TNF-α in response to eight different epitopes of LCMV. Fig.2D is a scatterplot showing that in mice subjected to treatment, a higher number of virus-specific CD8+ T cells has the ability to produce TNF-α. Fig.2E is a series of bar charts showing that blockade of PD-L1 also leads to increased viral control in the spleen, liver, lung, and serum.
Fig.3A is a graph showing the increase in specific in DbGP33-41 and DbGP276-286 T-cells (marked "GP33 and GP276") in mice with deficiency of CD-4 infected C1-13, which were treated with anti-PD-L1 (marked "αPD-L1") with 46 days to 60 days after infection compared to the control (marked "untx), which indicates that mice, which were treated with anti-PD-L1, contained approximately 7 times more specific in DbGP276-286 CD8+ T-cells Seles the NCI and approximately 4 times more specific in DbGP33-41 CD8+ T cells in the spleen, than mice not subjected to treatment. Fig.3B is a series of images of the experiment with flow cytometry, demonstrating an increased incidence of specific in DbGP33-41 and DbGP276-286 CD8+ T cells in the spleen in mice with deficiency of CD-4 infected C1-13, which were treated with anti-PD-L1 (marked "αPD-L1 Tx") with 46 days to 60 days after infection compared to control (marked "untx"). Fig.3C is a series of images of the experiment with flow cytometry, showing increased proliferation specific for DbGP276-286 CD8+ T cells in mice, which were treated with anti-PD-L1, when measured by BrdU incorporation and Ki67 expression. Fig.3D is a graph showing that mice with high levels of expansion of CD8+ T cells, a significant response in mononuclear cells of peripheral blood (PBMC), as shown by comparing specific in DbGP276-286 CD8+ T cells in PBMC compared with specific DbGP276-286 CD8+ T-cells in the spleen.
Fig.4A is a series of graphs showing the increase in production of IFN-γ specific in DbGP276-286 and DbGP33-41 CD8+ T-cells in mice, which were treated with anti-PD-L1, as compared with the control. Mice were treated with anti-PD-L1, there was a higher incidence of specific DbNP396-404, KbNP205-212, DbNP166-175 and DbGP92-101 CD8+ T cells. Fig.4B present the focus of a graph showing that in mice, which were treated with anti-PD-L1, 50% specific in DbGP276-286 CD8+ T cells produce IFN-γ, compared with 20% specific in DbGP276-286 CD8+ T cells from control mice. Fig.4C is a series of images of the experiment with flow cytometry, demonstrating that we were treated with anti-PD-L1 chronically infected mice produced higher levels of TNF-α than those immunized mice infected with LCMV Armstrong. Fig.4D is a graph showing that treatment with anti-PD-L1 mice infected with LCMV-C1-13, restores lytic activity ex vivo virus-specific T cells compared to untreated infected mice when measured using the test release51Cr. Fig.4E is a series of graphs showing the reduction of the titer of the virus in various organs after treatment with α-PD-L1 mice infected with LCMV-C1-13. Viral titres were reduced by approximately 3 times in the spleen, 4 times in the liver, 2 times at light, and 2 times in serum after treatment with anti-PD-L1 in 2 weeks compared to untreated mice.
Fig.5A is a series of images of the experiment with flow cytometry, showing surface expression of PD-1 with 10 tetramers HIV specific in dominant the m epitopes, towards the taxon With chronic HIV infection. The percentages represent the percentage of tetramer+cells that are PD-1+. Fig.5B is a series of graphs demonstrating that the percentage and MFI of PD-1 significantly positively regulated by HIV-specific CD8+ T-cells compared with the total population of CD8+ T cells (p<0,0001) when antiretroviral therapy is not treated individuals, and PD-1 is increased by the total population of CD8+ T cells in HIV-infected compared to HIV-seronegative controls (p=0,0033 and p<0,0001, respectively). The analysis included 120 tetramer of HIV strains from 65 HIV-infected individuals and 11 HIV-seronegative controls. Fig.5C is a series of graphs showing the average percentage and MFI of expression of PD-1 tetramer+the cells according to the specificity of the epitope. Fig.5D is a graph depicting the variation of the percentage of PD-1+cells at different specific epitopes populations of individuals with multiple defined answers. The horizontal line indicates the median of the percentage of HIV tetramer+PD-1+cells in each individual.
Fig.6A is a series of graphs showing that there is no correlation between the number of HIV-specific CD8+ T cells when measured by staining tetramers and VI is usnei load plasma, at the same time, there is a positive correlation between both the percentage and MFI of PD-1 tetramer+cells and viral load in plasma (p=0,0013 and p<0,0001, respectively). Fig.6B is a series of graphs showing that there is no correlation between the number of HIV tetramer+cells and CD4 count, at the same time, there is an inverse correlation between the percentage and MFI of PD-1 on HIV tetramer+cells and CD4 count (p=0,0046 and p=0,015, respectively). Fig.6C is a series of graphs showing that the percentage and MFI of PD-1 on the total population of CD8+ T cells was positively correlated with viral load in plasma (p=0,0021 and p<0,0001, respectively). Fig.6D is a series of graphs showing that the percentage and MFI of expression of PD-1 on the total population of CD8+ T cells was negatively correlated with CD4 count (p=0,0049 and p=0,0006 respectively).
Fig.7A is a series of images of the experiment with flow cytometry, demonstrating the typical phenotypic staining of B*4201 TL9-specific CD8+ T cells from individual SK222, of which 98% B*4201 TL9-specific CD8+ T cells are PD-1+. Fig.7B is a graph illustrating the total phenotypic data from individuals who have >95% of HIV-specific CD8+ T cells are PD-1+. From seven to 19 samples were analyzed for the each of these phenotypic markers. Horizontal mark indicates the median of the percentage of tetramer+PD-1+cells that were positive for the indicated marker.
Fig.8A is a series of images of the experiment with flow cytometry, showing typical test data on the proliferation of B*4201 positive individual. After 6 days of stimulation with peptide, the percentage of B*4201 TL9-specific CD8+ T cells increased from 5.7% to 12.4% in the presence of anti-PD-L1 blocking antibodies. Fig.8B is a line graph showing test data on the total proliferation, indicating a significant increase in the proliferation of HIV-specific CD8+ T cells in the presence of anti-PD-L1 blocking antibody (n=28, p=0,0006, paired t-test). Fig.8C is a histogram showing the different effects of the blockade of PD-1/PD-L1 on the proliferation of HIV-specific CD8+ T cells, based on individual patients. White bars indicate the degree of increase of the tetramer+cells in the presence of peptide, the black bars indicate the degree of increase of the tetramer+cells in the presence of peptide plus anti-PD-L1 blocking antibody. Individuals who have carried out tests CFSE for more than one epitope, indicated by symbols in the form of stars, square or triangle.
Fig.9a-9d are a diagram and a series of graphs showing synergic is th effect of therapeutic vaccines combined with the blocker PD-L1, the frequency of antigen-specific CD8-T cells and the titer of virus in chronically infected mice. Fig.9a is a schematic diagram of the experimental Protocol. Infected with clone 13 LCMV (CL-13) mice were vaccinated with cowpox virus wild-type (VV/WT) or vaccinia virus expressing the LCMV epitope GP33-41 (VV/GP33), 4 weeks after infection. At the same time, mice were treated 5 times every three days anti-PD-L1 or without him. Fig.9b is a series of images of the experiment with flow cytometry, demonstrating the frequency of GP33 - and GP276-specific CD8-T cells in PBMC after 1 week of treatment. The number represents the frequency of tetramer-positive cells compared to CD8-T-cells. Data represent results of three experiments. Fig.9c-9d are graphs of the frequency of GP33 - and GP276-specific CD8-T cells (Fig.9c) and the titers of the viruses (Fig.9d) in the blood after treatment. Changes in the number of tetramer-positive CD8-T cells and viral titres were observed in the blood dyeing by tetramers and analysis belascoaran respectively at the specified time point. The number of tetramer-positive CD8-T cells and viral titres presents in mice individually (top four panels) and in the summary view (bottom panel) after the introduction of VV/WT or VV/GP33 (straight line) and treatment with anti-PD-L1(shaded area). The dotted lines represent the limit of detection of viruses. The combined results of the three experiments.
Fig.10a-10d are graphs and digital images showing the increase in antigen-specific CD8-T cells and increased control of the virus in different tissues of mice treated with therapeutic vaccine in combination with blockade of PD-L1. Fig.10a is a series of images of the experiment with flow cytometry, demonstrating the frequency of GP33-specific CD8-T cells in various tissues 4 weeks after treatment. The number represents the frequency of tetramer-positive cells compared to CD8-T-cells. Data represent the results of two experiments. Fig.10b is a graph showing the dependence of the number of GP33-specific CD8-T cells in various tissues 4 weeks after treatment. Fig.10c is a set of histograms showing the titers of virus in these tissues through 2 (shaded bars) and after 4 (unfilled bars) weeks after treatment. The dotted lines represent the limit of detection of viruses. N=6 mice per group. The combined results of the two experiments. Fig.10d is a digital image of immunostaining of spleen antigens aLCMV (red) after 2 weeks of treatment. Magnification ×20.
Fig.11a-11d are charts and graphs, shows the possibility enhanced by the restoration of the function of exhausted CD8-T cells with therapeutic vaccines in combination with blockade of PD-L1. Fig.11a is a series of images of the experiment with flow cytometry, demonstrating the production of IFN-γ and degranulation of splenocytes of vaccinated mice 4 weeks after treatment. Splenocytes stimulated with the indicated peptides in the presence of αCD107a/b antibodies and then were co-stained for IFN-γ. The diagram is limited by the window for CD8-T cells and represent two independent experiments. Fig.11b is a graph showing the percentage of IFN-γ+CD107+cells compared to CD8-T-cells specific for each of the LCMV peptides from Fig.11a, summed for many of the mice (n=6 for each answer). Summarized results of the two experiments. Fig.11c is a series of diagrams showing the production of TNF-α CD8-T-cells capable of producing IFN-γ in vaccinated mice. After stimulation of splenocytes peptide GP33-41 or GP276-286 was the sort window for producing IFN-γ CD8-T cells, and then built chart IFN-γ (x axis) against TNF-α (y-axis). The top and bottom numbers on the diagram indicate the frequency of TNF-α+cells among IFN-γ+cells and the mean fluorescence intensity (MFI) of IFN-γ+cells, respectively. Data represent the results of two independent experiments. Fig.11d is a graph showing the percentage of TNF-α+cells on the relationship is to TNF-γ +cells for peptide GP33-41 or GP276-286 of Fig.11c, summed for many of the mice (n=6 for each response).
Fig.12a-12b are a series of diagrams showing that the effect of therapeutic vaccines in combination with blockade of PD-L1 changes the phenotype of antigen-specific CD8-T cells chronically infected mice. Fig.12a is a series of diagrams showing the phenotype of GP33 tetramer-specific CD8-T cells in PBMC at specified time points after treatment. Histograms were limited window for GP33+CD8-T cells. The frequency of the population expressing high levels of CD27 or CD127, specified in the form of interest on the charts. Numbers in histograms for Granzyme B represent MFI expression. Data represent results of three independent experiments. Fig.12b is a series of diagrams showing phenotypic changes GP33 tetramer-specific CD8-T cells in various tissues 4 weeks after treatment. Histograms were limited window for GP33+CD8-T cells. The frequency of the population expressing high levels of CD127 or PD-1, are shown as percentage charts. Numbers in histograms for Granzyme B and Bcl-2 represent MFI expression. Data represent the results of two independent experiments.
Fig.13a-13e are schematic diagram, charts and graphs that demonstrate synergistic is to function effectively therapeutic vaccine in combination with inhibitors of PD-L1 on restoration of function "useless" exhausted CD8 T cells. Fig.13a is a schematic diagram of the Protocol. In mice overburden CD4 T cells and then infected with clone 13 LCMV. Some mice were vaccinated with cowpox virus wild-type (VV/WT) or vaccinia virus expressing the LCMV epitope GP33-41 (VV/GP33), after 7 weeks after infection. At the same time, mice were treated 5 times every three days αPD-L1 or isotype. Two weeks after the initial treatment with antibodies, the mice were scored for analysis. Fig.13b is a series of images of the experiment with flow cytometry and a histogram showing the frequency of GP33-specific CD8-T cells in the indicated tissues 4 weeks after treatment. The number represents the frequency of GP33 tetramer-positive cells compared to CD8-T-cells. Summative also the frequency of GP33-specific cells compared to CD8-T cells in various tissues 2 weeks after treatment. Fig.13c is a series of images of the experiment with flow cytometry and a histogram showing the results of experiments in which splenocytes stimulated with GP33 peptide in the presence of antibodies αCD107a/b and then were co-stained for IFN-γ. Presents charts was limited window for CD8-T cells. The percentage of IFN-γ+CD107+cells compared to CD8-T-cells specific for peptide GP33, summed for many of the mice. Fig.13d pre which is a histogram of the percentage of IFN-γ +cells after stimulation with GP33 peptide in relation to the cells positive for Db-restricted GP33-41 tetramer, summed for many of the mice. Fig.13e is a histogram of the titers of virus in these tissues after 2 weeks of treatment. All graphs represent the results of two experiments, and all the summarized results of the two experiments combined (n=6 mice per group).
Fig.14a-14b are diagrams and graphs showing that blockade of signaling pathways PD1/PD-L1 increases the total number specific to the antigen of T cells with subsequent adoptive transfer mice - congenital media. Whole splenocytes adaptive transferred mice - congenital media with or without treatment with anti-PD-L1. Fig.14a is a series of diagrams of a typical flow cytometry at specific time points with a limited window for CD8+ T cells. Fig.14b is a graphic showing the kinetics of expansion Db-GP33-specific CD8 T cells in the peripheral blood of two independent experiments (n=4 animals per group).
Fig.15a-15e are diagrams and graphs showing that blockade of road PD-1/PDL1 after adoptive T-cell immunotherapy increases the production of cytokines, antigen-specific CD8 T-cells. Splenocytes were isolated on day 17 after transfer and analyzed for the expression of C is Takenov upon stimulation with antigenic peptide. Fig.15a is a series of diagrams of a typical flow cytometry showing the expression of IFNγ, determined by intracellular cytokine staining after 5 hours after stimulation of certain CD8 epitopes or controls without peptide. Fig.15b and 15d are diagrams showing a dual expression of TNFα or 107ab and IFNγ (statistical values in the quadrants represent the percentage of the window for CD8). Fig.15c and 15e represent a histogram of the percentage of cells producing IFNγ and also producing TNFα or 107ab (n=3 animals per group).
Fig.16A-16B are a graph and a chart showing elevated levels of cells secreting antibodies in mice infected with clone 13 LCMV. Total ASC levels were measured in mice chronically infected with LCMV after treatment with αPD-L1 using ELISPOT and CD138 staining. Fig.16A is a graph showing the total number of ASC in the spleen, the combined results of three independent experiments. Fig.16B is a series of charts demonstrating that the increase of cells secreting antibodies (ASC) in the spleen can be measured using the marker CD138. Shows a typical chart, ASC characterized as CD138+ and B220 low/intermediate (box limited to lymphocytes).
Fig.17 is a graph showing that treatment with anti-PD-L1 mouse is, chronically infected with LCMV, does not lead to higher levels of the ASC in the bone marrow. The total number of ASC counted in the spleen and bone marrow of mice chronically infected with LCMV, 14 days after treatment with anti(α)PD-L1 using ELISPOT. The line represents the geometric mean of the group.
Fig.18 is a graph showing that co-administration αPD-L1 and αCTLA-4 leads to a synergistic increase in ASC in the spleen. Mice chronically infected with LCMV, introduced αPD-L1, αCTLA-4 or both of the peptide within 14 days and ASC in the spleen was calculated using ELISPOT. The line represents the geometric mean of the group.
Fig.19A-19B are diagrams showing increased proliferation and activity of its germinative center b cells and CD4 T cells in mice, which were treated with αPD-L1. Fig.19A is a diagram analysis using flow cytometry of CD4 T-cells and b-cells showing elevated levels of Ki-67 after treatment αPD-L1. The results are restricted to Windows for either CD4 or b cells, as shown at the top of each column. Fig.19B is a series of diagrams showing an increased frequency of b cells expressing the PNA, and high levels of FAS, indicating increased activity of its germinative center in mice, which were treated with αPD-L1. Charts represent one typical d is agrama, summarizing the results of two separate experiments.
Fig.20A-20C are diagrams and graphs showing the expression of PD-1 on subpopulations of CD8 and CD4 T cells. Fig.20A is a series of images of the experiment with flow cytometry, demonstrating joint surface expression of PD-1 and various phenotypic markers among CD8+/CD3+ lymphocytes. Fig.20B is a series of graphs of different percentage of CD8+/CD3+ and (D) CD4+/CD3+ subpopulation of T cells that Express PD-1. Horizontal lines indicate the average percentage of PD-1 on T-cells, which are positive (unfilled circles) and negative (filled triangles) at the specified marker. Fig.20C is a series of graphs representing the phenotypic data of CD4+ T cells expressing PD-1, from one subpopulation.
Fig.21A-B are a diagram and graphs showing that PD-1 is expressed at higher levels on CD8 T cells specific for chronic infections. Fig.21A is a series of images of the experiment with flow cytometry, demonstrating the typical staining for PD-1 CD8 T cells specific for Epstein-Barr (EBV), cytomegalovirus (CMV), influenza virus, and cowpox virus. Specified geometric mean fluorescence intensity (GMFI) of expression of PD-1 among tetramer cells. Fig.21B is a graph showing the total data GMFI PD-1 on CD8 T cells from healthy volunteers (n=35), specific for EBV, CMV, influenza virus, and cowpox virus.
Fig.22A-C are diagrams and graphs showing that blockade by anti-PD-L1 enhances proliferation in vitro CD8 T cells specific for chronic infections. Fig.22A is a series of images of the experiment with flow cytometry, demonstrating lymphocytes that have been tagged with CFSE, and then were cultured for 6 days under these conditions. In the images reflected typical staining from individuals, positive against EBV and CMV. Fig.22B represents the histogram of responses that are specific for antigens of EBV, CMV, influenza virus, and cowpox virus, after blockade using anti-PD-L1 blocking antibodies. The columns indicate the degree of increase in the tetramer+cells in the presence of peptide plus anti-PD-L1 blocking antibody compared only with the peptide. Fig.22C is a linear graph showing the relationship between the degree of increase in the tetramer+cells after blockade by anti-PD-L1 antibody and the expression of PD-1 (before cultivation).
Fig.23B-23C are diagrams and graphs showing that CD8+ T cells specific for hepatitis C virus (HCV), Express PD-1, if the HRO is practical of HCV infection of human rights. Fig.23A is a typical graph from five patients with chronic HCV infection, demonstrating the expression of PD-1 on CD8+ T-cells specific for HCV. Figures in bold indicate the frequency of expression of PD-1 (x-axis) on CD8+ T-cells specific for HCV (y-axis). In the diagrams the figures in italics indicate the frequency of tetramer-positive cells among total CD8+ T cells. On the y-axis 1073 and 1406 indicate specific epitopes of HCV tetramer. Patients listed as "Pt" followed by the number of the patient. Cells were limited window for CD8+ lymphocytes. The diagrams are presented in logarithmic scale. Fig.23B represents the comparison of the expression of PD-1 on CD8+ T-cells from healthy donors (CD8 healthy), patients infected with HCV (HCV CD8), and CD8+ T-cells specific for HCV (HCV tet+). Fig.23C is a graph showing the expression of PD-1 on CD8+ T-cells specific for influenza virus (Flu tet+) from infected HCV (HCV+) and healthy donors (healthy), in comparison with the expression of PD-1 on CD8+ T-cells specific for HCV (HCV tet+). To compare differences in the expression of PD-1 in the same patient on total CD8+ T cells compared to CD8+ T cells specific for HCV, used the unpaired t-test.
Fig.24A-24D are diagrams and graphs showing that the frequency of CD8+ T cells expressing PD-1 in the liver is higher than in peripheral blood. Fig.24A represents the Wallpaper typical charts from five patients with chronic HCV infection, demonstrating the expression of PD-1 on total CD8+ T-cells from peripheral blood against them in the liver. The numbers in bold on the chart indicate the frequency of cells with expression of PD-1 among total CD8+ T cells in the box, limited to lymphocytes. The diagrams are presented in logarithmic scale. Fig.24B represents the comparison of the expression of PD-1 on CD8+ T-cells from peripheral blood against them in the liver in patients chronically infected with HCV. To compare differences in the expression of PD-1 in the same patients used a paired t-test. Fig.24C is a comparison of the expression of PD-1 on CD8+ effector-memory cells (TEM) from the peripheral blood against them in the liver. Subpopulations of memory were identified by differential expression of CD62L and CD45RA. Numbers in bold at the top of the charts indicate the frequency of cells in each quadrant. Cells were limited window for CD8+ lymphocytes. A subpopulation of TEMwas limited by the box (boxes), and the expression of PD-1 presents the histograms below. The dotted line represents the expression of PD-1 in naive CD8+ T-cells (used as negative population). Numbers in histograms indicate the frequency of cells expressing PD-1. Comparison of frequency of expression of PD-1 on CD8+ TEMthe ten cells of patients with chronic HCV infection are summarized in the following histograms. To compare the possible differences in the expression of PD-1 on CD8+ T EMfrom the peripheral blood and liver of the same patient used a paired t-test. Fig.24D is a typical graph from two patients with chronic HCV infection, showing the difference in CD127 expression on total CD8+ T-cells from peripheral blood and liver. Figures in bold indicate the frequency of CD127 expression on total CD8+ T-cells. Cells are limited window for CD8+ lymphocytes. The chart is presented in logarithmic scale. Summary comparison of CD127 expression on total CD8+ T-cells in the peripheral blood in relation to them in the liver is shown below charts FACS. For statistical analysis used paired t-test.
Fig.25 is a series of graphs and charts, showing that CD8+ T cells specific for HCV in the liver Express an exhausted phenotype. A typical graph of the expression of PD-1 and CD127 on CD8+ T-cells specific for HCV, from the peripheral blood and liver of two patients with chronic HCV infection. The first series of charts illustrates HCV tetramer-positive population (boxes). Numbers above the boxes indicate the frequency of tetramer-positive cells among CD3+ lymphocytes. The specificity of the epitope of the HCV tetramer are indicated on the y-axis (1073). The second and third rows of graphs show the expression of PD-1 and CD127 on CD8+ T-cells specific for HCV, from the peripheral blood and liver of two sick the chronic HCV infection. Figures in bold indicate the frequency of expression of PD-1 and CD127 on CD8+ T-cells specific for HCV. The chart is presented in logarithmic scale and limited window for CD3+ CD8+ lymphocytes. Below FACS diagram shows the summary data for comparison of expression of PD-1 (left) and the expression of CD127 (right) on total CD8+ T cells compared to CD8+ T-cells specific for HCV, from the peripheral blood (HCV tet+ PBMC) and compared with CD8+ T-cells specific for HCV from the liver. For comparison of expression of one and the same patient used paired t-tests.
Fig.26 is a series of charts demonstrating that blockade of road PD-1/PD-L1 enhances the expansion of antigen-stimulated T cells specific for HCV. PBMC labeled with CFSE, from two separate patients with HLA-A2 stimulated using related peptide antigen for 6 days in the presence of IL-2 and anti-PD-L1 antibody (upper panel) or anti-PD-1 antibody (lower panel). It is also shown restimulating control. Each quadrant shows the percentage of proliferating CFSE-low and CFSE-highly specific for HCV HLA-A2+ CD8+ T cells.
Fig.27A-27D are diagrams and graphs showing increased expression of PD-1 on CD8 T cells specific for human immunodeficiency virus monkeys (SIV) after infection SIV239. Fig.27A is a diagram showing the th expression of PD-1 on total CD8 T cells from normal macaques. Fig.27B is a graph showing the expression of PD-1 on total and specific for the capsid proteins of SIV CD8 T-cells from macaques infected SIV239. The analysis is performed on PBMC 12 weeks after infection with SIV. Fig.27C is a graph showing the amount of PD-1 positive cells from the total and specific for SIV CD8 T cells of normal and SIV infected macaques. Data for macaques infected with SIV, presents 12 weeks after infection. Fig.27D (last panel) is a graph showing the average total fluorescence intensity (MFI) expression of PD-1 on total and specific for SIV CD8 T cells of normal and SIV infected macaques.
Fig.28A-28B are a diagram and a graph, respectively, showing that blockade of PD-1 in vitro leads to increased expansion of SIV-specific CD8 T cells. PBMC from Mamu A*01 positive macaques that were infected SHIV89.6P, stimulated by the peptide P11C (0.1 ág/ml) in the absence and in the presence of anti-PD-1 blocking Ab (10 μg/ml) for 6 days. After three days of stimulation was added IL-2 (50 units/ml). After stimulation, cells were stained for surface expression of CD3, CD8 and Gag-CM9 tetramer. Estimulando cells (nostim) served as negative controls. Cells were limited window for lymphocytes, based more on scattering than CD3, and analyzed for the expression of CD8 and tetramer. Fig.28A is a typical FASC chart. The numbers on the diagram indicate the frequency of tetramer-positive cells as a percentage of total CD8 T cells. Fig.28B is a graph showing summary data from six macaques. Analyses were performed using cells obtained 12 weeks after infection. The degree of improvement was calculated as the ratio of the frequency of tetramer-positive cells in the P11C-stimulated cultures and in unstimulated cells.
Fig.29 is a series of graphs showing the kinetics of expression of PD-L1, PD-L2 and PD-1 in different cell types after infection with LCMV. Mice were infected with 2×106The BATTLE of the clone 13 (CL-13). The expression of PD-L1, PD-L2 and PD-1 in different cell types is shown in the bar graph at the specified time points after infection. Presents the mean fluorescence intensity (MFI) for the expression of PD-1 on the specified cell type.
Nucleic acid sequences and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids as defined in 37 C. F. R. 1.822. Shows only one chain after which outlinesthe each nucleic acid, but complementary circuit means enabled by any link to the chain. In the accompanying sequence listing:
SEQ ID NO:1 is an example of the amino acid sequence of PD-1 person.
SEQ ID NO:2 represents an example of the amino acid sequence of PD-1 mouse.
SEQ ID NO:3 is an example of the amino acid sequence of PD-L1 human.
SEQ ID NO:4 is an example of the amino acid sequence of PD-L2 man.
SEQ ID NO:5 through 12 are examples of the amino acid sequences of regions of the skeleton of a man.
SEQ ID NO:13-35 are examples of amino acid sequences of antigenic peptides.
SEQ ID NO:36-43 represent amino acid sequences of peptides to major histocompatibility complex.
SEQ ID NO:44 and SEQ ID NO:45 represent the amino acid sequences of the epitopes of T cells.
SEQ ID NO:46 is a examples of amino acid sequence variants of the PD-L2 man.
SEQ ID nos:47-52 are examples of amino acid sequences of antigenic peptides.
This disclosure relates to the use of antagonists PD-1 for the induction of an immune response, such as aimed at the tumor or chronic viral infection.
Unless otherwise noted, technical terms are used in accordance with the generally accepted use. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
To facilitate review of the various embodiments of this disclosure offers the following explanations of specific terms.
The change in the level of production or expression: change by either an increase or decrease of the level of production or expression of nucleic acid sequence or amino acid sequence (e.g., polypeptide, siRNA, microRNA, mRNA, gene) compared with the control level of production or expression.
Antisense, sense molecule and protogen: DNA has two antiparallel chains, 5'→3' chain is denoted as plus chain, and 3'→5' chain is denoted as a negative circuit. As RNA polymerase builds the nucleic acid in the direction of 5'→3' minus strand of DNA serves as the template for RNA in the transcription process. Thus, the RNA transcript must have the sequence complementary to the minus chain and identical plus chain (except for the replacement T U)
Antisense molecules are molecules that specifically hybridize or specifically complementary to any RNA or plus DNA chain. Semantic molecules are molecules that specifically hybridize or specifically complementary to the minus DNA strand. Molecules protogene are either antisense molecules, sense molecule directed to a DNA target. Antisense RNA (ASRC) is a molecule of RNA, complementary to the sense (coding) the nucleic acid molecule.
Amplification: when used in reference to nucleic acid, it denotes the method by which increases the number of copies of nucleic acid molecules in the sample or the sample. An example of amplification is the polymerase chain reaction, in which the biological sample taken from an individual, enter in contact with the pair of oligonucleotide primers under conditions that allow the passage of hybridization of the primers with the matrix nucleic acid in the sample. Primers under certain conditions, extended, dissociate from the matrix and then again parts are annealed, extended and dissociate with amplification of the copy number of nucleic acid. The product of amplification in vitro can be described by means of electrophoresis patterns of the restriction endonucleases, the, hybridization of oligonucleotides or ligating and/or sequencing nucleic acids using standard methods. Other examples of methods of amplification in vitro include amplification with the replacement chain (see U.S. patent No. 5744311); mistranscription isothermal amplification (see U.S. patent No. 6033881); amplification using a chain reaction of reparation (see application WO90/01069); amplification using ligase chain reaction (see patent EP-A-320308); amplification using ligase chain reaction with filling in the gaps (see U.S. patent No. 5427930); amplification using coupled ligase detection and PCR (see U.S. patent No. 6027889); and mistranscription amplification RNA first NASBATM(see U.S. patent No. 6025134).
Antibody: a polypeptide ligand comprising at least the variable region of the light chain or heavy chain immunoglobulin that specifically recognizes and binds an epitope (e.g., antigen, such as a tumor or viral antigen or its fragment). The antibody includes intact immunoglobulins and their variants and the parts are well known in the art, such as Fab' fragments, F(ab)'2fragments, single-chain Fv proteins ("scFv"), and stabilized by a disulfide bond Fv proteins ("dsFv"). Protein scFv is a hybrid protein in which the variable region light chain immunoglobulin and cook Belina region of the heavy chain of an antibody linked by a linker, while in dsFvs circuit subjected to mutation for the introduction of a disulfide bond to stabilize communication circuits. The term also includes genetically engineered forms such as hybrid antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (for example, bespecifically antibodies). Cm. also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rdEd., W. H. Freeman & Co., New York, 1997.
Typically, the immunoglobulin has a heavy and light chain. Each heavy and light chain contains a constant region and a variable area (also known as "domains"). The combination of variable regions of heavy and light chain specifically bind the antigen. Variable region light and heavy chains contain the field 'frame', interrupted by three hypervariable regions, also called "scopes that define complementarity" or "CDR". The length of the field frame and the CDR is defined (see Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, is hereby incorporated into this description by reference). The Kabat database is currently supported in the direct access mode. Area series frame of various light and heavy chains relatively conservative within the species. The area frame antibodies, which is a combined Kark the owls constituent light and heavy chains, used for positioning and alignment of the CDR in three-dimensional space.
CDRs are primarily responsible for binding to the epitope of the antigen. CDRs in each chain are usually referred to as CDR1, CDR2 and CDR3, numbered sequentially, starting with the N-Terminus, and also usually are identified by a circuit in which localizes specific CDR. Thus, CDR3 VHlocalized in the variable domain heavy chain antibodies, in which it occurs, whereas CDR1 VLis a CDR1 of the variable domain of the light chain antibodies, in which it occurs.
References to "VH"or "VH" refer to the variable region of the heavy chain of immunoglobulin, including such as Fv, scFv, dsFv or Fab. References to "VL"or "VL" refer to the variable region of the light chain of immunoglobulin, including such as Fv, scFv, dsFv or Fab.
"Monoclonal antibody" is an antibody produced by a single clone of b-lymphocytes or cell, in which transliterowany the genes for the light and heavy chains of the same antibodies. Monoclonal antibodies are produced using methods known to experts in the art, for example, by creating a hybridoma producing the antibody cells by fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies is A.
"Humanitarianly" immunoglobulin is an immunoglobulin, comprising the area frame from a person and one or more CDRs of an immunoglobulin is not from the person (animal, such as mouse, rat, or synthetic). Immunoglobulin is not from the person supplying the CDR is called the "donor" and the human immunoglobulin supplying frame is called the "acceptor". In one embodiment, in humanitariannet all immunoglobulin CDRs are derived from the immunoglobulin-donor. The presence of the constant regions is not required, but if present, they should be essentially identical to the constant regions of a human immunoglobulin, i.e., identical, at least about 85-90%, about 95% or more. Therefore, all parts gumanitarnogo immunoglobulin except, maybe, CDR, are essentially identical to the corresponding parts of natural sequences of human immunoglobulin. "Humanitariannet antibody" is an antibody comprising humanitarianlaw light chain and humanitarian heavy chain immunoglobulin. Humanitariannet antibody binds to the same antigen as the antibody-donor, which provides the CDR. Frame acceptor gumanitarnogo immunoglobulin or antibody may be a limited number of substitutions aminoxy the lot, taken from the frame of the donor. Humanized or other monoclonal antibodies may have additional conservative substitutions of amino acids, which are essentially no effect on antigen binding or other immunoglobulin. Humanized antibodies can be generated using methods of genetic engineering (for example, see U.S. patent No. 5585089).
"Neutralizing antibody" is an antibody that prevents any biological activity of the polypeptide such as a polypeptide PD-1. For example, a neutralizing antibody may interfere with the ability of the polypeptide PD-1 to reduce the immune response, such as cytotoxicity of T cells. In some examples, a neutralizing antibody may reduce the ability of the polypeptide PD-1 to reduce the immune response by approximately 50%, approximately 70%, approximately 90% or more. To assess the potentially neutralizing antibodies can be used by any standard method of measuring immune responses, including those described in this document.
Antigen: a compound, composition or substance that can stimulate the production of antibodies or T-cell response in an animal, including compositions that are injected animal or imbibed from him. The antigen interacts with the products of specific humoral or cellular component of the immune is istemi, including those induced by heterologous immunogenum. The term "antigen" includes all related antigenic epitopes. "Epitope" or "antigenic determinant" refers to a site on the antigen, which is responsible for the In - and/or T-cells. In one embodiment, T cells are responsible for the epitope, when the epitope prezentuetsya in connection with the MHC molecule. Epitopes can be formed or from neighboring amino acids, or amino acids that are not neighbors, staying close in the tertiary protein folding. Epitopes formed from neighboring amino acids, usually stored under exposure to denaturing solvents, whereas epitopes formed by tertiary packing, is usually lost in the processing of denaturing solvents. An epitope typically includes at least 3, and more usually at least 5, about 9 or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.
The antigen may be tissue-specific antigen, or the antigen-specific diseases. These terms are not exclusive, as tissue-specific antigen may be an antigen that is specific for the disease. Timespecific the ski antigen is expressed in a limited number of tissues, such as one fabric. Specific non-limiting examples of tissue-specific antigen are prostatespecific the antigen specific antigen of the uterus and/or specific antigen testes. Tissue-specific antigen can be expressed in more than one tissue, such as, but not limited to, an antigen that is expressed in more than one reproductive tissues, such as prostate tissue, and the tissue of the uterus. Antigen-specific disease, is waiting on the development of the disease. Specific non-limiting example of an antigen specific for the disease, is an antigen whose expression correlates with the formation of tumors or expects it. Antigen-specific disease, may be the antigen recognized by T-cells or b-cells.
Antigen presenting cell (APC): a cell that can present antigen associated with MHC molecules of class I or class II to T cells. APC include, but are not limited to, monocytes, macrophages, dendritic cells, b cells, T cells and Langerhans cells. T-cell, which represents the other antigen to T cells (including CD4+ and/or CD8+ T cells), is an antigen presenting T-cell (T-APC).
Associated or stably associated (oligonucleotide):oligonucleotide binds or stably binds to kleinova acid-target if a sufficient amount of the oligonucleotide forms a base pair or hybridizes with its nucleic acid target, so that you can determine this binding. Binding may be determined using either the physical or functional properties of the target:oligonucleotide complex. The binding between the target and the oligonucleotide may be determined using any method known to a person skilled in the technical field, including both functional methods, and evaluation methods of physical binding. For example, binding may be defined functionally by determining whether the binding of a detectable effect on the process of biosynthesis, such as gene expression, DNA replication, transcription, translation and the like.
Physical methods of determining binding of complementary strands of DNA or RNA are well known in the art and include methods such as the methods Gnkazy I or chemical Footprinting, delays in the gel and affinity cleavage, procedures, Northern blotting, dot-blotting and determination of light absorption. For example, one method that is widely used because of their simplicity and reliability, includes identifying changes in light absorption of a solution containing the oligonucleotide (or similar) and nucleic acid-target, at from 220 to 300 nm, while the temperature is and is slowly growing. If the oligonucleotide probe or similar contacts its target, there is a sudden increase in absorption at a characteristic temperature, because of the oligonucleotide (or similar) and target dissociate from one another or melt.
The linkage between the oligomer and its nucleic acid target is often characterized by the temperature (Tm) at which 50% of the oligomer dissociates from the target. Higher (Tmmeans the presence of a stronger or more stable complex compared to the complex with a lower (Tm).
Cancer or tumor: malignization neoplasma, which has been characteristic of malignancy with loss of differentiation, increased rate of growth, invasion of surrounding tissue with the ability to metastasize. Cancer of the reproductive system is a cancer primarily occurs in reproductive tissues such as the uterus, testes, ovaries, prostate, fallopian tube, or penis. For example, prostate cancer is malignization neoplasm that occurs in or out of the tissue of the prostate, and cancer of the uterus is malignization neoplasm that occurs in or out of the tissue of the uterus and cancer of the testes is malignization neoplasm that occurs in the testes. Residual cancer is a cancer that remains with the individual after I the th form of treatment, received by the individual for the reduction or destruction of thyroid cancer. Metastatic cancer is a cancer in one or more places of the body other than the place of origin of the original (primary) cancer which is metastatic cancer.
Chemotherapy; chemotherapy agents: when used in the present description any chemical agent with therapeutic suitability for the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasm and cancer, and diseases characterized by hyperplastic growth, such as psoriasis. In one embodiment, the chemotherapeutic agent is an agent for use in the treatment of neoplasm, such as a solid tumor. In one embodiment, the chemotherapeutic agent is a radioactive molecule. Specialist in the art can easily identify chemotherapeutic agent for use (for example, see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison''s Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nded., © 2000 Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer DS, Knobf MF, Durivage HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Immunogenic polypeptides disclosed in the present description of the research Institute, can be used in combination with an additional chemotherapeutic agents.
Reference level: the level of a molecule such as a polypeptide or nucleic acid, usually found in nature under certain conditions and/or for a specific genetic background. In certain embodiments, the control level of a molecule can be measured in a cell or in a sample that was not exposed, either directly or indirectly, processing. In some examples, the reference level may be a level in the cell not exposed to the agent, such as antagonist PD-1. In additional examples, the reference level may be the level of the individual, which was not introduced antagonist PD-1.
DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes, including ribonucleic acid (RNA)). The repeating units in the polymers of DNA are four different nucleotides, each of which includes one of the four bases, adenine, guanine, cytosine and thymine, are associated with sugar deoxyribose, is attached to the phosphate group. Triplets of nucleotides called codons) code for each amino acid in the polypeptide or stop signal. Ter is in "codon" is also used for the corresponding (and complementary) sequences of three nucleotides in mRNA, which is transcribed DNA sequence.
Unless otherwise specified, any reference to a DNA molecule is designed to enable reverse complementary DNA molecules. Except when the text of this description requires a single-stranded molecule, DNA molecule, although written with the image of only one chain cover both strands of double-stranded DNA molecule.
To encode:they say that polynucleotide encodes a polypeptide if it is in its natural state or after manipulation using methods well known to experts in the art, can be transcribed and/or broadcasted by obtaining mRNA to polypeptide and/or polypeptide or its fragment. The antisense chain complementary to such nucleic acid, and the coding sequence can be deduced from it.
Expression: the process by which information encoded genes, is converted into the structures present and operating in the cell. Expressed genes include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but not translated into protein (e.g., miRNAs, transfer RNA and ribosomal RNA). Thus, the expression of a target sequence such as a gene or promoter region of the gene can lead to the expression of the RNA, protein or both. The expression of a target sequence can be ingibirovany or strengthened (raised or lowered).
Sequence controlling the expression: nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence to which they are operatively connected. Sequence controlling the expression of the operatively linked nucleic acid sequence, when the sequence controlling the expression, supervise and regulate the transcription and, when it is intended broadcast nucleic acid sequence. Thus, the sequence controlling the expression can include appropriate promoters, enhancers, transcription terminators, the initiating codon (i.e., ATG) in front of the gene coding for protein, splicing signals, the elements to maintain the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "regulatory sequence" is intended to include at least those components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and sequences partners hybridization. The sequence is kontroliruyushchii expression, can include a promoter.
The promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to provide dependent promoter gene expression features controlled-specific cell-type, tissue-specific or inducible by external signals or agents; such elements may be localized in the 5'- or 3'-regions of the gene. Included as constitutive and inducible promoters (see, e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning occurs in bacterial systems, can be used inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (hybrid promoter ptrp-lac) and the like. In one embodiment, when cloning in cellular systems mammals can be used promoters derived from the genome of mammalian cells (such as the promoter metallothionein) or from mammalian viruses (such as the long terminal repeat of retrovirus; late promoter of adenovirus; the 7.5 K promoter of vaccinia virus). To ensure transcription of the nucleic acid sequences can also be used promoters produced by recombinant DNA or synthetic techniques.
Hetero is ulicny: originating from a single genetic source or species. Typically, the antibody that specifically binds to protein of interest, will not be specifically bind to a heterologous protein.
Cell-host cell in which the vector can breed and its DNA expressed. The cell can be prokaryotic or eukaryotic. The cell can be a cell of a mammal, such as human cells. The term also includes any descendants of the underlying host cell. It is clear that all descendants may not be identical to the parent cell, as can occur mutations that occur during replication. However, these descendants are enabled when you use the term "a host cell".
Immune response: the response of cells of the immune system such as b-cell, T-cell or monocyte to the stimulus. In one embodiment, the response is specific against a particular antigen (antigen-specific response"). In one embodiment, the immune response is a T-cell response, such as CD4+ response or CD8+ response. In another embodiment, the response is a b-cell response and leads to the production of specific antibodies.
"No response" in relation to immune cells includes a refractory immune cells to stimulation, such as stimulation via an activating receptor or a cytokine. The lack of response may occur, such as the er, due to exposure to immunosuppressive drugs or due to exposure to high doses of antigen. Used in the present description, the term "anergy" or "tolerance" includes refractory to stimulation, mediated by the activating receptor. This refractoriness is usually antigen-specific and continues after cessation of exposure cholerasuis antigen. For example, anergy in T cells (as opposed to lack of response) is characterized by the absence of production of cytokines (such as IL-2). Anergy of T cells occurs when T cells are exposed to antigen and receive the first signal (signal, mediated by the receptor of T cells or CD-3) in the absence of a second signal (co-stimulating signal). Under these conditions, repeated exposure of cells with the same antigen (even if the exposure is carried out in the presence of co-stimulating molecules) leads to failure of production of cytokines and, thus, the inability to proliferate. T cells in a state of anergy can, however, generate a response to unrelated antigens and can proliferate when cultured with cytokines (such as IL-2). For example, T cells in a state of anergy can also be identified by the absence of production of IL-2 by T lymphocytes when measured using ELISA or test on the proliferation of using indicator cleocin the second line. An alternative can be used reporter gene construct. For example, T cells in a state of anergy is not able to initiate transcription of the gene IL-2, inducible heterologous promoter under the control of the 5'-enhancer gene IL-2 or multimeric sequence AP1, which can be found in the enhancer (Kang et al. Science 257:1134, 1992). Antigen-specific T cells in a state of anergy may have reduced the cytotoxic activity of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even 100% compared with the corresponding control antigen-specific T-cell.
Immunogenic peptide: a peptide which comprises an allele-specific motif or other sequence, so that the peptide will bind to MHC molecule and induce a cytotoxic response of T-lymphocytes ("CTL") response cells (e.g., production of antibodies against the antigen from which occurred immunogenic peptide.
In one embodiment, the immunogenic peptides identified using sequence motifs or other methods, such as using a computer neural network or polynomial analysis known in the art. Typically, the algorithms to determine the "threshold bind peptides used to select peptides with glasses that give them a high probability of binding to set the authorized affinity and to be immunogenic. The algorithms are based either on the effects of binding MHC specific amino acid at a certain position, the effects of binding of the antibody specific amino acid at a certain position, or on the effects of binding of specific substitutions in the peptide containing the motif. In the context of the immunogenic peptide "conservative balance represents the balance that appears with significantly higher frequency than is to be expected in a randomized distribution in a certain position in the peptide. In one embodiment, the conservative residue is a by which the structure of the MHC can provide a point of contact with the immunogenic peptide.
Immunogenic peptides can also be identified by measuring their binding with specific MHC protein (e.g., HLA-A02.01) and by their ability to stimulate CD4 and/or CD8 when the presentation is in the context of the MHC protein.
Immunogenic composition: a composition comprising the immunogenic polypeptide or nucleic acid encoding the immunogenic polypeptide that induces a measurable CTL response against cells expressing the polypeptide, or induces a measurable response In cells (such as the production of antibodies that specifically bind the polypeptide) against the polypeptide. When using in vitro immunogenic composition m which may consist of a selected nucleic acid, vector that includes a nucleic acid, or immunogenic peptide. When used in vivo immunogenic composition should usually include nucleic acid vector comprising nucleic acid, and/or immunogenic polypeptide in a pharmaceutically acceptable carrier, and/or other agents. The immunogenic composition may optionally include adjuvant, antagonist PD-1, co-stimulating molecule or nucleic acid encoding a co-stimulating molecule. The polypeptide or nucleic acid encoding the polypeptide, can be readily tested for their ability to induce CTL using methods known in the art.
The inhibition or treatment of a disease: inhibiting the disease, such as tumor growth or persistent infection, refers to inhibiting the full development of the disease or reducing the physiological effects of the disease in the development process. In some examples, the inhibition or treatment of a disease refers to the reduction of symptoms of a tumor or infection by a pathogen. For example, cancer treatment can prevent the development of paraneoplastic syndrome in an individual, for which it is known that he has cancer, or to cause a decrease of the characteristic or symptom of cancer. In another embodiment, treatment of the infection may relate to the inhibition of development is whether the reduction of a symptom of infection. "Treatment" refers to therapeutic intervention that facilitates a sign or symptom of a disease or pathological condition related to the disease. Therapeutic vaccination refers to the introduction of the agent to an individual already infected with the pathogen. The individual may have no symptoms, so treatment prevents the development of the symptom. Therapeutic vaccines may also reduce the severity of one or more existing symptoms or to reduce the pathogen load.
Infectious disease: any disease caused by an infectious agent. Examples of infectious agents include, but are not limited to, viruses, bacteria, Mycoplasma and fungi. In the specific example is a disease caused by at least one type of infectious pathogen. In another example, the disease caused by at least two different types of infectious pathogens. Infectious diseases can affect any system of the body, can be acute (short-range) or chronic/persistent (long-term), can occur with or without fever can affect any age group and can overlap each other.
Viral infections usually occur after immunosuppression due to reactivation of viruses already present in the recipient. Specific examples of sustained viral infections including the Ute, but not limited to, cytomegalovirus (CMV) pneumonia, enteritis and retinitis; lymphoproliferative disease caused by Epstein-Barr (EBV); chickenpox/shingles (caused by varicella zoster virus, VZV); mycotic called NSV-1 and -2; encephalitis caused by HSV-6; hemorrhagic cystitis caused by BK virus; viral influenza; pneumonia caused by respiratory syncytial virus (RSV); AIDS (caused by HIV) and hepatitis A, B or C.
Additional examples of infectious virus include: Retroviridae; Picornaviridae (for example, poliovirus, hepatitis A virus; enteroviruses, Coxsackie virus human rhinoviruses, ECHO viruses); Calciviridae (such as strains that cause gastroenteritis); Togaviridae (for example, viruses equine encephalitis, rubella viruses); Flaviridae (for example, viruses, Dengue viruses, encephalitis viruses, yellow fever); Coronaviridae (for example, coronaviruses); Rhabdoviridae (for example, viruses, vesicular stomatitis, rabies viruses); Filoviridae (for example, viruses, Ebola); Paramyxoviridae (for example, parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus), Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (for example, viruses Hantaan, mangaverse, phlebovirus and nairovirus); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orbivirus and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviride (parvoviruses); Papovaviridae (papilloma viruses, viruses polyoma); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses); from the poxviridae (the virus of smallpox, cowpox viruses, poxviruses); and Iridoviridae (such as fever virus African swine); and unclassified viruses (e.g. the etiological agents of spongiform encephalopathies, the agent of Delta hepatitis (assumed to be, which is defective satellite of hepatitis B virus), the agents of hepatitis non-A, not-B (class 1=transmitted by internal; class 2=parenterally transmitted (i.e. hepatitis C); a virus Norwalk and related viruses, and astroviruses).
Examples of fungal infections include, but are not limited to: aspergillosis; thrush (caused by Candida albicans); cryptococcosis (caused by Cryptococcus and histoplasmosis. Thus, examples of infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.
Examples of infectious bacteria include: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (such as M. tuberculosis, M. avium, M. intracellular, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (group A Streptococcus), Streptococcus agalactiae (group B Streptococcus), Streptococcus (Viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogen the Yu Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira and Actinomyces israelli. Other infectious organisms (such as protozoa) include: Plasmodium falciparum and Toxoplasma gondii.
"Persistent infection is an infection in which an infectious agent (such as a virus, Mycoplasma, bacterium, parasite or fungus) is not displayed or is not eliminated from an infected host even after the induction of the immune response. Resistant infections can be a chronic infection, latent infection, or slow infection. Latent infection is characterized by the absence of demonstrable presence of infectious virus between episodes of recurrent disease. Chronic infection is characterized by a prolonged presence of infectious virus after primary infection and may include chronic or relapsing disease. Slow infection is characterized by a long incubation period with subsequent disease progression. Unlike latent and chronic infections slow the infection may not begin with the acute period of the propagation of the virus. While acute infections are relatively short (still is I from several days to several weeks) and viruses are eliminated from the body through the immune system, resistant infections may last, for example, in the months, years or even for life. These infections often recur over a long period of time, including the stage of silent and productive infection, without destruction of the cells or even with the induction of excessive damage to the host cells. Resistant infections often include stage as silent and productive infection without rapid destruction or even with the induction of excessive damage to the host cells. During sustained viral infections viral genome can be either stably integrated into the cellular DNA, or may be supported episomal. Persistent infection occurs during infection with these viruses, as viruses T-cell leukemia, Epstein-Barr, cytomegalovirus, herpes viruses, varicella zoster virus, measles virus, papovavirus, prions, hepatitis viruses, adenoviruses, parvoviruses and papilloma viruses.
Causing disease infectious agents (such as existing within specific cells of infected individuals) can also be defined at the host using standard methods even after resolution of the immune response. Mammal diagnosed as having a resistant infection in accordance with any standard method known in the art and described, for example, the U.S. patents No. 6368832, 6579854 and 6808710 and in publications of patent applications U.S.№№ 20040137577, 20030232323, 20030166531, 20030064380, 20030044768, 20030039653, 20020164600, 20020160000, 20020110836, 20020107363 and 20020106730, all included in the present description by reference.
"The relief of the symptom resistant infections" is the relief of any condition or symptom that is associated with persistent infection. Alternative relief of symptom resistant infections may include reduction of infectious microbial (such as viral, bacterial, fungal or parasitic) load the individual in relation to such load control, untreated. Compared with the equivalent control, untreated, such reduction or the degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% when measured using any standard method. It is desirable that persistent infection completely disappeared when defining any standard method known in the art, in this case, persistent infection is considered as cured. A patient who undergoes treatment of resistant infections, is such, has a doctor diagnosed this condition. The diagnosis can be established using any suitable means. Diagnosis and monitoring may include, for example, determining the level of microbial loads the key in a biological sample (for example, in biopsy material fabric, determination in blood or determination in the urine), determining the level of a surrogate marker of microbial infection in a biological sample, the definition of the symptoms associated with resistant infections, or determination of immune cells involved in the immune response typical of resistant infections (e.g., determination of antigen-specific T cells, which are in a state of anergy and/or functionally impaired). The patient, which prevents the development of resistant infections may be delivered or not delivered such a diagnosis. Specialist in the art should understand that these patients may be subjected to the same standardized tests as described above, or can be identified without testing, as having a high risk due to the presence of one or more risk factors (such as family history or exposure to an infective agent).
Selected: "isolated" biological component (such as a nucleic acid or protein or organelle) essentially separated or purified away from other biological components in the cell of the organism in which the component is in nature, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins, which are "isolated" include nucleic acids and proteins, is shelled by using standard cleaning methods. The term also embraces nucleic acids and proteins obtained by recombinant expression in a cell host, as well as chemically synthesized nucleic acids.
"Purified antibody" is at least 60% by weight of the proteins and naturally occurring organic molecules with which it is associated in nature. In some examples, the drug contains the antibody, such as a specific antibody against PD-1, PD-L1 or PD-L2, in the amount of at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 99% by weight. The purified antibody can be obtained, for example, by using affinity chromatography using recombinante produced protein or conservative motifs of peptide and standard methods.
Tagged: the designated compound or composition that is conjugated directly or indirectly to another molecule, to facilitate the determination of this molecule. Specific, non-limiting examples of labels include fluorescent labels, conjugates with enzymes and radioactive isotopes.
Lymphocytes: a type of white blood cell involved in the body's immune defenses. There are two main types of lymphocytes: b cells and T cells.
The major histocompatibility complex (MHC): group symbol, p is odnaznachno for coverage systems histocompatibility antigens described in different species, including leukocyte antigens ("HLA").
Mammal: this term includes both human and mammals, non-human. Similar, the term "individual" includes both human and veterinary medicine.
Neoplasia: abnormal cellular proliferation, which includes benign and malignant tumors and other proliferative disorders.
Oligonucleotide: a linear polynucleotide sequence of up to about 100 nucleotide bases in length.
Open reading frame (ORF): a sequence of nucleotide triplets (codons) encoding amino acids without any internal termination codons. These sequences are usually able to broadcast the peptide.
Operatively associated: the first nucleic acid sequence operatively linked to the second nucleic acid sequence when the first nucleic acid sequence is in functional communication with the second sequence of nucleic acid. For example, a promoter operatively linked to the coding sequence if the promoter affects the transcription or expression of the coding sequence. Typically operatively linked DNA sequences are contiguous and, where necessary to join d the e region, translated into protein in the same reading frame.
Pharmaceutically acceptable carriers: used pharmaceutically acceptable carriers are generally accepted. In Remington''s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery disclosed in the present description of the hybrid proteins.
In General, the nature of the medium must depend on a particular method of administration. For example, parenteral formulations usually consist of fluid, which include as a carrier pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol or the like. For solid compositions (such as powder, polylina, tablet or capsule form) conventional non-toxic solid carriers can include, for example, mannitol, lactose, starch, or magnesium stearate and pharmaceutical purity. In addition to biologically-neutral carriers, pharmaceutical compositions intended for insertion, can contain minor amounts of nontoxic auxiliary substances, such as moisturizing or emulsifying agents, preservatives and buferiruemoi pH agents, and the like, for example sodium acetate or arbitrageurs.
"Terap wtiches effective amount" is an amount of the composition or cells to achieve the desired effect in an individual, being treated. For example, this may be the number of antagonist PD-1, is required for induction of the immune response, inhibition of tumor growth or measured changes in the external symptoms of a tumor or resistant infections. With the introduction of the individual usually must use dosage, which in the target tissue (e.g., lymphocytes) to achieve concentrations, which, as shown, to cause the effect in vitro.
Polynucleotide: the term polynucleotide or nucleic acid sequence refers to a polymeric form of nucleotides of at least 10 bases in length. Recombinant polynucleotide includes polynucleotide, which are not directly adjacent to both coding sequences with which it is directly connected (one on the 5'end and one on the 3'-end) in the natural genome of the organism from which it comes. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an offline can replicate a plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., cDNA), independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of any of the nucleotide. The term includes single - and double-stranded forms of DNA.
Polypeptide: any chain of amino acids, regardless of length or post-translational modifications (e.g. glycosylation or phosphorylation). The polypeptide can be from 3 to 30 amino acids in length. In one embodiment, the polypeptide is from about 7 to about 25 amino acids in length. In yet another embodiment, the polypeptide is from about 8 to about 10 amino acids in length. In yet another embodiment, the peptide is 9 amino acids in length. In relation to polypeptides "includes" indicates that the molecule can be supplemented with additional amino acid sequence or other molecules, "consists essentially of" indicates that the molecule does not include the additional amino acid sequence, but may include other agents (such as labels or chemical compounds), and "is" indicates that the molecule does not include the additional amino acid sequence and additional agents.
Protein programmed death (PD)-1: a protein that forms a complex with the protein PD-L1 or PD-L2 and involved in immune response, such as costimulate T cells. Usually the protein PD-1 is essentially identical to natural PD-1 (wild type) (see, e.g., Ishida et al. EMBO J. 11:3887-3895, 1992, Shinohara et al. Genomics 23:704-706, 1994; and U.S. Pat the t U.S. No. 5698520, all included in the present description by reference in full). In some examples, when the signal PD-1 is reduced, for example, the cytotoxicity of CD8+ T cells as a result of reduced proliferation of T cells, cytokine production or clearance of the virus. Thus, the polypeptide PD-1 can reduce the cytotoxic activity of CD8+ T cells at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 100% compared to control levels when measured using any standard method.
Used in the present description, the term "activity" in regard to the polypeptide or protein PD-1 includes any activity that is inherent to natural protein PD-1, such as the ability to modulate the inhibitory signal in an activated immune cell in such a way, as by modulation of the probing nature of the ligand with the antigen presenting cell. Such modulation of the inhibitory signal in an immune cell leads to modulation of cell proliferation and/or survival of immune cells and/or secretion of cytokines by the immune cell. Protein PD-1 can also modulate co-stimulatory signal by competition with co-stimulatory receptor for binding of B7 molecules. Thus, the term "activity of PD-1" includes the ability of the polypeptide or protein PD-1 to contact their(MIS) natural(mi) ligand(s), the ability to modulate bone is ulatory or inhibitory signals to immune cells and the ability to modulate the immune response.
"Decrease in the expression or activity of PD-1" refers to the reduction of the level or biological activity of PD-1 with respect to the level or biological activity of the protein PD-1 in the control, such as not being treated with the individual or the raw sample. In specific examples, the level or activity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or even above 100% compared to the untreated control. For example, the biological activity of the protein PD-1 is reduced by decreasing the binding of the protein PD-1 with PD-L1, PD-L2, or both, thereby causing a decrease alarm PD-1 and, consequently, leading to an increase in the cytotoxicity of CD8+ T cells.
"Gene PD-1" is a nucleic acid that encodes a protein PD-1. "Hybrid gene PD-1" represents the coding region of PD-1, operatively linked to a second heterologous sequence nucleic acids. Hybrid gene PD-1 may include a promoter PD-1 or may include a heterologous promoter. In some embodiments, the second implementation the heterologous nucleic acid sequence is a reporter gene, i.e. a gene whose expression can be determined; reporter genes include, without limitation, genes encoding glucuronidase (GUS), luciferase, chloramphenicolchloramphenicol (CAT), green fluorescent protein (GFB), alkaline FOSFA the pelvis and beta-galactosidase.
The specific binding agent: an agent that binds essentially only with a specific target. Thus, the agent that specifically bind to PD-1, is an agent that specifically binds to a polypeptide PD-1, but not with unrelated polypeptides. In one embodiment, the specific binding agent is a monoclonal or polyclonal antibody that specifically binds to a polypeptide PD-1, PD-L1 or PD-L2.
The term "specifically binds" means in respect of an antigen, such as PD-1, the preferred Association of the antibody or other ligand, in whole or in part, cell or tissue bearing that antigen, but not with cells or tissues in which the antigen is absent. Of course, it is recognized that there may be a certain degree of non-specific interaction between the molecule and the cell or tissue, which is not a target. Nevertheless, specific binding can be defined as mediated by specific recognition of the antigen. Although selectively interacting antibodies bind the antigen, they can do so with low affinity. Specific binding leads to a much stronger Association between the antibody (or other ligand) and cells bearing the antigen, compared with the Association between the antibody (and the other ligand) and cells not bearing the antigen. Specific binding typically results in more than 2-fold, such as greater than 5-fold, more than 10-fold, or more than 100-fold, increase in the amount of bound antibody or other ligand (per unit time) to a cell or tissue bearing the polypeptide PD-1, compared to a cell or tissue with the absence of the polypeptide. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of formats immunotest suitable for selection of antibodies or other ligands that are specifically immunoreactive for a specific protein. For example, solid-phase immunotest ELISA are typically used to select monoclonal antibodies specifically immunoreactive protein. Cm. Harlow &Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988) for a description of the formats immunotest and conditions that can be used to determine specific immunoreactivity.
T-cell: a white blood cell that are required for the immune response. T-cells include, but are not limited to, CD4+T-cells and CD8+T-cells. CD4+T-lymphocyte is an immune cell, which on its surface is a marker, known as a "cluster of differentiation 4" (CD4). These cells, also known as helper T-cells help the organization shall izbyvat immune response, including responses antibody responses, T-killer cells. CD8+T-cells are a marker cluster of differentiation 8" (CD8). In one embodiment, CD8+ T-cell is a cytotoxic lymphocyte. In another embodiment, CD8+ cell is a suppressor T-cell. T-cells are "activated" when it can respond to a specific antigen of interest, presented on antigen presenting cells.
Translationa/transfusiona: translationa cell is a cell into which the introduced nucleic acid molecule using the methods of molecular biology. Used in the present description, the term transduction covers all the ways in which the nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation, plasmid vectors, and introduction deproteinizing DNA using electroporation, lipofection and bombardment by particles accelerated.
Vector: a nucleic acid molecule, which upon introduction into a cell-master creates thereby transformed cell host. The vector can include a nucleic acid sequence that allow it to replicate in the cell host, such as origin of replication. The vector may also include one or is more nucleic acids, coding of breeding marker and other genetic elements known in the art. Vectors include plasmid vectors, including plasmids for expression in gram-negative and gram-positive bacterial cells. Examples of vectors include vectors for expression in E. coli and Salmonella. Vectors include viral vectors, such as, but not limited to, retroviral, orthopoxvirus, avipoxviruses, fowlpoxvirus, capripoxvirus, simoxmorocco, adenoviral vectors, vectors based on herpes virus, alpha virus, baculovirus, virus Sindbis, cowpox virus and poliovirus.
If not explained otherwise, all technical and scientific terms used in this description have the same meaning as commonly understood by a specialist in the field of technology, belongs to this disclosure. The terms of the singular include references to the plural, unless the context clearly indicates otherwise. Similar, the word "or" is intended to include "and", unless the context clearly indicates otherwise. Additionally, it should be clear that all sizes of bases or dimensions of amino acids and all values of molecular weight or molecular weight data for nucleic acids or polypeptides are approximate and are provided for description. Although the practice is or inspection of this disclosure can be used in methods and materials, similar or equivalent to those described herein, suitable methods and materials are described below. The term "comprises" means "includes". All publications, patent applications, patents, and other references mentioned in the present description, are included in full as references. In the event of a conflict applicants will be guided by the present description, including an explanation of terms. In addition, the materials, methods and examples are illustrative only and are not considered limiting.
The methods disclosed in the present description, include the use of inhibitors path PD-1 antagonists PD-1). Molecules PD-1 are members of the superfamily of immunoglobulin genes. PD-1 person possesses an extracellular region containing domain of the immunoglobulin superfamily, a transmembrane domain and an intracellular region that includes immunoreceptor inhibitory motif-based tyrosine (ITIM) (Ishida et al., EMBO J. 11:3887, 1992; Shinohara et al., Genomics 23:704, 1994; U.S. patent No. 5698520). These characteristics are also typical of a large family of molecules, called immunoinhibitory receptors, which also includes gp49B, PIR-B and receptors, inhibiting killers (KIR) (Vivier and Daeron (1997) Immunol. Today 18:286). Without being bound to theory, it is believed that phosphorylated on tyrosinosis residues of the motif ITIM these receptor is in interacts with phosphatase, containing S112 (domain, which leads to inhibitory signals. A subclass of these immunoinhibitory receptor binds to molecules of the major histocompatibility complex (MHC), KIR and CTLA4 associated with B7-1 and B7-2.
Human PD-1 is a 50-55 kDa transmembrane receptor type I, which was originally identified in a line of T-cells subjected to induced activation of apoptosis. PD-1 is expressed on T-cells, B-cells and macrophages. Ligands of PD-1 are members of the B7 family - PD-ligand 1 (PD-L1, also known as B7-H1) and PD-L2 (also known as B7-DC).
In vivo PD-1 is expressed on activated T-cells, B-cells and monocytes. Experimental data bind interaction of PD-1 with its ligands with negative regulation of Central and peripheral immune response. In particular, the proliferation of T-cells of the wild type, but not T-cell deficiency PD-1, inhibited in the presence of PD-L1. In addition, mice with deficiency of PD-1 are autoimmune phenotype.
Example amino acid sequence of PD-1 person listed below (see also Ishida et al., EMBO J. 11:3887, 1992; Shinohara et al. Genomics 23:704, 1994; U.S. patent No. 5698520):
Example amino acid sequence of PD-1 mouse is presented below:
Additional amino acid sequence is lnasty disclosed in U.S. patent No. 6808710 and publications of patent applications U.S. No. 2004/0137577, 2003/0232323, 2003/0166531, 2003/0064380, 2003/0044768, 2003/0039653, 2002/0164600, 2002/0160000, 2002/0110836, 2002/0107363 and 2002/0106730, which is incorporated into this description by reference. PD-1 is a member of the superfamily of immunoglobulins (Ig), which contains a single Ig V-like domain in its extracellular region. The cytoplasmic domain of PD-1 contains two tyrosine from the most proximal to the membrane tyrosine (VAYEEL (see amino acids 223-228 SEQ ID NO:2) PD-1 mouse), localized within ITIM (immunoreceptor inhibitory motif-based tyrosine). The presence of an ITIM in PD-1 indicates that the function of this molecule is weakening alarm antigen-receptor with the involvement of cytoplasmic phosphatases. Protein PD-1 human and mouse are characterized by approximately 60% amino acid identity with the four conservative sites of potential N-glycosylation sites and residues that define the domain of the Ig-V ITIM in the cytoplasmic area and ITIM-like motif surrounding carboxykinase tyrosine (TEYATI (see amino acids 166-181 SEQ ID NO:2) of human and mouse, respectively), are also conservative orthologues in mouse and man.
PD-1 by its ability to bind PD-L1 belongs to the family of molecules CD28/CTLA-4. In vivo, as CTLA4, PD-1 is rapidly induced on the surface of T cells in response to anti-CD3 (Agata et al. Int. Immunol. 8:765, 1996). However, unlike CLA4 PD-1 also induced on the surface of b-cells in response to anti-IgM). PD-1 is also expressed on subpopulations of thymocytes and myeloid cells (Agata et al. (1996) above; Nishimura et al. (1996) Int. Immunol. 8:773).
Anergy of T cells is accompanied by induction of expression of PD-1. In the present description revealed that the cytotoxicity of T cells can be increased by injection into contact with T-cells with an agent that reduces the expression or activity of PD-1. More specifically, in the present description disclosed that the agent that reduces the expression or activity of PD-1, can be used to enhance the immune response, such as a viral antigen or a tumor antigen.
Without being bound to theory, the decrease in the expression or activity of PD-1 leads to increased activity of cytotoxic T-cells, thus increasing the specific immune response to an infectious agent. In order T cells responded to foreign proteins, antigenpresenting cells (APC) must submit two signals by a stationary T-lymphocytes. The first signal, which gives the specificity of the immune response, is transmitted through the receptor of T cells (TCR) after recognition of foreign antigenic peptide presented in the context of major histocompatibility complex (MHC). The second signal, called co-stimulating, induces proliferation of T-cells and the formation of their functionality. Costimulate is neither antigen-specific nor limited HC and is provided with one or more specific polypeptides on the cell surface, expressed by the APC. If T cells are stimulated only through T-cell receptor without additional co-stimulating signal, they are not reacting, moving into a state of anergy or die, which leads to negative modulation of the immune response.
Proteins CD80 (B7-1) and CD86 (B7-2) expressed on APC, are necessary co-stimulatory polypeptides. Whereas B7-2 plays a dominant role in the primary immune responses, B7-1 is positively regulated later in the immune response to prolonged primary responses of T cells or secondary co-stimulatory responses of T cells. The B7 polypeptides capable of providing immune cell co-stimulatory or inhibitory signals for the stimulation or inhibition of the responses of immune cells. For example, in the state of binding of co-stimulatory receptor PD-L1 (B7-4) induces costimulatory immune cells or, when present in soluble form inhibits costimulation immune cells. In the state of binding inhibitory receptor molecules PD-L1 can transfer immune cell inhibitory signal. Examples of members of the B7 family include B7-1, B7-2, B7-3 (recognizable by the antibody BB-1), B7h (PD-L1) and B7-4, and their soluble fragments or derivatives. Members of the B7 family are associated with one or more receptors on immune cell, such as CTL4, CD28, ICOS, PD-1 and/or other receptors, and depending on the receptor have the ability to transmit inhibitory signals or co-stimulatory signal to the immune cell.
CD28 is a receptor that is constitutively expressed on resting T-cells. After signaling through the T-cell receptor binding to CD28 and transmission co-stimulatory signal to induce the proliferation of T cells and the secretion of IL-2. CTLA4 (CD152), a receptor that is homologous to CD28, absent on resting T-cells, but its expression is induced after activation of T cells. CTLA4 plays a role in negative regulation of T-cell responses. ICOS, polypeptide, related to CD28 and CTLA4 involved in the production of IL-10. PD-1, the receptor is bound to PD-L1 and PD-L2, is also rapidly induced on the surface of T cells. PD-1 is also expressed on the surface of b-cells in response to anti-IgM) and subpopulations of thymocytes and myeloid cells.
The engagement of PD-1 (e.g., via cross-linking or aggregation) leads to the transfer of inhibitory signal in an immune cell, resulting in reduced immune responses, accompanied by the growth of anergy of the immune cells. The members of the family of PD-1 are associated with one or more receptors, such as PD-L1 and PD-L2 on antigen presenting cells. PD-L1 and PD-L2, both of which are polypeptide ligands of PD-1 person, are members of th is VA B7 polypeptides (see above). Each ligand PD-1 contains a signal sequence, domain, IgV, IgC domain, a transmembrane domain and a short cytoplasmic tail. In vivo these ligands, as shown, is expressed in placenta, spleen, lymph nodes, thymus and heart. PD-L2 is expressed also in the pancreas, lungs and liver, whereas PD-L1 is expressed in the liver of fetuses, activated T-cells and endothelial cells. Both ligand PD-1 is positively regulated by activated monocytes and dendritic cells.
Example amino acid sequence of PD-L1 (GENBANK®, no Deposit AAG18508 available from 4 October 2000) is presented below:
An example of the amino acid sequence of the precursor of PD-L2 (GENBANK®, no Deposit AAK153370 available from 8 April 2002) are presented below:
An example of a variant amino acid sequence of the precursor of PD-L2 (GENBANK®, no Deposit Q9BQ51 available from 12 December 2006) is presented below:
Antagonists PD-1 include agents that reduce the expression or activity of PD ligand 1 (PD-L1) or ligand 2 PD (PD-L2) or reduce the interaction between PD-1 and PD-L1 or the interaction between PD-1 and PD-L2. Examples of the compounds include an antibody (such as antibody against PD-1, an antibody against PD-L1 and antibody against PD-L2), forefront of the crystals of Rnci (such as molecules of Rnci anti-PD-1, Rnci anti-PD-L1 and Rnci anti-PD-L2), antisense molecules (such as antisense RNA, an anti-PD-1, antisense RNA, an anti-PD-L1 and antisense RNA, an anti-PD-L2), dominant negative proteins (such as a dominant negative protein PD-1, a dominant negative protein PD-L1 and dominant negative protein PD-L2) and small molecule inhibitors.
Antagonist PD-1 is any agent that has the ability to decrease the expression or activity of PD-1 in the cell. Expression or activity of PD-1 is reduced at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to such expression or activity in control. Examples of lower activity comprise at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or to the complete lack of detectable activity. In one example, the control is a cell that has not been treated with antagonist PD-1. In another example, the control is a standard value or the cell contacted by the agent, such as the media, known as do not have influence on the activity of PD-1. Expression or activity of PD-1 can be determined using any standard method known in this on the region of the equipment, including those described herein. Optional antagonist PD-1 inhibits or reduces the binding of PD-1 with PD-L1, PD-L2, or both.
Antibodies that specifically bind PD-1, PD-L1 or PD-L2 (or their combination) are used in disclosed in the present description methods. Antibodies include monoclonal antibodies, humanized antibodies, deimension antibodies and hybrid proteins-immunoglobulins (Ig). Polyclonal antibodies anti-PD-1, anti-PD-L1 or anti-PD-L2 can be obtained by a person skilled in the technical field, for example, by immunization of a suitable individual (such as an object veterinary) ligand PD-1 or PD-1 immunogen. The titer of anti-PD-1, anti-PD-L1 or anti-PD-L2 in immunized individuals can be tracked over time using standard methods, such as enzyme-linked immunosorbent assay (ELISA) using immobilized ligand or PD-1 polypeptide PD-1.
In one example, the molecules are antibodies that specifically bind PD-1, PD-L1 or PD-L2 (or their combination) can be isolated from the mammal (e.g., serum) and further purified using methods known to the person skilled in the art. For example, antibodies can be purified using chromatography with protein And for separation of IgG antibodies.
Cells produc is the dominant antibody can be obtained from the individual and used to produce monoclonal antibodies using standard methods (see Kohler and Milstein, Nature 256:495-49, 1995; Brown et al., J. Immunol. 127:539-46, 1981; Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77 96, 1985; Gefter, M. L. et al. (1977) Somatic Cell Genet. 3:231 36; Kenneth, R. H. in Monoclonal Antibodies: A New Dimension In Biological Analyses. Plenum Publishing Corp., New York, N. Y. (1980); Kozbor et al. Immunol. Today 4:72, 1983; Lerner, E. A. (1981) Yale J. Biol. Med. 54:387-402; Yeh et al., Proc. Natl. Acad. Sci. 76:2927-31, 1976). In one example immortalizing cell line (typically a myeloma) is drained to lymphocytes (typically splenocytes) from a mammal immunized with PD-1, PD-L1 or PD-L2, and conduct screening culture supernatants obtained hybridoma cells to identify hybridoma producing a monoclonal antibody that specifically binds with a polypeptide of interest.
In one embodiment, upon receipt of hybridoma immortalitya cell line such as a myeloma cell line) comes from the same mammal species as the lymphocytes. For example, mouse hybridoma can be created by the merger of lymphocytes from mice immunized with peptide PD-1, PD-L1 or PD-L2, with immortalizing cell line mouse. In one example uses the myeloma cell line mouse, which is sensitive to culture medium containing gipoksantin, aminopterin and thymidine ("HAT-environment"). Liu is th number of myeloma cell lines can be used as a partner for the merger in accordance with standard methods, including, for example, myeloma line P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Agl4, which are available from the American type culture collection (ATCC), Rockville, Md. HAT-sensitive myeloma cells from mice can be fused with mouse splenocytes using polyethylene glycol ("PEG"). This is followed by a selection of hybridoma cells resulting from the merger, using the HAT, which destroys naslite (and unproductively fused) myeloma cells. Hybridoma cells producing interesting monoclonal antibody can be determined, for example, through screening supernatants hybridoma cultures for the production of antibodies that bind the molecule PD-1, PD-L1 or PD-L2, for example, using an immunological test such as the enzyme linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)).
Alternatively of obtaining hybrid screening of monoclonal antibodies, monoclonal antibody that specifically binds PD-1, PD-L1 or PD-L2 can be identified and selected by screening a recombinant combinatorial library of the immunoglobulin (such as a library of antibodies in the format of phage display) with PD-1, PD-L1 or PD-L2 for selection of members of a library of antibodies, which specifically bind the polypeptide. Kits for generating and screening libraries of phage display are rodaje (for example, but not limited to, Pharmacia and Stratagene). Examples of methods and reagents particularly suitable for use when creating and screening libraries of phage display can be found, for example, in U.S. patent No. 5223409; PCT publication no WO 90/02809; PCT publication no WO 91/17271; PCT publication no WO 92/18619; PCT publication WO 92/20791; PCT publication no WO 92/15679; PCT publication no WO 92/01047; PCT publication WO 93/01288; PCT publication no WO 92/09690; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; Hoogenboom et al., Nucleic Acids Res. 19:4133-4137, 1991.
Amino acid sequence of antibodies that bind PD-1, is disclosed, for example, in patent publication U.S. No. 2006/0210567, which is incorporated into this description by reference. Antibodies that bind PD-1, disclosed in patent publication U.S. No. 2006/0034826, which is also included in the present description by reference. In some examples, the antibody specifically binds PD-1 ligand or PD-1 or PD-2 with an affinity constant of at least 107M-1such as at least 108M-1at least 5×108M-1or at least 109M-1.
In one example, determine the sequence of fields that determine the specificity of each CDR. Remains outside the CDR (websites, not in contact with the ligand), replace. For example, in any of the sequences as in the table above, not less than one, two or the ri amino acids can be replaced. Obtaining hybrid antibodies, which include area of the frame from one antibody and region CDR is from a great antibodies is well known in the art. For example, humanized antibodies can be obtained in the usual way. The antibody or antibody fragment can be humanitarianly immunoglobulin with areas of complementarity determining (CDR) from the donor monoclonal antibody, which binds PD-1, PD-L1 or PD-L2, and frames of variable regions of the heavy and light chains from the frames of the heavy and light chain acceptor human immunoglobulin. Usually humanitarianly immunoglobulin specifically binds to PD-1, PD-L1 or PD-L2 with an affinity constant of at least 107M-1such as at least 108M-1at least 5×108M-1or at least 109M-1.
Humanized monoclonal antibodies can be obtained by transferring donor areas, complementarity determining (CDR) of the variable regions of the heavy and light chains of the immunoglobulin donor mouse (such as PD-1, PD-L1 or PD-L2) in the variable domain of human rights and subsequent replacement of residues of human rights in areas of the frame, when you want to save the affinity. The use of components antibodies derived from humanized monoc the national antibodies eliminates potential problems associated with the immunogenicity of the constant regions of the donor antibody. Methods of producing humanized monoclonal antibodies are described, for example, Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci. U. S. A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; Singer et al., J. Immunol. 150:2844, 1993. The antibody may be of any isotype, but in some embodiments, the implementation of the antibody is an IgG, including, but not limited to, IgG1, IgG2, IgG3and IgG4.
In one embodiment, the sequence of frame variable regions of the heavy chain gumanitarnogo immunoglobulin may be at least about 65% identical to the sequence of frame variable regions of the heavy chain of the donor immunoglobulin. Thus, the sequence of frame variable regions of the heavy chain gumanitarnogo immunoglobulin may be at least about 75%, at least about 85%, at least about 90% or at least about 95% identical to the sequence of frame variable regions of the heavy chain of the donor immunoglobulin. The area of the skeleton of man and mutations that can be made to areas of the frame gumanitarnogo antibodies known in this on the region of the technique (see, for example, in U.S. patent No. 5585089, which is included in the present description by reference).
Examples of antibodies are LEN and 21/28 CL. The sequence of frames of the heavy and light chains are known in the art. Examples of frames light chain Mab LEN man have the following sequence:
Examples of frames heavy chain Mab 21/28' CL person have the following sequence:
Antibodies such as murine monoclonal antibodies, hybrid antibodies and humanized antibodies include full-sized molecules as well as fragments thereof, such as Fab, F(ab')2and Fv, which include the variable region of the heavy chain and light chain and are able to bind a specific epitope determinants. These antibody fragments retain some ability to selectively contact the antigen or receptor. These fragments include:
(1) Fab, the fragment which contains a monovalent antigennegative fragment of the antibody molecules, can be obtained by hydrolysis of whole antibody with the enzyme papain to obtain intact light chain and a portion of one heavy chain;
(2) Fab', the fragment of the antibody molecules, which can be obtained by treating whole antibody with pepsin, followed by reduction with getting intechnology chain and parts of one heavy chain; molecule antibodies have two Fab' fragment;
(3) (Fab')2fragment of an antibody, which can be obtained by treating whole antibody with the enzyme pepsin without subsequent recovery; and (Fab')2is a dimer of two Fab' fragments held together by two disulfide bonds;
(4) Fv, a fragment created using genetic engineering, containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and
(5) single-chain antibody (scFv), defined as a molecule that is created through genetic engineering, containing the variable region of light chain, the variable region of the heavy chain associated with a suitable polypeptide linker as a genetically created single-stranded hybrid molecules.
Methods of creating such fragments are known in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). In some examples, the variable region comprises the variable region of the light chain and the variable region of the heavy chain expressed as individual polypeptides. Antibody Fv typically be 25 kDa and contain a complete antigennegative site with three CDRs of each heavy chain and each light chain. To obtain these antibodies VHand VLcan be expressed with two and the individual constructs of nucleic acids in the cell host. If VHand VLexpressed not in contact, chain Fv antibodies are usually held together by non-covalent interactions. However, these circuits have a tendency to dissociation when breeding, so methods have been developed for the cross-linkage chains using glutaraldehyde, intramolecular disulfide bond or a peptide linker. Thus, in one example, Fv can be a Fv, stabilized by disulfide bonds (dsFv), where variable region of the heavy chain and the variable region of the light chain are chemically linked by disulfide bonds.
In an additional example, the Fv fragments comprise chain VHand VLassociated peptide linker. These single-stranded antigennegative proteins (scFv) are obtained by constructing a structural gene comprising DNA sequences encoding domains of the VHand VLassociated with the oligonucleotide. Structural gene inserted into the expression vector, which is then injected into the cell host, such as E. coli. Recombinant cell hosts synthesize single polypeptide chain with a linker peptide connecting the two domains V. Methods of producing scFvs are known in the art (see Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol.2, page 97, 1991; Bird et al., Science 242:423, 1988; U.S. patent No. 4946778; Pack et al., Bio/Technology 11:1271, 1993; Sandhu, above).
Antibody fragments m which may be obtained by proteolytic hydrolysis of the antibody or by expression of the DNA the coding fragment in E. coli. Antibody fragments can be obtained by hydrolysis of whole antibody with pepsin or papain using conventional methods. For example, antibody fragments can be obtained by enzymatic cleavage of antibodies with pepsin to obtain 5S fragment denoted (Fab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds, obtaining monovalent 3,5 S Fab' fragments. Alternative enzymatic cleavage using pepsin directly produces two monovalent Fab' fragments and an Fc fragment (see U.S. patent No. 4036945 and U.S. patent No. 4331647, and related links; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology, Vol.1, page 422, Academic Press, 1967; and Coligan et al. in sections 2.8.1-2.8.10 and 2.10.1-2.10.4).
Can use other methods of splitting antibodies, such as separation of heavy chains with the formation of monovalent fragments of light-heavy chain, additional cleavage of fragments, or other enzymatic, chemical or genetic methods, as long as the fragments bind to the antigen that is recognized by the intact antibody.
Specialist in the art should POPs avati, that can be obtained conservative variants of antibodies. Such conservative variants used in fragments of antibodies, such as dsFv fragments or scFv fragments should retain amino acid residues required for the correct installation of regions VHand VLand stabilization between them, and should keep the charging characteristics of the residues in order to maintain a low pI and low toxicity of the molecules. To increase the output in the areas of VHand VLcan be done amino acid substitutions (such as not more than one, no more than two, not more than three, not more than four or five amino acid substitutions). Table-conservative amino acid substitutions, representing functionally similar amino acids are well known to the person skilled in the art. The following six groups are examples of amino acids that are considered as being conservative substitutions for one another:
1) alanine (A), serine (S), threonine (T);
2) aspartic acid (D), glutamic acid (E);
3) asparagine (N), glutamine (Q);
4) arginine (R), lysine (K);
5) isoleucine (I), leucine (L), methionine (M), valine (V); and
6) phenylalanine (F), tyrosine (Y), tryptophan (W).
Thus, the specialist in the art can easily explore an amino acid sequence of interest and is tetela, to determine the position of one or more amino acids from the summary tables presented above, to identify conservative replacement and to provide a conservative variant, using well-known molecular biology techniques.
Effector molecules, such as therapeutic, diagnostic part or parts to determine, can be attached to the antibody that specifically binds PD-1, PD-L1 or PD-L2, using a variety of methods known to experts in this field of technology. Can be used as covalent and non-covalent methods of joining. The procedure for the accession of the effector molecule to the antibody varies in accordance with the chemical structure of the effector. Polypeptides typically contain a variety of functional groups such as carboxylic acid group (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available for interaction with a suitable functional group on the antibody with the resulting binding effector molecules. An alternative antibody derivatized to expose or attach additional reactive functional groups. The derivatization may involve attaching any of a number of linker molecules such as available from Pierce Chemical Company, Rockford, IL. The linker can be any of the Molek is a, used to connect antibodies with effector molecule. The linker is capable of forming covalent bonds with both the antibody and the effector molecule. Suitable linkers are well known to experts in the art and include, but are not limited to, carbon linkers with a linear or branched chain, heterocyclic carbon linkers or peptide linkers. When the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine) or to the amino - and carboxyl group of the alpha carbon terminal amino acids.
Nucleic acid sequences encoding the antibodies can be obtained using any suitable method, including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as phosphocreatine method of Narang et al., Meth. Enzymol. 68:90-99, 1979; fosfodiesterzy method of Brown et al., Meth. Enzymol. 68:109-151, 1979; diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; solid-phase phosphoramidite method described by Beaucage &Caruthers, Tetra. Letts. 22(20):1859-1862, 1981, for example, using automated synthesizer, as described in Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984; and the method with the solid substrate p is an awning U.S. No. 4458066. When chemical synthesis is produced by single-stranded oligonucleotide. It can be converted into double-stranded DNA using hybridization with a complementary sequence, or by polymerization of a DNA polymerase using the same chain as the matrix. Specialist in the art should know that while chemical synthesis of DNA is usually limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.
Illustrative sequence encoding a nucleic acid encoding the antibody that specifically binds PD-1, PD-L1 or PD-L2 can be obtained by using the methods of cloning. Examples of suitable methods cloning and sequencing, and instructions sufficient for the guidance of the experts in this field of technology, to carry out many tasks on cloning presented in Sambrook et al. above, Berger and Kimmel (eds.) above and Ausubel above. Product information from manufacturers of biological reagents and laboratory equipment also provides useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemca-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San Diego, CA) and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to the person skilled in the technical field.
Nucleic acids can also be obtained using the methods of amplification. Methods of amplification include polymerase chain reaction (PCR), ligase chain reaction (LCR), the amplification system based on transcription (TAS), the system self-sustaining replication sequence (3SR). A great variety of cloning methods, host cells and methods of amplification in vitro is well known to specialists in this field of technology.
In one example, the antibody used is obtained by inserting cDNA, which encodes the variable region from an antibody that specifically binds PD-1, PD-L1 or PD-L2, to a vector, which comprises the cDNA encoding the effector molecule (EM). Insert make the variable region and EM is read in the frame so that is produced by a single continuous polypeptide. Thus, the encoded polypeptide contains a functional Fv region and functional area EM. In one embodiment, cDNA encoding a detectable marker (such as an enzyme), are ligated with a scFv so that the marker is localized to the carboxyl end of the scFv. In another example, the designated marker is localized on aminocore scFv. In an additional example, cDNA encoding op is delaimy marker, are ligated with the variable region of the heavy chain of the antibody that specifically binds PD-1, PD-L1 or PD-L2, so that the marker is localized to the carboxyl end of the variable region of the heavy chain. Variable region of the heavy chain may then lagerbuchse with the variable region of the light chain of the antibody that specifically binds PD-1, PD-L1 or PD-L2, using disulfide bonds. In one example, cDNA encoding the marker, are ligated with the variable region of the light chain of the antibody that specifically binds PD-1, PD-L1 or PD-L2, so that the marker is localized to the carboxyl end of the variable region of the light chain. Variable region light chain may then lagerbuchse with the variable region of the heavy chain of the antibody that specifically binds PD-1, PD-L1 or PD-L2, using disulfide bonds.
After selection and cloning of nucleic acids encoding the antibody or functional fragment of a protein can be expressed in the generated recombinant cell, such as bacteria, plant cells, yeast, insect cells and mammalian. One or more DNA sequences encoding the antibody or functional fragment can be expressed in vitro using DNA transfer into a suitable cell host. The cell can be prokaryotic or eukaryotic. The term is also on the includes all descendants of the underlying host cell. It is clear that all descendants may not be identical to the parent cell, as can occur mutations that occur during replication. Methods stable transfer, which means that the foreign DNA is maintained in the master, known in this technical field.
Polynucleotide sequences encoding the antibody or functional fragment, can be operatively linked to sequences controlling the expression. The sequence controlling the expression, operatively linked with a coding sequence are ligated so that the expression of the coding sequence is achieved under conditions compatible with the sequences controlling the expression. Sequence controlling the expression include, but are not limited to, appropriate promoters, enhancers, transcription terminators, the initiating codon (i.e., ATG) in front of the gene encoding the protein, splicing signal for introns, elements, maintains the correct reading frame of that gene for the correct translation of mRNA, and stop codons.
Polynucleotide sequences encoding the antibody or functional fragment can be inserted in the expression vector, including, but not limited to, a plasmid, virus or other media with which to monominterface for insertion or incorporation of sequences and can be expressed in either prokaryotes, or eukaryotes. Hosts can include organisms bacteria, yeast, insects and mammals. Methods the expression of DNA sequences with eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors that can be expressed and replicated in the host, well known in this technical field.
Transformation of the host cell with recombinant DNA may be carried out using traditional methods, which are well known in the art. When the host is prokaryotic, such as E. coli, competent cells which are capable of capturing DNA can be obtained from cells collected after the exponential phase of growth and then processed by a method with CaCl2using procedures well known in the art. Alternatively can be used MgCl2or RbCl. Transformation can also be carried out after the formation of protoplast host cell, if this is desirable, or using electroporation.
When the host is a eukaryote, can be used such methods of transfection of DNA as a co-precipitation with calcium phosphate, conventional mechanical procedures such as microinjection, electroporation, insertion of plasma is water, encapsulated in liposomes, or viral vectors. The eukaryotic cells can also be cotransformation with polynucleotide sequences encoding the antibody or functional fragment, and a second foreign DNA molecule that encodes a breeding phenotype, such as gene timedancing herpes simplex. Another method is to use a eukaryotic viral vector, such as a virus 40 monkeys (SV40) or the human papilloma virus bull, temporary infection or transform eukaryotic cells and expression of the protein (see, for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Specialist in the art can easily use the expression system, such as used plasmids and vectors for the production of proteins in cells, including cells of higher eukaryotes, such as cell lines, COS, CHO, HeLa and myeloma cell line.
Isolation and purification of recombinante expressed polypeptide can be carried out using conventional methods, including preparative chromatography and immunological separation. After expression of the recombinant antibodies can be purified according to standard procedures of the art, including precipitation with ammonium sulfate, affinity column chromatography, column chromatography and the like (see generally R. Scoes, Protein Purification, Springer-Verlag, N. Y., 1982). In the present description discloses essentially pure composition with homogeneity, at least about 90 to 95%, and 98 to 99% or more homogeneity can be used for pharmaceutical purposes. After cleaning, partially or to homogeneity as desired, if the polypeptides will be used therapeutically, they should be essentially free of endotoxin.
Methods expression of single-chain antibodies, including single-chain antibodies from bacteria such as E. coli, and/or re-styling to a suitable active forms described and is well known and applicable to the antibodies disclosed in the present description. Cm. Buchner et al., Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huse et al., Science 246:1275, 1989, Ward et al., Nature 341:544, 1989, all included in the present description by reference.
Often functional heterologous proteins from E. coli or other bacteria isolated from the Taurus inclusions, and this requires solubilization using strong denaturing agents and subsequent re-installation. When solubilization stage, as is well known in the art, for dissociation of disulfide bonds must be present reducing agent. An example of a buffer with a regenerating agent is: 0.1 M Tris pH 8, 6M guanidine, 2 mm EDTA, 0.3 M DTE (dithioerythritol). Re-oxidation of thesis is henych relations can be carried out in the presence of thiol reagents of low molecular weight in the reduced and oxidized form, as described in Saxena et al., Biochemistry 9:5015-5021, 1970, is hereby incorporated into this description by reference, and especially as described by Buchner et al., above.
Resaturate is usually achieved by breeding (for example, 100-fold) denatured and restored the protein in the buffer for re-styling. An example of a buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mm oxidized glutathione (GSSG) and 2 mm EDTA.
In the modification of Protocol purification of double-stranded antibody heavy and light chains separately solubilizers and restored and then combined in the solution for re-styling. An example of the resulting output is such that when the mixing of these two proteins in the molar ratio is not exceeded such molar excess of one protein over the other, as 5-fold. Preferably after completion of the redox shuffling add to the solution for re-laying the excess of oxidized glutathione or other oxidizing compounds of low molecular weight.
In addition to recombinant methods disclosed in the present description antibodies and functional fragments can also be created in whole or in part using standard peptide synthesis. Solid phase synthesis of polypeptides of less than about 50 amino acids in length can be carried out by attaching the C-terminal aminoxy is lots sequences to the insoluble substrate, followed by the sequential addition of the remaining amino acids in the sequence. Methods solid-phase synthesis are described by Barany &Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol.2: Special Methods in Peptide Synthesis, Part A. pp.3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, 111, 1984. Proteins of greater length can be synthesized by condensation of amino - and carboxyl ends of the shorter fragments. Methods for formation of peptide bonds by activating carboxyterminal end (such as through the use of the consolidating reagent N,N'-dicyclohexylcarbodiimide) are well known in the art.
B. Inhibitory nucleic acid
Inhibitory nucleic acid that reduces expression and/or activity of PD-1, PD-L1 or PD-L2 can also be used in methods disclosed in the present description. In one embodiment, a small inhibitory RNA (siRNA) for interference or inhibition of expression of the target genes. Nucleic acid sequence encoding PD-1, PD-L1 or PD-L2, are disclosed in GENBANK®, no Deposit NM_005018, AF344424, NP_079515 and NP_054862.
Typically, miRNAs create by splitting relatively long molecules of double-stranded RNA by Dicer enzymes or DCL (Zamore, Science, 296:1265-1269, 2002; Bernstein et al., Nature, 409:363-366, 2001). The animal and plant miRNAs are going in RISC and control ribonucleotides RISC activity, specific sequence, thereby rivada to the cleavage of mRNA or other RNA target molecules in the cytoplasm. At the core of miRNAs controlling histone associated with heterochromatin and DNA methylation, which leads to transcriptional silence individual genes or large chromatin domains. miRNAs PD-1 commercially available, for example, from Santa Cruz Biotechnology, Inc.
In the present disclosure proposes RNA that is suitable for interference or inhibition of expression of the target genes, the RNA includes double-stranded RNA from about 15 to about 40 nucleotides, containing from 0 to 5 nucleotide 3'- and/or 5'-overhangs on each chain. The RNA sequence is essentially identical to part of the mRNA or transcript of the target genes, such as PD-1, PD-L1 or PD-L2, for which the desired interference or inhibition. For the purposes of this disclosure the RNA sequence "essentially identical" to specific parts of mRNA or transcript of the target genes, for which the desired interference or inhibition differs by no more than about 30 percent and in some embodiments, the implementation of no more than approximately 10 percent of the specific part of the mRNA or transcript of the target genes. In specific embodiments, the implementation of the RNA sequence completely identical to a specific part of the mRNA or transcript of the target genes.
Thus, miRNAs disclosed in the present description, include double-stranded RNA from approx the tion 15 to about 40 nucleotides and 3'- or 5'-overhangs, with length from 0 to 5 nucleotides on each chain, where the sequence of double-stranded RNA is essentially identical (see above) part of the mRNA or transcript of a nucleic acid encoding PD-1, PD-L1 or PD-L2. In the specific examples of double-stranded RNA contains from about 19 to about 25 nucleotides, for example, 20, 21 or 22 nucleotides, is essentially identical to the nucleic acid encoding PD-1, PD-L1 or PD-L2. Additional examples of double-stranded RNA contains from about 19 to about 25 nucleotides, 100% identical to the nucleic acid encoding PD-1, PD-L1 or PD-L2. It should be noted that in this context "about" refers only to an integer. In one example, "approximately" 20 nucleotide refers to a nucleotide 19 to 21 nucleotides in length.
In regard to the ledge on the double-stranded RNA the length of the projection is independent from the two circuits, so that the length of one projection does not depend on the length of the ledge on the other chain. In specific examples, the length of the 3'- or 5'-ledge is 0 nucleotides of at least one chain, and in some cases 0 nucleotides on both chains (hence, dsrnas with blunt ends). In other examples, the length of the 3'- or 5'-ledge is from 1 to 5 nucleotide nucleotides on at least one chain. More specifically, in some examples, the length of the 3'- or 5'-ledge of the pillar is t 2 nucleotides on at least one chain or 2 nucleotides on both chains. In the specific examples molecule dsrnas has 3'overhangs of 2 nucleotides on both chains.
Thus, in one embodiment, the proposed RNA double-stranded RNA contains 20, 21 or 22 nucleotides and the length of the 3'-ledge is 2 nucleotides on both chains. In embodiments, the implementation proposed in the present description RNA, double-stranded RNA contains approximately 40-60% adenine+uracil (AU) and about 60-40% guanine+cytosine (GC). More specifically, typical examples of double-stranded RNA contains approximately 50% AU and approximately 50% GC.
This document also describes RNA, which additionally include at least one modified ribonucleotide, for example, in the semantic chains of double-stranded RNA. Specific examples of the modified ribonucleotide is in a 3'ledge at least one circuit, or more specifically in a 3'ledge semantic chain. Specifically considered that examples of modified ribonucleotides include ribonucleotides, which include detectable label (e.g., fluorochrome, such as rhodamine or FITZ), analogue of thiophosphate nucleotide, deoxyribonucleotide (considered as a modified, because the basic molecule is a ribonucleic acid), 2'-fluorouracil, 2'-aminouracil, 2'-aminotetralin, 4-thiouracil, 5-bromouracil, 5-iodouracil, 5-(3-am is naalil)uracil, inosine or 2'O-Me-nucleotide analogue.
Antisense molecules and ribozymes for PD-1, PD-L1 and PD-L2 are also used in the disclosed in the present description method. Antisense nucleic acids are DNA or RNA that are complementary to at least part of a specific mRNA molecule (Weintraub, Scientific American 262:40, 1990). In the cell, the antisense nucleic acids hybridize with the corresponding mRNA, forming a double-stranded molecule. Antisense nucleic acids interfere with the mRNA, as the cell will not translate mRNA that is double-stranded. Preferred antisense oligomers of about 15 nucleotides, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target cell, producing PD-1, PD-L1 or PD-L2. Using methods with antimyeloma sequences for inhibition of in vitro translation of genes is well known in the art (see, for example, Marcus-Sakura, Anal. Biochem. 172:289, 1988).
Antisense oligonucleotide can be, for example, from 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. The antisense nucleic acid can be constructed using chemical synthesis and enzymatic reactions ligation using procedures known in the art. E.g. the measures molecule antisense nucleic acid can be chemically synthesized using natural nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, for example, can be used phosphorothioate derivatives, and substituted acridine nucleotides. Examples of modified nucleotides that can be used to generate the antisense nucleic acid include, inter alia, 5-fluorouracil, 5-bromouracil, 5-florouracil, 5-iodouracil, gipoksantin, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyl, dihydrouracil, beta-D-galactosidase, inosine.
The use of the oligonucleotide for killing transcription is known as the triplex strategy as oligonucleotide wrap-around double-stranded DNA, forming triplex helix. Therefore, these triplex connections may be to recognize a unique site on a chosen gene (Maher, et al., Antisense Res. and Dev. 1(3):227, 1991; Helene, C, Anticancer Drug Design 6(6):569, 1991). This type of inhibitory oligonucleotide is used also disclosed in the present description is Etowah.
Also used ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to the DNA restriction endonucleases. By modification of the nucleotide sequences that encode these RNAS, it is possible to create molecules that recognize specific nucleotide sequences in an RNA molecule and cleave them (Cech, J. Amer. Med. Assn. 260:3030, 1988). The main advantage of this approach is that, since they are specific in terms of consistency, inactivated only mRNAs with specific sequences.
There are two basic types of ribozymes, namely ribozymes type Tetrahymena (Hasselhoff, Nature 334:585, 1988) and type "hammer head". Ribozymes type Tetrahymena recognize sequences that are four bases in length, while ribozymes type "hammer head" learn sequences of 11-18 bases in length. The longer recognizable sequence, the greater the probability that the sequence will be seeing each other only in the type of the mRNA target. Therefore, the ribozymes of the hammer head type is preferred ribozymes type Tetrahymena to inactivate specific type of mRNA, and recognizable sequence of 18 bases preferably shorter in nahemah sequences.
Various delivery systems are known and can be used for the introduction of miRNAs and other inhibitory molecules of nucleic acids as therapeutic agents. Such systems include, for example, encapsulation in liposomes, microparticles, microcapsules, nanoparticles, recombinant cells Express therapeutic(s) molecule(s) (see, for example, Wu et al, J. Biol. Chem. 262, 4429, 1987), construction of a therapeutic nucleic acid as part of a retroviral or other vector, and the like.
C. low Molecular weight inhibitors
Antagonists PD-1 is composed of molecules, which are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Screening methods that determine the reduction in the activity of PD-1 (such as the definition of cell death), suitable for identification of compounds from various sources on the activity. Initial screening can be performed by using a mixed library of compounds, various other compounds and libraries of compounds. Thus, it can be identified molecules that bind PD-1, PD-L1 or PD-L2 molecules that inhibit the expression of PD-1, PD-L1 and/or PD-L2, and molecules that ing berout activity of PD-1, PD-L1 and/or PD-L2. These low molecular weight compounds can be identified from combinatorial libraries, natural products or other libraries of low molecular weight compounds. In addition, the antagonist PD-1 can be identified compounds from commercial sources, as well as from commercially available analogues identified inhibitors.
The precise source of test extracts or compounds is not critical for the identification of antagonists PD-1. Accordingly, antagonists of PD-1 can be identified virtually any number of chemical extracts or compounds. Examples of such extracts or compounds that can be antagonists PD-1, include, but are not limited to, extracts of plants, fungi, prokaryotes or animals, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Also available are numerous methods for the exercise of arbitrary or direct synthesis (for example, poluentes or total synthesis) of any number of chemical compounds, including, but not limited to, compounds based on saccharides, lipids, peptides, and nucleic acids. Libraries of synthetic compounds commercially available from Brandon Associates (Merrimack, N. H.) and Aldrich Chemical (Milwaukee, Wis.). Antagonists PD-1 can be identified from Bible the tech synthetic compounds, which are commercially available from a number of companies, including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N. J.), Brandon Associates (Merrimack, N. H.), and Microsource (New Milford, Conn.). Antagonists PD-1 can be identified from a library of rare chemicals, such as the library, which is available from Aldrich (Milwaukee, Wis.). Antagonists PD-1 can be identified from libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FIa.) and PharmaMar, U. S. A. (Cambridge, Mass.). Natural and obtained synthetically produced libraries and compounds are easily modified through conventional chemical, physical and biochemical methods.
Used connections can be found in numerous chemical classes, though typically they are organic compounds, including low molecular weight organic compounds. Low molecular weight organic compounds having a molecular weight of more than 50, while less than about 2500 daltons, for example less than about 750, or less than about 350 daltons, can be used in disclosed in the present description methods. Examples of classes include heterocycles, peptides, saccharides, steroids and the like. Connections can be modified to increase the EF is aktivnosti, stability, pharmaceutical compatibility and the like. In some embodiments, the implementation of the used compounds have a Kd for PD-1, PD-L1 or PD-L2 is less than 1 nm, less than 10 nm, less than 1 μm, or less than 1 mm.
D. Options peptide PD-1 as antagonists
In one embodiment, variants of the protein PD-1, which function as antagonists can be identified by screening combinatorial libraries of mutants, such as point mutant or truncated mutants of the protein PD-1 to identify proteins with antagonistic activity. In one example, the antagonist is a soluble protein PD-1.
Thus, a library of variants of the PD-1 may be generated using combinatorial mutagenesis at the level of nucleic acids and encoded by library of genetic diversity. Library of variants of the PD-1 can be obtained, for example, using enzymatic ligating a mixture of synthetic oligonucleotides in the genetic sequence, such as a set of degenerate potential sequences of PD-1 expressed in individual polypeptides, or alternatively, as a set of larger hybrid proteins (such as, for phage display) containing the set of sequences of PD-1.
There are various methods that can be used for receiving the Oia libraries of potential variants of the PD-1 from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic gene then Legerova into a suitable expression vector. Using a set of degenerate genes allows you to prepare in one mixture, all of the sequences encoding the desired set of potential sequences antagonists PD-1. Methods for the synthesis of degenerate oligonucleotides is well known in the art (see, for example, Narang, et al., Tetrahedron 39:3, 1983; Itakura et al. Annu. Rev. Biochem. 53:323, 1984; Itakura et al. Science 198:1056, 1984).
In addition, libraries of fragments of the coding sequence of the protein PD-1 can be used to create a set of fragments of PD-1 for screening and subsequent selection of variants of the antagonist PD-1. In one embodiment, a library of coding sequence fragments can be generated by treating the double-stranded PCR fragments coding sequence of PD-1 nuclease under conditions when single-stranded gap occurs only about once per molecule, denaturing the double-stranded DNA renaturation of DNA with the formation of double-stranded DNA which can include sense/antisense pairs from different products arising after breaks, removing single-stranded parts of the newly formed duplexes by treatment of n is Azoy S1 and ligation of the library of the received fragments into the expression vector. Using this method can be obtained expression library that encodes N-terminal, C-terminal and internal fragments of PD-1 in various sizes.
In the art there are several methods for screening gene products of combinatorial libraries generated by point mutations or truncation, and screening cDNA libraries for gene products having a selected property. Such methods are adaptable for rapid screening of the gene libraries generated using combinatorial mutagenesis protein PD-1. The most widely used methods, which are suitable for the analysis of high-throughput screening of gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells received by the library of vectors and expression of the combinatorial genes under conditions in which the definition of a desired activity facilitates the selection of the vector encoding the gene whose product was determined. To identify antagonists of PD-1 can be used returnable group mutagenesis (REM), in combination with the methods of screening (Arkin and Youvan, Proc. Natl. Acad. Sci. USA 89:7811-7815, 1992; Delagrave et al., Protein Eng. 6(3):327-331, 1993).
In one embodiment, for analysis of a library of variants of the PD-1 can be used tests based on cellular technologies. In the example, library of expression vectors can be transliterowany cell line, which usually synthesizes and secretes a PD-1. Transfetsirovannyh cells are then cultivated so that is secreted PD-1 or a specific variant of PD-1. The effect of expression of the mutant on the activity of PD-1 in supernatant cells can be determined, for example, using any of the functional test. Plasmid DNA can then be extracted from the cells in which the activity of PD-1 zingiberone, and individual clones are additionally characterized.
As antagonists of PD-1 can also be used peptidomimetics. Peptide analogs are commonly used in the pharmaceutical industry as ones drug with properties similar to those of the matrix peptide. These types of ones connections are typically designed with the help of computer molecular modeling. Peptide mimetics that are structurally similar to therapeutically used peptides, can be used to produce equivalent therapeutic or prophylactic effect. Typically, peptidomimetics are structurally similar to the polypeptide-sample (for example, a polypeptide that has a biological activity of PD-1), but has one or more peptide bonds, optionally substituted connections-CH2NH-, -CH S-, -CH2-CH2-, -CH.=.CH- (CIS and TRANS), -COCH2-, -CH(OH)CH2and-CH2SO-. These peptide bond may be replaced by methods known in the art (see, for example, Morley, Trends Pharm. Sci. pp. 463-468, 1980; Hudson et al. Int. J. Pept. Prot. Res. 14:177-185, 1979; Spatola, Life Sci. 38:1243-1249, 1986; Holladay, et al. Tetrahedron Lett. 24:4401-4404, 1983). Peptide mimetics can be obtained economically, to be stable and may have an increased half-life or suction. Prokachivanie of peptidomimetics usually involves the covalent addition of one or more labels, directly or through a spacer (such as amide group) for noninteracting(im) position(s) on peptidomimetic, which predicted data for the quantitative assessment of structure-activity and/or molecular modeling. Such noninteracting provisions are usually provisions that do not form direct contacts with the macromolecule(s) with which(s) associated peptidomimetic for the induction of therapeutic effect. The derivatization of peptidomimetics should not significantly interfere with the desired biological or pharmaceutical activity peptidomimetic.
In disclosed in the present description methods can also be used dominant negative protein or nucleic acid encoding a dominant negative protein that interferes with the biological and the effectiveness of PD-1 (i.e. in the binding of PD-1 with PD-L1, PD-L2, or both). Dominant negative protein is any amino acid molecule having a sequence that is at least 50%, 70%, 80%, 90%, 95% or even 99% identical to sequence of at least 10, 20, 35, 50, 100, or 150 amino acids of the wild-type protein, which corresponds to a dominant negative protein. For example, a dominant negative PD-L1 has such a mutation is that it binds to PD-1 more strongly than native PD-1 (wild type), but does not activate any way of cell signaling through PD-1.
Dominant negative protein may be inserted in the expression vector. The expression vector may be a non-viral vector or a viral vector (e.g., retrovirus, recombinant associated with adenovirus viral or recombinant adenoviral vector). Alternative dominant negative protein can be directly introduced in the form of recombinant protein systemically or in the infected area, for example, using the methods of microinjection.
Polypeptide antagonists can be produced in prokaryotic or eukaryotic cells-owners as a result of expression of polynucleotides encoding amino acid sequence, frequently as part of a larger polypeptide (hybrid protein, for example, with ras or enzyme). Alternate is but such peptides can be synthesized by chemical methods. Methods the expression of heterologous proteins in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art (see Maniatis et al. Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N. Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Kaiser et al., Science 243:187, 1989; Merrifield, Science 232:342, 1986; Kent, Annu. Rev. Biochem. 57:957, 1988).
The peptides can be obtained, for example, by direct chemical synthesis, and used as antagonists of the interaction of PD-1 ligand. The peptides can be obtained in the form of modified peptides with ones parts, attached through covalent bonds to the N-end and/or C-Termini. In certain preferred embodiments, implementation, or carboxylic or aminocore, or both chemically modified. The most common modification of the terminal amino and carboxyl groups are acetylation and amidation, respectively. In various embodiments of may be introduced aminobenzene modifications, such as acylation (e.g., acetylation) or alkylation (e.g., methylation), and carboxykinase modifications such as amidation, as well as other modifications of the end parts, including cyclization. Certain aminobenzene and/or carboxykinase modifications and/or UD is inane peptide relative to the sequence of the skeleton can give the best physical, chemical, biochemical and pharmacological properties, such as increased stability, enhanced effectiveness and/or efficiency, resistance to serum proteases, desirable pharmacokinetic properties, and others.
Methods of treatment: the introduction of the antagonist PD-1 individual
In the present description provides methods to treat a variety of infections and types of cancer. In these methods treat or prevent infection or cancer, or facilitate their symptom by introducing the individual a therapeutically effective amount of the antagonist PD-1. The individual may be any mammal, such as human, Primate, mouse, rat, dog, cat, cow, horse and pig. In some examples, the individual is a Primate, such as a person. In additional examples, the individual are rodents, such as mouse.
In some embodiments, the implementation of the individual there is a risk of infection. The individual at risk of developing infection is an individual that has no infection, but it can be infected infectious agent of interest. In additional examples, the individual has an infection such as persistent infection. The individual with persistent infection can be identified by standard methods appropriate to a person skilled in the art, such to the to the doctor.
In some examples, the individual has a stable infection by infection with bacteria, virus, fungus or parasite. Usually resistant infections in contrast to acute infections effectively not disappear when the induction of the host immune response. Infectious agent and the immune response to reach equilibrium so that the infected individual remains with the infection for a long period of time without necessarily symptoms. Resistant infections include, for example, latent, chronic and slow infection. Persistent infection occurs during infection with these viruses, as viruses T-cell leukemia, Epstein-Barr, cytomegalovirus, herpes viruses, varicella zoster virus, measles virus, papovavirus, prions, hepatitis viruses, adenoviruses, parvoviruses and papilloma viruses.
In a chronic infection, the infectious agent can be identified in the body during all periods of time. However, the signs and symptoms of the disease may be present or absent for an extended period of time. Examples of chronic infections include hepatitis B (caused by hepatitis B virus (HBV) and hepatitis C (caused by hepatitis C virus (HCV)), infections caused by adenovirus, cytomegalovirus, Epstein-Barr, herpes simplex virus 1, herpes simplex virus 2, herpes virus 6 human is ka, varicella-zoster virus, hepatitis B virus, hepatitis D, human papilloma virus, parvovirus B19, a virus polyoma BK, virus polyoma JC, measles virus, rubella virus, human immunodeficiency virus (HIV), the virus I T-cell leukemia human and virus II T-cell leukemia person. Parasite resistant infections can result from infection with Leishmania, Toxoplasma, Trypanosoma, Plasmodium, Schistosoma and Encephalitozoon.
During latent infection infectious agent (such as a virus) is, apparently, inactive and dormant, so that the individual is not always manifested signs or symptoms. When latent viral infection, the virus remains in equilibrium with the host for extended periods of time before re-emergence of symptoms; however, existing viruses cannot be detected before the onset of reactivation of the disease. Examples of latent infections include infections caused by herpes simplex virus (HSV)-1 (herpetic fever), HSV-2 (genital herpes) and virus VZV varicella-zoster (chickenpox/herpes Stripping).
Slow infections infectious agents graduale grow in number over a very long period of time, during which no significant signs or symptoms of the disease. Examples of slow infections include AIDS (call the by HIV-1 and HIV-2), tumors in animals caused by lentiviruses and prions.
In addition, resistant infections often occur as a late complication of acute infections. For example, subacute sclerosing panencephalitis (SSPE) may occur after acute measles infection, or regressive encephalitis can result from infectious diseases rubella.
In one non-limiting example, the individual can be diagnosed with persistent infection with chlamydia after determining the type of chlamydia in a biological sample from the individual using PCR analysis. Mammals that have undiagnosed persistent infection, do not need treatment in accordance with this disclosure. Microbial agents that can cause persistent infection include viruses (such as human papilloma virus, hepatitis virus, human immunodeficiency virus and herpes), bacteria (such as Escherichia coli and Chlamydia spp.), parasites such as Leishmania spp., Schistosoma spp., Trypanosoma spp., Toxoplasma spp.) and mushrooms.
In addition to the compound that reduces the expression or activity of PD-1, the individual being treated may also be given the vaccine. In one example, the vaccine may include adjuvant. In another example, the vaccine may include Primerose booster immunization. The vaccine may be a dead heat vaccine, attenuated VA is the CIN or subunit vaccine. The individual already infected with the pathogen, can be treated with a therapeutic vaccine, such as an antagonist PD-1 and antigen. The individual may be asymptomatic disease, so that treatment prevents the development of the symptom. Therapeutic vaccines may also reduce the severity of one or more existing symptoms or to reduce the pathogen load.
In some examples, methods of treatment, the individual is administered a therapeutically effective amount of the antagonist PD-1 in combination with viral antigen. Non-limiting examples of suitable viral agents include, inter alia: antigens HA, NA, M, NP and NS of the influenza virus; p24, pol, gp41 and gpl20 of HIV; proteins F and G metapneumovirus (hMNV); proteins El, E2 and proteins frame of the hepatitis C virus (HCV); El, E2 and proteins frame of Dengue viruses (DEN 1-4); protein Ll of human papilloma virus; peptide gp220/350 and EBNA-3A virus Epstein-Barr; glycoprotein gB, glycoprotein gH, pp65, IEl (exon 4) and ppl50 cytomegalovirus (CMV); epitopes of IE62 peptide and glycoprotein E of varicella-zoster virus (VZV); epitopes of glycoprotein D of herpes simplex virus. Antigenic polypeptides may be polypeptides, existing in the natural viral isolates in animals or humans, or can be created to enable one or more amino acid substitutions compared to the natural (pathogenic or not) in isolation. Examples of the antigen is in are listed below:
In additional embodiments, the implementation of the individual has a tumor. The method includes the introduction to the individual a therapeutically effective amount of the antagonist PD-1, thereby carrying out the treatment of the tumor. In some examples, a therapeutically effective amount of a tumor antigen or a nucleotide encoding a tumor antigen, also administered to the individual. Antagonist PD-1 and tumor antigen or a nucleotide encoding a tumor antigen, can be administered simultaneously or sequentially.
Introduction antagonist PD-1 leads to reduction of the size, distribution or metastatic potential of a tumor in the individual. The efficiency is determined in connection with any known method for diagnosing or treating a particular cancer.
Tumor (also called "cancer") include solid tumors and leukemias. Examples of tumors include tumors listed in table 2 (together with the known tumor antigens associated with these types of cancer).
Examples of tumors and their tumor antigens
|Acute myelogenous leukemia|
|Chronic myelogenous leukemia||WT1, PRAME, PR1, proteinase 3, elastase, cathepsin G|
|Myelodysplastic syndrome||WT1, PRAME, PR1, proteinase 3, elastase, cathepsin G|
|Acute lymphoblastic leukemia||PRAME|
|Chronic lymphocytic leukemia||Survivin|
|Multiple myeloma||New York esophageal 1 (NY-Eso1)|
|Malignization melanoma||MAGE, MART, tyrosinase, PRAME, GP100|
|Breast cancer||WT1, Herceptin|
|Prostate cancer||Prostatespecific antigen (PSA)|
|Cancer of the colon||Removeability antigen (CEA)|
|Renal cell cancer (RCC)||The interesting examples of tumor antigens include those listed below in table 3:|
Specific non-limiting examples are angioimmunoblastic nodular lymphoma or Hodgkin lymphoma with a predominance of lymphocytes. Angioimmunoblastic lymphoma (AIL) is an aggressive (rapidly progressing) type of T-cell non-Hodgkin lymphoma characterized by enlarged lymph nodes and hypergammaglobulinemia (elevated antibodies in the blood). Other symptoms may include skin rash, fever, weight loss, positive test Kumba or night sweats. This cancer usually occurs in adults. Patients usually are aged 40-90 years (median 65) and are more often men. When progression AIL can develop hepatosplenomegaly, hemolytic anemia and the polyclonal hypergammaglobulinemia. The skin is involved in approximately 40-50% of patients.
Nodular Hodgkin lymphoma with a predominance of lymphocytes is an In-cell neoplasm, which, obviously, comes from its germinative center b cells with mutated nonfunctional genes of immunoglobulins. Similar to angioimmunoblastic lymphoma, the neoplastic cells are connected with a network of follicular dendritic cells. Express the PD-1 is observed in T-cells, closely associated with neoplastic CD20+ cells in nodular Hodgkin's disease with predominance of lymphocytes, with a pattern similar to that observed in CD57+ T-cells. CD57 identified as another marker of T cells associated with its germinative center, as CXCR5, data that support the conclusion that the neoplastic cells in nodular Hodgkin's disease with predominance of lymphocytes have a close relationship with T-cells associated with its germinative center.
Expression of interest to the tumor antigen can be determined at the level of protein or nucleic acid by any method known in the art. For example, to determine the gene expression can be used in the analysis Northern hybridization using probes that specifically recognize one or more of these sequences. An alternative expression is measured using tests based on PCR with reverse transcription, for example, using primers specific for the differentially expressed gene-sequences. The expression is also determined at the protein level, for example, by measuring levels of the peptides encoded described in this document gene products, or measuring their activity. Such methods are well known in the art and include, for example, immunotest n the basis of antibodies to proteins, encoded by those genes. Any biological material can be used to determine/quantitative assessment of protein or activity.
In one example, the individual had previously been diagnosed with cancer. In additional examples, the individual had undergone previous treatment for cancer. However, in some examples, the individual has not been previously diagnosed with cancer. The diagnosis of a solid tumor can be done through the identification of the investigated mass, although it may be done by other means, such as radiological diagnosis or ultrasound. Cancer treatment may include surgery or may include the use of chemotherapeutic agents, such as docetaxel, vinorelbine gemcitabine, capecitabine, or combinations of cyclophosphamide, methotrexate and fluorouracil, cyclophosphamide, doxorubicin and fluorouracil, doxorubicin and cyclophosphamide; doxorubicin and cyclophosphamide with paclitaxel; doxorubicin and then CMF (cyclophosphamide, epirubicin and fluorouracil). In addition, treatment may include the use of radiation.
In some examples, the individual is administered a therapeutically effective amount of the antagonist PD-1. The individual is also administered a therapeutically effective amount of a tumor antigen or a nucleic acid that encodes the antigen. The introduction may be the simultaneous or sequential.
For the treatment of an individual with persistent infection or cancer of the interested individual is administered a therapeutically effective amount of the antagonist PD-1. In one example, a therapeutically effective amount of the antagonist PD-1 is a biologically active dose, such as dose, which should induce an increase in CD8+ T-cell cytotoxic activity with increased specific immune response against the infectious agent. Preferably, the antagonist PD-1 had the ability to decrease the expression or activity of PD-1 specific antigen immune cells (such as T-cells, such as CD8+ T cells) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 100% below the control level without treatment. The level of activity of PD-1 in immune cells measured by any method known in the art, including, for example, analysis by immunoblotting, immunohistochemistry, ELISA and analysis Northern-blotting. Alternative biological activity of PD-1 was measured by assessing the binding of PD-1 with PD-L1, PD-L2, or both. The biological activity of PD-1 is determined according to its ability to increase CD8+ T cell cytotoxicity, including, for example, cytokine production, clearance of the infectious agent and the proliferation of specific antigen CD8+ T-cells. Preferably, the agent that reduced the t of the expression or activity of PD-1, could improve the immune response, specific infectious agent or tumor by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 100% above the reference level without treatment. The agent of the present invention, therefore, is any agent that has one or more of these forms of activity. Although the agent preferably is expressed in CD8+ T-cells, it should be understood that any cell that can influence the immune response against resistant infections, is also included in the methods of the invention include, for example, B-cells.
Optional individual is administered one or more additional therapeutic agents. Additional therapeutic agents include, for example, antiviral compounds (e.g., vidarabine, acyclovir, ganciclovir, valganciclovir, nucleoside analog - nucleoside reverse transcriptase inhibitor (NRTI) (for example, AZT (zidovudine), ddI (didanosine), ddC (zalcitabine), d4T (stavudine) or 3TC (lamivudine)), non-nucleoside reverse transcriptase inhibitors (NNRTIS) (eg, nevirapine or delavirdine), proteiny inhibitor (saquinavir, ritonavir, indinavir or nelfinavir), ribavirin or interferon), antibacterial compounds, antifungal compounds, antiparasitic compounds, anti-inflammatory compounds, antineoplastic agent (chemotherapeutic what agents or analgesics.
The additional therapeutic agent is administered before, concurrently or after administration of the antagonist PD-1. For example, the antagonist PD-1 and the additional agent are administered in separate compositions that are separated from each other in time at least 1, 2, 4, 6, 10, 12, 18 or more than 24 hours. Optional additional agent together with an antagonist PD-1. When an additional agent is in another song, you can use different routes of administration. The agent is administered in doses known to be effective for the treatment, inhibition or prevention of infection with this agent.
The concentration of antagonist PD-1 and the additional agent are dependent on a number of factors, including the introduction, the localization of the treatment, the physiological condition of the mammal and other injected drugs. Thus, therapeutic dose can be chitravati to optimize safety and efficacy and are within the competence of a person skilled in the art. Determining the appropriate dose and mode of administration in a particular situation is within the competence of a person skilled in the technical field.
Optional individual additionally get a vaccine that induces an immune response against human immunodeficiency virus (HIV), tuberculosis, influenza or hepatitis C. Typical vaccines are described, for example, Berzofsky et al.(J. Clin. Invest. 114:456-462, 2004). If desired, the vaccine is injected with Primerose booster injection or with adjuvants. The vaccine may also be a tumor vaccine, such as a therapeutically effective amount of a tumor antigen. In some embodiments, the implementation of the individual is administered a therapeutically effective amount of the antigenic polypeptide, such as viral or tumor antigen.
The individual can enter a therapeutically effective amount of a tumor antigen or a nucleic acid that encodes a tumor antigen. Polynucleotide include recombinant DNA, which is included in the vector can replicate autonomously plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or is in the form of a separate molecule such as a cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of any of the nucleotide. The term includes single-stranded and double-stranded forms of DNA.
Was designed a number of viral vectors, including virus polyoma, i.e., SV40 (Madzak et al., 1992, J. Gen. Virol, 73:1533-1536), adenovirus (Berkner, 1992, Cur. Top. Environ. Immunol, 158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia et al., 1992, J. Virol, 66:4407-4412; Quantin et al., 1992, Proc. Nad. Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:24-256), the smallpox virus (Mackett et al., 1992, Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top. Environ. Immunol, 158:91-123; On et al., 1990, Gene, 89:279-282), the herpes viruses, including HSV and EBV (Margolskee, 1992, Curr. Top. Environ. Immunol., 158:up 67-90; Johnson et al., 1992, J. Virol, 66:2952-2965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol, 1:337-371; Fresse et al., 1990, Biochem. Pharmacol, 40:2189-2199), viruses sindbis (H. Herweijer et al., 1995, Human Gene Therapy 6:1161-1167; U.S. patent No. 5091309 and 52217879), alpha viruses (S. Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377) and retroviruses birds (Brandyopadhyay et al., 1984, Mol. Cell Biol, 4:749-754; Petropouplos et al., 1992, J. Virol., 66:3391-3397), mouse (Miller, 1992, Curr. Top. Environ. Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol., 54:401-407) and man (Page et al., 1990, J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739). In the art also known baculovirus (multicore poladroid.net virus Autographa californica; AcMNPV) vectors, and they are commercially available (for example, from PharMingen, San Diego, Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).
In one embodiment, the viral vector is enabled polynucleotide encoding a tumor antigen or viral antigen. Suitable vectors include retroviral vectors, orthopoxvirus vectors, avipox vectors, fowlpox vectors, capripox vectors, swiaczny vectors, adenoviral vectors, vectors, herpes virus, alphavirus vectors, baculovirus vectors, vectors in the eng sindbis, the vectors of the smallpox virus and poliovirus vectors. Specific illustrative vectors are poxvirus vectors, such as the smallpox virus, fowlpox virus and highly attenuated smallpox virus (MVA), adenovirus, baculovirus, and the like.
Used poxviruses include Orthodoxy, swiaczny, avidoxy and carboxy viruses. Orthopox includes smallpox, ectromelia and raccoonpox. One of the examples used ortodoxa is vaccinia. Avipox includes fowlpox, smallpox Canaries and smallpox pigs. Capripox includes smallpox goats and sheep pox. In one example, sipox is a pox swine. Examples of poxvirus vectors for expression are described, for example, in U.S. patent No. 6165460, which is included in the present description by reference. Other viral vectors that can be used include other DNA viruses such as herpesviruses and adenoviruses, and RNA viruses, such as retroviruses and poliovirus.
In some embodiments, the implementation of the antagonists PD-1 is administered in a quantity sufficient to increase the cytotoxicity of T-cells, such as CD8+ T cells. Increase T-cell cytotoxicity leads to increased immune response and suppression of resistant infections or to reduce sign or symptom of cancer. An increased immune response can be measured, for example, to increase the proliferation of IMM is the R cells, such as T - or B-cells, increased production of cytokines and increased clearance of the infectious agent or reducing the severity of the tumor. Thus, the method can lead to mitigating one or more symptoms associated with persistent infection or tumor. Thus, the introduction of the antagonist PD-1 inhibits sustainable infection, inhibits the growth/tumour size or mitigates one or more symptoms associated with persistent infection or tumor by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to an individual without treatment.
Treatment is effective if the treatment leads to a clinically useful result, such as reduction of the infectious agent or reducing the severity of a tumor in the individual. When prophylactic treatment is "effective" means that the treatment retards or prevents infection from the source. Efficiency can be determined using any known method of diagnosis or treatment of specific infections or tumors.
Thus, the methods include the introduction to the individual a pharmaceutical composition that includes a therapeutically effective amount of the antagonist PD-1. An effective amount of a therapeutic compound, such as an antibody can be, for example, from about 0.1 mg/kg to about 150 mg/kg Effective doses will vary, as clear specification of what elistan in the art, depending on the route of administration, the use of filler and joint injection with other therapeutic means, including the use of other anti-infective agents or therapeutic agents for the treatment, prevention or relief of a symptom of a specific infection or tumor. For a sick person suffering from (or having a risk of development of infection or tumor, use of therapeutic regime using standard methods.
Antagonist PD-1 is administered to such an individual using methods known in the art. You can use any antagonist PD-1, such as disclosed in the present description. In addition, you can use more than one antagonist PD-1. Antagonist PD-1 can be entered locally or systemically. For example, the antagonist PD-1 injected oral, rectal, nasal, topical, parenteral, subcutaneously, intraperitoneally, intramuscularly or intravenously. Antagonist PD-1, you can enter a prophylactically or after detection of infection or tumor. Antagonist PD-1 does not necessarily represent the views of the components of the mixture of therapeutic drugs to treat the infection. Examples of formulations suitable for parenteral administration include aqueous solutions of the active agent in isotonic saline, 5% glucose or other standard pharmaceutically acceptable filler. The quality is TBE pharmaceutical excipients for delivery of therapeutic compounds used as standard solubilizing agents, such as PVP or cyclodextrins.
Described in the present description, therapeutic compounds are compositions for other routes of administration using traditional methods. For example, the antagonist PD-1 are in capsule or tablet for oral administration. Capsules can contain any of the standard pharmaceutically acceptable substances such as gelatin or cellulose. Tablets can be prepared in accordance with conventional procedures by compressing mixtures of therapeutic compound with a solid carrier and a lubricant agent. Examples of solid carriers include starch and sugar bentonite. Antagonist PD-1 can be entered in the form of pellets with a hard shell or capsule containing a binder, such as lactose or lures, traditional filler and tabletroute agent. Other formulations include ointments, suppositories, paste, spray, patch, cream, gel, absorbable sponge or foam. Such compounds are obtained by methods well known in the art.
In addition, antagonists of PD-1 can be administered by implantation (either directly in the body (e.g., intestine, or liver), or subcutaneously) solid or biodegradable matrix which slowly releases the connection to the neighboring and surrounding tissue of the individual. For example, for the treatment of gastrointestinal infections is Obedinenie you can enter systemically (for example, intravenous, rectal or oral) or topically (for example, directly into the tissue of the stomach). Alternative impregnated antagonist PD-1 briquette or biodegradable tampon is placed in direct contact with the tissue of the stomach. Antagonist PD-1 slowly released in vivo by diffusion of the drug from the swab and the destruction of the polymer matrix. Another example is the treatment of infections of the liver (i.e., hepatitis) by infusion into the bloodstream liver solution containing antagonist PD-1.
When therapeutic compound is a nucleic acid encoding the antagonist PD-1, a nucleic acid can be entered in vivo to promote expression of the encoded protein, by constructing it contains as part of a suitable expression vector nucleic acid and its introduction in such a way that it becomes intracellular (as when using a retroviral vector, by direct injection, by bombardment of the particles by coating with lipids or cell surface receptors or transfiziologii agents or by its introduction in connection with homeobox-like peptide which is known that it enters the nucleus (see, e.g., Joliot, et al., Proc. Natl. Acad. Sci USA 88:1864-1868, 1991), and the like. Alternative therapeutic nucleic acid is injected intracellular and include vdnk host cell for expression, by homologous recombination or leave episomal.
For local DNA introduction you can use the standard vectors for gene therapy. Such vectors include viral vectors, including vectors derived from viruses of hepatitis C replication defect (such as HBV and HCV), retroviruses (see PCT publication no WO 89/07136; Rosenberg et al., N. Eng. J. Med. 323(9):570-578, 1990, adenovirus (see Morsey et al., J. Cell. Biochem., Supp. 17E, 1993), adeno-associated virus (Kotin et al., Proc. Natl. Acad. Sci. USA 87:2211-2215, 1990), herpes simplex virus defective replication (HSV; Lu et al., Abstract, page 66, Abstracts of the Meeting on Gene Therapy, Sept. 22-26, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1992), and any modified versions of these vectors. You can use any other delivery system, which provides for the transfer of nucleic acids into eukaryotic cells in vivo. For example, nucleic acid can be packaged in liposomes, such as cationic liposomes (lipofectin), mediated by receptors of delivery systems, non-viral vectors based on nucleic acids, membranes of red blood cells or microspheres (such as microparticles; see, for example, U.S. patent No. 4789734; U.S. patent No. 4925673; U.S. patent No. 3625214). Can be administered as naked DNA.
As for inhibiting nucleic acids, therapeutically effective amount is an amount that can cause clinically desired result, such as decreased gene product PD-1 subject to l is the significance of the animal. This number can be determined by a person skilled in the art. Dose for any particular patient depends on many factors, including the size of the patient, the surface area, age, the particular compound to be introduction, sex, time and route of administration, General health status and the simultaneous introduction of other drugs. Doses can vary, but a preferred dosage for intravenous administration of DNA is approximately 106 to 1022 copies of the DNA molecule.
Typically, the plasmid is administered to the mammal in an amount of from about 1 nanogram to about 5000 micrograms of DNA. Preferably, the composition contains from about 5 ng to about 1000 micrograms of DNA from 10 ng to 800 micrograms of DNA from 0.1 micrograms to 500 micrograms of DNA from 1 to 350 micrograms micrograms of DNA from 25 micrograms to 250 micrograms of DNA or from 100 micrograms to 200 micrograms of DNA. Alternate introduction of recombinant adenoviral vectors encoding the antagonist PD-1, the mammal can be carried out at a concentration of at least 105, 106, 107, 108, 109, 1010 or 1011 plaque-forming units (PFU).
In some embodiments, the implement for the treatment of neurological infections antagonist PD-1 can be administered intravenously or intrathecally (e.g., by direct infusion into the cerebrospinal liquid is awn). For the local introduction of impregnated connection briquette or biodegradable tampon is placed in direct contact with the tissue of the Central nervous system (CNS). The compound or mixture of compounds are slowly released in vivo by diffusion of the drug from the briquette and the destruction of the polymer matrix. The alternate connection is administered by infusion into the brain or cerebrospinal fluid using standard methods. For example, in the drilled hole of the skull is placed a hollow ring corkscrew with a catheter for use as input. Access fluid from a reservoir connected to the catheter, is provided with a needle or probe is inserted through the upper part of the hollow ring corkscrew. The Assembly of the catheter (described, for example, in U.S. patent No. 5954687) ensures the promotion of the flow of liquid suitable for transferring fluids at a certain place or out of it, on the brain, next to it or inside the brain to ensure the administration of medication through a certain period of time.
In additional embodiments, the implementation for heart infections antagonist PD-1 can be delivered, for example, in the tissue of the heart (such as the myocardium, pericardium, or endocardium) by direct intracardiac injection through the chest, or by using standard methods based on the percutaneous catheter under fluoroscopic the civil control. Thus, the antagonist PD-1 can be directly injected into the tissue or can be infusional through the stent or catheter, which is inserted into the body cavity. To establish connection, you can use many of the coronary perfusion catheters or catheters. Alternative antagonist PD-1 cover or impregnate with a stent, which is placed in the vessel of the heart.
Lung infections can be treated, for example, by introducing antagonist PD-1 through inhalation. Compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, such as a gas, such as carbon dioxide, or a nebulizer.
Specialist in the art should understand that the treated patients could be subjected to the same tests for the diagnosis of resistant infections in an individual or could be identified without checking as an individual with a high risk due to the presence of one or more risk factors (such as exposure to infectious agent exposure with an infected individual, genetic predisposition or presence of pathological conditions that predispose to secondary infections). Reduction of symptoms of resistant infections or damage may include, but are not limited to, alleviation of symptoms, reducing the extent of disease, stabilization (without the adsene) state of disease, delay or slowing of disease progression and mitigation of or the temporary relief of painful conditions. Treatment can be done at home under the close supervision of a health worker or health care hospital.
Methods of measuring the immune response after treatment with the use disclosed in the present description of the methods well known in the art. The activity of T-cells can be assessed, for example, using tests that detect the production of cytokines, tests measuring the proliferation of T-cells, tests measuring the clearance of microbial agent and tests measuring the cytotoxicity of CD8+ T-cells. These tests are described, for example, in U.S. patent No. 6808710 and publications of patent applications U.S.№№ 20040137577, 20030232323, 20030166531, 20030064380, 20030044768, 20030039653, 20020164600, 20020160000, 20020110836, 20020107363, and 20020106730, each of which is incorporated into this description by reference.
Optional ability antagonist PD-1 to increase the cytotoxicity of CD8+ T-cells is assessed through tests, which measure the proliferation of CD8+ T-cells (e.g., thymidine incorporation, by analyzing BrdU and staining for markers of cell cycle (e.g., Ki67 and CFSE), described, for example, Dong et al. Nature (5:1365-1369, 1999). In one example, cell proliferation track in culture treated T-cells expressing PD-1 antagonist PD-1 on pervi the resultant activation signal, as described above, and3H-thymidine. The level of proliferation of T-cells is determined by thymidine incorporation.
Cytotoxicity of CD8+ T-cells can also be assessed in tests lysis (such as test selection51Cr or tests that determine the allocation perforin or granzyme), tests that detect activation of caspases, or in tests that measured the clearance of microbial agent from an infected individual. For example, the amount of virus in a biological sample from an infected individual (e.g., serum, spleen, liver, lung or tissue in which the virus is genotype) can be measured before and after treatment.
Can be also measured the production of cytokines, such as IFNγ, TNF-α and IL-2. For example, purified T cells were cultured in the presence of the antagonist protein PD-1 and primary activation signal. The levels of different cytokines in the supernatant can be determined using a sandwich enzyme-linked immunosorbent assays or other traditional tests described in, for example, Dong et al. Nature (5:1365-1369, 1999).
If desired, the effectiveness of the antagonist PD-1 assessed for its ability to induce costimulation T-cells. For example, a method of costimulate T-cells in vitro involves contact purified T-cells that Express PD-1, with the first or primary activation signal in the form of monoclonal antibodies is against CD3 or forblog ether or antigen in complex with MHC class II. The ability of the compounds being considered as an agent to decrease the expression or activity of PD-1 and, therefore, to provide a secondary or co-stimulating signal required for modulation of the immune function of these T-cells can be analyzed using any of the traditional tests are well known in the art.
Response B-cell antagonist PD-1 can be estimated using specific LCMV ELISA, ELISPOT for plasma cells, test for B-cell memory phenotype of B-cells and analysis centers reproduction using immunohistochemistry.
Treatment methods: adoptive immunotherapy
In the present description discloses the methods of treatment, an interested individual, such as an individual with persistent viral infection or tumor. Methods include the introduction of a therapeutically effective amount of cytotoxic T-cells specific antigen of interest, such as a viral antigen or a tumor antigen, and a therapeutically effective amount of the antagonist PD-1.
Disclosed in the present description are intended to improve the immune response, such as strengthening the immune system of the individual. The introduction of purified specific antigen T-cell and antagonist PD-1, as disclosed in the present description, should able to improve the t of the individual to overcome pathological conditions, such as infectious disease or a tumor, by directing the immune response against a pathogen (such as a virus or fungus) or neoplasm. Thus, by cleaning and creation of purified populations of selected specific antigen T cells of the individual ex vivo and injection of therapeutic quantities of such cells to increase the immune response of the individual recipient. The introduction of a therapeutically effective amount of the antagonist PD-1 also enhances the immune response of the recipient.
In the present description provides methods of inducing the recipient's immune response to any antigen. The recipient can be any interested individual, including individuals with chronic infection, such as viral or fungal infection, or a person with a tumor. Such infections described above.
Infections in people with immune deficiency is a common problem for recipients allotransplantation stem cells and the recipient of transplants of organs from permanent immunosuppression. Resulting from deficiency of T-cell infection in these individuals typically associated with re-activation of viruses already present in the recipient. For example, after infection the majority of viruses of herpes group (such as CMV, EBV, VZV, HSV) are dormant and remain in a depressed state due to T-cell is. However, the immunosuppression of patients using traditional modes dormant viruses can again be activated. For example, immunosuppression, patients would be re-CMV activation, reactivation of Epstein-Barr (EBV), which causes the tumor B cells (EBV lymphoproliferative disease), and reactivation of BK virus that causes hemorrhagic cystitis. Additional examples of T-cell immunodeficiency serve HIV infection and congenital immunodeficiency. These viral infection and re-activation may be the result of suppression of immunity in individuals.
In several versions of the implementation of the recipient of a transplant of bone marrow is provided an immune response against the tumor. Antitumor immunity can be provided to the individual by the introduction of a specific antigen by T-cells that recognize tumor antigen. This introduction to the recipient should enhance the immune response of the recipient to the tumor through supply of T-cells, which are aimed at the tumor antigen of interest, learn it and immunological interact with him.
In one example, the method includes the selection of the donor population of donor cells, including T cells (such as mononuclear cells peripheral blood), and contacting the population don is rsky cells, including T-cells, with a population of antigen presenting cells (APC) from a donor who presenterat interest antigen, optionally in the presence of PD-1 results in the population of donor cells, including activated donor CD4+and/or CD8+T cells with a reduced number alloreactive T-cells that recognize the antigen of interest. The recipient is administered a therapeutically effective amount of a population of activated donor CD4+and/or CD8+T-cells, thereby providing the recipient's immune response to any antigen. The introduction of purified specific antigen T-cells may increase the ability of the individual to overcome pathological conditions, such as infectious disease or a tumor, by directing the immune response against a pathogen (such as a virus or fungus) or neoplasm. As a result, the recipient is provided an immune response against the antigen of interest.
In several implementations, the method also includes an introduction to the individual a therapeutically effective amount of the antagonist PD-1. The introduction of the antagonists PD-1 described in detail above.
To create a population of T-cells specific for this antigen of interest, can be used any antigenic peptide (such as immunogenic fragment) and from terenowego antigen. In the art there are many such antigenic peptides, such as viral and tumor antigens (see, for example, table 1-2). The present disclosure is not limited to the use of specific antigenic peptides. Specific examples of antigenic peptides from antigens of interest include, but are not limited to, antigens, which are viral, fungal or tumor antigens, such as shown in table 1. In the art known for more antigenic peptides (for example, see Novellino et al., Cancer Immunol. Immunother. 54(3):187-207, 2005, and Chen et al., Cytotherapy, 4:41-8, 2002, both incorporated into this description by reference).
Although in table 1 revealed specific fragments of a full-sized antigens of interest, a specialist in the art should understand that disclosed in the present description methods can also be used with other fragments or full-size protein. In one example, the antigen of interest is an "immunogenic fragment" full sequence of the antigen. "Immunogenic fragment" refers to a part of the protein, which when presenting cell in the context of MHC molecules can test the activation of T-cells to activate T cells against expressing the protein of the cell. Typically, such fragments that are bound by MHC molecules of class I, represent the Wallpaper is a sequence of from 8 to 12 amino acids of a full length antigen, although, of course, you can use a longer fragments. In some examples, the immunogenic fragment is a fragment that can specifically bind an MHC molecule on the surface of APC without additional processing sequence of the epitope. In some examples, the immunogenic fragment is a sequence of 8-50 amino acid sequence of full-length antigen, for example from 8 to 20 amino acids of 8 to 15 amino acids, from 8-12 amino acids, from 8 to 10 amino acids, or 8, 9, 10, 11, 12, 13, 14, 15 or 20 consecutive amino acids from a sequence of full-length antigen. In some examples, the cells APC incubated with the immunogenic fragment under conditions sufficient for specific binding of immunogenic fragment with MHC molecules on the APC surface, without the need for intracellular processing.
In one example, the antigen comprises a peptide antigen of interest, with the amino acid sequence containing the motif binding molecule HLA individual. Such motifs are well known in the art. For example, HLA-A2 is a common allele in the human population. Connecting the motive for this molecule includes peptides of 9 or 10 amino acids containing leucine or methionine in the second position and valine or leucine in the last position is (see the examples above). Peptides that include such motifs can be obtained by any means known in the art (such as recombinant, chemical and so on). Knowing the amino acid sequence of the antigen of interest, you can determine the sequence of immunogenic fragments with predicted binding with MHC with published programs. For example, to predict epitopes associated with any tumor, virus or fungus antigen using common methods to use the program to bind HLA motif on the Internet (Bioinformatics and Molecular Analysis Section-BIMAS). Interested antigens (or a full-length proteins, or immunogenic fragments) can then be obtained and cleaned using standard methods. For example, epitopes of interest or full-antigens can be obtained by recombinant or chemical synthesis using standard methods. Substantially purified peptide preparation should give only the main strip in non-polyacrylamide gel. In other examples, the antigen of interest includes crude viral lysate.
In one example, the antigen of interest is associated with a tumor antigen, and amino acid sequences containing motifs binding to HLA represent such p is coherence, which determine subdominant or hidden epitopes. Such epitopes can be identified by a lower relative affinity of binding to the HLA molecule compared to other epitopes in the molecule or in comparison with other molecules that bind to the HLA molecule.
Through the study of antigen analogues with single substitutions of amino acids and sequencing of endogenous associated subjected to natural processing of peptides identified critical residues, which correspond to the motifs required for specific binding molecules with HLA antigen (see, e.g., Southwood et al., J. Immunol. 160:3363, 1998; Rammensee et al., Immunogenetics 41:178, 1995; Rammensee et al., J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, Curr. Opin. Immunol. 6:13, 1994; Sette and Grey, Curr. Opin. Immunol. 4:79, 1992). Moreover, x-ray crystallographic analysis of complexes of HLA-peptide revealed pockets inside peptidase slit of HLA molecules that provide accommodation residue peptide ligands specific for allele way; these residues in turn determine the ability to bind HLA peptides in which they are located. (See, for example, Madden, Annu. Rev. Immunol. 13:587, 1995; Smith et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994; Jones, Curr. Opin. Immunol. 9:75, 1997; Brown et al., Nature 364:33, 1993.)
The desired antigen is chosen on the basis of the individual subject treated. For example, if an individual need is tsya in increased antiviral or antifungal immunity, choose one or more related virus or fungus antigens are targets. Illustrative of interest antigens from viruses include, among others, the antigens of the Epstein-Barr (EBV), hepatitis C virus (HCV), cytomegalovirus (CMV), simple herpes virus (HSV), BK virus, JC virus and human immunodeficiency virus (HIV). Illustrative of interest antigens from fungi include antigens fromCandida albicans,Cryptococcus, Blastomyces and Histoplasma or other infectious agent. In another example, the individual needs to be increased antitumor immunity. Illustrative of interest antigens from tumors include WT1, PSA, PRAME. Illustrative antigens of interest are listed in tables 1 and 2. In some examples, the antigen of interest includes, and viral antigen and a tumor antigen, a fungal antigen, and a tumor antigen or viral antigen, a fungal antigen, and a tumor antigen.
For the treatment of an individual with a tumor of interest to a tumor antigen selected on the basis of the expression of tumor protein recipient. For example, if a recipient has a tumor of the breast, choose the antigen is a tumor of the breast, and if the recipient has prostate cancer, then choose the antigen prostate tumors, etc., table 2 lists illustrative of the tumor and associated with tumor antigens, to the which you can use to obtain the purified specific antigen, T-cells, you can enter an individual with a specific tumor. However, the specialist in the art should understand that the same or other tumors can be treated with additional tumor antigens.
In one example, the specific antigen T cells that recognize tumor antigen, is administered in a therapeutically effective amount to an individual who has been or is to be entered allograft or autograft stem cells or which were vaccinated with tumor antigen. For example, you can enter a therapeutically effective amount of specific antigen by T-cells that recognize one or more tumour-related antigens, e.g., at least interesting of the antigens listed in tables 1 and 2.
In the specific examples, when the recipient has a tumor or when he has or when they must be received allograft, donor specific antigen tumor T cells and antagonist PD-1 is administered in a therapeutically effective amount of post-transplant allograft stem cells to prevent, reduce or delay recurrence of the tumor or to treat malignant relapse. Peeled specific antigen T cells can enter the individual after the reduction. In adempiere recipient Vaccinium interested tumor antigen, purified specific antigen T cells, purified from the recipient and then re-introduced to the recipient with a therapeutically effective amount of the antagonist PD-1 to enhance the immune response of the recipient against the tumor.
The introduction of a therapeutically effective amount of specific antigen by T-cells and a therapeutically effective amount of the antagonist PD-1 can be used prophylactically to prevent recurrence of the tumor in the recipient or for the treatment of tumor recurrence. Such specific antigen T cells can cause cell death, containing associated with a tumor antigen, or to help other immune cells.
In a separate example, the recipient has a tumor and he has or he should get allograft stem cells to restore immunity. After irradiation of the bone marrow or the introduction of cytotoxic drugs, which destroyed or otherwise violated the bone marrow, enter at least two types of donor specific antigen T-cells in a therapeutically effective amount; specific antigen T cells, which recognize associated with the virus antigen (or associated with fungus antigen), and specific antigen T cells that specifically recognize associated the tumor antigen. In addition, the individual is administered a therapeutically effective amount of the antagonist PD-1. This introduction can be used for the induction of antitumor effect and antiviral effect (such as antiviral effect).
To obtain population-specific antigen to T-cells for introduction to the interested individual populations of cells, including T cells, may come in contact with antigen presenting cells (APC) such as dendritic cells or T-APC, for the presentation of the antigen of interest. In some embodiments, the implementation of the corresponding T-cells (such as lymphocytes or PBMC) treated with antagonist PD-1 and add to APC, presenting one or more antigens of interest, and incubated under conditions sufficient to provide for the interaction between antigen presenting APC and T-cells, obtaining specific antigen T-cells. Processing of responding T-cell antagonist PD-1 can be carried out simultaneously with the contacting or APC. Processing antagonist PD-1 can also be made directly before contact with the APC.
Thus, in the present description provides methods to obtain enriched populations of specific antigen by T-cells. Usually T-APC presenterat antigens to T cells and induce limited MHC response class I (CD8+ T-cells)and class II (CD4+ T-cells) in the restricted type. A typical response of T-cells is the activation and proliferation. Thus, there is a population, which includes T-cells that specifically recognize the antigen of interest. Thus, the individual, such as an individual with a chronic infection or tumor, you can enter a therapeutically effective amount of this cell population to obtain an immune response.
Usually cells APC and T cells are autologous. In specific non-limiting examples of cells APs and corresponding T-cells taken from the same individual. However, the APC and the responding T-cells can be synergistic. APC can be used for presentation of the antigen population of autologous T-cells. Specialist in the art it should be clear that antigenic peptides that bind to MHC molecules of class I and II, can be obtained ex vivo (for example, instead of the processing of a full length protein in the cell) and to provide interaction (such as binding) molecules MHC class I and II on the cell surface. Usually APC presenterat antigen in the context of both class I and class II MHC.
In one example, the desired antigen, incubated with APC, is a hybrid protein which comprises the amino acid sequence of the antigen of interest (such as a sequence of 8-50 amino acids, for example the settlement of egovernance from 8 to 15 or 8 to 12 amino acids of the antigen of interest). Thus, the number of binding MHC epitopes can be included in one antigenic polypeptide, or can be used as a trimer in the same chain, where each trimer contains a molecule MHC class I, b2 microglobulin and interest antigenic peptide (see Nature 2005; V. 436, R. 578). In some examples use only one antigen, but in other embodiments implement the use of more than one antigen, for example at least 2 different antigen, at least 3 different antigen, at least 4 different antigen, at least 5 different antigens, at least 10 different antigens, at least 15 different antigens, at least 20 different antigens or even at least 50 different antigens.
In some examples, the antigen of interest is a full-antigenic amino acid sequence (such as full-fungal antigen, a tumor antigen or viral antigen such as a viral lysate or full-size cathepsin G). In additional examples, you can use one or more antigens of the infective agent. Some examples are interested in full-antigen is expressed APC.
APC can be obtained using methods known to the person skilled in the art (see Melenhorst et al., Cytotherapy 7, supp.1, 2005; Melenhorst et al., Blood 106:671a, 2005; Gagliardi et al., Int. Immunol. 7:1741-52, 1995, included in this is e description as a reference). In one example, to obtain T-APC monocytes peripheral blood donor activated with IL-2 and antibodies that specifically binds CD3 (such as OKT3) for about three days or more, for example from about one to two weeks, for example at approximately from seven to ten days.
It has been observed that in the presence of presentiamo antigen T cells that recognize the antigen will bind to the antigen presenting cells (APC), presenting antigen of interest, stronger than cells that are not specific antigen (and thus not linked specific antigen). In a separate example of a specific antigen, the T cells are selected by exposure of APCs with the target peptide antigen (such as target associated with viral or tumor antigen) against which must be sent to the desired T cells, in the presence of antagonist PD-1, so that the APC presents antigen in connection with the major histocompatibility complex (MHC) class I and/or class II. For example, APC can be exposed with a sufficient quantity of interest antigen sufficient for the occupation of MHC molecules on the APC surface (for example, occupies at least 1% of MHC molecules, for example at least 5%, at least 7.5% or at least 10%) and can stimulate a preference for the equipment linking T-target cells in the presence of antagonist PD-1 APC, presenting antigen of interest (compared to the APC, which is not presenterat antigen of interest). The population of T-cells, such as population, which was premirovan antigen of interest, and then incubated with APC optional in the presence of antagonist PD-1, such as an antibody that specifically binds PD-1, for the preferred cell activation with obtaining thus a population of cells enriched in the desired T-cells that recognize the antigen of interest.
T-cells, such as those found in populations of PBMC or lymphocytes can be incubated with one or more antigens of interest is not necessarily in the presence of antagonist PD-1 to obtain a population of T-cells, which premirovan in relation to one or more antigens of interest. T-cells can be premirovat using any method known in the art. In some examples, PBMC or lymphocytes incubated in the presence of purified target peptide antigen optionally in the presence of antagonist PD-1. In some examples, the antigen of interest is a viral or tumor antigen, such as, but not limited to, one or more antigens of interest, listed in table 1. The desired antigen can be in purified form, such as chemically synthesized peptide. In other primarykeyname antigen is in the crude form, such as crude lysate, for example viral lysate.
The amount of antigen used for premirovany T-cells, can easily be determined using methods known in the art. Usually, if the antigen used in purified form, use approximately 1 to 10 μg/ml peptide. When using viral lysate can be used is approximately 0.1 to 100 μl of the lysate, for example approximately 75 µl. When using the T-APC can be used approximately 4-6 million T-APC presenting antigen of interest, for every 40-60 million T-cells (or lymphocytes, or PBMC).
In a separate example, the lymphocytes will primesouth in vitro by incubation with soluble antigen or viral lysate for 5-7 days under conditions that provide premirovanii T-cells. Viable T cells secrete, for example, by centrifugation in ficoll-hipace with getting in premirovany T-cells. If this is desirable, viable bromirovannye T cells can be re-premirovat one or more times, for example, by incubation with antigen for 5-7 days in the same conditions that were used for the first premirovany, and select viable T cells.
In another example, the cells will primesouth in vivo by inoculation of individual antigen, for example, in the form of a vaccine. In this example, T-cells, the floor is obtained from the individual after immunization, already premirovany. For example, cells or PBMC obtained from an individual, then incubated with APC in the presence of antagonist PD-1, as described in the present description, without the need for additional premirovanii.
The method may additionally include receiving APC, which presenterat antigen of interest. For example, APC can be incubated with a sufficient quantity of one or more different peptide antigens under conditions sufficient for the presentation of the target peptide (peptides) on the surface of APC. This creates a population of APC, which presenterat interest antigen on the MHC molecules on the surface of APC. Disclosed methods are not limited to specific methods of presentation are interested antigen on the surface of the APC.
Antigens can also be expressed APC either naturally, or due to the insertion of a gene containing a DNA sequence that encodes a target protein (antigen). Nucleic acid encoding the desired antigen can be introduced into the T cells in the form of messenger RNA, or by using a vector such as an expression vector for mammalian or viral vectors (e.g. adenovirus, poxvirus or retroviral vectors). Polynucleotide encoding the antigen of interest include recombinant DNA, which is a standalone replicated plasmid or virus, or is that included in the genomic DNA of eukaryotes, or which exists as a separate molecule, independent of other sequences. Nucleic acid encoding the desired antigen can also be entered using electroporation, lipofection or based on calcium phosphate transfection.
Was designed a number of viral vectors, including polyoma, i.e., SV40 (Madzak et al., 1992, J. Gen. Virol., 73:1533-1536), adenovirus (Berkner, 1992, Cur. Top. Environ. Immunol., 158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Nad. Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256), the smallpox virus (Mackett et al., 1992, Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top. Environ. Immunol., 158:91-123; On et al., 1990, Gene, 89:279-282), the herpes viruses, including HSV, CMV and EBV (Margolskee, 1992, Curr. Top. Environ. Immunol., 158:up 67-90; Johnson et al., 1992, J. Virol., 66:2952-2965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199), viruses sindbis (H. Herweijer et al., 1995, Human Gene Therapy 6:1161-1167; U.S. patent No. 5091309 and 52217879), alpha viruses (S. Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377) and retroviruses birds (Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J. Virol., 66:3391-3397), mouse (Miller, 1992, Curr. Top. Environ. Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol., 54:401-407) and man (Page et al., 1990, J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739). In the art Izv the STN also baculovirus (Autographa californica multinuclear poladroid.net virus; AcMNPV) vectors, and they can be obtained from commercial sources (such as PharMingen, San Diego, Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).
In one embodiment, polynucleotide encoding antigen of interest include viral vector for transportation in APC. Suitable vectors include retroviral vectors, orthodoxie vectors, avipox vectors, fowlpox vectors, capripox vectors, swiaczny vectors, adenoviral vectors, vectors, herpes virus, alphavirus vectors, baculovirus vectors, vectors of virus sindbis, the vectors of the smallpox virus and poliovirus vectors. Specific illustrative vectors are poxvirus vectors, such as the smallpox virus, fowlpox virus and highly attenuated smallpox virus (MVA), adenovirus, baculovirus, and the like.
Used poxviruses include Orthodoxy, swiaczny, avidoxy and carboxy viruses. Orthopox includes smallpox, ectromelia and raccoonpox. One of the examples used ortodoxa is smallpox. Avipox includes fowlpox, smallpox Canaries and smallpox pigs. Capripox includes smallpox goats and sheep pox. In one example, sipox is a pox swine. Examples of poxvirus vectors for expression are described, for example, in U.S. patent No. 6165460, which is included in the present description by reference. Other viral vectors, which is s can be used, include other DNA viruses such as herpesviruses and adenoviruses, and RNA viruses, such as retroviruses and poliovirus.
Suitable vectors are disclosed, for example, in U.S. patent No. 6998252, which is included in the present description by reference. In one example, a recombinant poxvirus, such as a recombinant virus of smallpox, synthetically modify by inserting a hybrid gene containing the regulatory sequences of the virus or DNA sequence is functionally equivalent flanking DNA sequences, which in natural conditions do not coexist with flanking regulatory sequences of DNA virus that encode the antigen of interest. The recombinant virus containing such hybrid gene, effective for expression of the antigen. In one example, the vector of the virus of smallpox includes (A) a segment consisting of (i) a first DNA sequence that encodes the antigen, and (ii) the poxvirus promoter, where the promoter of the poxvirus is adjacent to a DNA sequence that encodes an antigenic polypeptide, and carries out its transcriptional control; and near the specified segment (B) DNA from a nonessential region of the poxvirus. The viral vector can encode a marker selection. In one example, poxvirus includes, for example, gene timeintensity (see U.S. patent No. 6998252, which is included in toadie description as a reference).
The population of APC, which presenterat antigen(s) in sufficient quantity, incubated with T cells (such as lymphocytes or PBMC) optionally in the presence of an effective amount of the antagonist PD-1 under conditions sufficient to facilitate binding of antigen presenting APC with T-cells that are specific immunological interact with antigen (specific antigen T-cells). Use a sufficient number of APC expressing the antigen with MHC in a quantity sufficient to stimulate increased binding of the desired T cells with APC. In some examples, at least 20% APC presenterat desired antigen on the MHC molecules on the APC surface, for example at least 30% of the APC, at least 40% of the APC, at least 50% of APC or at least 60% of the APC. The optimal number of added T-cells can vary depending on the used number of APC. In some examples use the ratio of T-cell: APC, equal to at least 6:1, for example at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 16:1, at least 20:1 or even at least 50:1.
To increase the number of specific antigen by T-cells can be stimulated cell proliferation, e.g., by incubation in the presence of the cytokine, such as IL-2, IL-7, IL-12 and IL-15. To icesto added cytokine is sufficient to stimulate the adoption and proliferation of T-cells and can be determined using traditional methods. In some examples, the number of added IL-2, IL-7, IL-12 or IL-15 of about 0.1-100 IU/ml, for example at least 1 IU/ml, at least 10 IU/ml or at least 20 IU/ml
After binding of sufficient specific antigen T cells with APC receive T cells that are specific recognize antigen of interest. This creates a population enriched (e.g., purified) specific antigen T-cells that are specific antigen of interest. In some examples, the resulting population of T-cells that are specific in relation to the antigen of interest has at least 30% purity, for example at least 40% purity or even at least 50% purity. Purity populations of T-cells specific against the antigen can be assessed using methods known to the person skilled in the technical field.
In one example, during stimulation of the proliferation of specific antigen by T-cells cells can be calculated to determine the number of cells. After reaching the desired number of cells determine the purity. Purity can be determined, for example, using markers located on the surface of specific antigen by T-cells, simultaneously with the assessment of the production of cytokines after antigen recognition, such as in alferon (IFN)γ, the tumor necrosis factor (TNF)α, interleukin (IL)-2, IL-10, transforming growth factor (TGF)β1 or IL-4. Usually specific antigen T cells are positive for the marker CD3, along with a marker for CD4 or CD8 and IFNγ (which is specific for activated T-cells). For example, to identify (and sorting, if this is desirable) populations of cells that are positive for CD3, CD4 or CD8 and IFN-γ, we can use the sorting of fluorescently activated cells (FACS) using differentially stained with anti-CD3, anti-CD4, anti-CD-8, and anti-IFN-γ. Briefly, stimulated by a specific antigen T cells incubated in the presence of anti-CD3, anti-CD4, anti-CD-8, and anti-IFN-γ (each contains the distinguished attached fluorophore) in a period of time sufficient for binding of the antibody with the cells. After removal of unbound antigen cells analyzed by FACS using traditional methods. Specific antigen, T-cells are those cells that are positive for IFN-γ and positive or CD8 positive for CD4. In some examples, the resulting population of antigenic T-cell has a purity constituting at least 30% relative to the total population of CD4+ or CD8+ positive cells, for example at least 40% pure, at least 50% pure, at least 60% purity or on the same at least 70% purity compared with the total population of CD4 positive or CD8 positive cells.
In another example, the method additionally includes determining the cytotoxicity of specific antigen by T-cells. Methods for determination of cytotoxicity known in the art, such as test selection51Cr (see, for example, Walker et al. Nature 328:345-8, 1987; Qin et al. Acta Pharmacol. Sin. 23(6):534-8, 2002; included in this description by reference).
Specific antigen T cells can be subjected to one or more rounds of selection to increase the purity of the specific antigen to T-cells. For example, purified specific antigen T cells, obtained as described above, again incubated with APC that presents the desired antigen, in the presence of antagonist PD-1 under conditions sufficient to facilitate binding of APC with purified specific antigen T-cells. Received specific antigen T cells can be stimulated to proliferation, for example, using IL-2. Usually obtained specific antigen T cells that are specific immunological interact with the antigen of interest, are cleaner after subsequent rounds of stimulation with APC compared with cells obtained from one round of selection. In one example, the resulting population of purified specific antigen T-cells have the t at least 90% purity relative to all CD3+ cells, for example at least 95% purity, or at least 98% purity. In a separate example of the obtained population of purified specific antigen T-cell has at least 95% purity relative to all available CD4+ cells, for example at least 98% purity. In another example, the resulting population of purified specific antigen T-cell has at least 90% purity relative to all CD3+ cells, for example at least 93% purity.
The present disclosure also provides therapeutic compositions that include enriched (e.g., peeled) specific antigen T cells and antagonist PD-1. In some examples, the obtained enriched population specific antigen T-cell specific antigen of interest) is placed in a dosage form for administration to the needy in this individual. Antagonist PD-1 is also in the dosage form for administration to the needy in the individual.
In one example, the resulting population of purified specific antigen T-cell has at least 30% purity relative to all CD3+ cells, for example at least 40% pure, at least 50% pure, at least 80% purity or even at least 90% purity. In a separate example of the obtained population of imennyh specific antigen T-cell has at least 30% purity relative to all CD3+ cells, for example, at least 40% pure, at least 50% pure, at least 80% pure, at least 90% pure, at least 95% purity, or even at least 98% purity. In another example, the resulting population of purified specific antigen T-cell has at least 50% purity relative to all CD3+ cells, for example at least 60% pure, at least 75% pure, at least 80% pure, at least 90% purity or even at least 93% purity. Multiplied and selected specific antigen T cells can be checked for the presence of Mycoplasma, sterility, endotoxin and quality controlled by function and clean before storing at low temperature or before the infusion of the recipient.
The individual is administered a therapeutically effective amount of specific antigen by T-cells. Specific non-limiting examples, a therapeutically effective amount of purified specific antigen T-cells include purified specific antigen T cells, administered in a dose from about 1×105cells per kilogram of the individual to approximately 1×109cells per kilogram of the individual, for example from about 1×106cells / kg to about 1×108cells / kg, for example from p is blithedale 5×10 6cells / kg to about 75×106cells per kilogram, for example, approximately 25×106cells per kilogram or approximately 50×106cells per kilogram.
Peeled specific antigen T cells can be introduced in one or multiple doses by the decision of the doctor. For example, cells can be entered at intervals of approximately two weeks, depending on the desired response and the response received. In some examples, upon receiving the desired response additional introduction of a specific antigen by T-cells do not produce. However, if the recipient manifest one or more symptoms associated with infection or the presence or growth of the tumor, at this point you can enter a therapeutically effective amount of specific antigen by T-cells. The introduction may be local or systemic.
Disclosed in the present description, peeled specific antigen T cells can enter with a pharmaceutically acceptable carrier, such as saline. Antagonist PD-1 can be formulated in pharmaceutically acceptable carrier, as described above. In some examples with specific antigen, T-cells and antagonist PD-1 is administered with other therapeutic agents. Other therapeutic agents can be entered before, during, and after centuries the Denia specific antigen T-cells depending on the desired effect. Illustrative therapeutic agents include, but are not limited to, antimicrobial agents, Immunostimulants, such as interferon-alpha, chemotherapeutic agents, or a peptide vaccine of the same antigen used for stimulation of T-cells in vitro. In some example compositions containing purified specific antigen T cells, also include one or more therapeutic agents.
The disclosure is illustrated by the following non-limiting examples.
Inhibition of paths PD-1 in mice with chronic infection with antibodies against PD-L1
To study the effect of chronic viral infection on the function of CD8+ T-cells used mice infected with different strains of the virus lymphocytic choriomeningitis (LCMV). The strain of LCMV Armstrong causes an acute infection that is terminated within 8 days, leaving a long-lived population of highly functional resting CD8+ T-cell memory. Strain C1-13 LCMV, on the contrary, causes a sustained infection in the host, different viremia that lasts up to 3 months. The virus remains in certain tissues indefinitely, and specific antigen T cells become functionally defective. CD8+ T cells DbNP396-404 physically disappear, and CD8+ T cells DbGP33-41 and DbGP276-286 sohranjajut is, but lose the ability to proliferate or secrete antiviral cytokines such as IFN-γ and TNF-α.
Mice C57BL/6 were obtained from the National cancer Institute (Frederick, MD). Mice were infected intravenously (in/in) 2×106PFU LCMV-Cl-13. Depletion of CD4 produced by injection of 500 µg GK1.5 in SFR on the day of infection and on the next day after infection. Immunized with LCMV get infection in mice/br 2×105PFU LCMV Armstrong.
Genetic analysis using microarrays was performed on FACS purified naïve specific DbGP33-41 P14 transgenic CD8+ T-cells specific against DbGP33-41 CD8+ T-cell memory obtained from immunized with LCMV Armstrong mice, and specific DbGP33-41 or DbGP276-286 CD8+ T-cells obtained from mice infected with LCMV Cl-13 C-depleted CD4+. The allocation of RNA and gene expression analysis using microarrays was performed as described in Kaech et al., (Cell 111:837-51, 2002). mRNA PD-1 expressives at a high level in exhausted CD8+ T cells compared to CD8+ T-cell memory (Fig.1A). Moreover, PD-1 expressively on the surface of CD8+ T cells in infected LCMV C1-13 mice, but was absent on the surface of CD8+ T-cells after clearance of LCMV Armstrong (Fig.1B). In chronically infected mice also at a higher level expressively one of the ligands of PD-1, PD-L1, the majority of lymphocytes and APC is compared with uninfected mice. Thus, the preservation of viral antigen and depletion of CD8+ T-cells occur simultaneously with the induction of the expression of PD-1.
To test the hypothesis that blockade of road PD-1/PD-L1 can restore the function of T-cells and to enhance viral control during chronic LCMV infection, congenitally path PD-1/PD-L1 was destroyed during chronic LCMV infection using blocking antibodies against αPD-L1. A blocking monoclonal antibody against PD-L1 was administered intraperitoneally (br) for each 3 day mice infected with LCMV C1-13 (200 µg monoclonal antibody IgG2b rat against PD-L1 mouse (clone 10F.5C5 or 10F.9G2)), from 23 to 37 days after infection. On the 37th day in mice treated was approximately 2.5 times more specific in relation to DbNP396-404 CD8+ T-cells and 3 times more specific in respect of DbGP33-41 CD8+ T-cells compared to control mice without treatment (Fig.2A). Induction of proliferation was specific for CD8+ T-cells, as CD4+ T-cells in the liver was approximately the same in mice subjected and not subjected to treatment (~6×104 IAbGP61-80 CD4+ T-cells in the spleen).
Besides the increased proliferation of CD8+ T-cells, inhibition of signaling PD-1 also caused increased production of antiviral cytokines in specific virus CD8+ T-cells. Was determined products of IFN-γ and IFN-α CD8+ T cells in response to the eight different CTL epitopes. The average response was 2.3 times higher in mice treated compared with mice without treatment (Fig.2B and 2C). After treatment was also observed 2-fold increase in the frequency of occurrence producing TNF-α cells (Fig.2D). Removing the virus is also accelerated because it was removed from the serum, spleen and liver of the treated mice. Lower titres were observed in the lung and kidney (~10-fold) to 37 days after infection (14 days after start of treatment) in the treated mice. However, in mice not subjected to treatment, we detected significant levels of virus in these tissues (Fig.2E). The titres in serum and tissue homogenates were determined using Vero cells as described in Ahmed et al. (J. Virol. 51:34-41, 1984). The results indicate that the antagonist PD-1 increases the proliferation of CD8+ T-cells and virus removal, thus, indicate that inhibition of signaling PD-1 restores the function of the CD8+ T-cells. Moreover, inhibition of signaling PD-1 also increases the responses of B-cells, because the number of secreting specific LCMV antibody cells in the spleen were increased (>10-fold) after treatment.
CD4+ T cells play a key role in the formation and maintenance of the responses of CD8+ T-cells. In this sense, CD8+ T cells, bromirovannye in the absence of CD4+ T-cells (so-called "helpless" CD8+ T-cells), the e is able to form a normal immune responses. Moreover, chronic LCMV infection is more severe in the absence of CD4+ T-cells. Accordingly, helpless T cells encountered during infection LCMV-C1-13, are even more profound functional impairment compared with T-cells occurred in the presence of CD4+ T-cells. Specific DbNP396-404 CD8+ T-cells are removed to undetectable levels, and DbGP33-41 and DbGP276-286 CD8+ T cells completely lose their ability to secrete IFN-γ and IFN-α.
CD4+ T cells were depleted during infection LCMV-C1-13, and mice were treated by treatment with antibodies against PD-L1 with 46 60 day after infection. Specific LCMV CD4+ T cells was not detected according to intracellular staining of IFN-γ before or after processing. In mice treated with treatment, there were approximately 7 times more DbGP276-286 CD8+ T-cells and 4 times more DbGP33-41 CD8+ T cells in the spleen compared with the control not treated mice (Fig.3A). A number of specific virus CD8+ T-cells in the spleen was also increased (Fig.3B). This increase is specific virus CD8+ T-cells in the treated mice was explained by increased proliferation, as detected by BrdU incorporation. In mice not subjected to treatment, 43% DbGP276-286 CD8+ T-cells incorporated BrdU at an intermediate level and 2% was incorporated BrdU at a high level, whereas in mice treated with 50% DbGP276-286 CD8+T-cells incorporated BrdU at an intermediate level and 37% had incorporated BrdU at a high level. Analysis using BrdU was performed by injecting 1 mg/ml BrdU in drinking water during processing, and staining produced in accordance with the Protocol of the manufacturer (BD Biosciences, San Diego, CA). Moreover, the treated mice had a higher percentage of CD8+ T-cells expressing associated with the cell cycle protein Ki67 (60% vs. 19% in mice without treatment, Fig.3C). Response to treatment CD8+ T-cells in PBMC was characteristic only for mice with a high level of expansion of CD8+ T-cells.
Inhibition of PD-1 also increased the production of antiviral cytokines in helpless, exhausted specific virus CD8+ T-cells. After processing, the number of DbGP33-41 and DbGP276-286 CD8+ T-cells producing IFN-γ were significantly increased (Fig.4A), while the treated mice were also detected a greater number of CD8+ T-cells specific DbNP396-404, KbNP205-212, DbNP166-175 and DbGP92-101 (Fig.4A). 50% specific DbGP276-286 CD8+ T-cells from the treated mice are able to produce IFN-γ, compared with 20% specific DbGP276-286 CD8+ T-cells in the control not treated mice (Fig.4B). The levels of IFN-γ and TNF-α produced specific DbGP276-286 CD8+ T cells from treated mice were, however, lower compared to the fully functional specific DbGP276-286 memory cells (Fig.4C).
Inhibition of PD - also increased lytic activity helpless, exhausted specific virus CD8+ T-cells. Lytic activity of specific virus CD8+ T-cells ex vivo was determined after treatment with test selection51Cr (Wherry et al., 2003. J. Virol. 77:4911-27). The titres decreased about 3 times in the spleen, 4 times in the liver, 2 times at light, and 2 times in serum after 2 weeks of treatment compared with mice not treated (Fig.4E).
These results thus demonstrate that blockade of road PD-1 violates the tolerance of peripheral CTL to chronic viral infection and that exhausted CD8+ T cells, deprived of support from CD4+ T-cells are not irreversibly inactivated.
The introduction of antiviral vaccines and antagonist PD-1
One approach to maintaining T-cell responses in persistent infection is therapeutic vaccination. The rationale for this approach is that endogenous antigens may be not optimal or immunogenic form during chronic viral infection, and that the provision of immunogen in the form of a vaccine can provide a more effective incentive for specific virus T - and B-cells. When using a model of chronic LCMV mice were administered recombinant smallpox virus expressing the LCMV epitope GP33, as terap ticheskoj vaccine (VVGP33), that led to a moderate increase in responses of CD8+ T-cells in some chronically infected mice. Four of the nine chronically infected mice that received therapeutic vaccine, showed a positive response, whereas none of the control mice was not significantly improve the immune response against GP33. The combination of this therapeutic vaccination with an inhibitor of PD-1 answers specific LCMV T-cells were maintained at a higher level than any single treatment, and the effect of the combined treatment was superior to the additive effect.
Inhibition of paths PD-1 in chronically infected mice using Rnci PD-1
Interfering RNA (Rnci) are able to suppress gene expression in mammalian cells. Long double-stranded RNAS (dsrnas) are introduced into cells, and then they are subjected to processing to small silencingly RNAS (miRNAs), which are directed against specific mRNA molecules or small groups of mRNA. This methodology is particularly suitable in situations where antibodies do not function. For example, Rnci can be used in situations where a unique splicing variants produce a soluble form of PD-1 and CTLA-4.
Silence RNA for PD-1 is inserted into a commercially available expression vector for miRNAs, such as E. cressionnie pSilencer vectors TMor adenoviral vectors (Ambion, Austin, TX). These vectors are then injected into contact with depleted T-target cells in vivo or ex vivo (see example 4 below).
Ex vivo rejuvenation of exhausted T-cells
Specific virus CD8+ T cells isolated from chronically infected with LCMV-Cl-13 mice using magnetic beads or centrifugation in a density gradient. Transfetsirovannyh CD8+ T-cells are introduced into contact with a monoclonal antibody directed against PD-L1, PD-L2 or PD-1. As described in example 1 inhibition of paths PD-1 leads to the rejuvenation of CD8+ T-cells. Accordingly, when, for example, increased proliferation of CD8+ T-cells and production of cytokines. These rejuvenated CD8+ T cells again introduce infected mice, and the amount of virus measured as described in example 1.
In vitro screening of new anti-aging CD8+ T-cell connections
Compounds that modulate the path PD-1, can be identified using screening tests in vivo and in vitro on the basis of their ability to pay depletion of CD8+ T-cells in chronic viral infection.
Exhausted CD8+ T-cells from mice chronically infected with LCMV-C1-13, and then enter into contact with the test compound. A number of antiviral cytokines (such as IFN-γ or TNF-α), isolated from contact with T-cells, and the measured for example, using ELISA or other quantitative method and compare with the amount, if any, antiviral cytokine selected from the depleted T-cells, not exposed to the test compound. The increase in the number of antiviral cytokine allocated-treated cells, compared with the amount in untreated cells identifies the compound as an antagonist PD-1, suitable to modulate the activity of T-cells.
In vivo screening of new anti-aging CD8+ T-cell connections
Exhausted CD8+ T cells obtained from mice chronically infected with LCMV-C1-13. Infected mice intravenously injected test the connection. A number of antiviral cytokines (such as IFN-γ or TNF-α), which is secreted in the serum subjected and not subjected to the treatment of mice, measured, for example, using ELISA or other quantitative method and compare. The increase in the number of antiviral cytokine detected in the serum of the treated mice compared with its number of non-treated mice, identifies the test compound as an antagonist PD-1. Alternative before and after treatment, the test compound can be determined the titer of virus (e.g., serum titer of the virus).
Chimpanzees as the model for immunotherapy stable HCV infection
Chimpanzees serve as a model of stability of HCV in humans. Defects in T-cell immunity, leading to lifelong persistence of the virus include insufficiency of specific HCV CD4+ T-helper cells, and impaired or altered activity of CD8+ T-effector cells. Persistently infected chimpanzees treated with antibodies against CTLA-4, PD-1 or a combination of both. Determine the effectiveness of the blockade of inhibitory pathways in combination with vaccination using recombinant structural and non-structural HCV proteins and the ability of such strategies to increase the frequency of occurrence and duration of life specific virus T-cell memory. Failure of T-cell immunity is extremely specific in relation to HCV in persistently infected humans and chimpanzees. Examine the blood and liver of infected chimpanzees on the expression of CTLA-4, PD-1, BTLA and their ligands and the presence of Treg cells. Antiviral activity can then be restored by introducing a humanized chimpanzee monoclonal antibodies that block signaling through these molecules.
Persistently infected chimpanzees treated humanitarianism antibodies αCTLA-4 (MDX-010, Medarex) or antibodies αPD-1. The initial dose of MDX-010 is 0.3 mg/kg, followed by 2 weeks 1.0 mg/kg and then a three-week intervalli, 10, 30 mg/kg After treatment with antibodies against congenerous molecules necessary to determine the humoral and cellular immune responses, as well as the level of HCV RNA. Samples collected at 1, 2, 3, 5 and 8 weeks and then at monthly intervals. Samples include: 1) serum analysis of transaminases, antibodies, neutralizing antibodies against HCV and answers cytokines, 2) plasma for viruses and the evolution of the genome, 3) PBMC for in vitro measurements of the immune system, expression and function of co-stimulating/inhibiting receptors, 4) fresh (not fixed) to the liver for excretion intrahepatic lymphocytes and RNA and 5) fixed (formalin/enclosed in paraffin) liver for histological and immunohistochemical analysis. Collect regional lymph nodes in 2 or 3 time points to assess the expression congenerous molecules and splicing variants using immunohistochemical and molecular methods. Tests to assess the efficacy and safety of this treatment should be carried out as described in the present description.
To determine potentiates whether vaccination with antigens of HCV therapeutic effect of antibodies against PD-1, chimpanzees are treated as follows: 1) intramuscular immunization with recombinant glycoproteins E1 and E2 envelope (in Freund MF59) and other proteins (centre plus NS 3, 4 and 5, made with ISCOMS) at 0, 4 and 24 weeks is s; 2) intramuscular immunization with the vaccine used in 1), but added together with antibodies αCTLA-4 (30 mg each per kg of body weight intravenously at 0, 4 and 24 weeks, when introducing the vaccine); 3) identical 2) except that instead of the antibody CTLA-4 using antibodies αPD-1 (or BTLA); 4) identical groups 2 and 3 except that in addition to the vaccine uses a combination of antibodies to CTLA-4 and PD-1 (or BTLA). After immunization during 1 year follow specific HCV T - and B-cell responses.
The markers evaluated in HCV-tetramer+ and total T-cells in this assay include markers of differentiation (e.g., CD25, CD69, CD38 and HLA-DR), survival/proliferation (e.g., bcl-2 and Ki67), the potential cytotoxicity (e.g., granzyme and perforin) and receptors cytokines (CD122 and CD127). Between the level of the chemokine IP-10 before treatment and response to PEG-IFN-γ/ribavirin, there is an interesting correlation. The level of IP-10 was measured to study the potential correlation between negative regulatory pathways or specific HCV T-cell responses and the level of IP-10. The expression of inhibitory receptors and ligands on PBMC assessed using flow cytometry.
Immunoablative PD-1 in reactive lymphoid tissue
The materials of judicial practice were obtained from the Brigham & Women's Hospital, Boston, MA correspondence is established in the institution rules. All diagnoses were based on histological and immunophenotypic features described in the system of classification of lymphomas of the world health organization (Jaffe ES, et al. 2001), and in all cases the diagnostic material was checked by hematopathology.
Immunoablative PD-1 was performed on formalin fixed prisoners in paraffin sections of tissue after the restoration of antigen processing in a microwave oven in 10 mm citrate buffer, pH 6,0 previously described monoclonal antibody against PD-1 person (2H7; 5) using the indirect method, avidin-Biotin-horseradish peroxidase and manifestations of dyeing by diaminobenzidine, as described previously (Jones D, et al. 1999; Dorfman DM, et al. 2003). Cases were considered as immunoreactive against PD-1, if at least 25% of the neoplastic cells showed positive staining. To confirm the specificity of staining staining of PD-1 compared with staining with control antibody mouse isotype IgG, diluted to the same concentration of protein in all investigated cases.
For staining fixed in formalin prisoners in paraffin samples reactive lymphoid tissue, thymus and the number of cases of b-cell and T-cell lymphoproliferative disorders used a monoclonal antibody 2H7 against PD-1. In samples of tonsil, showing jet-ISM is the link, including follicular hyperplasia, mainly small subpopulation of lymphocytes in the breeding centers revealed cytoplasmic staining of PD-1 with a rare PD-1-positive cells detected in mitlitary T-cell zones. The nature of the staining of PD-1 in the centers of reproduction was virtually identical to the nature of the staining antibody against CD-3, a marker of all T-cells, whereas the antibody against CD-20, the marker of all B-cells were stained with the vast majority of B-cell centers reproduction. Similar results were observed on histological sections reactive lymph nodes and spleen. Staining of PD-1 was not observed in the adult thymus.
Immunoablative PD-1 in prisoners in paraffin sections of tissue by B-cell and T-cell lymphoproliferative disorders
Investigated a number of B-cell and T-cell lymphoproliferative disorders on the subject of expression of PD-1; the results are summarized in table 4. Examined forty-two cases of B-cell lymphoproliferative disorders on the subject of expression of PD-1, including typical cases of lymphoblastic leukemia/lymphoblastic lymphoma predecessors of B-cells, as well as a number of lymphoproliferative disorders of Mature B-cells, including a number of B-cell non-Hodgkin's lymphomas of follicular origin, including 6 cases of follicular lymph is s and 7 cases of Burkitt lymphoma. In any B-cell lymphoproliferative the violation was not observed staining of PD-1. In some cases there were neoplastically reactive lymphoid tissue, which was observed pattern of staining of PD-1, was discovered in the amygdala and other reactive lymphoid tissues, noted above.
Similarly in 25 cases jackinsky lymphoma, including 11 cases of classical hodgskins lymphoma and 14 cases jackinsky lymphoma with a predominance of lymphocytes, has not been demonstrated staining of PD-1 in neoplastic cells. Interestingly, all 14 cases jackinsky lymphoma with a predominance of lymphocytes T-cells surrounding the neoplastic CD20-positive L&H cells were immunopositive against PD-1, similar to the nature of the staining observed for CD57+ T-cells in Hodgkin's lymphoma with a predominance of lymphocytes. These PD-1-positive cells represented a subpopulation of the existing total population of CD3+ T-cells.
Investigated a number of T-cell lymphoproliferative disorders on the subject of expression of PD-1; the results are summarized in table 4. Cases of lymphoblastic leukemia/lymphoblastic lymphoma of precursor T-cells, neoplasma immature T-cells were negative against PD-1, as the cases of neoplasm peripheral posttympanic T-cells, including T-cell prolymphocytic leukemia is, lymphoma peripheral T-cells, unspecified anaplastic both lymphoma and T-cell leukemia/lymphoma in adults. On the contrary, in all 19 cases angioimmunoblastic lymphoma there were pockets of PD-1-positive cells that were also immunoreactive against markers for all T-cells, such as CD-3. PD-1-positive cells were consistently found in the pockets of networks breeding CD21+ follicular dendritic cells (FDC), which is a characteristic feature angioimmunoblastic lymphoma.
General methods of studying the expression of PD-1 on specific HIV CD8+ T-cells
For the experiments described in examples 11-14, the following methods were used.
Individuals: study Participants with HIV infection monophyletic taxon C were recruited from clinics for outpatients McCord Hospital, Durban, South Africa, and St. Mary's Hospital, Mariannhill, South Africa. Peripheral blood was obtained from 65 individuals of this group, and all subjects prior analysis had not received antiretroviral therapy. Individuals were selected for inclusion in the study based on the expression of HLA alleles, corresponding to the ten tetramera class I, which is s were constructed (see below). The average number of viruses in the cohort was 42800 copies HIV-1 RNA/ml plasma (range 163-750000), and the median absolute CD4 was $ 362 (range 129-1179). Information on the duration of infection was not available. All individuals gave written informed consent to the study, which was approved by the Supervisory committees of local institutions.
Designing antibodies against PD-1 and PD-L1 Monoclonal antibodies against PD-L1 (29E.2A3, IgG2b mouse) and PD-1 person (EH12, mouse IgGl) were obtained as described previously and have been shown to block the interaction of PD-1:PD-L1.
Tetrameric MHC class I: this study used ten tetramers HIV MHC class I, synthesized as previously described (Altman JD, et al. 1996): A*0205 GL9 (p24, GAFDLSFFL; SEQ ID NO:1), A*3002 KIY9 (integrase, KIQNFRVYY; SEQ ID NO:2), B*0801 DI8 (p24, DIYKRWII; SEQ ID NO:3), B*0801 FL8 (Nef, FLKEKGGL; SEQ ID NO:4), B*4201 RM9 (Nef, RPQVPLRPM; SEQ ID NO:5), B*4201 TL9 (p24, TPQDLNTML; SEQ ID NO:6), B*4201 TL10 (Nef, TPGPGVRYPL; SEQ ID NO:7), B*4201 YL9 (RT, YPGIKVKQL; SEQ ID NO:8), B*8101 TL9 (p24, TPQDLNTML; SEQ ID NO:9) and Cw0304 YL9 (p24, YVDRFFKTL; SEQ ID NO:10).
Staining with tetramers of HLA class I and phenotypic analysis: freshly isolated mononuclear cells from peripheral blood (PBMC, 0.5 million) were stained on tetramer for 20 minutes at 37°C. Cells then once washed with phosphate buffered saline (SFR), besieged and directly stained fluorescently the anatomist (FITZ), conjugated with anti-CD8 (Becton Dickinson), conjugated with phycoerythrin anti-PD-1 (clone EH 12) and ViaProbe (Becton Dickinson). Cells then were incubated for 20 minutes at room temperature, once washed SFR and resuspendable 200 ál SFR with 1% paraformaldehyde and took on activated fluorescence of the case (FACSCaliburTM, Becton Dickinson). On FACSCaliburTMreceived at least 100,000 events.
Tests proliferation of CFSE: One million freshly isolated PBMC were washed twice in SFR, besieged and resuspendable in 1 ml of 0.5 μm carboxyfluorescein diacetate Succinimidyl of ester (CFSE, Molecular Probes) for 7 minutes at 37°C. Cells were washed twice in SFR, resuspendable in 1 ml of medium R10 (RPMI 1640 with the addition of glutathione, penicillin, streptomycin and 10% serum embryo calves (FCS) were placed in one well of a 24-well plate. A pilot study showed that the final concentration of peptide 0.2 ág/ml provides optimal proliferative responses, so each test used the final concentration of peptide in the hole. The negative control wells contained only PBMC in the environment, or PBMC in medium with purified anti-PD-L1 (10 μg/ml), and positive control wells stimulated with 10 µg/ml phytohemaglutinin (PHA). After 6-day incubation in the incubator at 37°C. cells were washed in 2 ml SPR and were stained with conjugated the PE tetramera MHC class I, ViaProbe (Becton Dickinson) and antibodies anti-CD8-APC. Cells were extracted on a FACSCalibur and analyzed using CellQuest® software (Becton Dickinson). Cells were passed at ViaProbe with a limited window for CD8+ lymphocytes. The fold increase in tetramer+ cells was calculated by dividing the percentage of cells CD8+ tetramer+ in the presence of the peptide on the percentage of cells CD8+ tetramer+ in the absence of peptide stimulation.
Statistical analysis: correlation Analysis of Spearman, the Mann-Whitney test and paired t-test was made using GraphPad Prism Version 4.0 a. All tests were two-sided, and as was considered significant values p with p<0,05.
The expression of PD-1 on specific HIV CD8+ T-cells
Was synthesized by a panel of 10 tetramers of MHC class I-specific in relation to the dominant monophyletic taxon C virus HIV-1 epitopes CD8+ T-cells, on the basis of the predominant HLA alleles and the frequency of the targeted epitopes in Gag, Nef, RT, integrase and providing direct visualization of the expression of PD-1 on the surface of these cells. Was conducted HLA high resolution throughout the group, and for the study was selected subgroup of 65 individuals not receiving antiretroviral therapy, based on the expression of candidate HLA alleles. We examined a total of 120 individual epitopes, and in Fig.5A shows representative okra is ivanie ex vivo PD-1 on HIV tetramer+ cells. The expression of PD-1 is easily distinguishable on these tetramer+ cells and it was significantly higher than in the General population of CD8 T-cells from the same individuals (p<0,0001); in turn, the expression of PD-1 and tetramer+ CD8+ T-cells, and on cells of the total population of CD8+ T-cells was significantly higher than that of serologically HIV-negative controls (Fig.5B). For eight of the ten tested tetramers was identified at least one individual whose level of expression of specific antigen CD8+ cells was 100% (Fig.5C). PBMC from 3 to 25 individuals were stained for each response tetramer of HIV with median levels of expression in the range from 68% to 94% tetramer+ cells (Fig.5C). These data were further confirmed by analysis of mean fluorescence intensity (MFI) of PD-1 and tetramer+ cells and the total population of CD8+ T-cells (Fig.5B, C).
Then it was determined whether there is evidence specific to epitopes differences in terms of levels of expression of PD-1 in individuals with multiple detectable responses. Of the 65 surveyed individuals 16 individuals were from 3 to 5 tetramer-positive responses each. The expression of PD-1 was almost identical and close to 100% for each of the analysed response for three of the sixteen individuals; however, the remaining 13 individuals were identified distinct patterns of exp is ASCII PD-1 depending on the epitope (Fig.5D). These data indicate that expression of PD-1 can be differentiated using existing simultaneously specific epitopes CD8+ T-cells from one individual that may be consistent with recent data indicating that the epitope-specific differences in antiviral efficacy (Tsomides TJ, et al. 1994; Yang O, et al. 1996; Loffredo JT, et al. 2005).
The relationship between the expression of PD-1 and disease progression of HIV
There was correlation between the expression of PD-1 on specific HIV CD8+ T-cells and the amount of virus in plasma and CD4+ cells, each of which is a prognostic factor in the progression of HIV disease. In accordance with previous studies, the ratio between the number of tetramer-positive cells and the contents of the virus or the number of CD4+ cells did not show any significant correlation (Fig.6A, B). On the contrary, there was a significant positive correlation with the content of the virus and the percentage and MFI of expression of PD-1 positive HIV tetramer cells (p=0,0013 and p<0,0001, respectively, Fig.6A). There was also an inverse correlation between CD4 counts and the percentage and MFI of PD-1 positive HIV tetramer cells (p=0,0046 and p=0,0150 respectively, Fig.6B). As tested tetramera, apparently, is only part of the specific pop the regulation of CD8+ T-cells in these individuals, investigated the relationship between expression of PD-1 on all CD8+ cells and these parameters. There were significant positive correlations between the contents of the virus and the percentage and MFI of expression of PD-1 on the total population of CD8+ T-cells (p=0,0021 and p<0,0001, respectively, Fig.6C), and were also observed an inverse correlation between the rate of CD4+ cells and the percentage and MFI of expression of PD-1 on the total population of CD8+ T-cells (p=0,0049 and p=0,0006, respectively, Fig.6D). In the same group tested the expression of PD-1 on specific CMV CD8+ T-cells in 5 individuals, and these cells expressionalism significantly lower PD-1 compared with a specific HIV CD8 T-cells (median 23% of CMV tetramer+ PD-1+, p=0,0036), and this expression was not different from the expression in the main part of CD8+ T-cells in these individuals, which indicates that that high expression of PD-1 is not the same feature of all specific virus CD8+ T-cells. These data suggest that an increased amount of antigen in chronic HIV infection causes increased expression of PD-1 on CD8+ T-cells, and they correspond to the data for mice with chronic LCMV infection, in which the expression of PD-1 is associated with the functional exhaustion of CD8+ T-cells (Barber DL, et al. 2005). Moreover, they provide the first clear link in a broader study that includes an analysis of the number of epitopes between the special the ranks against HIV CD8+ T-cells and the contents of the virus or CD4 counts.
The relationship between the expression of PD-1 and the condition and function of CD8 T-cell memory
The expression of PD-1 was then analyzed in the context of a number of additional phenotypic markers associated with the condition and function of CD8 T-cell memory, including CD27, CD28, CD45RA, CD57, CD62L, CD127, CCR7, perforin, Grasim B and Ki67 (Fig.7). Representative colour on these markers on B*4201 TL9 tetramer+ cells from one individual are shown in Fig.7A, and is summarised for 13 individuals shown in Fig.7B. These studies were limited to those answers tetramers, which more than 95% of positivity PD-1, as multiparameter flow cytometry with more than 4 colors was not available in KwaZulu Natal. HIV tetramer+ PD-1+ cells Express a high level of CD27 and Grasim B, very low levels of CD28, CCR7, and intracellular Ki67, low CD45RA and perforin and at the intermediate level of CD57 and CD62L (Fig.7B). These data indicate that specific HIV PD-1+ T cells are effector/effector memory phenotype and are consistent with previous reports of displaced maturation-specific HIV CD8+ T-cells. In addition, conducted the sequencing of the virus to determine whether these cells escape from immune response. Audited 45 these tetramer-positive responses only 5 viral epitopes were distinguished by the camping from the consensus sequence monophyletic taxon C South Africa that indicates that these cells have a weak selection pressure in vivo.
Previous experiments in mice with LCMV model showed that blockade of in vivo interaction of PD-1/PD-L1 by infusion of anti-PD-L1 blocking antibody leads to increased functionality specific LCMV CD8+ T-cells according to the results of measuring the production of cytokines, killer ability, ability to proliferation and, most significantly, reducing the amount of virus. A brief (12-hour) specific antigen stimulation in vitro freshly isolated PBMC from 15 HIV+ individuals in the presence or absence of 1 μg/ml purified anti-PD-L1 antibodies did not cause increase production IFN-γ, TNF-α or IL-2.
The effect of blockade of road PD-1/PD-L1 on the proliferation of specific HIV CD8+ T-cells
Because specific HIV CD8+ T-cells also exhibit impaired ability to proliferation (2004), it was determined whether blockade of PD-1/PD-L1 to improve this function in vitro. Representative data for B*4201-positive individual is shown in Fig.8A. Incubation of freshly isolated CFSE labeled PBMC only with the environment or with the environment with anti-PD-L1 antibody resulted in maintaining populations of specific B*4201-TL9 CD8+ T-cells (1.2% of CD8+ T-cells), which remained CFSEhi six days in culture. Stimulat what I CFSE labeled PBMC for 6 days a peptide TL9 resulted in a 4.8-fold expansion CFSElo B*4201 TL9 tetramer+ cells, whereas stimulation of CFSE labeled PBMC with peptide TL9 in the presence of anti-PD-L1 blocking antibodies were additionally increased the proliferation of specific TL9 cells, which led to the 10.3-fold increase in tetramer+ cells. Tests proliferation of CFSE were held on 28 samples in the presence and in the absence of purified blocking antibodies against PD-L1 human. There was a significant increase in proliferation specific for HIV CD8+ T-cells in the presence of peptide plus a blocking antibody against PD-L1 compared to the rate of proliferation after stimulation by a single peptide (Fig.8B; p=0,0006, paired t-test). The fold increase in tetramer+ cells in the presence of blocking antibodies against PD-L1, depending on the individual and the epitope of the particular individual (Fig.8C), which again suggests the presence of a specific epitope from differences in the degree of functional depletion of these responses.
Therapeutic vaccination in combination with blockade of inhibitory path PD-1 synergistically improves the immune control of chronic viral infection: a conceptual study of combinatorial therapeutic vaccine
Functional damage to T-cells, including the production of cytokines, cytolysis and cell specific antigen, T-cells, is a defining characteristic of persons is the pursuit of many chronic infections. Vaccine T-cell immune response observed during many different resistant infections pathogens, including HIV, HBV, HCV, and TB in humans. Inactivation of T-cells during chronic infection could be correlated with the magnitude and stability of the antigen content and to arise from breach of the signals, proximal with respect to the receptor of T-cells, increasing inhibitory proteins or reduction of co-stimulating proteins and defects in the auxiliary or cytokine signals. The defect in depleted T-cells is the primary cause of the inability of the owner to destroy the resistant pathogen. During chronic infection in malnourished specific virus CD8 T-cells increased two key inhibitory protein: PD-1 and CTLA-4. Blockade in vivo PD-1 increases the number and function of specific virus CD8 T-cells and causes a reduction of the virus.
Modern strategies of vaccination chronic viral infections has several disadvantages. Specifically, effective support for the antiviral response of CD8 T-cells is not observed after therapeutic vaccination. In addition, high concentrations of virus and low proliferative potential of responding T-cells during chronic infection, likely limit the effectiveness of therapeutic vaccination. Thus, it is important to develop is trategie therapeutic vaccination for effective support of endogenous T-cell response of the host to control chronic infection.
To determine the efficiency of the antagonist PD-1 in combination with a therapeutic vaccine used a well-known model of chronic infection induced by infection with clone 13 LCMV. As a therapeutic vaccine used vaccinia virus expressing the epitope of LCMV GP33, for monitoring specific epitope immune response CD8 T-cells. Therapeutic vaccine combined with antibody against PD-L1 to block inhibitory ways to study synergistic effects in relation to the proliferation of specific antigen CD8 T-cells and removal of resistant virus.
In these experiments we used the following methods:
Mouse and infection: Mice C57BL/6 (females aged 4 to 6 weeks) were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were kept free from pathogens vivarium in accordance with the rules of the NIH for the care of animals. To initiate chronic infections of mice infected with 2×106The BATTLE of the clone 13 LCMV (CL-13), as described previously. The cultivation of the virus and the tests with plaques to determine the titers of virus were described previously.
In vivo blockade of antibody and therapeutic vaccination: two Hundred micrograms of rat antibodies against PD-L1 mouse (10F:9G2) was administered intraperitoneally every third day, starting 4 weeks after infection with CL-13. At the time of the first introduction and the ti-PD-L1 were injected intraperitoneally with 2×10 6The BATTLE of recombinant vaccinia virus expressing the epitope GP33-41 (VV/GP33), as a therapeutic vaccine or vaccinia virus wild-type (VV/WT) as a control vaccine.
The selection of lymphocytes: lymphocytes were isolated from the tissues and blood, as described previously. The liver and lung were perfesional chilled on ice SFR removing for isolation of lymphocytes.
Flow cytometry: peptide tetrameric class I MHC received and used, as described previously. All antibodies were obtained from BD Pharmingen except for antibodies to granzyme B (Caltag), Bcl-2 (R&D Systems) and CD127 (eBioscience). All surface staining and intracellular cytokines were performed as described (Barber et al., Nature 439:682, 2006). To identify the degranulation splenocytes were stimulated for 5 h in the presence brefeldin, monensin, anti-CD107a-FITZ and anti-CD107b-FITZ.
Confocal microscopy: from mice were isolated spleen and frozen in OCT (TissueTek). From these blocks were prepared 10-20 mm cryostate slices and fixed in chilled on ice acetone for 10 minutes. For immunofluorescence assay, the sections were stained with the following antibodies: ER-TR7 for detection of reticular cells (Biogenesis, Kingston, NH) and the polyclonal serum of Guinea pigs against LCMV. Color visualized using Alexa Fluor-488 goat against Ig rat and Alexa Fluor-568 goat against Ig Guinea pigs (Molecular Probes) and analyzed the using confocal microscopy (Leica Microsystems AG, Germany). Images were obtained using ImageJ (National Institutes of Health) and Photoshop (Adobe Systems Inc.).
The results showed that the combination of therapeutic vaccines with antibody against PD-L1 provides a synergistic effect on the proliferation of specific antigen CD8 T-cells and destruction of resistant virus. Therapeutic vaccine could effectively maintain functionally restored population of CD8 T-cells by blocking the inhibitory path PD-1/PD-L1. When combined therapeutic vaccination was systematically achieved increased proliferation of specific antigen CD8 T-cells and accelerated the control virus (Fig.9A-9D and Fig.10A-10D). Combined therapeutic vaccine stimulates a dramatic increase in functionally active CD8 T-cells (Fig.11A-D). In addition, a therapeutic vaccine using a vector that expresses a specific epitope, during the siege of paths PD-1/PD-L1 enhances the proliferation of CD8 T-cells specific for the epitope encoded by the vector (Fig.9 and 11). An increased level of expression of CD127 observed at specific antigen CD8 T-cells in the group treated with the combined vaccine, reflects the emergence of long answers of the memory cells, while reduced levels of expression of PD-1 and granzyme B correlate with the removal of resistant virus (Fig.12A-12B).
There were synergistically the effect of therapeutic vaccines connected with blockade of PD-L1 on restoration of function "helpless" exhausted CD8 T-cells (see Fig.13A-13E). In mice decreased the number of CD4 T-cells and then infected with clone 13 LCMV. Some mice were vaccinated with cowpox virus wild-type (VV/WT) or vaccinia virus expressing the LCMV epitope GP33-41 (VV/GP33) at 7 weeks after infection. In the same period, mice were treated with αPD-L1 5 times every three days or its isotype. Two weeks after starting treatment with antibodies, the mice were scored for analysis. The results are shown in Fig.13A. Investigated the frequency of occurrence of specific GP33 CD8 T-cells (Fig.13b). Splenocytes stimulated with GP33 peptide in the presence of antibodies αCD107a/b and then were co-stained for IFN-γ. The graphs were obtained by limiting the window for CD8 T-cells (Fig.13c). Was defined as the percentage of IFN-γ+cells after stimulation with GP33 peptide from cells positive for a limited Db-GP33-41 tetramer (Fig.13d), and the titer of virus (Fig.13e). The results demonstrate the synergistic effect of the vaccine, which is connected with blockade of PD-1.
These results show that the combination of the blockade negative regulatory pathway and maintenance of CD8 T cells during chronic infection can be used in the creation of therapeutic vaccines to improve the response of T-cells in patients with chronic infections or C is kachestvennymi neoplasms. Therapeutic interventions, such as the use of antagonist PD-1 that support the responses of T-cells and reduce the amount of virus that could increase survival in the absence of disease and to reduce transmission of the virus. Effective therapeutic vaccination could be used in chronic viral infections and resistant bacterial, and parasitic infections. This strategy is also suitable for the treatment of malignant tumors.
The increased T-cell immunotherapy through blockade path PD1/PDL1
It is important to develop strategies for treatment and destruction of chronic viral infections such as human immunodeficiency virus and hepatitis C. the CDC recently reported that over one million Americans live with HIV, which exemplifies the need for more effective therapy. It is important to determine how inhibitory signaling in respect of lymphocytes may contribute to the ability of the pathogen steadily to escape from the immune response of the host.
It was shown that the inhibitory immunoreceptor PD-1 (family member B7/CD28 co-stimulating receptors) and its ligand (PD-L1) sharply increased during States of chronic infection with the virus of lymphocytic choriomeningitis (LCMV). Additional studies using the LCMV model showed that blockade path PD1/PDL1 considerable which increases endogenous antiviral response of CD8 T-cells during the late phase of chronic infection, when CD8 T-cells are depleted. Exhausted T cells are functionally defective and do not organize an effective immune responses when meeting with the antigen. However, blockade path PD1/PDL1, apparently, draws the exhaustion and restore functional ability. The data suggest that these effects are well preserved beyond the immediate period of treatment with anti-PDL1.
The following experiments were conducted to (1) assess the ability of anti-PDL1 to increase the proliferation and viability of antiviral CD8 T cells after adoptive transfer of immune (memory cells) of mice splenocytes (media) with congenital infection, (2) to assess the functional activity of the specific virus CD8 T-cell memory, which were reproduced in the blockade of PD1/PDL1, and (3) to determine the expression of various differentiation markers in specific virus CD8 T-cells, which were reproduced in the blockade of PD1/PDL1.
The role of path PD-1 was assessed in a well-developed model of cytomander chronic viral infection. Described in the present description model parallel model of T-cell cytomander tumors in relation to immunological barriers that limit the applicability of these therapies (such as distorted or suppressed T-cell/antitumor responses). the mice infected neonatal or in utero LCMV, not organized endogenous specific LCMV immune responses, and throughout their lives they continue to have high levels of infectious LCMV in the blood and all tissues. These animals are congenital media and largely tolerant to the pathogen. When adoptive transfer of splenocytes from LCMV immune mice congenital media is moved immune memory cells rapidly undergo reproduction and provide powerful immune response against the virus. Approximately 2/3 of the animals receiving the adoptive cytomander, cope with the task complete removal of infection in the transfer of high doses of splenocytes.
In these experiments we used the following materials and methods:
Mouse and infection: female mice B57BL/6 aged 4-6 weeks were obtained from Jackson Laboratory (Bar Harbor, Maine). Acute infection caused by intraperitoneal injection of 2×105PFU LCMV Armstrong. Mice-congenital media were propagated at Emory University (Atlanta, GA) from a colony originating from infected neonatal mice (104The BATTLE of the clone 13 LCMV, inside the brain).
Adoptive immunotherapy and in vivo blockade of antibody. Allocated 40×106whole splenocytes from LCMV-immune mice (after 30-90 days after infection) and transferred them intravenous 6-12-week-old mice vehicles LCMV. Within 1 day after adoptive immunotherapy on every 3rd day were injected with 200 micrograms of antibody rats against PD-L1 mouse (10F.9G2).
Flow cytometry and tetramer staining. Tetrameric H-2Db class I MHC, kompleksirovanii with GP33-41LCMV was obtained as described previously. All antibodies were obtained from BD/Pharmingen (San Diego, CA). Mononuclear cells from peripheral blood and splenocytes were isolated and stained as described previously. Data were obtained using the flow cytometer FACSCaliburTM(BD) and analyzed using the program FlowJoe (Tree Star Inc. Ashland, OR).
Staining of intracellular cytokines: For staining of intracellular cytokines 106the splenocytes were cultured in the presence or absence of the indicated peptide (0.2 ág/ml) and brefeldin a for 5-6 hours at 37°C. After staining for markers of cell surface permeabilities and stained for intracellular cytokines using drug Cytofix/Cytoperm (BD/Pharmigen).
The results were as follows:
Therapy with antibody against PD-L1 increases the number of specific virus CD8 T-cells: Mononuclear cells from peripheral blood (PBMC) were isolated from treated and not treated animals at 7, 11, 15, 22 and 35 days. Cell specific epitope DbGP33 was determined by staining with tetramer. In two independent experiments, it was found that in animals receiving therapy with antibody against PD-L1 during the first 15 days after adoptive transfer, poyavlyal the ü significantly more specific LCMV CD8 T-cells after normalization to the number of D bGP33-positive cells per million PBMC (Fig.14). These data confirm the role of path PD-1/PD-L1 in ensuring a certain part of the suppression of proliferation of T-cell memory. Moreover, these results suggest that therapeutic inhibition of this pathway could enhance the development and maintenance of a secondary immune response that occurs after adoptive transfer in conditions of chronic infection with high concentrations of antigen.
Blockade of PD-1/PD-L1 enhances the functionality specific antigen CD8 T-cells: Splenocytes were isolated from treated and not treated animals on day 17 after adoptive transfer and analyzed for the expression of inflammatory cytokines (IFN-gamma and TNF-alpha) or CD107ab (associated with lysosomes membrane protein, LAMP). It was found that the expression of IFN-gamma is increased in animals treated with a blocking antibody against PD-L1, compared to animals without treatment at all certain CD8 epitopes (Fig.15a). In addition, post-treatment antibody against PD-L1 was also increased joint expression of IFN-gamma and TNF-alpha (Fig.15B-15E). These data indicate that adoptive transferred splenocytes memory, breeding in conditions of blockade of PD-L1, functionally superior in terms of production of inflammatory cytokines and release of cytolytic granules compared to what splenocyte from animals not treated.
Answers mouse B-cells during the blockade of PD-1
To determine whether increases whether blockade of PD-1 responses of B-cells during chronic LCMV infection, were conducted the following experiments. Responses and B-cells and T-cells are important for the control of chronic LCMV infection, therefore improving the response of B-cells in chronically infected with LCMV mice may contribute to the decrease in the content of the virus and increasing function of T-cells.
In these experiments we used the following materials and methods:
Mouse and virus: female mice B57Bl/6 at the age of four to six weeks were obtained from Jackson Laboratory (Bar Harbor, Maine). Before infection in mice with chronic LCMV reduced CD4 T-cells by the introduction of antibodies gk1.5. Previous data show that the introduction of 500 µg gk1.5 in the days -2 and 0 before the introduction of the virus leads to a decrease in CD4 T-cells in the spleen and lymph nodes at 95-99% with slow recovery of CD4 T-cells for 2 to 4 weeks. Mice received 2×106The BOUT of strain clone 13 LCMV intravenously at day 0 for the initiation of chronic infection. Virus titres were determined using a 6-day blastophaga test on Vero cells.
The definition of ASC using ALISPOT: from suspensions of single cells in spleen and bone marrow erythrocytes were removed by treatment 0,84% NH4Cl and resuspending in RPMI-dobavki 5% FCS. Secreting antibodies cells were identified location of the cells in 96-well plates to a nitrocellulose filter bottom Multiscreen HA (Millipore). The tablet was pre-coated with 100 μl of 5 μg/ml antibody goat against IgG+IgM+IgA mouse (Caltag/Invitrogen) overnight at 4°C. the Tablets were then washed 3×SFR/0.2% tween, then 1×SFR and blocked for 2 hours in RPMI+10% FCS to prevent nonspecific binding. The blocking medium was replaced with 100 μl of RPMI 5% FCS, and the tablet was added 50 μl of serial three-fold dilutions of 1×107cells/ml. the plates were incubated for 6 hours at 37°C and 5% CO2. The cells were removed, and the tablets were washed 3× SFR and 3× SFR/0.2% tween. The wells were then covered biotinylating goat antibodies against mouse IgG (Caltag/Invitrogen), diluted 1/1000 in SFR/0.2% tween/1% FCS and incubated overnight at 4°C. the Second antibody was removed, and the tablets were washed 3×SFR/0.2% tween. Cells were then incubated with avidin-D-HRP (Vector), diluted 1/1000 in SFR/0.2% tween/1% FCS for one hour at room temp. The tablets were washed 3×SFR/0.2% tween and 3×SFR, and expression was performed by adding 100 μl of chromogenic substrate of horseradish peroxidase-H2O2. The substrate was obtained by adding 150 ál of freshly prepared solution AEC (10 mg 3-amino-9-ethylcarbazole (ICN) per ml, dissolved in dimethylformamide (Sigma) to 10 ml of 0.1 M sodium acetate buffer pH of 4.8, filtering otogakure membrane with pore size of 0.2 mm and immediately before use by adding 150 ml of 3% H 2O2. After 3-5 minutes appeared grainy red spots, and the reaction was stopped by washing with tap water. Spots were counted using a stereomicroscope, equipped with a vertical white light.
Determination of total bone marrow cells: in order To calculate the total response ASC in the bone marrow response of bone marrow cells of two hips were multiplied by a factor of 7.9, since distribution studies59Fe showed that 12.6% of all bone marrow mouse localized in two United hips. No difference was found in ASC activity of bone marrow cells between the femur, tibia, humerus, rib or the sternum. Usually two thighs get from 2×107up to 2.5×107total bone marrow cells.
Flow cytometry: Directly conjugated antibodies were obtained from Pharmingen (anti-B220, anti-CD4, anti-CD138, anti-CD95, anti-Ki67, anti-IgD biotinylated) or Vector labs (PNA). Streptavidin-APC was obtained from Molecular Probes. All staining was performed at 4°C in STR with the addition of 1% FCS and 0.1% sodium azide. Cells then were fixed in 2% formaldehyde (in SFR) and were analyzed on a FACS Calibur using CellQuest (BD Biosciences).
Statistical analysis: Tests were performed using Prism 4.0 (GraphPad, San Diego, CA). Statistical analysis was performed using bilateral unpaired T-test with 95%confidence interval.
The total number of secreting antibody cells in the spleen increased after in vivo blockade of PD-1: mice infected with clone 13 LCMV, treated with antibody (α) against PD-L1 within approximately 60 days after infection. Mice were administered 200 μg αPD-L1 on every third day for two weeks. On day 14 of treatment αPD-L1 mice were scored, and the number of secreting antibody cells in the spleen was measured using ELISPOT and staining with flow cytometry. In three separate experiments in mice treated with αPD-L1, were shown to significantly increased levels secreting antibody cells (ASC) in the spleen (p=0,011) compared with mice without treatment (Fig.16a). ASC differentiation of B-cells in the spleen by suppressing marker of B-cells B220 and CD138 expression (syndecan-1). In accordance with the results of ELISPOT in infected mice treated with αPD-L1, has increased the number of B220low/intCD138+ cells (Fig.16b).
Treatment of chronically infected with LCMV mice αLD-L1 does not lead to an increased level of ASC in the bone marrow. Determined, increase Lee also secreting antibody cells in the bone marrow at the time of treatment αPD-L1. The most long-lived plasma cells found in the bone marrow, and these plasma cells are important for long-term maintenance of antibody levels in serum. Chronically infected with LCMV we who she was treated with αPD-L1 within approximately 60 days after infection. On day 14 of treatment αPD-L1 measured the levels of ASC in the spleen and bone marrow using ELISPOT. Although two weeks after treatment in the spleen were increased amount ASC, changes in the number of ASC in the bone marrow at this time was not (Fig.17).
Joint treatment of chronically infected with LCMV mice αPD-L1 and118αCTLA-4 leads to a synergistic increase the level of ASC in the spleen. Next investigated whether blockade of signaling other molecules negative regulation, CTLA-4, to increase the effect observed in the blockade of PD-1. Suppose that binding of CTLA-4 with B7 and competes with positive co-stimulating molecule CD28, and/or gives the right signals, counter TCR. Mice infected with clone 13 LCMV, treated, or αPD-L1 or αCTLA-4, or both, or left without treatment, and after two weeks of treatment was measured levels secreting antibody of cells using ELISPOT. Although treatment αCTLA-4 had no detectable effect on the level of ASC, joint treatment αPD-L1 and αCTLA-4 led to a synergistic increase in ASC compared with the increase observed in the isolated treatment of αPD-L1 (Fig.18).
Increased proliferation of B-cells and CD4 T-cells and the activity centers of reproduction treated with αPD-L1 mice: Analysis of flow cytometry populations of the spleen in chronic mice treated with αPD-L1 showed elevated levels of cell proliferation by increased okrasivaniju-67, and CD4 T-cells and B-cells. B-cells in the active area of the centre of reproduction can be identified in the spleen at the high level of PNA staining and FAS. After treatment αPD-L1 there was a significant increase in the frequency of occurrence PNA+FAS+ B cells compared to the control without treatment (Fig.19a-19b).
The expression of PD-1 on T-cells
CD8 T cells are essential for the control of many chronic infections. As disclosed in the present description, these CD8 T-cells become depleted after chronic antigenic stimulation, which is characterized by the induction hypoproliferative status and loss of the ability to produce antiviral cytokines. Exhausted T cells have high expression protein 1 programmed death (PD-1), and PD-1 also stimulates the activation of T-cells and can be activated by ligands of PD-1, PD-L1 and PD-L2. In the present description revealed that the inhibitory path PD-1 is an important mediator depletion of CD8 T cells during chronic viral infection in mice. Specific virus CD8 T cells maintained a high level of expression of PD-1 in response to chronic infection, but not in response to infection, which successfully eliminated. Blockade of the interaction of PD-1/PD-L1 led to increased proliferation of T-cells, production of antiviral cytokines and reducing the concentration of the virus.
Was made in the dena assessment Express whether specific for chronic infections CD8 T-cells of human PD-1 and increases if the blockade of PD-1 responses of CD8 T-cells. In this study (1) determined the pattern of expression of PD-1 on subpopulations of mononuclear cells of peripheral blood (PBMC): CD4, CD8, B cells, NK, monocytes, DC; (2) determined the phenotype of CD4 and CD8 T-cells that Express PD-1; (3) determined the expression of PD-1 on specific chronically persisting antigen [virus Epstein-Barr (EBV) and cytomegalovirus (CMV)] and quickly remove antigen (influenza and vaccinia (smallpox); and (4) determined the effect of blockade of the interaction of PD-1/PD-L1 on the proliferation of specific antigen cells.
In these studies used the following materials and methods:
Blood samples: peripheral blood Samples were obtained from 36 healthy individuals who were seropositive against viruses EBV, CMV, influenza or mad cow pox. These individuals were selected on the basis of their expression of HLA allele corresponding to tetramers class I HLA, specific proteins of viruses EBV, CMV, influenza or mad cow pox. PBMC were isolated from blood samples from the environment Department of lymphocytes (Cellgro, Herndon, VA).
Antibodies, peptides and tetramer: received conjugated with phycoerythrin antibody against PD-1 person (EH12, IgG1 mouse) and unconjugated antibody against PD-L1 che is ovecka (29E.2A3, IgG2b mouse). Directly conjugated antibodies were obtained from Beckman Coulter, San Diego, CA (anti-CD3, CD11a, CD27, CD28, CD38, CD45RA, CD57, CD62L and granzyme-B), BD Pharmingen, San Diego, CA (CD8, CD95, CD195, HLA-DR, Ki-67 and perforin) and R&D systems, Minneapolis, MA (CCR7). Peptides were obtained in the lab. peptide synthesis in Emory University, Atlanta, GA. Plasmid constructs expressing HLA-A2, -B7, and B8 were kindly provided by Tetramer Core Facility, Atlanta, GA, and APC labeled peptide tetrameric class I MHC-bearing epitopes CTL EBV (HLA-A2-GLCTLVAML (SEQ ID NO:36), HLA-B8-RAKFKQLL (SEQ ID NO:37) and FLRGRAYGL (SEQ ID NO:38)), CMV (HLA-A2-NLVPMVATV (SEQ ID NO:39), HLA-B7-TPRVTGGGAM (SEQ ID NO:40)), influenza (HLA-A2-GILGFVFTL (SEQ ID NO:41)) and cowpox (HLA-A2-CLTEYILWV (SEQ ID NO:42) and KVDDTFYYV (SEQ ID NO:43)).
Immunophenotyping and proliferation of the CFSE: Samples of whole heparinized human blood (200 μl) were stained with antibodies or tetramine and then analyzed (Ibegbu et al., J Immunol. 174:6088-6094, 2005) on a FACS Calibur using CellQuest or flowing LSRII cytometer using FACSDiva software (BD Immunocytometry Systems). For CFSE assays PBMC (2x106/ml) were carefully washed and marked a 3 µm carboxyfluorescein diacetate Succinimidyl ester (CFSE, Molecular Probes) at room temperature in the dark for 5 min (see, for example, Weston and Parish, J Immunol. Methods 133:87-97, 1990). Labeled with CFSE PBMC stimulated with either a single peptide (1 μg/ml) or peptide with the antibody against PD-L1 (10 µg/ml). Control cultures contained either PBMC, PBMC with the antibody p is otiv PD-L1 or PBMC from control by isotype antibody (IgG2b, 10 µg/ml). After 6-day incubation at 37°C. cells were washed and stained on the extracellular tetramera, along with antibodies against CD3 and CD8.
The results were as follows:
The pattern of expression of PD-1 in PBMC subpopulations: the Expression of PD-1 was studied on subpopulations of PBMC of healthy individuals. It was found that CD8+ T cells, CD4+ T-cells and monocytes (CD14+) Express PD-1 at a high level, B-cells (CD20+) Express PD-1 at a low level, and NK cells (CD56+) and DC (CDl1c+) does not Express PD-1.
PD-1 is expressed predominantly effector T-cells, memory CD8 and CD4: CD8 T cells from normal healthy individuals examined for joint expression of PD-1 and various phenotypic markers associated with the state of differentiation and function (Fig.20A). In General, naive and with the phenotype of Central memory CD8 T cells Express PD-1 only at a low level, while CD8 T cells, which Express various markers associated with effector/effector memory/or depletion phenotype, also Express PD-1 at a high level (Fig.20B). These data suggest that PD-1 is expressed predominantly CD8 T-cell effector memory. A similar trend was observed in the studies by applicants CD4 T-cells (Fig.20C).
PD-1 is increased on a sustainable specific antigen CD8 T-cell of the memory: To assess are specific for chronic infections CD8 T cells increased the expression of PD-1 was compared to the expression of PD-1 on CD8 T-cell memory, specific chronic stable viruses (EBV and CMV), and specific for acute viral infections (influenza and vaccinia (smallpox) T-cells in 36 healthy individuals by staining for specific viruses EBV, CMV, influenza and vaccinia (smallpox tetramer (Fig.21A-21B). In Fig.21A presents a representative PD-1 GMF1 CD8 T-cells specific against viruses EBV, CMV, influenza and vaccinia virus. It was found that the expression of PD-1 on specific EBV CD8 T-cells was increased compared with the specific influenza viruses (p=0,0335) and cowpox (p=0,0036) CD8 T cells (Fig.21A-21B). Similarly, specific CMV CD8 T cells expressed PD-1 more frequently than cells specific against influenza viruses (p=0,0431) and cowpox (p=0,019), (Fig.21A-21B). These results suggest a correlation between the expression of PD-1 and exposure to antigen.
Blockade by anti-PD-L1 enhances the proliferation of chronic stable specific virus CD8 T-cells was evaluated in improving the blockade of PD-1 sustainable answers specific virus CD8 T-cells are similar to the results observed in mice. Labeled with CFSE cell is stimulated by each of the peptides, specific viruses EBV, CMV, influenza and vaccinia virus in the presence or in the absence of antibodies against PD-L1. After 6 days compared the percentage of cells tetramer+CFSEloand cells CD8+ CFSEloin cultures that were stimulated only by the peptide, and cultures that were stimulated with peptide and then blocked with anti-PD-L1. In Fig.22A shows representative graphs of flow cytometry with the proliferation of specific CMV and EBV CD8 T-cells. The combined data for CMV - (n=5), EBV (n=6), influenza (n=2) and vaccinia - (n=2) seropositive individuals is shown in Fig.22B. Blockade of the interaction of PD-1/PD-L1 antibody against PD-L1 led to increased proliferation specific for EBV and CMV CD8 T-cells, while specific influenza virus and cowpox CD8 T cells did not show proliferation after blockade by anti-PD-L1. These results show that in the presence of peptide plus a blocking antibody against PD-L1 is a 3.5-fold increase in the frequency of occurrence of specific EBV and CMV CD8 T-cells in comparison with the stimulation of a single peptide. We evaluated whether the proliferation of specific antigen CD8 T-cells after blockade of antibody against PD-L1 with the expression of PD-1 on these cells. The data show a positive correlation between the expression of PD-1 and proliferation spec is specific against the antigen CD8 T-cells (p=0,0083) (Fig.22C).
Infiltrated into liver cells in chronic HCV infection humans are exhausted phenotype with high expression of PD-1 and low expression of CD127
The following experiments documented that chronic HCV infection peripheral specific HCV T cells Express PD-1 at a high level and that the blockade of the interaction of PD-1/PD-L1 leads to increased capacity for proliferation. It is important that intrahepatic specific HCV T-cells not only Express PD-1 at a high level, but also Express a low number of receptor-alpha IL-7 (CD127) - depletion phenotype, which was specific for the antigen of HCV and was localized in the liver, the site of viral replication.
Currently there is no vaccine to prevent HCV infection, and the only licensed therapy, interferon alpha (IFNα), alone or in combination with nucleoside analogue ribavirin, costly, associated at best with only a 50% rate of clearance for the predominant genotype (genotype 1) and is complicated by significant side effects. The lack of effective treatment options against HCV emphasizes the need for effective interventions to strengthen or Supplement the natural immune response, which in themselves or in combination the AI with antiviral drug therapy can prevent the deleterious effects of HCV infection.
Currently, little is known about the expression of PD-1 and its role in the depletion of T-cells during chronic HCV infection, especially in place of active infection, liver. The present study was undertaken to better understand the phenotype of T-cells in HCV infection by measuring the expression of PD-1 on specific antigen CD8+ T-cells and in the liver and in peripheral blood of patients with chronic HCV infection.
In these studies used the following materials and methods:
Individuals: the study included seventeen patients with chronic HCV infection (positive HCV antibodies and HCV PCR), negative for HIV antibody screening. Before inclusion in the study all patients had not received antiviral therapy of HCV. Seven of the fifteen patients were positive for HLA-A2 according to FACS analysis. Characteristics of patients are summarized in table 5.
Testing for HCV antibodies, determination of virus genotyping: Testing for HCV antibodies using the ELISA was performed using instructions of the kit manufacturer (Abbott Diagnostics, Abbott Park, Ill; Bio-Rad Laboratories, Hercules, CA). Quantitative determination of HCV virus was made using analysis FROM real-time PCR (Roche Molecular Systems, Alameda, CA). Genotyping of HCV were made using analysis of RT-PCR in Rea is enom time (Abbott Diagnostics, Abbott Park, Ill) and using the analysis probe strips (LIPA) (Bayer Diagnostics, Research Triangle Park, NC).
Mononuclear cells of peripheral blood From each patient was collected blood (50-70 ml) with anticoagulants EDTA and heparin and used either directly for FACS staining, or to highlight PBMC. PBMC were isolated by density gradient Ficoll-Paque PLUS (Amersham, Oslo, Norway), washed twice SFR and either analyzed immediately or stored frozen in medium containing 90% serum embryo calves (Hyclone) and 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO).
Liver biopsy: liver Tissue was obtained either by needle biopsy under ultrasound control, either through fluoroscopies method through the jugular vein and immediately placed in medium RPMI-1640 (Gibco) containing 10% serum embryo calves (Hyclone, Logan, UT) for immunological analyses. Another slice was fixed in formalin for histological study.
The allocation of intrahepatic T-cells: a sample of liver biopsy specimen obtained in the medium RPMI-1640 (Gibco, Carlsbad, CA) containing 10% serum embryo calves (Hyclone, Logan, UT), washed three times with the same medium for removing debris cells and RBC. Selection infiltrated liver lymphocytes was performed using the automated mechanical system disaggregation (Medimachine, Becton Dickinson, San Jose, CA). The sample was inserted into a 50 μm Medicon and stable and in Medimachine and process for 15 seconds. Disaggregated cells were removed with a syringe through a port of the syringe. Medicon washed twice with RPMI medium (Gibco, Carlsbad, CA) containing 10% serum embryo calves (Hyclone, Logan, UT), to ensure maximum yield of cells. Cells were immediately used for FACS staining.
Antibodies, tetrameric HLA-A2 and flow cytometry: Cells were stained with labeled FITZ, PE, PerCP and APC propecial monoclonal antibodies or tetramine in accordance with manufacturer's instructions and was carried out by flow cytometry using FACS Calibur (Becton Dickinson, San Jose, CA). FACS data were analyzed using FlowJo (Treestar). Used the following monoclonal antibodies from BD Pharmingen (BD Biosciences, San Jose, CA): anti-CD8 PerCP and anti-CD45RA APC. Anti-CD62L FITZ, CD3 FITZ and CD127 PE were obtained from Beckman Coulter (Fullerton, CA). Anti-PD-1 PE conjugated antibody (clone EH12) was obtained as described (Dorfman et al., Am. J. Surg. Pathol. 30:802-810, 2006). HLA-A2 tetramer were specific for these epitopes CD8+ T-cells: HCV 1073: CINGVCWTV (SEQ ID NO:44); HCV-1406: KLVALGINAV (SEQ ID NO:45). Collecting flow cytometry was performed on FACSCaliberTM(BD Biosciences, San Jose, CA), and analysis was performed using FlowJo (v8.1.1).
Prokachivanie CFSE and blockade of antibody: 10×06PBMC were washed SFR and noted 3 μm CFSE (Molecular Probes). The cell concentration was brought to 1×106cells/ml and cultured in the presence of 2 μg/ml of peptide A2-HCV 1073 (CINGVCWTV, SEQ ID NO:44).On day 3 after stimulation was added 10 u/ml IL-2. In each test included restimulating control. Specific blocking antibodies (anti-PD-Ll; clone # 29E and anti-PD-1; clone # EH12 (Dofman et al., above)) was added to cell cultures at a concentration of 10 μg/ml at the time of stimulation. Cells were incubated for 6 days, were collected and stained with surface antibodies and tetramera and were analyzed by flow cytometry.
Statistical analysis: results were presented in graphs and analyzed using GraphPad Prism (v4). Internal comparison for the same patient was performed using paired t-tests. Comparison between patients was performed using unpaired t-tests.
The results were as follows:
The expression of PD-1 on specific HCV antigen CD8+ T-cells: Researched seventeen patients with HCV infection (all HIV negative) (table 5). At fifteen patients taking samples and blood and liver to phenotype analysis flow cytometry, and all patients before inclusion in the study were not subjected to treatment with pharmacological antiviral therapy. Seven patients of the group were positive for HLA-A2 to identify population specific HCV CD8+ T-cells in peripheral staining of the HLA tetramer (table 5). These specific HCV CD8+ T cells were analyzed for expression of PD-1 (Fi is.23A). The level of expression of PD-1 on total CD8+ T-cells in peripheral blood from healthy donors did not differ significantly from the level in the General pool of peripheral CD8+ T-cells from HCV infected patients (Fig.23B). On the contrary, most of the specific HCV tetramer-positive CD8+ T-cells taken from the peripheral blood were PD-1-positive (mean 85%, SEM, standard error of the mean, 3,6) (Fig.23A) with significantly higher expression compared with the General population of CD8+ T-cells (p<0,0001) (Fig.23B). Also investigated the expression of molecules differentiation, costimulation, movement, and effector functions at specific antigen CD8+ T-cells. Specific HCV tetramer-positive cells exhibit a memory phenotype (high CD11a, low CD45RA), markers of early differentiation (high CD27, high CD28, intermediate expression of CCR7 and CD62L) and low levels of mediators of effector functions granzyme B and perforin. Interestingly, these HCV tetramer-positive T cells in peripheral blood expressed at a high level of CD127 (α-chain of the receptor for IL-7), a phenotypic marker, the expression of which is at a low level indicates the impaired differentiation of T-cell memory.
To determine whether the phenotype of CD8+ T-cells by flow phranchesko infection, Flu-specific T cells was investigated in five ZV the world HLA-A2+ donors, who were not infected with HCV. The percentage of peripheral Flu tetramer+ CD8+ T-cells that Express PD-1, was equal to 49% (SEM 14,1) (Fig.23C). Using tetramer analysis of five of the seven HLA-A2-positive chronic HCV patients were identified Flu-specific CD8+ T cells. The percentage of Flu-specific T cells expressing PD-1, these chronically infected HCV patients did not differ significantly from the same population from healthy donors (Fig.23C). Importantly, since five of the seven HLA-A2 patients with HCV were detected Flu-specific CD8+ T cells, it was possible to conduct an internal comparison for each patient PD-1 to T-cells specific for phranchesko (Flu) and chronic (HCV) infection. The difference between the expression of PD-1 Flu-specific and HCV-specific T-cells was significant (Fig.23C). The percentage of specific HCV CD8+ T cells expressing PD-1 (mean 83%, SEM 6,4), was higher than the percentage of PD-1+ specific Flu CD8+ T-cells (49%, SEM 12,3) (p=0,048) (Fig.23C).
The expression of PD-1 on peripheral blood lymphocytes and lymphocytes infiltrated in the human liver: Peripheral blood and liver biopsy specimens from fifteen patients chronically infected with HCV, were analyzed for the expression of PD-1. A representative analysis of flow cytometry in five patients is shown in Fig.24A. While in the peripheral blood of 27% (SEM 3,4) CD8+ T-cells were PD-1+, in pécs is neither the frequency of these cells was increased by half (57%, SEM 3,6) (Fig.24B). Thus, the liver is enriched in cells expressing PD-1 at a high level. While naïve cells must Express at a high level and CD62L and CD45RA, in the liver the majority of CD8+ T-cells were CD62L low/CD45RA low, which corresponds to a memory phenotype (Fig.24C). A special analysis of this population and memory in the liver, and in the periphery showed that the expression of PD-1 in the liver was increased compared to the periphery (Fig.24C). These data suggest that the increase in the percentage of cells expressing PD-1 on intrahepatic T-cells, caused not only by the absence of a naive population in this compartment. Instead, there is a preferential enrichment in the liver of PD-1+CD8+ T cells effector memory (CD62L low/CD45RA low) compared with peripheral blood (Fig.23C).
The expression of CD127 on peripheral blood lymphocytes and lymphocytes infiltrated in the human liver: IL-7 is required for the maintenance of CD8+ T memory cells (Kaech et al., Nat Immunol. 4:1191-8, 2003), and the alpha chain of its receptor, CD127, reduced by specific antigen T-cells during steady LCMV infections and herpes virus gamma (see, for example, Fuller et al., J. Immunol 174:5926-30, 2005). This loss of CD127 during chronic infection correlates with impaired production of cytokines, increased susceptibility to apoptosis and reduced ability in specific from what Oseni virus CD8+ T-cell memory to persist in the host. Accordingly, relief from acute infection of hepatitis B virus (HBV) correlates with increased expression of CD127 and simultaneous loss of expression of PD-1 (Boettler et al., J. Virol 80:3532-40, 2006). Interestingly, in patients with chronic HCV only 20% (SEM 4,8) total peripheral CD8+ T-cells were CD127-negative, but in hepatic CD8+ T-cell infiltrates this percentage was significantly increased to 58% (SEM 4,4) (Fig.24D). Thus, the liver is enriched in cells expressing an exhausted phenotype with a predominance of cells with high PD-1 and low CD127. These data suggest that infiltration in the liver CD8+ T-cells in patients with chronic HCV does not reflect the phenotypic population of peripheral CD8+ T-cells. During the course of HIV infection, when the virus infects T-cells and monocytes in the peripheral blood, low levels of CD127 associated with functional defects of T-cell or T-cell memory (Boutboul et al., Aids 19:1981-6, 2005). In this study, the compartmentalization of cells in the liver, indicating their exhausted phenotype, suggesting that the phenotype is closely related to stable replication of the virus.
The expression of PD-1 and CD127 on specific HCV antigen CD8+ T-cells in the liver: we investigated two patients with HLA-A2 group also had detectable staining on tetramer specific HCV population in the liver (Fig.25). The expression of PD-1 and CD127 were directly compared on specific HCV tetramer-positive CD8+ T-cells in the liver and on the periphery of these individuals. Specific HCV CD8+ T cells from the periphery were mostly PD-1-positive (mean 85%, SEM 3.6) and CD127-positive (mean 84%, SEM 4,0), while specific HCV CD8+ T cells were predominantly PD-1-positive (average 92%), but only rarely CD127-positive (average 13%) (Fig.25). In place of virus replication, apparently, multiplication CD127-negative cells expressing PD-1 at a high level. What peripheral specific antigen CD8+ T-cells Express CD127 otherwise than in intrahepatic compartment, could be associated with a level or exposure time of antigen needed to reduce CD127. During LCMV infection of mice under the conditions of exposure of the continued level of antigen in chronic infection CD127 steadily decreased, while brief exposure to LCMV antigen using GP33 only temporarily suppressed the expression of CD127 and did not cause depletion of T-cells (Lang et al., Eur J. Immunol. 35:738-45, 2005). There was also a dependence violation of the expression of CD62L and CD127 on the availability of antigen and exposure time, while the remaining antigen caused a steady decline and CD62L and CD127 (Bachmann et al., J. Immunol 175:4686-96, 2005). Apart from theory, with chronic HCV infection a few specific HCV CD8+ T cells detected in the periphery may not be exhibited continuously with dostat cnym to maintain a low level of CD127 amount of antigen. Thus, T-cells can "believe" that the virus was removed.
Blockade of PD-1/PD-L1 leads to increased reproduction of specific HCV tetramer-positive CD8+ T-cells: the Information collected on the population of patients, suggest that blockade of the interaction of PD-1/PD-L1 antibody against PD-L1 or PD-1 increases the proliferative capacity of specific HCV T-cells (Fig.26). The addition of blocking antibodies in the presence of IL-2 and HCV-specific peptide resulted in a fourfold increase breeding specific HCV T-cells, as shown by tracking the frequency of CD8+ T-cells, labeled carboxyfluorescein Succinimidyl complex ester (CFSE)lowthe tetramer, after stimulation related peptide for 6 days.
The results show that at the site of infection, the liver, the frequency of specific HCV CD8+ T cells expressing PD-1, is high. Secondly, most of the specific HCV CD8+ T-cells from the peripheral blood of patients with chronic HCV infection expresses CD127 at a high level. The phenotype of T-cells during chronic HCV infection was characterized by examining the expression of molecules PD-1 is associated with impaired effector function and depletion of T-cells. The results show that most of the specific HCV T-cells in the intrahepatic compartment of expr ssirum PD-1, but devoid of CD127, the phenotype corresponding to the depletion of T-cells. Thus, antagonists PD-1 are useful as therapeutic agents for the treatment of HCV infection.
Blockade of PD-1 induces the propagation of SIV-specific CD8 T-cells in vitro
Antiviral CD8 T cells play a crucial role in the control of HIV/SIV. The Central role of CD8 T-cells was shown re-emergence of the virus in temporary reductions in vivo SIV infected macaques. In accordance with this modern strategies of vaccines designed to induce high frequency of antiviral CD8 T-cells, included the introduction makaka pathogenic SHIV and SIV (see, for example, Barouch et al., Science 290, 486-92 (2000); Casimiro et al., J. Virol 79, 15547-55 (2005)).
And function, and the frequency of antiviral CD8 T-cells are important for the control of chronic viral infections such as HIV (Migueles et al. Nat Immunol. 3, 1061-8, 2002) and the virus lymphocytic choriomeningitis (LCMV). Effective antiviral CD8 T cells possess a number of functional properties, including the ability to produce various cytokines, cytotoxic potential and high proliferative potential and low apoptosis. In chronic viral infections specific virus CD8 T cells are depleted, which is associated with the loss of many of these functions (Zajac et al., J. Exp. Med. 188, 2205-13, 1998). Similarly, it was shown that the function of the I-specific HIV CD8 T-cells from individuals with progressive disease damaged. These CD8 T cells can produce cytokines, such as IFN-γ, but impaired production of IL-2, a cytokine that is important for the proliferation and survival of T-cells; expression of perforin (Appay et al., J. Exp. Med. 192, 63-75, 2000), molecules that are important for cytolytic function; and proliferative capacity, a property that is credited with a crucial role in the control of HIV (see, for example, Harari et al., Blood 103, 966-72, 2004) and SIV. Specific HIV CD8 T cells Express PD-1 at a high level, and this expression is directly proportional to the level of viremia. Temporary blockade of the interaction between PD-1 and PD-L1 in vitro restores the function of specific HIV T-cells.
Investigated the expression of PD-1 on specific SIV CD8 T-cells after infection of macaques with pathogenic SIV239. The results show that specific SIV CD8 T cells Express PD-1 at a high level and that the blockade of road PD-1:PDL-1 in vitro leads to increased multiplication of these cells.
The results were as follows:
Increased expression of PD-1 on specific SIV CD8 T-cells after infection SIV239: To understand the role of the expression of PD-1 and its Association with infection control SIV investigated the expression level of PD-1 on CD8 T cells from normal healthy and SIV infected macaques. A significant proportion (40-50%) of the total CD8 T-cells from normal healthy macaques expressed the PD-1 (Fig.27A). The expression of PD-1 was mainly limited by memory cells and was absent on naive CD8 T-cells. A similar pattern of expression of PD-1 was also observed in the total CD8 T-cells from SIVmac239 infected macaques (Fig.27B and C). However, the majority (>95%) specific SIV Gag CM9 CD8 T-cells were positive for the expression of PD-1, and in a significant proportion of these cells the expression of PD-1 was further enhanced (MFI 580) compared to the total CD8 T-cells (MFI 220) (Fig.27D). Together, these results show that a significant proportion of CD8 T-cells from normal and SIV infected macaques expresses PD-1 and that the level of expression of PD-1 on specific SIV CD8 T-cells are additionally increased.
In vitro blockade of PD-1 leads to increased reproduction specific SIV CD8 T-cells: To investigate the impact of blockade of PD-1 on the function of specific SIV CD8 T-cells was performed tests of proliferation in the presence and in the absence of blocking antibodies against molecules PD-1 person, which cross interacts with PD-1 macaques. PBMC from Mamu A*01-positive rhesus, which was infected by pathogenic immunodeficiency virus monkeys and humans 89.6 P (SHIV 89.6 P), stimulated by the peptide Pl1C (epitope Gag-CM9) in the absence and in the presence of blocking Ab against PD-1 in six days. At the end of stimulation was assessed by the frequency of Gag CM-9 tetramer-anthropological measurement is different cells. Estimulando cells served as negative controls. As can be seen in Fig.28A-28B, stimulation with peptide P11C resulted in approximately 4-80-fold increase in the frequency of tetramer-positive cells. In addition, four of the six macaques tested stimulation with peptide P11C in the presence of blocking Ab against PD-1 resulted in approximately 2-4-fold further increase in the frequency of tetramer-positive cells compared with stimulation with peptide P11C in the absence of blocking antibodies.
These results indicate that blockade of PD-1 increases the proliferative capacity of specific SIV CD8 T-cells in SIV infected macaques.
The role of PD-L2
Two ligand PD-1 differ in their expression patterns: PD-L1 is expressed constitutively and is increased to large values and on hematopoietic and nonhematopoietic cells, whereas PD-L2 is expressed inducible only on dendritic cells (DC) and macrophages. Although some studies evaluating the role of PD-L2 in the activation of T-cells, showed inhibitory function of PD-L2, in other studies it was reported that PD-L2 stimulates the proliferation of T-cells and production of cytokines by them. To assess the role of PD-L2 in the immune response T-cells have studied the kinetics of expression of PD-L2 cells of various types ex vivo after deg. of infection is of LCMV Armstrong (Fig.29). In contrast to the expression of PD-L1, expression of PD-L2 limited to DC for a very short period (day 1-4 after infection). This result suggests that the expression of PD-L2 is closely linked with the regulation of DC and leads to regulation of activated T-cells.
PD-1 is expressed by the majority of CD8 T-cell effector memory in the blood of healthy people
Investigated the expression of PD-1 on CD3+/CD8+ T-cells from the blood of healthy adults. In human blood 20-60% of CD8 T cells expressed PD-1. Checked the link between the state of differentiation of T-cells and the expression of PD-1. On the basis of the patterns of expression of CD45RA and CCR7 CD3+/CD8+ T cells were divided into subpopulations naive, Central memory (TCM), effector memory (TEMand finally differentiated effector (TEMRA). PD-1 is not expressively naive T-cells and approximately one-third TCMand TEMRA. In contrast, 60% of TEMexpressed PD-1. These data show that the majority of TEMisolated from the blood of healthy adults, expresses PD-1.
Based on these analyses, the T cells were divided into a number of populations based on the expression of CD45RA and CCR7. Was discovered additional link between the expression of CD45RA and expression of PD-1. Specifically, CCR7-/CD8+ T-cells with the lowest expression of CD45RA contained the greatest proportion of PD-1+ glue is OK. In conclusion, PD-1 mainly expressively TEMto a lesser extent, TEMRAand TCMand not expressively naive CD8 T-cells. These data illustrate that a large part of TEMCD8 T-cells in healthy adults expresses PD-1.
To further characterize the properties of PD-1+ CD8 T-cells was studied by a joint expression of PD-1 and several markers of differentiation of T-cells. The majority of PD-1+ CD8 T-cells carrying the markers associated with familiarity with the antigen and effector/effector memory differentiation. For example, CD11a+/CCR7-/CD62L-/CD45RA-/KLRGl+/Grasim B+/perforin+ CD8 T cells were enriched for expression of PD-1. In contrast, CD8 T cells naive phenotype (CD11a-/CCR7+/CD62L+/CD45RA+/KLRG1-) expressed PD-1 at a low level. Thus, PD-1 mainly expressively on familiar with antigen CD8 T cells with effector/effector memory properties.
PD-1 is expressed by the majority of CD4 T cells effector memory in the blood of healthy people
Then examined the expression of PD-1 among CD3+ CD4+ T-cells. In the blood of healthy adults thirty percent CD4 T cells expressed PD-1. Similar to CD8 T cells, naive CD4 T cells weakly expressed PD-1. While a minority of TCMCD4 T cells expressed PD-1, expression of PD-1 was predominantly enriched among TEMCD4 T-cells (5%).
To further characterize the properties of CD4 T-cells that expressed PD-1, was investigated CD4+/CD3+ T-cells from the blood of healthy individuals on the subject of joint expression of PD-1 and several markers of differentiation of T-cells. Similar to CD8 T cells, the expression of PD-1 was enriched in CD4 T-cells with effector/effector memory phenotype, including CD62L-, CD95+, CD45RA-, CCR7 - and CCR5+ cells.
PD-1 is expressed stronger on CD8 T-cells specific against infections EBV and CMV in humans
To check whether correlates the expression of PD-1 with prolonged viral antigen, compared the expression of PD-1 on specific viruses EBV, CMV, influenza and vaccinia (smallpox CD8 T-cells. Specific EBV and CMV CD8 T cells expressed PD-1 at a high level. On the contrary, specific influenza virus CD8 T-memory cells expressed PD-1 at an intermediate level, and specific vaccinia virus CD8 T cells expressed PD-1 at a low level. Thus, CD8 T-memory cells specific for chronic infections (EBV and CMV), expressed PD-1 at a higher level than acute (influenza and vaccinia) infections. These results indicate that CD8 T cells specific for chronic infections (EBV and CMV), expressed PD-1 at a higher level than is strych infections (influenza virus and cowpox). CD8 T cells specific against the most common chronic infections can Express PD-1 at a high level.
Blockade by anti-PD-L1 enhances the proliferation of CD8 T cells specific against infections EBV and CMV in humans
Blockade of inhibitory path PD-1 leads to increased clonal expansion of specific HIV CD8 T-cells after stimulation in vitro. Because CD8 T cells specific against common chronic infections, also Express PD-1, was examined whether blockade of road PD-1/PD-L1 to increase the proliferation of CD8 T-cells specific for EBV, CMV, and cowpox virus (acute infection, giving PD-1 CD8 T-cell memory). Lymphocytes isolated from the blood of individuals with CD8 T-cells specific for EBV, CMV, or VV, were CFSE labeled and cultured for 6 days in different conditions. As expected, incubation of freshly isolated mononuclear cells of peripheral blood (PBMC) only with medium or medium with antibody against PD-L1 did not cause the proliferation of specific virus CD8 T-cells. Stimulation of PBMC for 6 days peptides from viruses has led to the division of tetramer+ CD8 T-cells. However, peptide stimulation of PBMC in the presence of blocking antibodies against PD-L1 is additionally increased the division of specific EBV and CMV CD8 T-cells, which led the greater the ratio of expansion in comparison with a single peptide. Increased fission induced by a blocking antibody against PD-L1, varied between individuals and even between epitopes from a particular individual. Moreover, blockade of PD-1 did not lead to increased expansion specific cow pox and influenza CD8 T-cells. The degree of increase of fission induced by a blocking antibody against PD-L1 in culture, could be related to the number of PD-1 expressed specific antigen CD8 T-cells before stimulation. These data suggest that the expression of PD-1 on CD8 T-cells specific for chronic infections, inhibits their proliferative capacity upon antigenic stimulation.
Prolonged blockade of PD-L1 enhancesthe proliferation of CD8 T cells specific for chronic infections
When stimulated in vitro addition of blocking PD-L1 antibody led to increased division among CD8 T-cells specific for EBV and CMV. mAb against PD-L1 was added once (day 0), and proliferation was assessed at the end of the six-day cultivation period. Treatment of mice with anti-PD-L1 in vivo included multiple injection of blocking antibodies. Moreover, in these studies on mice, blockade of PD-L1 in vivo led to a rapid increase in the expression of PD-1 among CD8 T-cells specific against the chronic is the first viral antigen. For these reasons, it was tested whether re-additive anti-PD-L1 to stimulated cultures of T-cells further increase proliferation. The addition of mAb against PD-L1 in 0, 2 and 4 days of cultivation led to even greater accumulation of CD8 T-cells specific for EBV than a single addition of mAb on day 0. Similar data were observed for CD8 T-cells specific against CMV. These data suggest that prolonged blockade of signaling PD-1 is able to optimize the ability to increase the number of CD8 T-cells specific for chronic antigens.
It should be clear that the precise details of the described methods or compositions can be varied or modified without departing from the essence of the described invention. Applicants claim the right to all such modifications and variations that fall within the scope and essence of the following claims.
1. The method of treatment of an individual with a stable infection by a pathogen or tumor, comprising an introduction to the individual a therapeutically effective amount of the antagonist peptide programmed death (PD-1) and a therapeutically effective amount of the antigenic molecule from a pathogen or tumor, respectively,
2. The method according to p. 1, where the antagonist PD-1 is an antibody that specifically binds PD-1, or an antibody that specifically binds to the ligand-1 peptide programmed death (PD-L1) or combinations thereof.
3. The method according to p. 1, where the individual is characterized by a weakened immune system.
4. The method according to p. 1, where the antagonist PD-1 is an antibody against PD-1 or the antibody against PD-L1.
5. The method according to p. 4, wherein the antibody that specifically binds PD-1, represents (1) a monoclonal antibody or functional fragment, (2) humanitariannet EN is Italo or its functional fragment, or (3) hybrid immunoglobulin protein.
6. The method according to p. 4, wherein the antibody that binds to PD-L1 represents (1) a monoclonal antibody or functional fragment, (2) humanitariannet antibody or functional fragment or (3) hybrid immunoglobulin protein.
7. The method according to p. 1, where the individual has a stable infection by a pathogen and the method includes the introduction of viral antigen or nucleic acid encoding a viral antigen to the individual, thereby making the treatment of resistant infections in the individual.
8. The method according to p. 1, where the individual has no symptoms, so treatment prevents the development of symptoms.
9. The method according to p. 1, where the individual has a tumor and where the method includes the administration to an individual specified tumor antigen or of a specified nucleic acid that encodes a tumor antigen, thereby carrying out the treatment of tumors.
10. The method according to p. 9, where the antagonist PD-1 is an antibody that specifically binds PD-1, or an antibody that specifically binds to the ligand-1 peptide programmed death (PD-L1) or combinations thereof.
11. The method according to p. 10, where the antibody that specifically binds PD-1, represents (1) a monoclonal antibody or functional fragment, (2) humanitariannet antibody or functional fragment or (3) hybrid immunoglobulin protein.
12. the manual on p. 10, where the antibody that binds to PD-L1 represents (1) a monoclonal antibody or functional fragment, (2) humanitariannet antibody or functional fragment or (3) hybrid immunoglobulin protein.
13. The method according to p. 9, where a tumor antigen PRAME is a, WT1, survivin, cyclin D, cyclin E, proteinase 3 and its peptide PR1, neutrophil elastase, cathepsin G, MAGE, MART, tyrosinase, GP100, NY-Eso-1, Herceptin, removeability antigen (CEA) or prostatespecific antigen (PSA).
14. The method according to p. 1, where the virus is a human immunodeficiency virus or hepatitis virus.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, more specifically to MUC1 cytoplasmic domain peptides, and can be used in the anticancer therapy. A method for inhibiting MUC1-positive cancer cell in an individual involves administering into an individual the MUC1-peptide of the length of at least 6 sequential MUC1 residues and no more than 20 sequential MUC1residues and containing the sequence CQCRRK, wherein the amino terminal cysteine from CQCRRK is closed at its NH2 terminal by at least one amino acid residue, which shall not conform with the native transmembrane sequence MUC-1. Alternatively, there can be used MUC-1 peptide of the length of at least sequential MUC1 residues and no more than 20 sequential MUC1 residues, which contains the sequence CQCRRK with all amino acid residues of the above peptide being D-amino acids.
EFFECT: invention enables inhibiting MUC1oligomerisation effectively and inducing the tumour cell apoptosis and the tumour tissue necrosis in vivo.
80 cl, 16 dwg, 1 tbl, 3 ex
Improved amino acid sequences against il-6r and polypeptides which contain thereof for treatment of il-6r associated diseases and disorders // 2539798
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of biochemistry, in particular to single variable domain, aimed against IL-6R, to polypeptide and construction, directed against IL-6R, containing said single variable domain, as well as to methods of obtaining them. Disclosed are nucleic acids, coding said single variable domain, polypeptide and construction, as well as genetic constructions, containing said nucleic acids. Described are host cells and host organisms, containing said nucleic acids. Invention also deals with composition for blocking interaction of IL-6/IL-6R, containing effective quantity of described single variable domain, polypeptide, construction, nucleic acid or genetic construction. Also disclosed is method of prevention and/or treatment of at least one of diseases or disorders, associated with IL-6, IL-6R, complex IL-6/IL-6R and/or signal pathways, in which IL-6, IL-6R or complex IL-6/IL-6R is involved and/or biological functions and reactions, win which IL-6, IL-6R or complex IL-6/IL-6R takes part with application of described single variable domain, polypeptide, construction or composition.
EFFECT: invention makes it possible to block interaction of IL-6/IL-6R effectively with increased affinity and biological activity.
25 cl, 70 dwg, 56 tbl, 61 ex
SUBSTANCE: invention refers to biotechnology, in particular to tumour-specific promoters, and can be used in the anti-cancer therapy. There are constructed the broad-spectrum tumour-specific promoters providing the therapeutic gene expression inside a cancer cell. The invention also involves expression cassettes, expression vectors, pharmaceutical compositions, methods of treating cancer and using the expression cassettes and vectors.
EFFECT: promoters of the present invention provide a high expression level of the operatively linked therapeutic gene in the cancer cells of different origin, wherein the normal cell expression is absent or low.
29 cl, 19 dwg, 4 tbl, 20 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to specific compounds or their therapeutically acceptable salts presented in the patent claim and representing sulphonyl benzamide derivatives. Besides, the invention refers to a pharmaceutical composition and a method of treating bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, stomach cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, T-cell or B-cell lymphoid process, melanoma, myelogenic leukaemia, myeloma, oral cancer, ovarian cancer, non-small-cell lung cancer, prostate cancer, small-cell lung cancer, spleen cancer with the above composition containing an excipient and a therapeutically effective amount of the sulphonyl benzamide derivative or its therapeutically acceptable salt.
EFFECT: preparing the new pharmaceutical composition.
5 cl, 45 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to novel compounds of formula Ia, their stereoisomers or pharmaceutically acceptable salts, inhibiting JAK kinase activity. Compounds can be applied in treatment of inflammatory diseases, such as rheumatoid arthritis, psoriasis, contact dermatitis, in treatment of autoimmune diseases, such as lupus, multiple sclerosis, neurodegenerative diseases, such as Alzheimer's disease, etc. In formula Ia R1 represents H; R2 represents -OR4, -NR3R4- or -NR3S(O)2R4; R3 represents H or C1-C6alkyl, where said alkyl is optionally substituted with ORa; R4 represents H, C1-C6alkyl, -(C0-C5alkyl)(C3-C6cycloalkyl), -(C0-C5alkyl)(C4-C5heteroaryl), where heteroaryl contains 1-2 nitrogen atoms as heteroatoms, or -(C0-C5alkyl)(C6aryl), where said alkyl is optionally substituted with group R8 and said aryl, cycloalkyl and heteroaryl are optionally substituted with group R9; or R3 and R4, taken together with nitrogen atom, which they are bound to, form C3heterocyclyl, containing 1 nitrogen atom as heteroatom, optionally substituted with group R13; Z represents -NR5R6; R5 represents H; R6 represents H, C1-C10alkyl, -(C0-C5alkyl)(C4-C5heterocyclyl), where heterocyclyl contains oxygen atom as heteroatom, -(C0-C5alkyl)(C3-C8cycloalkyl), -(C0-C5alkyl)(C3-C5heteroaryl), where heteroaryl contains 1 nitrogen atom or 1 oxygen atom or contains 2 atoms, selected fromoxygen, nitrogen and sulphur, as heteroatoms, -(C0-C5alkyl)(C6aryl), where said alkyl is optionally substituted with group R10, and said aryl, cycloalkyl, heteroaryl and heterocyclyl are optionally substituted with group R11; R7 represents H; R8 and R10 each independently represents halogen or ORa; R9 independently represents -CN, -CF3, halogen, -C(O)ORa, -C(O)NRaRb, -(C0-C5alkyl)NRaRb, -(C0-C5alkyl)ORa, -(C0-C5alkyl)SRa, -O[C(Ra)2]1-3O-, C1-C3alkyl, optionally substituted with F, -(C0-C5alkyl)(C3-C6cycloalkyl), optionally substituted with group oxo or F, -(C0-C5alkyl)C3-C6heterocyclyl, where heterocyclyl contains 1-2 heteroatoms, selected from atoms of oxygen and nitrogen, and where heterocyclyl is optionally substituted with halogen or C1-C3alkyl, -(C0-C5alkyl)C6aryl, optionally substituted with halogen, or -(C0-C5alkyl)C4-C5heteroaryl, where heteroaryl contains 1 nitrogen atom or 1 oxygen atom or contains 2 atoms, selected from atom of oxygen, nitrogen and sulphur as heteroatoms, and where heteroaryl is optionally substituted with or C1-C3alkyl; R10 independently represents halogen or ORa. Other values of radicals are given in the invention formula.
EFFECT: obtaining pharmaceutically acceptable salts, inhibiting JAK kinase activity.
15 cl, 4 tbl, 452 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to compounds of formula I , II or IV , wherein the radical values W, V, Ra, Rb, X, L, Rt, A are presented in the patent claim.
EFFECT: declared compounds identify and bind the CA-IX protein; they can contain a radioactive element for radionuclide imaging or therapeutic application.
27 cl, 1 tbl, 5 dwg, 25 ex
SUBSTANCE: treating locally advanced oropharyngeal cancer is ensured by a radiation therapy in the mode of dynamic dose fractionation. The radiation therapy is started by supplying a fraction dose of 2.4 Gy. After 2 days of treatment gap, the patient is exposed to total fractions at a fraction dose of 3.6Gy for three days. Each session is precede by placing high-structure hydrogel matrix of sodium alginate under a patient's tongue with metronidazole 150mg and bilberry 20-35mg pre-introduced into the matrix, for 4-5 hours twice every 1-2 hours. The two following sessions of the exposure at a fraction dose of 2.4 Gy are preceded by placing the matrix once under the tongue for 4-5 hours. After 2 days of treatment gap, the following 5 sessions of the radiation therapy are performed at a fraction dose of 2.4 Gy to a cumulative dose of 30Gy. Colegel-DNA-Ch high-structure disk is preliminary placed under the tongue for 4-5 hours.
EFFECT: method enables avoiding the compulsory gaps of the radiation therapy by reducing a rate of severe local radiation reactions, and provides the target delivery and accurate dosage of metronidazole administered into the patient's body leading to the partial death of well-oxygenated cells and re-oxygenation of hypoxic tumour cells.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to compounds of formula or its therapeutically acceptable salts, wherein A1 represents furyl, imidazolyl, isothiazolyl, isoxazolyl, pyrazolyl, pyrrolyl, thiazolyl, thiadiazolyl, thienyl, triazolyl, piperidinyl, morpholinyl, dihydro-1,3,4-thiadiazol-2-yl, benzothien-2-yl, banzothiazol-2-yl, tetrahydrothien-3-yl, [1,2,4]triazolo[1,5-a]pyrimidin-2-yl or imidazo[2,1-b][1,3]-thiazol-5-yl; wherein A1 is unsubstituted or substituted by one, or two, or three, or four, or five substitutes independently specified in R1, OR1, C(O)OR1, NHR1, N(R1)2, C(N)C(O)R1, C(O)NHR1, NHC(O)R1, NR1C(O)R1, (O), NO2, F, Cl, Br and CF3; R1 represents R2, R3, R4 or R5; R2 represents phenyl; R3 represents pyrazolyl or isoxazolyl; R4 represents piperidinyl; R5 represents C1-C10alkyl or C2-C10alkenyl each of which is not specified or specified by substitutes specified in R7, SR7, N(R7)2, NHC(O)R7, F and Cl; R7 represents R8, R9, R10 or R11; R8 represents phenyl; R9 represents oxadiazolyl; R10 represents morpholinyl, pyrrolidinyl or tetrahydropyranyl; R11 represents C1-C10alkyl; Z1 represents phenylene; Z2 represents piperidine unsubstituted or substituted by OCH3, or piperazine; both Z1A and Z2A are absent; L1 represents C1-C10alkyl or C2-C10alkenyl each of which is unsubstituted or substituted by R37B; R37B represents phenyl; Z3 represents R38 or R40; R38 represents phenyl; R40 represents cyclohexyl or cyclohexenyl; wherein phenylene presented by Z1 is unsubstituted or substituted by the group OR41; R41 represents R42 or R43; R42 represents phenyl, which is uncondensed or condensed with pyrrolyl, imidazolyl or pyrazole; R43 represents pyridinyl, which is uncondensed or condensed with pyrrolyl; wherein each cyclic fragment presented by R2, R3, R4, R8, R9, R10, R38, R40, R42 and R43 is independently unsubstituted or substituted by one or more substitutes independently specified in R57, OR57, C(O)OR57, F, Cl CF3 and Br; R57 represents R58 or R61; R58 represents phenyl; R61 represents C1-C10alkyl; and wherein phenyl presented by the group R58 is unsubstituted or substituted by one or more substitutes independently specified in F and Cl.
EFFECT: invention refers to a pharmaceutical composition containing the above compounds, and to a method of treating diseases involving the expression of anti-apoptotic Bcl-2 proteins.
7 cl, 2 tbl, 48 ex
Agent for ehrlich tumour inhibition // 2538716
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to pharmacology and oncology, particularly to a new antitumour agent. Beta-ethyldiphacyl is presented to be used as an agent of an epithelial tumour (carcinoma) inhibition. There is provided to administer Ethyldiphacyl in doses of 15-50 mg/kg both before, and after the tumour grafting; the therapeutic course is up to 2 months after the tumour grafting with administrations following every 6-7 days; or preventive administration is performed 2-24 hours before the tumour grafting.
EFFECT: technical effect consists in prolonging the life of mammals suffering the from the tumour by 30%; the life is not burdened with the disease progression.
4 cl, 1 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: what is presented is a group of inventions concerning treating and preventing a disease dependent on mTOR (mammalian target of rapamycin) kinase and representing cancer or malignant growth. The group involves a pharmaceutical combination for the above application containing 5-(2,6-dimorpholin-4-ylpyrimidin-4-yl)-4 trifluoromethylpyridin-2-ylamine or its salt and the mTOR inhibitor everolimus; using it for preparing a drug preparation for the same application and a pharmaceutical composition containing the above combination.
EFFECT: synergetic effect of the antitumour action of 5-(2,6-dimorpholin-4-ylpyrimidin-4-yl)-4 trifluoromethylpyridin-2-ylamine and everolimus.
7 cl, 2 dwg
Improved amino acid sequences against il-6r and polypeptides which contain thereof for treatment of il-6r associated diseases and disorders // 2539798
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of biochemistry, in particular to single variable domain, aimed against IL-6R, to polypeptide and construction, directed against IL-6R, containing said single variable domain, as well as to methods of obtaining them. Disclosed are nucleic acids, coding said single variable domain, polypeptide and construction, as well as genetic constructions, containing said nucleic acids. Described are host cells and host organisms, containing said nucleic acids. Invention also deals with composition for blocking interaction of IL-6/IL-6R, containing effective quantity of described single variable domain, polypeptide, construction, nucleic acid or genetic construction. Also disclosed is method of prevention and/or treatment of at least one of diseases or disorders, associated with IL-6, IL-6R, complex IL-6/IL-6R and/or signal pathways, in which IL-6, IL-6R or complex IL-6/IL-6R is involved and/or biological functions and reactions, win which IL-6, IL-6R or complex IL-6/IL-6R takes part with application of described single variable domain, polypeptide, construction or composition.
EFFECT: invention makes it possible to block interaction of IL-6/IL-6R effectively with increased affinity and biological activity.
25 cl, 70 dwg, 56 tbl, 61 ex
Siglec-15 antibody // 2539790
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and immunology. What is described is a pharmaceutical composition used for treating and/or preventing pathological bone metabolism and containing this antibody. The invention can be used in medicine.
EFFECT: antibody and its functional fragment specifically recognising human Siglec-15 and possessing the osteoclast inhibitory activity are described.
73 cl, 57 dwg, 4 tbl, 33 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and immunology. What is described is a recovered human antibody or its antigen-binding fragment. The antibody binds to human interleukin-4 alpha-receptor (hlL-4R). There are also described a nucleic acid molecule coding this antibody, an expression vector, a host cell, a method for producing such antibody and a therapeutic composition containing this antibody.
EFFECT: presented group of inventions can be used in medicine for treating asthma and atopic dermatitis.
15 cl, 3 tbl, 3 ex
SUBSTANCE: invention relates to biochemistry.
EFFECT: method of identifying a candidate substance that inhibits hepsin activation of pro-macrophage-stimulating protein (pro-MSP) is provided.
10 cl, 10 dwg, 1 tbl, 1 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of immunology and biotechnology. Claimed is monoclonal antibody or its functional fragment, where said antibody and fragment bind with activated protein C and inhibit anticoagulant activity, but do not bind and do not inhibit activation of inactivated protein C, where said antibody is obtained by immunisation of mammal by APC and screening of binding ability of said antibody with APC, but not with protein C. Also described is pharmaceutical composition for treating diseases associated with anticoagulation activity of APC, including said antibody in effective amount and pharmaceutically acceptable carrier. Claimed are: method of inhibiting anticoagulation activity of activated protein C in subject, method of inhibiting amidolytic activity of activated protein C in subject, method of treating subject, requiring blood coagulation; method of treating subject with haemophilia; method of modulating haemostasis in subject; as well as method of modulating thrombogenesis in subject, which include introduction of effective quality of said antibody to subject. In addition, described is method of treating subject with sepsis, including introduction of effective quality of said antibody and activated protein C.
EFFECT: invention makes it possible to obtain monoclonal antibody or its functional fragment, where said antibody and fragment bind with activated protein C and inhibit anticoagulation activity, but do not bind and do not inhibit activation of inactivated protein C.
17 cl, 11 dwg, 6 ex
Method of treating multiple sclerosis // 2539034
SUBSTANCE: present invention refers to biotechnology, more specifically to granulocyte-macrophage colony-stimulating factor (GM-CSF) antagonists, and can be used in medicine. The invention consisting in using the GM-CSF specific antibody in treating or preventing multiple sclerosis in the patients with multiple sclerosis.
EFFECT: invention enables delaying the onset of multiple sclerosis recurrences.
9 cl, 5 dwg, 8 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to immunology. There are presented: an antibody binding to interleukin-17 (IL-17) characterised by 6 CDR of a light and heavy chain, as well as a coding nucleic acid and a vector for expression of the above antibody. What is described is a pharmaceutical composition for treating a patient with multiple sclerosis, rheumatoid arthritis, psoriasis, Crohn's disease, chronic obstructive pulmonary disease, asthma, graft rejection on the basis of the above antibody. What is disclosed is a method for preparing the antibody by means of expressing the respective nucleic acid and recovering the antibody from a cell culture or a cell culture supernatant.
EFFECT: using this invention provides the antibody with IC50 twice as much as shown by in vitro IL-6 and IL-8 neutralisation as compared to the known NVP-AIN-497 antibody, which binds human IL-17A and IL-17F that can find application in medicine in therapy of various inflammatory diseases.
9 cl, 6 tbl, 11 ex
Humanised antibody // 2538709
SUBSTANCE: group of inventions refers to medicine, namely to ophthalmology, and can be used for treating ocular diseases associated with amyloid-beta related pathological abnormalities/changes in the visual system tissues. That is ensured by administering a pharmaceutical composition, which contains a therapeutically effective amount of a humanised antibody or antigen-binding fragment, wherein the humanised antibody or its fragment is able to bind amyloid-beta. Presented are preventing, treating or relieving symptoms of an ocular disease, reducing the plaque load of retinal ganglion cells, diagnosing the ocular disease and diagnosing a predisposition to the ocular disease, prolonging the patient's sensitivity when treating with the pharmaceutical composition for treating the ocular disease.
EFFECT: group of inventions provides the effective treating of the above ocular pathology by using the composition containing the high-specific antibodies, which specifically recognise and bind to specific epitopes of various β-amyloid proteins.
20 cl, 18 dwg, 9 tbl, 18 ex
Therapy of tumours with application of antibody to vascular endothelial growth factor and antibodies to human epithelial growth factor receptor type 2 // 2538664
SUBSTANCE: group of inventions relates to medicine and deals with application of antibody to HER2 and/or antibody to VEGF for preparation of medication intended for reduction of metastases in treatment of breast cancer, characterised by super-expression of HER2 receptor protein in patient, unsusceptible to preceding therapy with application of antibody to VEGF, in which treatment includes introduction to patient of therapeutically effective amount of antibody to HER2 and antibody to VEGF, with antibody to VEGF representing bevacizumab, and antibody to HER2 representing trastuzumab. Group of inventions also deals with application of antibody to HER2 for reduction of metastases in method of breast cancer treatment and application of antibodies to VEGF for reduction of methastases in method of breast cancer treatment.
EFFECT: combination of bevacizumab and trastuzumab makes it possible to achieve prevention of metastases formation.
12 cl, 1 ex, 2 dwg, 4 tbl
Cd138-targeted cell agents and using them // 2537265
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and immunology. What is presented is a genetically engineered antibody able to bind human CD138. There are described its heavy and light chain sequences, as well as their CDR.
EFFECT: presented antibody can be used as an ingredient of a pharmaceutical composition for treating tumours.
13 cl, 21 dwg, 5 tbl
Antibody igg with affinity binding with respect to antigenic complex cd3, recombinant nucleic acids encoding antibody light and heavy chain, method for preparing system, method for preparing antibody, method for treatment of patient // 2244720
FIELD: genetic engineering, immunology, medicine.
SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.
EFFECT: improved preparing methods, valuable medicinal properties of antibody.
33 cl, 5 dwg, 1 ex