Stable and soluble antibodies, inhibiting tnfα
SUBSTANCE: invention relates to biotechnology and represents stable and soluble scFv-antibody and Fab-fragment, specific with respect to TNFα, which contain specific sequences of light chain and heavy chain, optimised as to stability, solubility, in vitro and in vivo TNFα binding and low immunogenity. Also claimed are antibody-coding DNA-sequence, vectors, host-cell, as well as method of obtaining antibody with application of said cells. In addition claimed are therapeutic and diagnostic compositions and stable water pharmaceutically ready form based on antibodies, as well as method of treating TNFα-associated disease.
EFFECT: invention can be efficiently used for diagnostics and treatment of TNFα-associated disorders.
61 cl, 18 dwg, 5 tbl, 5 ex
The technical field to which the invention relates.
This invention relates to an optimized antibodies and derivatives of antibodies that bind to tumor necrosis factor alpha (TNFα) and block the function of tumor necrosis factor alpha and applicable for the diagnosis and/or treatment, prevention or reduction of the intensity of symptoms TNFα-related diseases; their coding sequences, acquisition, and application of a pharmacologically suitable compositions.
The level of technology
The tumor necrosis factor alpha (TNFα, also known as cachectin) is a naturally-occurring mammalian cytokine produced by numerous cell types, including monocytes and macrophages in response to endotoxin or other stimuli. TNFα is a major mediator of inflammatory, immunological and pathophysiological reactions (Grell, M., et al. (1995) Cell, 83: 793-802).
Soluble TNFa is formed by the cleavage of the transmembrane protein precursor (Kriegler, et al. (1988) Cell 53: 45-53) and Assembly of secreted polypeptides 17 kDa soluble in homotrimeric complexes (Smith, et al. (1987), J. Biol. Chem. 262: 6951-6954; for reviews see TNF Butler, et al. (1986)Nature 320: 584; Old (1986), Science 230: 630). These complexes then bind to receptors found on the various cells. Linking produces a number of Pro-inflammatory EF is known, including: (i) the release of other proinflammatory cytokines, such as interleukins (IL) IL-6, IL-8 and IL-1, (ii) the release of matrix metalloproteinases and (iii) positive regulation of expression of endothelial adhesion molecules, further amplifying the inflammatory and immune cascade atregional leukocytes in extravascular tissue.
A large number of disorders associated with elevated levels of TNFα, and many of them are essential from a medical point of view. It was shown that TNFα is positively regulated in several human diseases, including chronic diseases, such as rheumatoid arthritis (RA), inflammatory disorders of the digestive tract, including Crohn's disease and ulcerative colitis, sepsis, congestive heart failure, bronchial asthma and multiple sclerosis. Mouse transgenic against human TNFα, produce high levels of TNFα constitutive and develop spontaneous destructive polyarthritis similar to RA (Keffer et al. 1991, EMBO J. 10, 4025-4031). Thus, TNFα called proinflammatory cytokine.
TNFα is well established now as a key factor in the pathogenesis of rheumatoid arthritis (RA)is a chronic, progressive and debilitating disease characterized mnogoslovnym inflammation of the destruction of the joints, with systemic symptoms of malaise and fatigue. RA often leads to chronic synovial inflammation with frequent progression to the destruction of articular cartilage and bone. Increased levels of TNFα detected in synovial fluid and peripheral blood of patients suffering from rheumatoid arthritis (RA). With the introduction of blocking TNFα agents for patients suffering from RA, they reduce inflammation, improve symptoms and delay the damage to the joint (McKown et al. (1999), Arthritis Rheum. 42: 1204-1208).
Physiologically TNFα is associated with protection against specific infections (Cerami et al. (1988), Immunol. Today 9:29). TNFα is released by macrophages that have been activated by lipopolysaccharides of gram-negative bacteria. As such, TNFα is, apparently, an endogenous mediator of paramount importance, participating in the development and pathogenesis of endotoxin (bacterial-toxic) shock associated with bacterial sepsis (Michie, et al. (1989), Br. J. Surg. 76: 670-671; Debets et al. (1989), Second Vienna Shock Forum, p.463-466; Simpson, et al. (1989) Crit. Care Clin. 5: 27-47; Waage et al. (1987) Lancet 1: 355-357; Hammerle et al. (1989) Second Vienna Shock Forum, p.715-718; Debats et al. (1989), Crit. Care Clin. 17: 489-497; Calandra et al. (1990), J. Infect. Dis. 161: 982-987; Revhaug et al. (1988), Arch. Surg. 123: 162-170).
It was found that, as in the case of other organ systems, TNFα plays a key role in the Central nervous system, in particular in inflammatory and autoimmune on what uchenykh nervous system, including multiple sclerosis, Guillain-Barre syndrome and severe pseudoparalysis the male, and in degenerative disorders of the nervous system, including Alzheimer's disease, Parkinson's disease and Huntington's disease. TNFα is also involved in the violations related systems retina and muscles, including retro-bulbar neuritis, dermatomyositis, amyotrophic lateral sclerosis and muscular dystrophy, as well as in lesions of the nervous system, including traumatic brain injury, acute spinal cord injury and stroke.
Hepatitis is another TNFα-related inflammatory disorder, which among other precipitating factors may be due to viral infections, including Epstein-Barr, cytomegalovirus, and hepatitis a-E. the Hepatitis causes acute inflammation of the liver in the portal and lobed areas with subsequent fibrosis and progression of tumors.
TNFα may mediate cachexia in cancer, which causes the greatest morbidity and mortality (Tisdale M.J. (2004), Langenbecks Arch Surg. 389: 299-305).
The key role of TNFα in inflammation, cellular immune responses and pathology of many diseases, has led to the search of TNFα antagonists.
TNFα is an important cytokine that systemic blockade which carries the risk of increased frequency and heavy the tee this horizon manifest itself clinically infections, in particular, reactivation of latent tuberculosis, and possibly other risks, including the induction of lymphoma, demyelinating disease and heart failure.
One class of TNFα antagonists for the treatment of TNFα-mediated diseases, are antibodies or antibody fragments that specifically bind TNFα and thereby inhibit their function. The use of anti-NFα antibodies showed that TNFα blockade can reverse the effects attributed to TNFα, including the reduction of IL-1, GM-CSF, IL-6, IL-8, and adhesion molecules and tissue destruction (Feldman et al. (1997), Adv. Immunol. 1997: 283-350).
Antibodies directed against TNFα, have been proposed for the prevention and treatment of endotoxic shock (Beutler et al. (1985) Science:234, 470-474). The use of anti-TNFα antibodies in the treatment of septic shock is discussed Bodmer et al., (Critical Care Medicine, 21:441-446, 1993), Wherry et al., 1993 (Critical Care Medicine, 21:436-440) and Kirschenbaum et al., 1998 (Critical Care Medicine, 26:1625-1626).
A method of treating neurodegenerative disease in man by the introduction of monoclonal anti-TNFα antibody or TNFα-binding fragment has been described in US 2003147891.
WO 0149321 describes the use of TNFα antagonists, including anti-TNFα antibodies, for the treatment of neurological and related disorders caused by TNFα. It provides a method of treatment for these diseases by the introduction of a TNFα antagonist.
WO 03047510 describes different is the IPA monoclonal and engineered antibody, directed against TNFα, receive them, compounds containing them, and their application in medicine.
Antibodies useful for therapies TNFα-mediated diseases, usually are either monoclonal antibodies (mAb)produced by hybrid technology from a natural source, or engineered antibodies. The last or correspond to natural-occurring antibody in the sense that they contain a full-sized heavy and light chains, or meet Fab-fragments, which can be also created from natural antibodies proteolytic cleavage, or single-chain scFv-antibodies, in which fragments of variable regions of heavy and light chains are linked peptide linker.
Both heavy and light chains of the antibodies contain constant and variable domains. Because antibodies are immunogenic, the number of sequences similar to the sequences of human rights, the antibody often increase in so-called "hybrid antibody that contains a constant region of human IgG and variable regions corresponding to the sequences of the antibodies of the animal, in most cases, mouse antibodies with the desired specificity. Then these variable regions can be further adapted so that they are more similar to typical human antibody is a, through mutagenesis that leads to "humanitarianlaw" antibody. In yet another alternative approach, only the antigen-binding part, i.e. complementarity determining areas (CDR) of the variable regions of mouse antibodies, together with a framework of human antibodies, which leads to "CDR-grafted antibody.
Monoclonal antibodies against TNFα have been described in the prior art. Meager et al., 1987 (Hybridoma 6:305-311) describe mouse monoclonal antibodies against recombinant TNFα. Shimamoto et al., 1988 (Immunology Letters 17:311-318) describe the application of mouse monoclonal antibody against TNFα in preventing endotoxic shock in mice.
US 5919452 describes chimeric anti-NFα-antibodies and their use in the treatment of pathologies associated with the presence of TNFα.
The use of anti-TNFα antibodies in the treatment of RA and Crohn's disease is discussed in Feldman et al., 1998 (Transplantation Proceedings 30:4126-4127), Adorini et al., 1997 (Trends in Immunology Today 18:209-211) and Feldman et al., 1997 (Advanced Immunology 64:283-350). Antibodies to TNFα used in such therapies are usually chimeric antibodies, such as antibodies, are described in US 5919452.
US 2003187231 describes humanized anti-NFα antibodies, at least one CDR region is not man that improved binding characteristics. In addition, in the International patent application WO 92/11383 described recombinant antibodies, including CDR-grafted the antibodies, specific against TNFα. Rankin et al. (1995) (British J. Rheumatology 34:334-342) describe the use of such CDR-grafted antibodies in the treatment of rheumatoid arthritis (RA).
WO 9211383 describes recombinant, humanitariannet CDR-grafted antibody specific against TNFα, which is produced from a mouse monoclonal antibody E, hTNFI, hTNF3 or 101.4, and describes the preparation and use of these antibodies in the diagnosis and/or therapy TNFα-related disorders.
Among specific inhibitors of TNFα, which became commercially available only recently, monoclonal, chimeric mouse-human antibody directed against TNFα (infliximab, Remicade™; Cenrtocor Corporation/Johnson & Johnson), has demonstrated clinical efficacy in the treatment of RA (Elliot et al. 1994, Lancet 344:1105-1110; Mani et al. (1998), Arthritis & Rheumatism 41:1552-1563). Infliximab has also demonstrated clinical efficacy in the treatment of inflammatory disorders of the digestive tract, Crohn's disease (Baert et al. 1999, Gastroenterology 116: 22-28).
US 22002037934 describes the treatment of hepatitis introduction of anti-TNFα antibodies, such as infliximab.
US 6428787 describes the treatment of neurological and TNFα-related diseases anti-TNFα-antibody, including infliximab, CDP571 and D2E7.
D2E7 (italicum), monoclonal anti-NFα-human antibody (Abbot) was developed for the treatment of RA and Crohn's disease (WO 9729131). Celltech develo who develops CDP571 (EP 0626389), humanitariannet anti-NFα-IgG4-antibody, for the treatment of Crohn's disease and CDP870, a fragment gumanitarnogo monoclonal anti-NFα-antibody, for the treatment of RA. The local introduction of these antibodies for the treatment of localized disorders described in US 2003185826.
Many single-chain (scFv) antibodies were generated against many different antigens, in particular, due to the fact that they can be easily selected on the high binding ability of using, for example, techniques such as phage display or ribosome display. In addition, scFv-antibodies can be produced in microbial systems that are associated with lower costs in comparison with obtaining a therapeutic full-length antibody.
In addition to the conventional extracellular and in vitro applications, scFv were also successfully used for intracellular applications (Worn et al., 2000, JBC, 28; 275(4):2795-2803; Auf der Maur et al. 2002, JBC, 22; 277 (47):960-966); thus, they developed the scFv directed against intracellular antigens. Usually intracellular expression of functional scFv is limited by their instability, insolubility and the tendency to form aggregates. For this reason, have been successfully developed screening system in vivo antibody scFv, which are particularly soluble and stable under reducing conditions typical of the intracellular environment (n is an example, nucleus, cytoplasm), using the so-called "Quality Control" screening (Screening quality control) (WO 0148017); Auf der Maur et al. (2001), FEBS Lett. 508:407-412; Aus der Maur et al. (2004), Methods 34:215-224), and they resulted in the identification of particularly stable and soluble frame sequences of scFv for such purposes (WO 03097697). In addition, these frames reveal extremely high levels of expression and enhanced properties stability and solubility at the natural oxidizing conditions in the extracellular environment. Thus, these favorable biophysical and biochemical properties lead to favorable outputs obtain and allow you to use these fragments of antibodies directed against specific antigens, locally and/or systemically as a protein therapeutic agents in specific therapeutic areas. As scFv antibodies, and Fab fragments, as opposed to full-sized antibodies, do not have the Fc-part, which are identified by Fc-receptor of monocytes, such as natural killer cells, they do not cause antibody-dependent cell-mediated cytotoxicity (ADCC) and, therefore, do not cause non-specific toxicity due to binding to Fc-receptors on cells that are not targets.
Thus, there is a need for new, effective forms of antibodies for the treatment of TNF is associated disorders, such as rheumatoid arthritis (RA), in particular, treatments that can provide a stable, regulated local therapy introduction with a low degree of side effects. This invention provides antibodies, compositions and methods for effective and continuous treatment of inflammatory arthritis and other TNFα-mediated disorders or pathophysiological mechanisms, in particular various forms of pain.
All publications and references cited here, is enabled thereby by reference in their entirety.
Disclosure of inventions
Thus, the main purpose of this invention is the provision of stable and soluble antibody or derivative of the antibody, which specifically binds TNFα in vitro and in vivo. In a preferred embodiment, the specified derived antibody is a scFv-antibody or Fab fragment.
Now to implement these and additional objectives of this invention, which will become more apparent as the continuation of the description, the specified antibody, or a derived antibodies are characterized by signs, namely, that they contain a variable domain light chain having the sequence of SEQ ID NO:1 or derived from the sequence SEQ ID NO:1, which is combined with the variable domain of the heavy chain having the sequence is SEQ ID NO:2 or derived from the sequence SEQ ID NO:2, and in the case of a derived sequence this sequence has a maximum of 5 changes in the frame specified VLdomain and/or up to a maximum of 9 changes in the frame specified VHdomain.
The preferred embodiment of the present invention is the indicated antibody, or a derived antibodies where one or more amino acid changes introduced in any of the provisions in this frame, preferably in one or more positions selected from positions 4, 46, 65, 67, 70 and 83 VLdomain and/or in one or more positions selected from the group provisions 11, 16, 28, 43, 48, 68, 70, 71, 72, 73, 76, 77, 79, 93 and 112 VH-domain. More preferably, at least one of these transformations leads to the amino acids present in SEQ ID NO:3 for VLand/or SEQ ID NO:4 for VHand even more preferably maximally present 13 transformations in General.
Most preferably, the specified antibody or derivative of the antibody containing VL-domain sequence SEQ ID NO:1 and/or VHdomain having the sequence of SEQ ID NO:2 or produced from the sequence SEQ ID NO:2, or VL-domain sequence SEQ ID NO:11 and VHdomain SEQ ID NO:4. If VH-domain antibodies of the present invention contains VLdomain SEQ ID NO:1, in the preferred embodiment, assests the tion, VHsequence produced from SEQ ID NO:2 such that the phenylalanine at position 68 is substituted by alanine, leucine, isoleucine or valine. Additional changes in the VHare optional. scFv antibodies of this type are shown in SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.
In another preferred embodiment of the present invention, the specified antibody, or a derived antibodies derived from antibodies with VL-the sequence SEQ ID NO:1 and VH-the sequence SEQ ID NO:2 and contains at least one amino acid residue, which is made of at least one of the CDR in the residue present in the corresponding CDR VL-the sequence SEQ ID NO:5 and/or VH-the sequence SEQ ID NO:6 or SEQ ID NO:25.
In one very preferred embodiment of the present invention, at least one of the CDR group CDR2 VL, CDR3 VL, CDR2 VHor CDR3 VHconverted into the corresponding CDR VL-the sequence SEQ ID NO:5 and/or VHsequence 25 or SEQ ID NO:6.
Most preferably, the specified antibody or derivative of the antibody containing the following combinations of VL/VHsequences:
VLSEQ ID NO:7/VHSEQ ID NO:2,
VLSEQ ID NO:8/VHSEQ ID NO:2,
VLSEQ ID NO:1/VHSEQ ID NO:9,
VLSEQ ID NO:1/VHSEQ ID NO:25,
VLSEQ ID NO:1/VHSEQ ID NO:28,
VLSEQ ID NO:1/VHSEQ ID NO:29,
VLSEQ ID NO:26/VHSEQ ID NO:30,
VLSEQ ID NO:27/VHSEQ ID NO:30.
In another preferred embodiment, the antibody or derivative of the antibody of the present invention have specificity against human TNFα. Preferably the binding of an antibody characterized Byd~100 nm or less. More preferred is an antibody with Kd10 nm or less, and most preferred is an antibody with Kd1 nm or less.
Derived antibodies in accordance with this invention are, for example, Fc-merge, merge with the toxin, merge with enzymatic activities of different formats, such as Manantial, dianthicola, linear antibodies, single-chain antibodies, bespecifically fragments of antibodies, in particular, scFv and Fab fragments.
Another preferred object of this invention is a scFv-antibody domains VLand VHwhich is connected by means of a linker, preferably in the form of VL-linker-VH-the sequence SEQ ID NO:10.
Another preferred object of this invention is a scFv-antibody produced from SEQ ID NO:40 (TV-A). This antibody can be obtained by mutagenesis and contains three or fewer mutations in the frame, CDR and/or linker sequences. Predpochtite is) this scFv-antibody has the sequence of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38.
Another preferred object of this invention is Fab-fragment containing the VL-the domain that is fused with the constant region of the Kappa chain of human Ig and VH-domain, which merged with SN domain of human IgG, whereby these two fused polypeptide connected megamachines by a disulfide bridge.
In another aspect of this antibody or derivative of the antibody, for example, a fragment of the antibodies of the present invention is labeled or chemically modified.
This invention also provides a DNA sequence encoding any of the antibodies or derivatives of the antibodies of the present invention, as well as cloning or expressing the vectors containing the specified DNA sequence. In addition, provided with suitable a host cell transformed by the specified DNA sequence, which is preferably E. coli, a yeast cell or a cell of a mammal.
In addition, provided is a method of obtaining antibodies or derivatives of the antibodies of the present invention, involving the cultivation of a host cell transformed with DNA encoding any of the above antibodies or derivatives of antibodies, under conditions that make the possible synthesis of the indicated antibody or derivative of the antibody, and removing the specified molecules from the specified culture. Preferably the method provides scFv-antibody or Fab fragment, purified from E. coli.
Another aspect of the present invention is the use of antibodies or derivatives of antibodies provided by the present invention as a diagnostic tool for in vitro diagnosis and/or pharmaceutical agent. This application is particularly preferred in the context of any associated with TNFα state.
The invention also includes a composition comprising the antibody or derivative of the antibody of the present invention in combination with a pharmaceutically acceptable carrier, diluent or excipient, and this composition is intended for use as a drug for the treatment of TNFα-related diseases.
In an additional aspect, the invention provides a combined preparation containing the antibody or derivative of the antibody of the present invention, preferably with the second connection, which is not an antibody or derivative antibody specific against TNFα.
In another aspect of this invention, a vector containing a DNA sequence encoding a scFv-antibody of the present invention, used for gene therapy.
Treatment of TNFα-associated Zabol the requirements is achieved by blocking TNFα due to the strong interaction of TNFα with this antibody or derivative of the antibody. Preferably, it is assumed the treatment of autoimmune, acute or chronic inflammatory conditions, cancer-related diseases, pain, neurological and neurodegenerative disorders, infectious diseases and cardiovascular diseases.
Brief description of drawings
The invention will be better understood, and goals, other than the above objectives will become apparent upon consideration of the following detailed description. Such description makes reference to the accompanying graphic material.
Figure 1 shows a scheme of scFv antibodies with sequences of TV and TV In defining the boundaries of the range of most frequent variations. Asterisks indicate the position in which the allowed amino acid changes in the framework of the antibodies of the present invention. Amino acids, the following CDR (CDR highlighted in grey), can be used in the corresponding CDR.
Figure 2 shows a rough diagram to represent the Fab-fragment.
Figure 3 shows the production yield of scFv by expression in E. coli. A. Electrophoresis LTO-polyacrylamide gel expressed proteins. C. Analytical gel filtration TV And and TB-wt, showing excellent solubility TV-A.
Figure 4 shows the comparison of the different affinity of scFv antibodies to TNFα determined using ELISA.
Figure 5 shows the inhibition of cytotoxicity, inducirowannoj TNFα person, in mouse fibroblasts L929. A. Dependent on the concentration inhibition TV And in comparison with TB-wt and infliximab with values IC50. C. Comparison of TV-And-derivatives in respect of block TNFα-induced cytotoxicity. C. comparison of the formats of scFv and Fab TV-A.
Fig.6 shows the effect of antibody treatment induced TNFα human joint swelling in rats (experiment: 5.3., experiment 1).
Fig.7 shows the pattern of point estimates for histopathological evaluation of inflammation.
Fig shows the effect of antibody treatment induced TNFα human inflammation of the joint in rats (experiment: 5.3., experiment 1).
Fig.9 shows the effect of antibody treatment induced TNFα human joint swelling in rats (experiment: 5.3., experiment 2).
Figure 10 shows the effect of antibody treatment induced TNFα person for the joint inflammation in rats (experiment: 5.3., experiment 2).
11 shows the stability of the TV And in various body fluids.
The implementation of the invention
It was found that the antibody or derivative of the antibody containing the frames identified in the so-called screening quality control (Quality Control) (WO 0148 017), are characterized by high stability and/or solubility and, therefore, can be also applicable in the context of extracellular applications, such as neutral is isace TNFα. This invention provides antibodies or derivatives of antibodies, characterized by a high stability and solubility, which specifically recognize and bind TNFα and, therefore, are suitable for blocking the function of TNFα in vivo. These antibodies or derivatives of antibodies differ in a special frame made using screening with the "quality control" for antibodies with particularly stable and soluble frames regardless of their antigen-binding site, which have been described in EP 1479694. If the frames used in this screening are the skeletons of human antibodies, they can be considered as non-immunogenic frames for applications in man. CDRs of these antibodies of the present invention are identical to CDR or produced from a CDR of mouse monoclonal antibodies Di62 (Doring et al., 1994), which specifically binds to human TNFα with high affinity (Kd=0.4 nm) and can block the binding of TNFα to its receptor. In addition, Di62 inhibits induced human TNFα cytotoxicity in L929 mouse cells. Understandable stage grafting CDRs of the mouse antibody, undoubtedly, the most suitable acceptor skeleton of a man with an undefined antigen-binding properties, where the specified frame has a VL-the sequence SEQ ID NO:5 and VHthe sequence is SEQ ID NO:6, moreover, these sequences are related (GGGGS)4-linker (SEQ ID NO:10), led to scFv-antibody sequence:
moreover, the indicated scFv-antibody called the per-Cent. TV-gave good yields in the expression of the protein (figa), but was not able to specifically bind TNFα (figa).
Thus, to obtain the antibody or derivative of the antibody, which is (i) sufficiently specific regarding the binding of TNFα, (ii) sufficiently soluble for efficient obtaining and purification and to block TNFα in vivo, (iii) stable enough for use as a pharmaceutical agent without rapid degradation and (iv) sufficient non-immunogenic, looking for the best compromise between solubility and the best antigen-binding properties of variation of this framework and these CDR. This invention provides the sequence for VLand VHthat are optimized in terms of combinations of criteria (i-iv). scFv-antibody containing the specified VL(SEQ ID NO:1, linked (GGGGS)4-linker with a specified VH(SEQ ID NO:2), called the per-A. the sequence of the TV And presents SEQ ID NO:40. This antibody is still quite stable and soluble to obtain a satisfactory outcome, with the expression and purification from E. coli (figa), and it is not aggregated (f is GF). Characteristics of binding to TNFα are excellent, with Kd0.8 nm.
This invention also describes a VLand VHsequences generated from the sequences presented in the TV And in a variety of ways. First, it was found that point mutations in positions up to five positions in the frame VLand/or provisions to nine provisions in the frame of the VHare acceptable, in particular, point mutations that make these frames a more TV-like, i.e. more similar to SEQ ID NO:3 for VLor SEQ ID NO:4 for VH. scFv-antibody containing VL-domain sequence SEQ ID NO:11 and VH-domain sequence SEQ ID NO:4, associated (GGGGS)4-linker, called TV-R46L, as it differs only in position 46 VLfrom the per-Cent. unlike TV this antibody still has good binding properties against TNFα (Kd~100 nm). This suggests that the amount of change in the TV IN R46L regarding TV And represents the upper limit for changes in the framework of the variable domain.
In a preferred embodiment of the present invention in the frames of the VLand/or VHTV And introduce only one or dvuhochkovy mutations. Preferred residues of frame mutations are at positions 4, 46, 65, 67, 70 and 83 to VLand in the provisions of 11, 16, 28, 43, 48, 68, 70, 71, 72, 73, 6, 77, 79, 93 and 112 to VH. These positions are numbered according to the numbering in the list of sequences. Amino acid substitutions are preferably either "conservative"or such that the replacement amino acids are more similar or preferably even identical with the corresponding amino acids present in the sequence of TV Century, for Example, amino acid A76 VHin the TV And can be changed at 176, 176 as is present in the TV, but it can also be changed to another amino acid with similar, i.e. non-polar side chain, such as V or L. This is an example of "conservative" substitutions of amino acids. Families of amino acid residues having similar side chains suitable for "conservative" substitutions discussed in this context were identified in this area, including basic side chains (K, R, N), acidic side chains (D, E), uncharged polar side chains (Q, N, S, T, Y, C), nonpolar side chains (G, A, V, L, I, P, F, M, W), beta-branched side chains (T, V I) and aromatic side chains (Y, F, W, H). Preferred conservative change is the change in VLat position 83, in which V is replaced, or F (SEQ ID NO:26)or A (SEQ ID NO:27). However, in SEQ ID NO:32 nonconservative change in VLis V83E, which combined with the change in CDR1, i.e. N31D, and the V Hchange V79A. Other extraordinary option TV And is a variant of SEQ ID NO:33, a conservative change F68L in the VHassociated with VLlinker bearing an R in position 2 instead of G.
Very preferred substitutions of single amino acids are R65S or Y67S in the VLand K43Q or F68 on V, L or And VH. Very preferred double substitutions are F70L/L72R or A76I/S77G in the VH. scFv-antibodies containing the sequence of per-And with these changes, detect the inhibition of TNFα-induced cytotoxicity in L929 cells. The results of some of which are shown in figv. Their sequences are as follows:
SEQ ID NO:18=TV-AND H_K43Q TV-And-N)
SEQ ID NO:19=TV-AND H_F68V (TB-A-H68)
SEQ ID NO:20=TV-AND H_F70L/L72R (TV And N/72)
SEQ ID NO:21=TV-AND H_A76I/S77G (TV And N/77)
SEQ ID NO:22=TV-AND L_L46R (TV And L46)
SEQ ID NO:23=TV-AND L_R65S (TV And L65)
SEQ ID NO:24=TV-AND L_Y67S (TV And L67).
In the preferred embodiment, any of the above VHdomains can be combined with any of the above VLdomains.
In another preferred embodiment of the present invention VLand VHdomains TV and TV-shuffled In such a way that VLdomain TV-a (SEQ ID NO:1) is merged with VHdomain TV-In (SEQ ID NO:4) or VLdomain TV-In (SEQ ID NO:4) is merged with VHdomain TV-a (SEQ ID NO:2). In a very preferred variations is the implementation of these shuffled version received in scFv, are connected by a (GGGGS)4the linker sequence SEQ ID NO:10, resulting scFv-antibodies TV-AV (SEQ ID NO:12) or TV-VA (SEQ ID NO:13), respectively. Specified (GGGGS)4the linker may have the amino acid substitution of glycine to a more hydrophilic, i.e. polar or even charged amino acid that can do more soluble antibody. Among these variations, it is preferable variation of the sequence GRGGS-(GGGGS)3(SEQ ID NO:39).
In the framework of the present invention is the combination of the VLor VHdomain TV-b-like sequences with VHor VLthe domain TV-And-similar sequences, resulting TV-In/TV-And-such indicate that these sequences are closer to one than to another sequence.
In another preferred embodiment of the present invention, one or more modified amino acids in CDR-region VL- and/or VHsequences of per-And in such a way that they coincide with the corresponding amino acids present in the selected sequences SEQ ID NO:5 VLand/or SEQ ID NO:6 or SEQ ID NO:25 VH. Very preferred are changes in one of the CDR VL(CDR2 VLor CDR3 VLand/or CDR VH(CDR2 or CDR3), with the most preferred changes lead to p is sledovatelnot SEQ ID NO:7 or SEQ ID NO:8 V Land/or sequences of SEQ ID NO:25 or SEQ ID NO:9 VHrespectively.
In another preferred embodiment of the present invention VL-the sequence SEQ ID NO:26 or SEQ ID NO:27 combine with VH-the sequence SEQ ID NO:30. In yet another preferred embodiment of the present invention VL-the sequence SEQ ID NO:1 together with VHsequences SEQ ID NO:28 or SEQ ID NO:29.
Usually any of the above VLsequences can be combined with any of the described VHsequences.
The objects of this invention are antibodies and antibody fragments, in particular VLor VH-polypeptides, single-chain antibodies (scFv) or Fab fragments. In the case of scFv antibodies selected VL-the domain can be associated with the selected VHdomain in any orientation by a flexible linker. Suitable linker current level of technology consists of a repeated amino acid sequence GGGGS or their variants. In a preferred embodiment of the present invention use (GGGGS)4the linker SEQ ID NO:10 or its derivative SEQ ID NO:39, but there are also options of 1-3 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used for the present invention, described Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. Immunother. 50:51-59. The location can be either VL-linker-VHor VH-linker-VLand the first orientation is the preferred orientation.
In the case of Fab-fragments, selected the variable domains of the light chain VLmerge with the constant region of the Kappa-chain Ig, whereas the appropriate variable domains of the heavy chain VHmerge with the first (N-terminal) constant domain IN human IgG. In an exemplary embodiment of the present invention, κ-domain has the sequence of SEQ ID NO:14, and the domain SN used to construct Fab fragment has the sequence of SEQ ID NO:15. Figure 2 shows an example of the Fab-fragment, in which the use of the VLand VHthe domain TV-And thus that VLdomain directly linked to the constant domain of a human Kappa, which leads to the following sequence:
and VHdomain merged with the first constant domain (SN), which leads to the following sequence:
On the C-end is formed miaocheng disulfide bridge between two constant domains.
Antibodies or derivatives of the antibodies of the present invention may have an affinity in relation to human TNFα with a dissociation constant is din the range of 0.8-10000 nm. In a preferred embodiment of the present invention Kdequal to ≤10 nm. The affinity of antibodies against the antigen can be determined experimentally using a suitable method (Berzofsky et al. "Antibody-Antigen Interactions", in Fundamental Immunology, Paul, W.E., Ed, Raven Press: New York, NY (1992); Kuby, J. Immunology, W.H. Freeman and Company: New York, NY) and described in these references methods.
In one aspect of this invention, antibodies or derivatives of antibodies, in particular scFv-antibodies or Fab fragments are labeled. Detective tagging TNFα-specific antibody, or a derived antibodies can be performed by linking it to an enzyme for use in enzyme immunoassay (EIA) or enzyme-linked immunosorbent analysis (ELISA), which are well known to the person skilled in the art (e.g., Current Protocols in Immunology, Coligan et al. Eds, John Wiley & Sons, 2005).
Through radioactive labeling of TNFα-specific antibodies or derivatives of antibodies can be used to detect TNFα using radioimmunoassay (RIA) (see, for example. Work et al., Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, N.Y. (1978). The radioactive isotope can be detected using a gamma counter or a scintillation counter or radioautography. Especially applicable isotopes are3H,131I35S14With and preferably
Antibodies or derivatives of the antibodies of this invention can also be tagged with fluorescent aiming compounds, such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalic aldehyde and fluorescamine, or chemiluminescent compounds, such as luminal, isoluminol, aromatic ester acridine, Gridneva salt of imidazole and oxalate ester.
Protocols for labeling and detection are well known to the person skilled in the art. For example, they are available from Using Antibodies: A Laboratory Manual: Portable Protocol NO. I (Harlow, E. and Lane, D., 1998).
Labeled antibodies or derivatives of the antibodies of the present invention is applicable for diagnostic purposes, in particular for detection of TNFα in a biological sample taken from the patient. Can be used with any sample containing TNFα, for example, biological fluids, such as blood, serum, lymph, urine, inflammatory exudate, cerebrospinal fluid, amniotic fluid, extract or homogenate tissue and the like, or histological specimens for detection in situ.
Definitions: the Term "pharmaceutical finished form" refers to preparations which are in such form that allows the biological activity of the antibody or derivative of the antibody to be unambiguous EF is objective and which contains no additional components, which are toxic to the subjects, which must be entered in this finished form. "Pharmaceutically acceptable excipients (carriers, adjuvants) are excipients that are acceptable for administration to a subject, the mammal to provide an effective dose of the active ingredient.
"Stable" finished form is the form in which the antibody or derivative of the antibody, essentially retains its physical stability and/or chemical stability and/or biological stability after storage. Various analytical methods of determining the stability of proteins are available in this area and are discussed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at the selected temperature for a selected period of time. Preferably this finished form is stable at room temperature (approximately 30°C.) or at 40°C for at least 1 month and/or stable at about 2-8°C for at least 1 year - for at least 2 years. In addition, this finished form is preferably stable after freezing (for example, up to -70°C) and thawing of this form.
The antibody or derivative of an antibody "retains its f the physical stability" in a pharmaceutical finished form, if it does not detect signs of aggregation, precipitation and/or denaturation after visual examination of color and/or transparency, or when measuring the scattering of UV light, or by using gel-filtration chromatography.
The antibody or derivative of an antibody "retains its chemical stability" in finished pharmaceutical form, if chemical stability at a particular time is such that it is believed that this protein still retains its biological activity as defined below. Chemical stability can be assessed by detection and quantification of chemically modified forms of this protein. Chemical modification may include modifying the size (e.g., size reduction), which can be estimated using, for example, gel filtration chromatography, electrophoresis LTO-PAG and/or assisted laser desorption / ionization using matrix/mass spectrometry, based on time-of-flight (MALDI/TOF MS). Other types of chemical changes include the change of the charge (for example, resulting from deliciouse), which can be measured, for example, ion-exchange chromatography.
The antibody or derivative of an antibody "retains its biological activity" in a pharmaceutical finished form, if the biological activity of this is ntitle at a particular point in time is within about 10% (within the errors of this analysis) from biological activity, exercise during cooking, when determining, for example, antigen-binding analysis. Other tests "biological activity" for the antibodies described in detail here below.
"Isotonic" means that interest the finished form is essentially the same osmotic pressure as human blood. Isotonic form will typically have an osmotic pressure of approximately 250-350 mOsm. Isotonicity can be determined, for example, using osmometry based on the pressure measurement (elasticity) pair, or osmometry based on freezing to ice.
"Polyol" is a substance with many hydroxyl groups and includes sugars (reducing and non), polyalcohols, xylytol and saharomiceta. Preferred here polyols have a molecular weight that is less than about 600 kDa (for example, is in the range from about 120 to 400 kDa). "Revitalizing sugar is sugar, which contains polyacetylene group, which can recover metal ions or react covalently with lysine and other amino groups in proteins, and "non sugar is sugar, which does not have these properties, a reducing sugar. Examples of reducing sugars are fruit is a, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Non sugars include sucrose, trehalose, sorbose, melezitose and raffinose. Examples of polyalcohols, xylytol are mannitol, xylitol, aritra, threitol, sorbitol and glycerin. As sharokina, they include L-gluconate and its metal salts. If it is desirable that the finished shape was stable after freezing-thawing, the polyol is preferably a polyol, which is not crystallized at the temperature of freezing (for example, -20°C), so that it destabilizes the antibody in a ready form. Non sugars such as sucrose and trehalose, are preferred in this invention, and trehalose is preferred over sucrose, due to the superior stability of the solution of trehalose.
In this context, a "buffer" means a buffered solution resists changes in pH due to the action of its components conjugate acid-base. The buffer of this invention has a pH in the range of about 4.5 to 6.0; preferably approximately from 4.8 to 5.5, and most preferably has a pH of approximately 5.0 to. Examples of buffers that can control the pH in this area include acetate (e.g. sodium acetate), succinate the th (for example, sodium succinate), gluconate, his-tag, citrate buffers and containing other organic acid buffers. When the desired is stable to freeze-thawing finished form, this buffer is preferably a phosphate buffer.
In a pharmacological sense, in the context of this invention, a "therapeutically effective amount" refers to an amount effective in the prevention or treatment of disorders, in relation to which the antibody or derivative of the antibody is effective. "The disease/disorder" is any condition that would benefit from treatment with this antibody or derivative of the antibody. This includes chronic and acute disorders or diseases including disorders and diseases, pathological conditions which predispose the mammal to the disorder.
"Preservative" is a compound that can be incorporated into the finished shape to substantially reduce bacterial action in it, and, consequently, to facilitate, for example, getting forms ready for multiple applications. Examples of potential preservatives include chloride octadecyltrimethylammonium chloride hexadecane, benzalkonium chloride (a mixture of chlorides of alkylbenzyldimethylammonium in which alkyl the s groups are groups with long chain) and chloride benzene. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkylarene, such as methyl - or propylparaben, catechin, resorcinol, cyclohexanol, 3-pentanol and m-cresol. The most preferred preservative is benzyl alcohol.
This invention also provides pharmaceutical compositions containing one or more antibodies or derivatives of antibodies, together with at least one physiologically acceptable carrier or excipient. The pharmaceutical compositions may contain, for example, one or more components from water, buffers (e.g., neutral buffered saline or phosphate buffered saline solution), ethanol, mineral oil, vegetable oil, dimethyl sulfoxide, carbohydrates (e.g. glucose, mannose, sucrose or dextrans), mannitol, proteins, adjuvants, polypeptides or amino acids such as glycine, antioxidants, chelat forming agents, such as EDTA or glutathione, and/or preservatives. As noted above, provided here, the pharmaceutical compositions can be (optional) included other active ingredients.
A medium is a substance that can be associated with the antibody or derivative of the antibody before administration to the patient, often for the purpose of control of what I stability or bioavailability of the compound. Carriers for use in such ready-made forms are typically biocompatible and may be biodegradable. Carriers include, for example, monovalent or polyvalent molecules such as serum albumin (such as human or cow), egg albumin, peptides, polylysin and polysaccharides, such as amylodextrin and polyamidoamine. Carriers include solid materials-media, such as granules and particles containing, for example, polylactate-polyglycolic, the copolymer poly(lactide-glycolide), polyacrylate, latex, starch, cellulose or dextran. The carrier can carry these compounds in a variety of ways, including the formation of covalent bonds (either directly or via a linker group), non-covalent interactions or the formation of a mixture.
The pharmaceutical compositions can be formulated for any suitable route of administration, including, for example, local, oral, nasal, rectal or parenteral administration. In some embodiments, the implementation, the preferred compositions are in a form suitable for oral administration. Such forms include, for example, pills, tablets, lozenges, pellets, aqueous or oil suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or sir is dust, or elixirs. In other embodiments, the implementation provided here compositions can be prepared in the form of a lyophilisate. The term "parenteral" includes in this context, subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, vnutriobolochechnoe and intraperitoneal injection, as well as any similar injection or infusion.
Compositions intended for oral use may be prepared according to any method known in this field for the preparation of pharmaceutical compositions, and may contain one or more agents, such as sweetening agents, improves the taste and odor agents, coloring agents, and preserving agents, to provide an attractive and suitable for food products. Tablets contain the active ingredient mixed with physiologically acceptable excipients which are suitable for the preparation of tablets. Such excipients include, for example, inert diluents (e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate), granulating and dezintegriruetsja agents (for example, corn starch, or alginic acid), binding agents (e.g. starch, gelatin or the Arabian gum) and lubricating agents (for example, steer the magnesium, stearic acid or talc). Tablets can be uncoated or they may be applied by known methods of coating to delay disintegration and absorption in the gastrointestinal tract and software supported by this action over a longer period of time. For example, there may be used material for temporary detention, such as glycerylmonostearate or glycerylmonostearate.
Ready-made forms for oral administration may be presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (for example, peanut oil, liquid paraffin or olive oil). Aqueous suspensions contain the antibody or derivative of the antibody in a mixture with excipients suitable for the preparation of aqueous suspensions. Such excipients include suspendresume agents (for example sodium carboxymethylcellulose, methylcellulose, gidropropilmetilzelluloza, sodium alginate, polyvinylpyrrolidone, tragacanth gum and Arabic gum); and dispersing or hydrating agents (e.g., natural-occurring phosphatides, such to the to lecithin, condensation products of ethylene oxide with aliphatic alcohols with long chain, such as heptadecafluorooctane, condensation products of ethylene oxide with partial esters produced from fatty acids and exit, such as monooleate of polyoxyethylenesorbitan, or condensation products of ethylene oxide with partial esters produced from fatty acids and anhydroglucitol, such as monooleate polyethylenimine. Aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl-p-hydroxybenzoate, one or more coloring agents, one or more improves the taste and odor agents and one or more sweetening agents such as sucrose or saccharin. Syrups and elixirs can be prepared with sweetening agents such as glycerin, propylene glycol, sorbitol or sucrose. Such forms may also contain one or more demulsifiers, preservatives, flavoring agents and/or coloring agents.
Oil suspensions can be prepared by suspendirovanie active ingredients in a vegetable oil (such as peanut oil, olive oil, sesame oil or cocoa butter) or in mineral oil such as liquid paraffin. Oily suspensions may contain a thickening agent, such as beeswax, hard paraffin or citylove the alcohol. Sweetening agents, such as described above agents, and improving the taste and odor agents can be added to provide good food oral medication. Such suspension may be preserved by the addition of an antioxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of aqueous suspension by the addition of water provide the active ingredient in a mixture with dispersing or wetting agent, suspenders agent and one or more preservatives. Suitable dispersing or moisturizing agents and suspendresume agents listed above as examples of the aforementioned agents. May also contain additional excipients, for example sweetening, improves taste and odor, and coloring agents.
Pharmaceutical compositions can also be in the form of emulsions of the type oil-in-water. The oil phase may be a vegetable oil (such as olive oil or peanut oil), mineral oil (e.g., liquid paraffin) or their mixture. Suitable emulsifying agents include natural-occurring gums (e.g., Arabian gum or tragacanth gum), natural-occurring phosphatides (e.g., soy lecithin, and esters or partial esters derived from fatty acids and exit), anhydrides (e.g., Mongolia sorbitan) and condensation products of partial esters, derived from fatty acids and exit, with ethylene oxide (for example, monooleate of polyoxyethylenesorbitan). The emulsion may also contain one or more sweetening and/or flavoring agents.
The pharmaceutical composition can be prepared in the form of sterile injectable aqueous or oily suspensions, in which the modulator, depending on the medium, and the concentration of either suspended or dissolved in the carrier. Such a composition can be prepared according to known in the field methods using suitable dispersing, wetting agents and/or suspendida agents, such as agents described above. Among the acceptable vehicles and solvents that may be used are water, 1,3-butanediol, ringer's solution and isotonic sodium chloride solution. In addition, as a solvent or suspendida environment can be used sterile, non-volatile oils. For this purpose, can be used any easy non-volatile oils, including synthetic mono - or diglycerides. In addition, fatty acids such as oleic acid, can be used in the preparation of injectable compositions, and that the carrier can be dissolved adjuvants such as local anesthetics, preservatives and/or bufferedio agents.
Pharm is septicemia compositions can be prepared in the form of sustained release (i.e. forms such as capsule, which does the slow release of the modulator after the introduction). Such compositions may generally be prepared using well known technology and to be entered, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired site of the target. Carriers for use in such ready-made forms are biocompatible, and may also be biodegradable; preferably, this finished form provides a relatively constant release rate of the modulator. The amount of antibody or derivative of the antibody contained in the form prolonged release depends, for example, from the site of implantation, the rate and expected duration of release and the nature of the diseases/disorders, subject to treatment or prevention.
The antibody or derivative of an antibody described herein are typically administered in an amount that achieves a concentration in body fluids (e.g. blood, plasma, serum, cerebrospinal fluid (CSF), synovial fluid, lymph, cellular interstitial fluid, tears or urine)that is sufficient for the detected binding to TNFα and prevention or inhibition of TNFα-related diseases/disorders. Dose is considered effective if it leads to significant benefits for the patient, as is written here. The preferred systemic doses are in the range of about 0.1 mg to about 140 mg/kg of body weight per day (about 0.5 mg to about 7 g per patient per day), and oral dose is usually about 5-20 times the intravenous dose. The amount of antibody or derivative of the antibody, which may be combined with materials native to receive the form of a single dose will vary depending on the treatment of the host and a particular way of introduction. Unit dosage forms will generally contain about 1 mg to about 500 mg of the active ingredient.
The pharmaceutical compositions may be packaged for treating conditions responsive to the antibody or derivative antibodies directed to TNFα. Packaged pharmaceutical compositions may include a container having an effective amount of at least one antibody, or a derived antibodies described herein and instructions (e.g., labeling)indicating that the data contained in the packaging composition should be used for the treatment of diseases/disorders that respond to this one antibody, or a derived antibodies after administration to the patient.
Antibodies or derivatives of the antibodies of this invention can also be chemically modified. Site is titlename modifying groups are polymers, for example optionally substituted polyalkenes, polyalkylene or polyoxyalkylene polymer with a straight or branched chain or a branched or unbranched polysaccharide. Such effector group may increase the period of existence of this antibody in vivo. Specific examples of synthetic polymers include optionally substituted with straight or branched chain poly(ethylene glycol) (PEG), poly(propylene glycol), poly(vinyl alcohol) or their derivatives. Specific natural occurring polymers include lactose, amylose, dextran, glycogen, or their derivatives. The size of the polymer may vary, if desired, but will usually be in the range of average molecular mass of 500 Da To 50,000 Da. For local use, when this antibody is designed for penetration into the tissue, the preferred molecular weight of this polymer is about 5,000 Da. This polymer molecule can be attached to the antibody, in particular the C-terminal side of the heavy chain Fab fragment, via covalently associated hinge peptide, as described in WO 0194585. Regarding attaching PEG-groups can be referenced in "Poly(ethyleneglycol) Chemistry, Biotechnological and Biomedical Applications", 1992, J. Milton Harris (ed). Plenum Press, New York and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York.
Receive what their finished form.
After receipt of interest antibody or derivative of the antibody, as described above, is prepared containing pharmaceutical finished form. The antibody, which should be cooked, not subjected to prior lyophilization, and interest in this invention the finished form is a water-ready form. Preferably, the antibody or derivative of the antibody in this finished form is a fragment of antibodies, such as scFv. A therapeutically effective amount of the antibody present in this finished form, is determined taking into account, for example, the desired volume of the doses and how (methods) introduction. The approximate concentration of the antibodies in this finished form was equal to approximately 0.1 mg/ml to about 50 mg/ml, preferably about 0.5 mg/ml, about 25 mg/ml and most preferably about 2 mg/ml, about 10 mg/ml
Prepare a water finished form containing the antibody or derivative of the antibody in a pH-buffered solution. The buffer of this invention has a pH in the range of about 4.5 to about 6.0 and preferably about 4.8 - approximately 5.5 and most preferably has a pH of 5.0. Examples of buffers that can control the pH in this range include acetate (e.g. sodium acetate), succinate (e.g., succinate is the atrium), gluconate, his-tag, citrate buffers and containing other organic acid buffers. The concentration of the buffer may be approximately 1 mm to approximately 50 mm, preferably about 5 mm to about 30 mm, depending, for example, from a particular buffer and the desired isotonicity of the finished form. The preferred buffer is sodium acetate buffer (approximately 10 mm), pH 5.0.
In this finished form include polyol, which acts as a regulatory isotonicity agent and can stabilize this antibody. In preferred embodiments, implementation, this finished form does not contain much salt, such as sodium chloride, sufficient to create isotonicity, as this may cause the antibody or derivative of the antibody to precipitate and/or may lead to oxidation at low pH. In preferred embodiments, the implementation of this polyol is a non sugar, such as sucrose or trehalose. The polyol is added in the finished form in amounts that may vary depending on the desired isotonicity of this form. Preferably, water-ready form is isotonic, and in this case, the appropriate concentration of polyol in the finished form are, for example, in the range of about 1% to about 15% weight/volume, preference is sustained fashion in the range of about 2% - approximately 10% weight/volume. However, there may be also suitable hypertonic or hypotonic ready form. The number of added polyol may also vary depending on the molecular weight of the polyol. For example, can be added to lower the number of monosaccharide (e.g., mannitol) in comparison with the disaccharide such as trehalose).
To the finished form of the antibody, or a derived antibodies to add surface-active substance. Exemplary surfactants include nonionic surfactants, such as Polysorbate (for example, Polysorbate 20, 80 and so on) or poloxamer (for example, poloxamer 188). The amount of added surfactant is such that it reduces the aggregation prepared as ready-to form antibody/derivative antibodies and/or minimizes the formation of particles in the finished form and/or reduces adsorption. For example, the surfactant may be present in the finished form in the amount of about 0,001% to about 0.5%, preferably about 0.05% to about 0.2% and most preferably about 0.01% to about 0.1%.
In one embodiment, this finished form contains the above agents (i.e., the antibody or derivative of the antibody, a buffer, a polyol poverhnosti-active substance) and is essentially, do not contain one or more preservatives, such as benzyl alcohol, phenol, m-cresol, chlorobutanol and benzenehexachloride. In another embodiment, in this finished form may include a preservative, in particular, when this form is ready mnogochasovoj ready form. The concentration of preservative may be in the range of about 0.1% to about 2%, most preferably about 0.5% to about 1%. One or more other pharmaceutically acceptable carriers, excipients or stabilizers, such as carriers, excipients and stabilizers described in Remington's Pharmaceutical Sciences 21stedition, Osol, A. Ed. (2006), can be included in the finished form, provided that they are not harmful influence on the desired characteristics of this form. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the used doses and concentrations, and include additional buffering agents; co-solvents; antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., complexes of Zn-protein); biodegradable polymers, such as polyesters; and/or forming a salt counterions, such as sodium.
Ready-made forms that must be used in vivo, due in order to be sterile. This is easily accomplished by filtration through sterile filtration membranes before preparing ready-made form or after cooking is finished.
The introduction of ready-made forms
The finished form is administered to the mammal in need of the treatment with this antibody, preferably a human, in accordance with known methods, such as intravenous bolus or by continuous infusion over a period of time, using intramuscular, intraperitoneal, intracerebroventricular, subcutaneous, intra-articular, vnutricinovialnoe, vnutriobolochechnoe, oral, local or inhalation methods. In preferred embodiments, the implementation of the finished form is administered to the mammal by intravenous introduction. For such purposes, this finished form may, for example, inetservices using a syringe or through the IV line.
The appropriate dosage ("therapeutically effective amount") of this antibody will depend, for example, from the subject to the treatment condition, the severity and course of this condition, regardless of injected whether this antibody for preventive or therapeutic purposes, previous therapy, the patient's clinical presentation and response to this antibody, the type of antibody and the judgment of the attending physician. Antibody, or a derived antibodies suitable brasavola the patient once or over a number of introductions, and it can enter at any time from diagnosis onwards. The antibody or derivative of the antibody can be administered as a single treatment or in combination with other drugs or therapies that are applicable in the treatment of the condition.
As a General proposal, a therapeutically effective amount of the antibody or derivative of the antibody will be in the range of about 0.1 to about 50 mg/kg of body weight of the patient, regardless of whether one or more injections, with a typical range of antibodies used is, for example, the range of about 0.3 to about 20 mg/kg, more preferably about 0.3 to about 15 mg/kg, with the introduction of once a day. However, they can be applied to other schemes. The progress of this therapy is easy to observe using conventional methods.
In another embodiment of this invention, there is provided a product containing a container that holds water for pharmaceutical finished form of the present invention, and optionally instructions for use. Suitable containers include, for example, bottles, vials and syringes. The container can be made of various materials such as glass or plastic. The approximate what ontaneda can be a glass vial on 3-20 ml for single use. Alternative for mnogochasovoj prepared this container may be a glass vial on 3-100 ml of This container holds the finished form, and the label on the container or attached to the container, can contain instructions for use. This product may additionally include other materials desirable from a commercial point of view or from the point of view of the user, including other buffers, diluents, filters, needles, syringes and inserts packaging with instructions for use.
Antibodies of the present invention
Antibodies or derivatives of the antibodies of this invention can be obtained using routine methods used in the field of recombinant genetics. If the sequences of polypeptides known cDNA encoding them, can be obtained by gene synthesis (www.genscript.com). These cDNA can be cloned into a suitable vector plasmid. After obtaining the DNA encoding the VL- and/or VH-the domain can be accomplished by site-specific mutagenesis, for example, by PCR using mutagenic primers, to obtain various derivatives. The best "starting sequence" can be selected depending on the number of changes desired in the VL- and/or VH-sequences. The preferred sequence are the Xia sequence TV-a and their derivatives, for example, scFv sequences or sequence Fab-fused peptide can be selected as the matrix for PCR-mediated mutagenesis and/or cloning.
Standard methods cloning and mutagenesis are well known to the person skilled in the art, can be used to attach linkers, domain shuffling or constructed of m to obtain Fab fragments. The main protocols that describe the General methods of this invention, are described in Molecular Cloning, A Laboratory Manual (Sambrook &Russel, 3rded. 2001) and in Current Protocols in Molecular Biology (Ausubel et al., 1999).
DNA sequence that carries the gene encoding the scFv polypeptide, or, in the case of Fab-fragments encoding either two separate gene or bicistronic operon containing these two genes for m VL-Cκ and VH-CH1 (figure 2), clone expressing in a suitable vector, preferably a vector with an inducible promoter. Care should be taken that every gene there would be a suitable binding site of the ribosome (RBS figure 2), which guarantees the broadcast. It should be understood that the antibodies of the present invention contain the above sequence, and does not consist of them. For example, the cloning strategy may require to be prepared by the construction, from which the antibody, in which the N-terminal side are the Dean or more additional residues. Specifically, methionine, originating from the starting codon, may be present in the target protein in those cases when he was not split after the broadcast. Most of the designs for scFv antibodies lead to additional alanine at the N-terminal side. In a preferred embodiment of the present invention is selected expressing vector for expression in periplasmic E. coli (Krebber, 1997). The specified vector contains the promoter before tsepliaeva signal sequence. Then, the coding sequence for a peptide antibodies merge in reading frame with tsepliaeva signal sequence. This makes it possible targeting of expressed polypeptide in bacterial periplasm, where this signal sequence is cleaved. Then the antibody stack. In the case of Fab-fragments as a fusion peptides VL-Cκ and merge VH-CH1 should be merged with the signal export. Once these peptides reach periplasm, forms a covalent bond S-S C-terminal cysteine. If it is preferable cytoplasmic expression of antibodies, these antibodies usually get in the high outputs from Taurus inclusions which can be easily separated from other cellular fragments and protein. In this case, these calf enable solubilizing under denaturing agent, such to the to guanidine hydrochloride (GndHCL), and then subjected to refolding using renaturation procedures, well known to specialists in this field.
Plasmids expressing the polypeptides of scFv or Fab, is introduced into a suitable host, preferably bacterial, yeast cell or a cell of a mammal, most preferably in a suitable strain of E. coli, for example JM83, for expression in periplasm or strain BL21 for expression in inclusion bodies. The polypeptide can be assembled from periplasm or may form a bullock inclusion, and can be purified using standard methods, such as ion exchange chromatography, reversed-phase chromatography, affinity chromatography and/or gel filtration, well-known experts in this field.
Antibodies or derivatives of the antibodies of this invention can be characterized in relation to yield, solubility and stability in vitro. Binding activity against TNFα, in particular in relation to human TNFα, can be tested in vitro using ELISA or surface plasma resonance (BIACore) using recombinant human TNFα, as described in WO 9729131, the latter method also allows to determine the dissociation constant koffthat should preferably be less than 10-3with-1. Values of Kd≤10 nm are preferred.
Not taliswoman activity in vivo antibody or derivative of the antibody of the present invention can be determined using analysis of cytotoxicity using L929. Recombinant human TNFα exerts cytotoxic activity against cultured fibroblastic cells mouse L929 dependent on concentration. This TNFα-induced cytotoxicity may also be inhibited by neutralizing TNFα antibodies (Doring, 1994). A preferred value IC50corresponding premaxillae concentration of inhibitor, which is ≤100 ng ml-1.
Since TNFα has proven pathophysiological role in various human diseases, in particular inflammatory diseases, immune and immuno-regulated disorders, infections, causing sepsis, endotoxin and cardiovascular shock, neurodegenerative diseases and malignant diseases, and because it is assumed that TNFα plays associated with diseases role in the ever-growing number of human diseases, it is difficult to give an exhaustive list of indications, which also guarantees a full representation of the spectrum of clinical applications of TNFα inhibitors in the future. Thus, antibodies or derivatives of the antibodies of this invention can be used for the treatment of diseases listed in the following directory, which should not be construed as a complete or exclusive list. Included also other diseases not mentioned specifically, are directly or posredovanje affected by TNFα.
Autoimmune or chronic inflammation
Chronic and/or autoimmune condition of inflammation in General, immune-mediated inflammatory disorders in General, inflammatory disease of the Central nervous system, inflammatory diseases affecting the eyes, joints, skin, mucous membranes, gastrointestinal tract, urogenital system, or lungs; state uveitis, retinitis, HLA-B27+ uveitis, a disease behceta, dry eye syndrome, glaucoma, Sjogren syndrome, diabetes (including diabetic neuropathy), insulin resistance, arthritis in General, rheumatoid arthritis, osteoarthritis, reactive arthritis and Reiter syndrome, juvenile arthritis, ankylosing spondylitis, multiple sclerosis, Guillain-Barre syndrome, severe psevdomatematicheskoe myasthenia gravis, amyotrophic lateral sclerosis, sarcoidosis, glomerulonephritis, chronic kidney disease, cystitis, psoriasis (including psoriatic arthritis), suppurative hydradenitis, panniculitis, pyoderma gangrenosum, SAPHO syndrome (synovitis, acne, pustular rash, hyperstat and octet), acne, sweet syndrome, utricularia vulgaris, Crohn's disease (including extraintestinal manifestations), ulcerative colitis, bronchial asthma, allergic pneumonitis, a common Allergy, allergic rhinitis, allergic sinusitis, chronic obstructive shall Olesen (COPD), the lung fibrosis, Wegener's granulomatosis, Kawasaki disease, giant cell arteritis diagnostics, vasculitis charge-Strauss, Nowotny polyarteritis, burns, disease, graft-versus-host reaction "host versus graft", the phenomena of rejection after organ transplantation or bone marrow, systemic and local state of vasculitis in General, systemic and discoid lupus erythematosus, polymyositis and dermatomyositis, scleroderma, pre-eclampsia, acute and chronic pancreatitis, viral hepatitis, alcoholic hepatitis.
Acute inflammation and/or prevention of postoperative and posttraumatic inflammation and pain
Prevention of postoperative inflammation in General, eye surgery (such as cataract surgery (replacement of the lens of the eye or glaucoma)and joint surgery (including arthroscopic surgery), surgery in the associated joint structures (e.g., ligaments), maxillofacial and dental surgery, minimally invasive cardiovascular procedures (e.g., percutaneous catheter coronaroplasty, RTCA, atherectomy, stent), laparoscopic and/or endoscopic intra-abdominal and gynecological procedures, endoscopic urological procedures (e.g., surgery of prostate gland, urethroscopy, cystoscopy, interstitial cystitis), peripheral the TES inflammation (warning) in General.
Neurological and neurodegenerative diseases
Alzheimer's disease, Parkinson's disease, Huntington's disease, bell's palsy, a disease of Creutzfeldt-Jakob disease.
Associated with cancer survivorship associated with cancer, inflammation associated with cancer, pain associated with cancer cachexia, bone metastases.
Acute and chronic forms of pain, regardless of whether they are Central or peripheral effects of TNFα and whether they are classified as inflammatory, nociceptive or neuropathic forms of pain; ischialgia, pain in the lower back, their carpal canal syndrome, complex regional pain syndrome (CRPS), gout, post herpetic neuralgia, fibromyalgia, local pain conditions, syndromes of chronic pain due to metastatic tumor, dysmenorrhea.
Bacterial, viral or fungal sepsis, tuberculosis, AIDS.
Atherosclerosis, coronary artery disease, hypertension, dyslipidemia, heart failure and chronic heart failure.
In a preferred embodiment of the present invention the treatment of osteoarthritis or uveitis or inflammatory bowel disease can be achieved with the use of antibodies or derivatives of the antibodies of the present invention.
This is the invention also provides a pharmaceutical composition, containing the antibody molecule or derivative of the antibody of the present invention in combination with pharmaceutically acceptable excipient, diluent or carrier.
The pharmaceutical composition should preferably contain a therapeutically effective amount of the antibody of the present invention, i.e. the number of the specified antibodies, which are necessary for the treatment, relief of symptoms or warning TNFα-associated disease or condition or symptoms detected therapeutic or preventive actions. A therapeutically effective dose can be determined either in tests on cell cultures or in animal models, usually rodents, rabbits, dogs, pigs or primates. The animal model can also be used to determine the appropriate concentration range and route of administration. A suitable animal model to monitor the actions of the antibody or derivative of the antibody of the present invention is a rat model for acute monoarthritis (Bolon et al. (2004), Vet. Pathol. 41:235-243). TNFα person is injected intra-articular knee joint of rats, resulting in acute, self-limited to monoarthritis in the injected joint. The bioactivity of anti-TNFα antibodies (derived) can be quantified by the decrease in TNFα-induced swelling and/or reduction of GIS the ideological parameters of inflammation.
Because antibodies or derivatives of the antibodies of this invention are vysokochastotnymi, high antibody concentrations (60 mg/ml or more) allow the use of small amounts of application.
The antibody or derivative of the antibody of the present invention can be used in any therapy where it is desirable to reduce the level of biologically active TNFα present in the body of man or animal. This TNFα may be in the blood flow in the body or be present at an undesirable high level, being localized in a particular area in the body. This invention provides methods of systemic and local applications in General, which include, but are not limited to, the following: oral administration, intravenous, subcutaneous, intramuscular, intra-articular, intra-vitreal, intradermal or intraparenchymatous injection, aerosol inhalation, local application to the skin, mucous membranes or eyes, systemic or local release by implantable Minnesota or local release using implantable forms/device that allows slow release, local application on the serosal surface, vnutriobolochechnoe or intraventricular application, oral administration in the form of compositions, which makes the possibility of the major adjustable intraluminal release in selected areas of the gastrointestinal tract, localized part of the release of suitable forms/devices (e.g. stents), local delivery into the bladder, localized intraluminal release (e.g., biliary tract, ureter) or local delivery through an endoscopic device or the release of contact lenses. The preferred application is a local application, such as intra-articular injection, or topical application, for example in the eye. For both the preferred applications of the antibody of the present invention must be in solution.
This invention also describes the use of antibodies or derived antibodies of this invention for the preparation of drugs for the treatment of TNFα-related diseases. In this case, the antibody or derivative of the antibody contained in therapeutic composition. The above composition is used as a drug, most preferably for the prevention or therapy associated with TNFα diseases.
In another aspect of scFv antibodies of this invention are used in gene therapy, in particular in adoptive cellular gene therapy. Autoimmune disorders are a spontaneous immune response aimed at your own fabric. Antigen-specific CD4+T-cells, antigenpresenting de is britnie cells (DC) are important mediators in the pathogenesis of autoimmune diseases and therefore, the ideal candidates for adoptive cellular gene therapy, ex vivo approach to therapeutic gene transfer. Using transduced by retrovirus cells and luciferase bioluminescence (Tarner et al., 2003, Ann. N.Y.Acad. Sci. 998:512-519) demonstrated that primary T-cells, hybridoma T cells and DC quickly and mostly return to sites of inflammation in animal models of multiple sclerosis, arthritis and diabetes. These cells transduced retroviral vectors that trigger the expression of various regulatory proteins such as interleukins and anti-TNF-scFv, take these immunoregulatory proteins in inflamed damage, providing therapy for experimental autoimmune encephalitis, collagen-induced arthritis and non-obese diabetic mice. Stable and soluble frames antibodies or derivatives of the antibodies of this invention are particularly suitable for intracellular delivery of the antigen, for example, when the antibody or derivative of the antibody expressed from the transgene carried a suitable retroviral vector. Adoptive cellular gene therapy leads to localized expression and secretion of anti-TNFα-scFv. Smith et al. (2003) showed that scFv produced from TNFα-neutralizing monoclonal antibodies (i) can neutralize the th TNFα in vitro and (ii) change the distribution of the expression of cytokines in mice, suffering from collagen-induced arthritis, locally in the joints, but not systematically. Alternative direct systemic or local injection of suitable vectors (e.g., viruses)that makes possible the continuous expression of anti-TNFα-scFv-antibodies, is considered as another possible approach is gene therapy.
The invention will be clearer with reference to the following examples. However, they should not be construed as limiting the scope of this invention. All literature and patent citations is incorporated herein by reference.
Experiment 1. Construction of scFv-antibodies
The source material for the generation of humanized antibodies against human TNFα or derivatives of these antibodies, such as single-chain fragments (scFv) or Fab fragments was mouse monoclonal antibody Di62. The sequence of the variable region of the light chain and the heavy chain described in Doring et al. (1994, Mol. Immunol. 31:1059-1067). The properties of monoclonal antibodies are also discussed in this publication. Briefly, Di62 specifically binds to human TNFα dependent on concentration. This antibody is a high-affinity antibody (Kd=0.4 nm) and can block St. the statements TNFα to its receptor. In addition, Di62 inhibits induced human TNFα cytotoxicity in L929 mouse cells.
Based on the published sequence Di62 designed in the form of a derived single-chain antibodies (scFv) in the orientation of VL-linker-VHin which the sequence of the linker consists of four repetitions of the four residues glycine and one serine residue (Gly4Ser)4. This scFv referred to as TB-wt, VLSEQ ID NO:16 and VHSEQ ID NO:17.
For humanization of this derived antibodies with the aim to make it more similar to the sequences of a person to minimize potential immunogenicity and b) make it more stable and soluble, CDR sequence TB-wt was grafted on a stable and soluble frame sequence of a human immunoglobulin (Auf der Maur et al. (2001), FEBS Lett. 508:407-412; Auf der Maur et al. (2004), Methods 34:215-224). A subset I of VL-Kappa and a subset I of VHperson identified as the nearest of the subfamily person. Suitable acceptor framework was chosen from a pool of VLand VHsequences of the human, were selected on the preferred biochemical and biophysical properties, such as stability and solubility, and expression properties (Auf der Maur et al. (2001), FEBS Lett. 508:407-412; Auf der Maur et al. (2004), Methods 34:215-224). Isolation and properties of these frames antibodies is written in WO 03097697/EP 1506236. From this pool as a suitable acceptor was identified frame single-chain antibodies with an undefined antigen-binding properties. This acceptor consists of domain VL-Kappa I human (SEQ ID NO:14) in combination with domain VHI human (SEQ ID NO:15). This acceptor framework called FW2.3. Among 81 balance VL-frame TB-wt and FW2.3 have 55 identical amino acid residues, which corresponds to 67% identity. Among 87 residues VH-frame TB-wt and FW2.3 have 55 identical residues, which corresponds to 63% identity. Both single-stranded derived antibodies have identical length CDR, except CDR3 VHthat is longer in FW2.3. The amino acid composition of residues in the CDR is different for both scFv. Describes various methods of humanization of the variable domains of the antibody (Riechmann et al. (1998), Nature 332:323-327; Padlan, E.A. (1991). Mol. Immunol. 28:489-498; Roguska et al. (1994), Proc. Natl. Acad. Sci. USA 91:969-973; Gonzales et al. (2005), Numor Biol. 26:31-43; Ewert, S., et al. (2004), Methods 34:184-199). First, spend a minimal approach, namely, conservative transferring all CDR loops mouse from TB-wt on FW2.3. The obtained scFv called TV-In and has a VL-the sequence SEQ ID NO:3 and VH-the sequence SEQ ID NO:4. Loop CDR in TB-wt is determined in accordance with the numbering scheme of Kabat (Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5thEd, Natl. Inst. Health, Dethesda, MD) and include the following about the of faultless (see figure 1):
CDR1: L24-L34 (the same numbering of Kabat)
CDR2: L50-L56 (the same numbering of Kabat)
CDR3: L89-L97 (the same numbering of Kabat)
CDR1: n-N (the same numbering of Kabat)
CDR2: Uw50-N (Kabat numbering Uw50-N)
CDR3: N-N (Kabat numbering N-N).
This antibody was able to bind efficiently TNFα (figa). The next stage was to determine what balance or what remains of these components must be replaced to optimize the properties of the obtained gumanitarnogo antibodies.
Since the replacement of amino acid residues of a person other amino acids should be minimized, since the introduction of alien amino acid sequence increases the risk of immunogenicity of the antibody, or a derived antibodies in humans (Gonzales et al. (2005), Tumor Biol. 26:31-43), designed several options. One of these options is here called TV-L46 (VLSEQ ID NO:11; VHSEQ ID NO:4) were designed to minimize the risk of immunogenicity, but still maintaining sufficient activity linking. This option is based on TV-In and includes a change of a single amino acid at position 46 in the VL, namely R→L. This amino acid is located within the upper core light chain and takes part in the interface of the dimer. She participates in the creation of conformal the L-CDR1 and affects the package V H/VL. It was reported that the leucine residue is preferred in this particular situation (PCT/US 03/19333). Unlike TV, scFv TB-B L46 saves a TNFα-specific binding (figa;d~100nm)).
To further enhance TNFα-binding activity of one or more additional changes made in one or more of VLresidues 4, 46, 65, 67, 70 and/or VHresidues 28, 43, 68, 70, 71, 72, 73, 76, 77. Here this option with substitutions at all positions referred to as TV And (VLSEQ ID NO:1; VHSEQ ID NO:2).
In addition, with regard to specific anti-TNFα antibodies of the present invention, competitive assays with peptides produced from L-CDR1 and L-CDR2, showed that both CDR loops are important for binding of these scFv antigen (Doring et al. (1994), Mol. Immunol. 31:1059-1067).
In the following experiments, aimed at minimizing the number of residues not a person required to save the link, and on the optimization of biophysical properties (stability and solubility), used systematic mutagenesis and shuffling of domains, which allow to determine the functional differences between mouse and humanitarianism VLand VH-domains.
By shuffling of domains got two options. The first option consisted of VLdomain from the TV And connected via a glycine-serine linker (SEQ ID NO:10) is V Hdomain TV-In that led to TV-AV (SEQ ID NO:12). The second option, TV VA, is the address of the first option, namely VLdomain TV-In in combination with VHdomain TV-a (SEQ ID NO:13).
Additional variants were generated by systematic mutagenesis TV-A. Figure 1 shows the comparison of sequences of the VLand VHsequences of TV and TV-Century In a whole frame 14 residues differ between TV and TV-In (asterisks). Only five of them, VL-the remains of 4 and 70 and VHremnants 28, 71 and 73, reveal only minor differences in the size and properties and, therefore, were not considered for mutagenesis at this point. The following provisions of the framework was replaced by the corresponding amino acids from the TV Century. These mutants TV And with one or two mutations are as follows:
|TV In N||K→Q||border||(SEQ ID NO:18)|
|TV And N||F→V||the outer loop VH||(SEQ ID NO:19)|
|TV And N/72||F→L, L→R||the outer loop VH||(SEQ ID NO:20)|
|TB-A H76/77||A→I, S→G||the outer loop VH||(SEQ ID NO:21)|
|TB-A L46||L→R||border||(SEQ ID NO:22)|
|TB-A L65||R→S||the outer loop VL||(SEQ ID NO:23)|
|TB-A L67||Y→S||the outer loop VL||(SEQ ID NO:24).|
Many factors can affect the immunogenicity of the antibody or derivative antibody (Gonzales et al. (2005), Tumor Biol. 26:31-43). To further reduce the content of immunoglobulin is not human variable regions gumanitarnogo scFv TB-A, mouse CDR2 and CDR3 loops of the VLand the mouse CDR2 loop of the VHreplaced the corresponding CDR loops person from FW2.3. The resulting construction is called here TB_L2 (SEQ ID NO:7), TB_L3 (SEQ ID NO:8) and TVN (SEQ ID NO:9), respectively.
cDNA encoding a murine single-chain version of monoclonal antibodies Di62 and two humanized versions of TV-and TB-A, generated by gene synthesis (www.genscript.com). All point mutations in other embodiments (TV-L46, TB-N, TB-N, TB-N/71, TB-A N75/76, TB-A-L46, TB-A L65, TB-A L67 TB-A V83F, TB-A V83A, TB-A D66G) who drove PCR-launch site-directed mutagenesis according to standard cloning procedures. Replacement of the murine CDR loops TB-A CDR loops person from FW2.3 were performed using PCR and cloning procedures that exist in this area. cDNA encoding all variations VHTV And described in SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, was obtained by synthesis of the complete genes.
Some even more preferred TB-A described in SEQ ID NO:31 - SEQ ID NO:38. It was found that these antibodies are particularly stable and soluble, as shown below in table I.
All scFv fragments cloned in expressing vector to obtain periplasmic E. coli (Krebber et al. (1997), J.Immunol. Methods 201:35-55).
In addition to the above-described single-stranded derived antibodies (scFv) corresponding Fab fragments were generated as follows. The selected variable domains of the light chain (VL) was merged with the constant region of the Kappa-chain Ig, whereas the appropriate variable domains of the heavy chain (VH) was merged with the first (N-terminal) constant domain (SN) IgG human. Both the constant domain of human amplified using PCR from cDNA libraries spleen person and received in the form of sequence SEQ ID NO:14 for κ and SEQ ID NO:15 for SN.
Experiment 2. The expression, release and stability of humanized scFv or Fab antibodies
Plasmids encoding TB-wt, its humanized derivative or Fab fragments were injected into a suitable strain of E. coli (for example, JM83) sexpression in periplasm. scFv variants also expressed in the form of Taurus include, for example, in E. coli strain BL21. Functional single-chain antibodies were obtained by refolding Taurus inclusion and subsequent treatment, for example, gel-filtration.
The outputs of expression after periplasmatic expression of scFv was in the range of 0.5 to 12 mg per liter of culture under standard laboratory culture conditions (environment dYT, with an induction time of approximately 3 h at 30°C with shaking at 200 rpm) using conventional shake flasks. Usually authors observed, as expected from the preliminary analysis of frames selected for the stability and solubility (Auf der Maur, et al. (2004), Methods 34:215-224)that the closer the sequence gumanitarnogo derived to the sequence of the acceptor framework (FW2.3), the higher the output obtained after expression in bacteria. For example, the output obtained from the expression of the per-In is much better than the output obtained from the expression of the per-A. In accordance with these discoveries, reducing the number of different amino acid residues present in the TV And had a positive effect on the outputs of expression (figa).
Another important characteristic of antibodies or derivatives of the antibodies of the present invention is their solubility. Figv shows the superiority of the frame of the TV And on donors the m frame (TB-wt) in respect of solubility in phosphate buffered saline. Analytical gel filtration TV And migrates predominantly in a Monomeric state (peak at 70 ml), whereas TB-wt shows a strong tendency to form aggregates (peak at 50 ml). In addition, the maximum solubility TV and some of its derivatives was estimated by precipitation using PEG (Athat D.H. et al., JBC. 1981, 256; 23.12108-12117). In short, the apparent solubility of the test proteins was measured in the presence of polyethylene glycol 3000 (PEG). Solubility curves were determined by measuring the protein concentration in the supernatant was centrifuged mixtures of protein-PEG. All curves showed a linear dependence of log S (mg/ml) at concentrations PEG (%, weight/volume). The maximum solubility (Smax) test protein was determined by extrapolation of the linear regression in the direction of 0% PEG (table). It was estimated that the per-And Smaxwas approximately 70 mg/ml All test proteins were found exclusively good solubility. The second approach for the evaluation of intermolecular attraction/repulsion TV-a (SEQ ID NO:40), TV And _G2R h_ Wednesday, February+F68L (SEQ ID NO:33) and a TV AND H_F68L (SEQ ID NO:35) in a concentration of 1 mg/ml in SFR (50 mm phosphate pH 6.5, 150 mm NaCl) was applied a method called chromatography samozaodrasle (SIC). In this way protein of interest immobilized on porous stationary phase is Packed into a column. The interaction between the free (mobile is the phase and the immobilized protein detects offsets in the amount of retention. The osmotic second virial coefficient B22protein, which is a measure of intermolecular attraction/repulsion, was calculated according to Tessier, PM et al. Biophys. J. 2002, 82: 1620-1632 (table). The more positive is the In22the lower intermolecular attraction test protein and, consequently, the higher the solubility. Due to the high similarity of sequences of test proteins, it is assumed that values of B22different proteins can be directly compared with each other.
|Table I: Properties of solubility derivatives TV And|
|Sequence||pI||Log Smax||The value of22(SIC)|
|TV AND _M48L/F68I||7,8||not determined.||not determined.|
|TB-A L_V83E H_V79A||to 6.58||not determined.||not determined.|
|TV And _G2R _F68L||8,2||1,91±0,09||of 1.59×10-3±5,9×10-5|
|TV AND H_K43R/F68I||7,8||1,86±0,02||1,28×10-3±3,0×10-4|
|TV AND H_F68L||7,8||1,88±0,07||of 1.06×10-4±2,9×10-5|
|TV AND _F68A||7,8||not determined.||not determined.|
|TV AND _F68V/F70L||7,8||not determined.||not determined.|
|TV AND _F70L||7,8||not determined.||not determined.|
Another relevant characteristic of antibodies or derivatives of the antibodies of the present invention is their high stability. The stability of proteins TV, TV-AND H_M48L/F68I (SEQ ID NO:31), TV And _G2R H_F68L (SEQ ID NO:33), TV AND H_K43R/F68I (SEQ ID NO:34) and a TV AND H_F68L SEQ ID NO:35) was evaluated by determining the temperature for the start of the deployment of protein by circular dichroism and light scattering at 218 and 292 nm (table II). In this experiment the TV And started to turn around at a temperature of 53°C, while its derivative is IN-AND H_M48L/F68I (SEQ ID NO:31), TV And _G2R H_F68L (SEQ ID NO:33), TV AND H_K43R/F68I (SEQ ID NO:34) and a TV AND H_F68L SEQ ID NO:35) was found increased heat (56°C and 58°C). All test proteins were found to be irreversible denaturation and was deposited after deployment, making it impossible to determine the melting temperature. To determine the average transition point in a reversible process deployment induced by guanidine hydrochloride (GdnHCL) to save unfolded proteins in solution. In this approach, the maximum emission of fluorescence was determined by using fluorometry for tracking deployment. In this production the TV And showed again good stability to the middle point of the conformational transition at 2.07 M GdnHCL. In accordance with the results of thermal deployment derivatives TV And _G2R H_F68L (SEQ ID NO:33) and a TV AND H_K43R/F68I (SEQ ID NO:34) showed increased stability, reflected higher average points conformational transition of 2.33 and 2.3 M GdnHCL, respectively.
|Table II: Properties of solubility derivatives TV And|
|Sequence||The beginning of denaturation (°C)||[GdnHCl] at the midpoint of conformational transition|
|TV AND _M48L/F68I||58||not determined.|
|TB-A L_V83E H_V79A||not determined.||not determined.|
|TV And _G2R _F68L||58||2,33 M|
|TV-AND-_F68A||not determined.||not determined.|
|TV AND _F68V/F70L||not determined.||not determined.|
|TV AND _F70L||not determined.||not determined.|
The stability of the TV And in human serum, human urine, vitreous fluid of the body of the pig and the fluid of the anterior chamber pigs was assessed by measurement of TNFα-binding activity of TV-And after incubation for three days at 37°C in the respective fluids of the body or in the buffer for analysis (TBSTM) as a positive control. TV And diluted in body fluids to a final concentration of 10 μm. After the of erida incubation series dilution of these samples were analyzed using ELISA for the determination of the binding constant K dTV And (11). When comparing samples of body fluids with the breakdown of the positive control TBSTM, offset Todin the direction of higher concentrations would indicate a reduction of active protein during the incubation period. However, in the experiments of the authors of this shift is not detected, which suggests that the number of fully active TV And remained constant in the analysis of each body fluids due to the high stability of this antibody.
Experiment 3: the Binding properties of humanized derivatives of antibodies
Binding properties of all humanized scFv variants were tested in ELISA on recombinant human TNFα. Dissociation constants (Kdfor all variants lie within the range of 0.8 - over 10000 nm. Apparently, there is an inverse correlation between the degree of homology with an acceptor framework of human rights and the affinity of the corresponding connecting component (figa). However, some options TV And containing mutations in TV-And-sequence, detect the levels of affinity in relation to human TNFα, which is comparable with the levels of affinity-per-A. In figure 4 shows two derivatives TV And with improved output expression (compare Figo), which show similar affinity with TV And when compared to ELISA.
TV And is a good example of an explicit trade-off is and output expression and affinity. As for affinity, was not detected significant differences between single-stranded format and format of the Fab-fragment of a TV-a (data not shown).
The affinity against TNFα and kinetics of binding was determined for TV And using surface plasma resonance (BIACore), which led to the dissociation constant Kd=0.8 nm, the rate of dissociation (koff=4,4×10-4with-1and velocity of the Association of kon=5×105with-1M-1.
Experiment 4. Analysis of cytotoxicity of L929
The function of antibodies or derivatives of antibodies in neutralization of TNFα in vivo can be tested by measuring the inhibition of TNFα cytotoxicity against cultured mouse fibroblasts L929 or alternative in the cells of maasarani person FOUND-1 (table III). Humanized scFv-derived Di62 show different effectiveness analysis of L929, as shown in figv. Some of scFv-derived detect the magnitude of the IC50(inhibitory concentration to obtain 50% inhibition) in the range of 5 ng/ml, whereas others do not apply in the analysis of L929. Data ELISA and the results from the analysis of L929 not always correlate with each other. However, the data FOUND-1 and the results of L929 correlate well with the only difference that required a much higher concentration of recombinant human TNFα and, therefore, also and this is tagonist to watch the action. Thus, the FOUND-1 was used to confirm the results of L929. For direct comparison test protein efficiency were expressed as relative values, normalized to the TV And (EC50X/EU50TV-A). Typically, however, the value of the IC50became again the higher, the closer was the sequence linking the component to the acceptor skeleton man (FW2.3). Figure 5 compares the effectiveness of various derivatives TV And in blocking induced TNFα cytotoxicity against fibroblastic of L929 mouse cells. The absorption at 450 nm correlates with the survival of cells.
TV-a and anti-hTNFα-IgG Infliximab® find similar magnitude IC50in the analysis of L929, while the effectiveness of TB-wt in blocking induced human TNFα cytotoxicity is significantly lower (figa). When comparing derivatives TV And on TV And in relation to their potential in blocking induced human TNFα cytotoxicity most of these derivatives, except per-N, have a reduced efficiency in L929 (pigv).
|Table III: Functional properties of derivatives TV And|
|Sequence||Relative effektivnosti 50X/EU50TV-a|
|Cells L929s||Cells FOUND-1|
|TV AND _M48L/F68I||1,1||1,6|
|TB-A L_V83E H_V79A||not determined.||not determined.|
|TV And _G2R H_F68L||0,8||1,3|
|TV AND _F68V/F70L||1,28||not determined.|
|TV AND _F70L||2,7||not determined.|
In accordance with the ELISA data, no significant differences in the ability to block TNFα-induced cytotoxicity between formats of scFv and Fab TV (figs). The value of the IC50for Fab-format TV And about two times higher than the value of the IC50for scFv-format TV (figs), the ve is Aetna, as a result of higher molecular mass Fab-fragment.
Experiment 5. Experiments on animals with derivative with anti-TNFα antibodies
5.1. Description of the experiment
For testing the efficiency derived anti-NFα antibodies (scFv and Fab) ESBATech in functionally neutralize the bioactivity of human TNFα in a situation in vivo used recently published rat model of acute monoarthritis. This model was described in detail Bolon et al. (see Bolon et al. (2004), Vet. Pathol. 41:235-243). Briefly, in this model of arthritis animal human TNFα injected intra-articular knee joint male Lewis rats. Injection of TNFα person causes an acute, self-limited to monoarthritis received an injection of the joint. Arthritis can be evaluated quantitatively by measuring the swelling of the joint and histological evaluation. Then the relevant bioactivity of TNFα antagonists can be quantified by the decrease in TNFα-induced swelling of the joint and/or reduction of histological parameters of inflammation.
5.2. Materials and methods
The experiment plan
These studies were designed to test the corresponding potential representative scFv-antibodies and Fab representative antibodies of the above number of commercially available antibody Infliximab (Remigade®) in respect of inhibition of TNFα bioactivity brow the ESA in a suitable model of arthritis animal. Beaulon and colleagues have previously shown that intra-articular injection of 10 micrograms of recombinant human TNFα in the knee joint of rats provokes acute self-limited monoarthritis, which can be quantified standard macroscopic and microscopic analysis. Thus, this animal model served as an ideal system to evaluate therapeutic actions of locally-provided derivatives of the antibodies. Two experiments were performed sequentially (.IV and V). 1) Research background the effectiveness of assessing the overall potential of these antibodies in blocking induced TNFα human monoarthritis; 2) study of the dependence of the response on the dose that evaluates the relative efficiency of the derivatives of these antibodies in comparison between them. Cytokines and derivatives of antibodies, in each case, provided one time in the form of separate injections, as described below. Used dose of a cytokine selected on the basis of the publication Bolon and employees, whereas the range of doses derived antibodies was determined on the basis of available data on cell cultures and on the basis of guesses based on knowledge. These experiments were conducted in accordance with the General guidelines for the care of animals.
Animals and animal welfare
Young adult male Lewis rats (6-7 weeks, 175-200 g) random about the time attributed to treatment groups (n=3 per group (cohort)) and contained in accordance with Bolon et al. (2004), Vet. Pathol. 41:235-243.
Instillation of cytokine and antibody
Anesthesia and injection of the cytokine was performed as described by Bolona and employees. In order not to exceed the total intra-articular injectable volume 50 microlitres, derived cytokines and antibodies were injected in two separate intra-articular injection, by means of which 10 micrograms of recombinant human TNFα were injected with 10 Microlitre sterile filtered phosphate buffered saline (SFR) and the appropriate dose of the respective antibodies were injected with 40 Microlitre sterilized by filtration phosphate buffered saline. Animals treated intraperitoneally Infliximab/Remigade®, derivatives were injected with antibodies for three hours before intra-articular injection of TNFα person. All animals treated with the insertion of intra-articular antibody, the appropriate dose of the antibodies were injected with five minutes before the injection of TNFα person. Control animals were injected with 10 microliters SR without TNFα person.
Infliximab/Remigade®used in these experiments, bought in recognized in medicine and Pharmacopoeia of the Swiss pharmacy. scFv and Fab (TV-A)antibodies specific against human TNFα, as well as raw frame scFv-antibodies, used as a nonspecific control experiment, the dose of the TVET expressed in E. coli and purified by standard methods. Contamination with endotoxins supported below ME on 10 mg of protein in all preparations, as component of lipopolysaccharide is a potent inducer of TNFα.
Recombinant human TNFα was purchased from PeproTech EC Ltd.
Measuring the diameter of the joint
Immediately before injection of the corresponding injected intraperitoneally or intra-articular derived antibodies or, in the case of control animals, before injection SFR or TNFα, the diameter of the knee joint, which must be made of injection was determined using a standard caliper. After 48 hours after the injection of TNFα (or SFR in control animals) and just before the killing of these animals is the diameter of the injected knee again measured and counted swollen joint by subtracting a value of the second diameter measurement of the magnitude of the first dimension diameter (6 and 9).
After 48 hours after the injection of TNFα (or SFR in control animals) animals were autonational. At necropsy, injected knee joints were separated from the foot and thigh, recorded intact by immersion in 70% ethanol and processed for standard staining with hematoxylin and eosin (HE), as described by Bolona and employees.
Histological grading analysis measurement of inflammation of the joints was performed, as described by Bolona and employees. Histopathologic criteria for the evaluation of inflammation of the joints used in accordance with the criteria Bolon and employees (Fig.7, 8 and 10).
In the first series of experiments representative of the input intra-articular scFv-antibody, TV, ESBATech, and the corresponding input intra-articular Fab-antibody ESBATech were compared for their ability to block the induction of acute monoarthritis with intra-articular and intraperitoneally injected Infliximab/Remigade® in accordance with table IV.
|Table IV: Scheme of the injection experiment 1|
|Group||TNFα (ICG) in SFR||Inhibitor||Dose (µg)|
|3 (n=3)||0||TV And scFv||180|
|4 (n=3)||10||TV And scFv||180|
|8 (n=3)||0||Infliximab (i.a.)||450|
|9 (n=3)||10||Infliximab (i.a.)||450|
|10 (n=3)||10||Infliximab (i.a.)||180|
|11 (n=3)||10||Infliximab (I.P. Pavlova.)||450|
|12 (n=3)||10||Infliximab (I.P. Pavlova.)||180|
The results obtained in respect of acts of treatment (processing) on the diameter changes of the knee joint (as an indicator of TNFα-induced swelling of the joints), presented on Fig.6. All antibodies completely blocked TFα-induced swelling of the joint.
For the evaluation of the action of the treatment (processing) on joint inflammation performed histological grade assessment slides with undyed cloth. Joint inflammation was assessed as a score using the following criteria (see Fig.7 in relation to representative examples of evaluation points):
Score 0 - normal
Point 1 - weak thickening of the synovial lining
Point 2 - the thickening of the synovial lining and mild thickening of the underlying pavement
Point 3 - the thickening of the synovial lining and moderate inflammation of the underlying pavement
The results obtained on the effect of treatment on histopathological evaluation of inflammation, shown in Fig.
Observed comparable effects of all treatments on the evaluation of histopathological inflammation.
In the second series of experiments compared the relative dependence on dose response estimate derived antibodies. Representative input intra-articular scFv-antibody TV And ESBATech and the corresponding input intra-articular Fab-antibody ESBATech experiment 1 was compared with the input intra-articular and intraperitoneally Infliximab/Remigade® and unrelated scFv-antibody, having binding activity against human TNFα in a wide range of doses that differ from the range of doses in experiment 1, in accordance with table V.>
|Table V. Scheme of injection experiment 2|
|Group||TNFα (ICG) in SFR||Inhibitor||Dose (µg)|
|3 (n=3)||10||an unrelated scFv-antibody||180|
|4 (n=3)||10||scFv-antibody TV And||156|
|5 (n=3)||10||scFv-antibody TV And||45|
|6 (n=3)||10||scFv-antibody TV And||11|
|7 (n=3)||10||Fab-antibody TV And||156|
|8 (n=3)||10||Fab-antibody TV And||45|
|9 (n=3)||10||Fab-antibody TV And||11|
|10 (n=3)||10||Infliximab (i.a.)||156|
|11 (n=3)||10||Infliximab (i.a.)||45|
|12 (n=3)||10||Infliximab (i.a.)||11|
|13 (n=3)||10||Infliximab (I.P. Pavlova.)||156|
|14 (n=3)||10||Infliximab (I.P. Pavlova.)||45|
|15 (n=3)||10||Infliximab (I.P. Pavlova.)||11|
The results obtained on the effect of treatment on the change in the diameter of the knee joint (as an indicator of the effect on TNFα-induced swelling of the joint, shown in Fig.9.
The results obtained on the effect of treatment on histopathological evaluation of inflammation, presented in figure 10.
In General, as repres ntative anti-NFα-sFv-antibody ESBATech, and representative anti-NFα-Fb-antibody ESBATech were highly effective in blocking induced TNFα human monoarthritis after local (intra-articular) introduction.
Although described here is preferred in the present embodiments of this invention, it should be clearly understood that this invention is not limited to them and may otherwise be carried out and be variously embodied within the following claims.
1. Stable and soluble antibody, or a derived antibodies that specifically bind TNFα, with the indicated antibody or derivative of the antibody contains a variable domain light chain (VL), having the sequence of SEQ ID NO:1, or derived from the sequence SEQ ID NO:1, which is combined with the variable domain of the heavy chain (VH), having the sequence of SEQ ID NO:2, or derived from the sequence SEQ ID NO:2, and in the case of a derived sequence this sequence has a maximum of 5 changes in the frame specified VLdomain and/or up to a maximum of 9 changes in the frame specified VHdomain, provided that the antibody or derivative of the antibody does not contain the sequence of SEQ ID NO:3 and SEQ ID NO:4.
2. The antibody or derivative of the antibody according to claim 1, where the change VLare in one or more spallogenic 4, 46, 65, 67, 70 and/or 83, and changes VHlocated in one of the positions 11, 16, 28, 43, 48, 68, 70, 71, 72, 73, 76, 77, 93 and/or 112.
3. The antibody or derivative of the antibody according to claim 1, where the change VLare in one or more of positions 4, 46, 65, 67, 70 and/or 83, and changes VHare in one or more of the provisions 11, 16, 28, 43, 48, 68, 70, 71, 72, 73, 76, 77, 79, 93 and/or 112.
4. The antibody or derivative of the antibody according to any one of the preceding paragraphs, in which at least one of these transformations leads to the amino acids present in SEQ ID NO:3 for VLand/or SEQ ID NO:4 for VH.
5. The antibody or derivative of the antibody according to claim 1, where VL-domain contains the sequence of SEQ ID:NO 1.
6. The antibody or derivative of the antibody according to claim 5, containing VH-domain sequence SEQ ID NO:2.
7. The antibody or derivative of the antibody according to claim 5, containing VHdomain, derived from the sequence SEQ ID NO:2, which F68 changed to A, L, I or V.
8. The antibody or derivative of the antibody according to claim 1 containing VL-domain sequence SEQ ID NO:11 and VH-domain sequence SEQ ID NO:4.
9. The antibody or derivative of the antibody according to claim 1, additionally containing at least one amino acid residue, at least one of the CDR, which turned into a residue present in the corresponding CDR VL-the sequence SEQ ID NO:5 and/or V -the sequence SEQ ID NO:6 or SEQ ID NO:25.
10. The antibody or derivative of the antibody according to claim 9, where at least one of the CDR group CDR2 VL, CDR3 VH, CDR2 VHor CDR3 VHconverted into the corresponding CDR VL-the sequence SEQ ID NO:5 and/or VH-the sequence SEQ ID NO:25 or SEQ ID NO:6.
11. The antibody or derivative of the antibody of claim 10 VL-the sequence SEQ ID NO:7 or SEQ ID NO:8 and VH-the sequence SEQ ID NO:2.
12. The antibody or derivative of the antibody of claim 10 VL-the sequence SEQ ID NO:1 and VH-the sequence SEQ ID NO:4, or SEQ ID NO:9, or SEQ ID NO:28, or SEQ ID NO:29.
13. The antibody or derivative of the antibody of claim 10 VL-the sequence SEQ ID NO:26 or SEQ ID NO:27 and VH-the sequence SEQ ID NO:30.
14. The antibody or derivative of the antibody according to claim 9, having specificity against human TNFα.
15. Derived antibody according to claim 1, which is a scFv-antibody, in which VLand VHdomains joined by a linker.
16. scFv-antibody indicated in paragraph 15 contains the location of the sequences VL-linker-VH.
17. scFv-antibody indicated in paragraph 15, where the linker has the sequence of SEQ ID NO:10 or produced from the specified sequence.
18. scFv-antibody on 17, where at least one G of the specified linker changed to a more polar or charged amino acid.
19. scFv-antibody on the .18, where the linker has the sequence of SEQ ID NO:39.
20. Derived antibody according to claim 1, which is a Fab fragment, in which VLdomain fused with the constant region of the Kappa chain of human Ig, VHdomain merged with SN domain of human IgG, and the two fused polypeptide connected megamachines by a disulfide bridge.
21. Derived antibody according to claim 1, which represents the scFv-antibody or Fab fragment, which are labeled or chemically modified.
22. The antibody or derivative of the antibody according to claim 1 as pharmaceuticals.
23. The DNA sequence encoding the antibody or derivative of the antibody according to any one of claims 1 to 20.
24. The cloning vector containing a DNA sequence according to item 23.
25. Expressing a vector containing a DNA sequence according to item 23.
26. Vector on A.25 as pharmaceutical agents for gene therapy applications in humans and/or animals.
27. Suitable a host cell transformed by expressing vector on A.25.
28. A host cell according to item 27, which is a prokaryotic or eukaryotic cell, particularly a cell of E. coli, yeast, plant, insect, or mammal.
29. The method of obtaining molecules are antibodies or derived antibody according to any one of claims 1 to 20, providing for the cultivation of a host cell according to item 27 or 28 in the conditions, the cat is who make possible the synthesis of the specified molecule antibodies and removing it from the specified culture.
30. Therapeutic composition for treating TNFα-related diseases containing the antibody according to any one of claims 1 to 21 in combination with a pharmaceutically acceptable carrier, diluent or excipient.
31. therapeutic composition according to item 30, where TNFα-associated disease is osteoarthritis, uveitis or inflammatory bowel disease.
32. Diagnostic composition for the diagnosis of TNFα-related diseases containing the antibody according to item 21.
33. Diagnostic composition for p, where TNFα-associated disease is osteoarthritis, uveitis or inflammatory bowel disease.
34. Combined preparation for the treatment of TNFα-related diseases containing the antibody according to any one of claims 1 to 21, together, at least with the second connection.
35. Combined preparation according to clause 34, where TNFα-associated disease is osteoarthritis, uveitis or inflammatory bowel disease.
36. Combined preparation according to clause 34, in which the second compound is not an antibody specific against TNFα.
37. Combined preparation for 34 or 35 for use as a medicine.
38. A method of treating TNFα-related diseases, where the antibody or derivative of the antibody according to any one of claims 1 to 21 administered locally or topically.p> 39. A stable aqueous pharmaceutical finished form containing a therapeutically effective amount of the antibody or derivative of the antibody according to any one of claims 1 to 21, not subjected to prior lyophilization, acetate buffer with a pH of approximately from 4.8 to 5.5, a surfactant and a polyol, where this finished form does not contain regulatory toychest amount of sodium chloride.
40. Ready form by § 39, which is isotonic.
41. Ready form by § 39 or 40, which is stable at a temperature of about 2-8°C for at least one year.
42. Ready form by § 39, which is stable after freezing and thawing of this form.
43. Ready form by § 39, which is stable at approximately 30°C for at least one month.
44. Ready form by § 39, where the polyol is a non-sugar.
45. Ready form for item 44, where non sugar is trehalose.
46. Ready form for item 44, where non sugar is sucrose.
47. Ready form by § 39, where the antibody is an antibody fragment.
48. Ready for p, where the antibody fragment is a scFv.
49. Ready form by § 39, where the antibody binds to TNFα.
50. Ready form by § 39, which is stable at a temperature of AP is sustained fashion 2-8°C for at least two years.
51. Ready form by § 39, where the concentration of antibodies in the form of approximately from 0.1 to 50 mg/ml
52. Ready form by § 39, where the surface-active agent is a Polysorbate.
53. Ready for p, where scFv binds to TNFα.
54. Ready form by § 39, where the acetate is present in amount of about 5-30 mm.
55. Ready form by item 54, where the acetate is present in the amount of 10-30 mm.
56. The finished form in item 53, optionally containing a preservative.
57. Ready for p, where the preservative is benzyl alcohol.
58. The finished form in item 53, where the antibody is present in the amount of approximately 30-50 mg/ml
59. Ready form by § 58, where the buffer is 10-30 mm sodium acetate at pH 5, the polyol is trehalose in an amount of about 2-10% weight/volume, surface-active agent is Polysorbate in an amount of about 0.01 to 0.1% volume/volume, where the finished form additionally contains benzyl alcohol as preservative, and where the finished form is stable at a temperature of about 2-8°C for at least two years.
60. A stable aqueous pharmaceutical finished form containing a therapeutically effective amount of the antibody or derivative of the antibody according to any one of claims 1 to 21, not subjected to preliminary leofiles the tion, acetate buffer with a pH of approximately from 4.8 to 5.5, a surfactant and a polyol, where this finished form does not contain regulatory toychest quantity of sodium chloride, and specified the finished form is contained in the product, which is a container.
61. A stable aqueous pharmaceutical finished form p, where the antibody is an antibody or derivative of the antibody according to any one of claims 1 to 21.
SUBSTANCE: invention represents immunoglobulins, in particular antibodies, which specifically bind to human interleukin 13 (hIL-13). Claimed are recombinant or transfected mammalian host cell, for cloning, as well as for expression of vectors, coding antibodies. Claimed are antibody-containing pharmaceutical composition, set, containing said pharmaceutical composition and pharmaceutical composition, containing monoclonal antibody against IL-4, such as pascolizumab. Claimed is method ob obtaining antibody, and applications of said antibodies.
EFFECT: antibodies can be used for treatment of various diseases to disorders, responsible for modulation of hIL-13 interaction with human IL-13 receptor.
51 cl, 29 dwg, 22 tbl, 7 ex
SUBSTANCE: invention relates to molecular pharmacology and specifically to a peptide which is part of an interleukine-15 (IL-15) sequence which can inhibit biological activity of the said molecule.
EFFECT: obtaining a peptide which inhibits T cell proliferation induced by IL-15, and apoptosis caused by tumour necrosis factor when bonding with the alpha subunit of the (IL-15R) receptor.
8 cl, 4 dwg, 5 ex
SUBSTANCE: there are offered versions of human IL-13 antibodies, including based on CDR antibody BAK278D6. There is described a based composition, and also isolated nucleic acid, a host cell for preparing antibodies and versions of the method for preparing antibodies. There is disclosed application of antibodies for preparing a drug and a composition for treating various diseases mediated by IL-13 activity. Application of the invention provides antibodies neutralising IL-13.
EFFECT: applicable in medicine for preparing a vaccine.
52 cl, 32 dwg, 7 tbl, 29 ex
SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.
EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.
14 cl, 1 tbl, 6 ex
SUBSTANCE: invention concerns immunology and biotechnology. There is offered human monoclonal antibody specific to TNF-alpha containing light and heavy chain with appropriate CDR3 sites. There are described versions thereof including those based on heavy and light chains and coded by human genes VH3-33 and A30VK1 or VH3-53 and L2VK3 respectively. There are disclosed: the method for estimating the TNF-alpha content in the patient's sample with using specified antibodies, and application of antibodies for preparing a medical product. There are described: compositions for diagnostics and treatment of the conditions associated with TNF-alpha activity on the basis of antibodies. There is disclosed coding nucleic acid, a cell for making said antibodies and the method for making said antibodies.
EFFECT: application of the invention ensured high-affinity neutralizing monoclonal antibodies with improved Kd and IC50 in comparison with Infliximab, Adalimumab or Etanercept that can find application in medicine for treatment and diagnostics of the diseases associated with TNF-alpha hyperactivity.
35 cl, 13 dwg, 36 tbl, 14 ex
SUBSTANCE: there are disclosed polypeptide variants containing Fc-areas IgG, having amino acid modifications providing changed effector functions Fc in specified polypeptides. There is disclosed composition for antibody targeting on antigen, containing the specified polypeptide. There is described method for preparing the specified polypeptide. Also, there are disclosed the methods for treating V-cell tumour or a malignant disease characterised by V-cell expression of CD20, treating chronic lymphocytic leukosis, relieving the symptoms of the V-cell controlled autoimmune disease, treating a angiogenesis-associated disorder, treating HER2-expressing cancer, treating LFA-1-mediated involvement, treating IgE-mediated involvement wherein specified methods imply introduction to the patient of the therapeutically effective amount of said polypeptide.
EFFECT: higher clinical effectiveness.
63 cl, 6 ex, 13 dwg, 10 tbl
SUBSTANCE: present invention relates to biotechnology. Proposed is an antibody against interferon-α/β-binding protein I or against its mutein, obtained through conservative substitution. Proteins, against which the antibody is directed, are extracted from urine and link interferon-α/β-binding protein I with Kd constant from 3.6x10-9 to 1.6x10-10 M.
EFFECT: use of the invention allows for detecting presence of interferon-α/β-binding protein I in different samples.
5 cl, 10 dwg, 6 tbl, 17 ex
FIELD: chemistry, biochemistry.
SUBSTANCE: claimed invention relates to field of biotechnology. Claimed is antibody against interferon-α/β-connecting protein II or against its mutein, obtained by conservative substitutions. Here protein, against which antibody is directed, is separated from urine and connects interferon-α/β-connecting protein II with constant Kd equal 2.12x10-9 M.
EFFECT: obtaining possibility to detect interferon-α/β-connecting protein II presence in different samples.
5 cl, 10 dwg, 6 tbl, 17 ex
FIELD: chemistry; medicine.
SUBSTANCE: claimed are polypeptide and respective polynucleotide zcytor17lig and molecules of antibody against human zcytor17. Human zcytor17lig is novel cytokine. Claimed invention also relates to methods of protein obtaining, its application for stimulation of immune reaction in mammal. Described is method of obtaining antibodies to said protein and respective antibodies.
EFFECT: polypeptides can be used in realisation of methods stimulation of immune system, proliferation and development of hemopoietic cells in vitro and in vivo.
17 cl, 3 dwg, 21 tbl, 47 ex
SUBSTANCE: full-size human monoclonal antibodies against MCP-1 antigene are obtained. Antibodies are used for MCP-1 level definition in patient sample, as well as in tumour and inflammatory disease treatment. Invention allows obtainment of monoclonal antibodies highly specific to MCP-1 antigene.
EFFECT: efficient antibody use for treatment of the said diseases and in diagnostic purposes.
16 cl, 18 dwg, 17 tbl, 11 ex
FIELD: biotechnology, medicine.
SUBSTANCE: Zalpha 11-ligand is isolated from cDNA library generated from activated cells of human peripheral blood that have been selected from CD3. Animal is inoculated with Zalpha 11-ligand and antibodies are prepared that are able to bind specifically with epitopes, peptides or polypeptides of Zalpha 11-ligand. Invention provides effective regulation and/or development of hemopoietic cells in vitro and in vivo. Invention can be used for preparing Zalpha 11-ligand and antibodies for it.
EFFECT: valuable properties of new cytokine.
18 cl, 5 tbl, 1 dwg, 55 ex
FIELD: medicine, immunobiology, pharmacy.
SUBSTANCE: humanized monoclonal antibody (monAb) or its fragments comprises heavy and/or light chain with the binding rate constant with AILIM 1.0 x 103 (1/M x s) and above, and the dissociation rate constant between monAb and AILIM 1.0 x 10-3 (1/s) or less. MonAb shows also a nucleotide sequence encoding variable region of light and/or heavy chain and corresponding amino acid sequences. Invention relates to DNA and it part encoding monAb or its fragments, and vectors comprising nucleotide sequences encoding antibody or its fragments. The humanized monAb can be prepared by using a genetically recombinant host. MonAb is comprised as a component of pharmaceutical compositions used for inhibition or induction of AILIM-mediated transfer of signal into cell for induction of antibody-dependent cytotoxicity against AILIM-expressing cell and others. Invention can be effective in treatment of different autoimmune diseases associated with AILIM-mediated transfer of co-stimulating signal. Invention can be used in medicine for treatment of diseases associated with AILIM-mediated transfer of co-stimulating signal.
EFFECT: valuable medicinal properties of antibody.
75 cl, 78 dwg, 14 ex
FIELD: biotechnology, immunology, molecular biology, medicine, pharmacy.
SUBSTANCE: invention describes the isolated human antibody or its antigen-binding fragment able to bind the human tumor necrosis factor (TNF-α). Amino acid sequence is given in the description. Invention discloses nucleic acid encoding heavy and light chain of isolated human antibody. Nucleotide sequences are given in the description. Invention describes recombinant vector expressing variable region of heavy and light chains of isolated human antibody, Chinese hamster ovary cells CHO dhfr- carrying vector. Invention discloses a method for synthesis of isolated human antibody. The isolated human antibody or its antigen-binding fragment can be used as an active component of pharmaceutical composition used in treatment of disturbances when activity of TNF-α is harmful. Using the invention allows neutralization of effect of TNF-α in case when its activity is harmful. Invention can be used in medicine.
EFFECT: valuable medicinal properties of antibody, improved method for synthesis.
17 cl, 11 dwg, 17 tbl, 4 ex
SUBSTANCE: obtained human antibody or its antigen-binding fragment specifically binds tumor necrosis factor hTNFα. The like antibodies show high affinity relative to hTNFα in vitro and in vivo. Antibodies according to the invention are taken as a full-length antibody or its antigen-binding fragment. The antibodies or their fragments are usable for detecting hTNFα and for inhibiting hTNFα activity in human beings suffering from a disorder in the case of which hTNFα activity is harmful.
EFFECT: wide range of applications of high affinity recombinant antibodies to hTNFα or their fragments of low dissociation kinetics.
15 cl, 11 dwg, 17 tbl
FIELD: immunology, biology.
SUBSTANCE: invention relates to variants of IL-1β-binding molecule having common functionally active sites (CDR sites) and may bind human IL-1β. Said molecules have neutralizing activity IC50 of approximately 50 pM and binding constant KD of approximately 30 pM. Amino acid sequence is described in description of present invention. Variants of DNA constructs encoding of heavy chain and light chain of IL-1β-binding molecule are disclosed. Expression vectors carrying at least one abovementioned nuclear acid and method for production of IL-1β-binding molecules by using the same also are described.
EFFECT: IL-1β-binding molecules against human IL-1β with high neutralizing activity and binding constant useful in suppression of HAMA response.
10 cl, 1 dwg, 3 tbl, 4 ex
FIELD: immunology, biotechnology.
SUBSTANCE: invention describes murine antibody and its humanized variant (CDP870) showing specificity to human tumor necrosis factor-alpha. Amino acid sequence is given in the description. Also, invention describes compounds showing affinity with respect to human tumor necrosis factor-alpha based on humanized antibody wherein lysylmaleimide group bound covalently with one or some methoxypoly(ethylene glycol) molecules by lysyl residue is joined to one of cysteine residues by C-end of heavy chain of the humanized antibody. Invention discloses DNA sequences encoding antibodies showing specificity to human tumor necrosis factor-alpha and variants if expression vectors involving indicated DNAs. Also, invention describes variants of a method for preparing a host-cell using expression vectors and variants of a method for preparing antibodies based on prepared host-cells. Invention discloses therapeutic compositions used in treatment of pathology mediated by tumor necrosis factor-alpha based on antibodies. Invention provides providing antibodies showing high affinity: 0.85 x 10-10 M for murine antibodies and 0.5 x 10-10 M for its humanized variant and low immunogenicity for human for humanized antibodies. Part of patients with improved ACR20 in administration of 5 and 20 mg/kg of CDP870 is 75% and 75% in 8 weeks, respectively. Half-life value of CDP870 in plasma is 14 days.
EFFECT: valuable biological and medicinal properties of antibodies.
58 cl, 24 dwg, 6 tbl, 1 ex
FIELD: gene engineering.
SUBSTANCE: recombinant fragmid DNA pHEN-TAB, containing unique human single-strand antibody gene is selected from constructed in vitro combinatorial phage library udder controlling of lactose operon promoter. Then Escherichia coli HB2151 cells are transformed with obtained fragmid DNA to produce recombinant bacterium strain Escherichia coli HB2151/pHEN-TAB as producer of human single-strand antibody capable of binding of human tumor necrosis factor alpha. Said antibody gas affinity constant of Kaf = 3.96±0.52x108 M-1.
EFFECT: new soluble human single-strand antibody scTAB against human tumor necrosis factor alpha with high affinity.
3 cl, 6 dwg, 6 ex
FIELD: biotechnology, immunology, molecular biology, pharmacy.
SUBSTANCE: invention describes variants of MCP-1-binding molecules. One of MCP-1-binding molecule comprises at least one variable region of immunoglobulin (VH) heavy chain comprising of hypervariable sites CDR1, CDR2 and CDR3 while other molecules comprises both light and heavy chains. Invention proposes DNA constructs encoding indicated MCP-1-binding molecules and expressing vector carrying at least one of these DNA constructs. Invention describes a method for preparing MCP-1-binding molecule. Invention discloses a method for treatment of disease or disorder mediated by MCP-1 or eotaxine-1 based on antibody raised to MCP-1 that binds eotaxine-1 by cross mode. Invention describes a pharmaceutical composition based on antibody raised to MCP-1 that binds eotaxine-1 by cross mode and used in treatment of disease or disorder mediated by MCP-1 or eotaxine-1 in a patient. MCP-1-binding molecules inhibit binding MCP-1 with its receptor. The full immobilized antibody is highly specific as far as it binds human recombinant MCP-1 with value KD = (43 ± 2.9) x 1012 and can be used in medicine.
EFFECT: valuable medicinal properties of antibodies, improved method of treatment.
13 cl, 5 dwg, 4 tbl, 2 ex
FIELD: biotechnology, immunology.
SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.
EFFECT: valuable biological and medicinal properties of antibodies.
53 cl, 4 tbl, 10 dwg, 2 ex
FIELD: biotechnology, immunology.
SUBSTANCE: disclosed are variants of chimerical anti-IL-6 antibodies based on mice CLB-8 antibody. Each antibody contains constant region from one or more human antibodies. Described are variants of nuclear acids encoding anti-IL-6 antibody, vectors and host cells. Developed is method for production of anti-IL-6 antibody by using nuclear acid or vector. Described are variants of composition for application in method for modulation of malignant disease or immune disorder mediated with IL-6. Developed is method for treatment or modulation of malignant disease or immune disorder mediated with IL-6.
EFFECT: variant of chimerical anti-IL-6 antibody with high affinity of mice anti-IL-6 antibody and reduced immonogenicity.
26 cl, 16 dwg, 1 tbl, 8 ex