Antibody or fragment thereof, having neutralising effect on hiv, but not on il2

FIELD: chemistry.

SUBSTANCE: invention pertains to biotechnology. Described is an anti-HIV monoclonal antibody or its functional fragment which has in the variable region of the H-(heavy) chain regions which determine complementarity, CDR1, CDR2 and CDR3, which have sequences given in the description, and CDR1, CDR2 and CDR3 regions with sequences given in the description in the variable region of the L-(light) chain. Expression vectors which code fragments of the heavy and light chain of the described antibody and host cells which are transformed by the said vectors are disclosed. A method for detecting the HIV strain and a method for individual passive immunotherapy are disclosed.

EFFECT: invention enables to obtain an antibody which enables neutralisation of HIV infection without an autoimmune side effect.

17 cl, 9 dwg, 2 tbl, 6 ex

 

The invention relates to the antibody or its fragment having neutralizing activity against HIV and is suitable for the treatment and/or prevention of HIV infection. The invention also relates to pharmaceutical compositions containing the indicated antibody for diagnostic composition and method of implementation of passive immunotherapy.

The human immunodeficiency virus (HIV) is a member of the family of lentiviruses that are related to retroviruses of animals, and is the causative agent of Acquired Immunodeficiency Syndrome (AIDS). To date, were identified and characterized at the molecular level two closely related type HIV type 1 (HIV-1) and type 2 (HIV-2).

The use of antiviral agents against HIV, such as inhibitors of RNA-dependent DNA polymerase (reverse transcriptase), has greatly improved the condition of patients suffering from AIDS. However, in most cases, therapeutic effectiveness of these drugs against AIDS is a partial or temporary, and, in addition, these drugs are toxic or inhibit the growth of hematopoietic cells and, consequently, inhibit the immune system, which had been a failure.

In this regard, is widely accepted that prevention programs AIDS and antivi usnow therapy using drugs (VALDISSERI, 2003, Nat. Med, 9:881) should be combined with effective antimicrobial agents and vaccines. But, as has been proven, the development and testing of these vaccines are associated with difficulties (LETVIN, et al. 2002, Annu. Rev. Immunol, 20:73; McMICHAEL & HANKE, 2003, Nat. Med., 9:874).

Mucosal surfaces are the primary site of penetration of HIV-1 (NICOLOSI, et al. 1994, J. Acquir. Immun. Defic. Syndr., 7:296). Transmission of HIV-1 can occur through contact of mucous surfaces infected with HIV-1 fluids, such as semen, colostrum, breast milk and cervical-vaginal secretions (CHERMANN, 1998, Am. J. Reprod. Immunol., 40:183; MILMAN & SCHARMA, 1994, AIDS, 10:1305).

Due to the interaction of HIV with the mucous surface, the component of the vaccine developed against HIV should cause the immune system of the mucosa to intervene in an early stage of virus transmission through mucous surface and interact with potential receptors.

It is recognized that antibodies, neutralizing the AIDS virus, can certainly play an important role in antiviral defense.

However, although it was possible to get neutralizing antibodies against the particular strain of virus grown in the laboratory, has failed so far to achieve similar success in the creation of antibodies that could neutralize a wide range of viral strains and which could be effective activity in vivo.

Echodo the EN protein gp41 of HIV, being the most conservative area in the shell of the virus is highly immunogenic glycoprotein.

From US 6 455 265 known that conservative and area antigenic determinants of retroviral envelope protein gp41 of HIV can cause malicious autoimmune phenomena due to the analogies of the three-dimensional structure and/or cross-reactivity towards certain parts of the protein of the human immune system, and, in particular, IL-2 (interleukin-2).

From WO 2005/010033 known recombinant loop proteins gp41 containing linker fragment in the connecting loop between the N - and C-helices gp41 and lacking or exhibiting reduced autoimmune side effects. Consequently, there is a need in the antibody, which allows you to neutralize HIV infection, and, in particular, HIV-1 infection, without autoimmune side effect.

There is a need in the antibody, which helps prevent and/or reduce HIV infection through mucous surface.

There is also a need to create antibodies for the manufacture of medicinal products intended for use in passive immunotherapy.

There is a need in the antibody that can be used for diagnostic purposes.

The invention aims at the satisfaction of vysheupomjanutoe fully or in part.

The inventors obtained the effective monoclonal antibody, which can satisfy the aforementioned needs.

In particular, the inventors identified a new Fab (fragment antigen binding - antigennegative fragment) IgA antibody containing the variable regions of H chain at least one area defining complementarity, (CDR complementary determining region selected from the CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence represented by SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, or their functional equivalents.

In particular, the newly identified Fab IgA antibodies can recognize recombinant loop protein gp41 or protein gp41 wild-type, but may not recognize the peptide, called P1, which is a recombinant protein loop gp41 or protein gp41 wild-type.

The peptide PI (SEQ ID NO 12) corresponds to, for example, amino acid sequence with 649 at 683 of the amino acid sequence of wild-type protein gp41, presented in SEQ ID N0 11 and obtained from HIV-1 strain Nhw.

For the first time identified the antibody also has the ability to inhibit transcytosis are activated HIV-1 and block infection of CD4+T-cells.

Thus, according to one aspect, the present invention relates to a monoclonal antibody or its fragment containing variable is blasti N-circuit at least one region, determining complementarity, (CDR)selected from the CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence represented by SEQ ID nos I, SEQ ID NO 2 and SEQ ID NO 3, or their functional equivalents.

According to another aspect, the present invention relates to a monoclonal antibody or its fragment that recognizes recombinant loop protein gp41, as defined below, and cannot recognize a peptide having the amino acid sequence represented by SEQ ID NO 12. As an example, recombinant loop protein gp41, which can be recognized by an antibody according to the invention, it is possible to produce recombinant loop protein gp41, having the amino acid sequence represented by SEQ ID NO 13.

Accordingly, the antibody according to the invention can also recognize the sequence of the wild-type protein gp41, which can exist in nature in different strains of HIV.

According to another aspect, the present invention relates to a monoclonal antibody or its fragment containing the variable region of the L chain (light (light) chain), containing at least one region, complementarity determining (CDR)selected from the CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence represented by SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, or their functional equivalents.

The term "fragment of an is and the body", when used in the present description, refers to an antibody that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence identical to the corresponding provisions in the natural sequence, and its biological properties are saved or not get worse. This fragment of the antibody may contain additional modifications such as insertion, deletion and/or substitution of amino acid residues and/or merge with other peptides or proteins to obtain chimeric proteins. The term "antibody fragment" may also cover different parts of the antibody, i.e. a constant, variable, heavy and light chain.

In the sense of the invention, the expression "functional equivalent" in relation peptide is intended to denote a peptide that has an amino acid sequence homologous or identical to another amino acid sequence, and which has and maintains or has similar biological properties in comparison with other specified peptide sequence. Typically, peptide analogues contain conservative amino acid substitutions and/or insertions and/or deletions) in comparison with the natural sequence.

According to another aspect, the present invention also relates to variable regions of H chain containing minority who she least one CDR, selected from the CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence represented by SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, or their functional equivalents.

According to another aspect, the present invention relates to variable regions of H chain that recognizes recombinant loop protein gp41, as defined below, for example, having the amino acid sequence represented by SEQ ID NO 13, and cannot recognize a peptide with the amino acid sequence represented by SEQ ID NO 12.

According to another aspect, antibodies of the invention or their fragments have the ability to neutralize HIV.

According to another aspect of the present invention also relates to nucleic acid sequences that encodes the antibody of the invention or its fragment and the expression vector and the cell host containing the sequence of nucleic acids.

According to another aspect, the present invention relates to pharmaceutical compositions containing as active agent an effective amount of an agent selected from the antibodies according to the invention or its fragment, the nucleic acid sequences according to the invention, the vector according to the invention or a host cell according to the invention and a suitable carrier.

According to another aspect, the present invention also Rel is referring to the diagnostic composition and method for the detection in vitro of HIV strain in the sample.

According to another aspect, the present invention also relates to a method of conducting passive immunotherapy of an individual susceptible to HIV infection, including the introduction of a therapeutically effective amount of at least one antibody according to the invention or its fragment.

ANTIBODIES

The antibody according to the invention refers to intact immunoglobulin or its fragment and, in particular, to its antigennegative site.

Immunoglobulin (Ig) represents a tetramer molecule consisting of two identical pairs of polypeptide chains, each pair has one "light" (L) (approximately 25 kDa) and one "heavy" (H) chain (about 50-70 kDa). Amino-terminal site of each chain includes a variable region (V) of from about 100 to 110 or more amino acids primarily responsible for antigen recognition. Carboxy-terminal site of each chain defines a constant region (S)primarily responsible for effector function. Human light chains are classified as κ and λ light chains. Constant region heavy chains are classified as µ, δ, γ, α, or ε, and define the type of antibody, as IgM, IgD, IgG, IgA, and IgE, respectively. Variable regions of each pair of light/heavy chain form the binding site of the antibody, so that the intact immunoglobulin, usually has at least the VA binding site.

In a particular embodiment of the invention, the monoclonal antibody according to the invention or its fragment may be an IgA, and more specifically, secretory IgA (S-IgA).

According to another embodiment, the monoclonal antibody according to the invention or its fragment can be a human antibody.

Immunoglobulin chain are characterized by the same General structure of relatively conservative frame regions (FR - framework regions), linked with free hypervariable regions, also called scopes that define complementarity, or CDR. CDR two chains of each pair are aligned by means of frame areas that can bind a specific epitope (antigennegative plot antibodies). As light and heavy chain contains, from N-Terminus to the C-end, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

According to one embodiment, the antibody according to the invention may contain variable regions of H chain at least one area defining complementarity, (CDR)selected from the CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence represented by SEQ ID nos I, SEQ ID NO 2 and SEQ ID NO 3, or their functional equivalents.

According to a specific embodiment, the variable region of the H-chain antibodies according to the invention or its fragment may contain as CDR regions of a field definition is allowing complementarity, CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence represented by SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, or their functional equivalents.

In another embodiment, the antibody according to the invention represents a variable region of H chain, as previously defined. In particular, the variable region of the H-chain according to the invention may contain at least one CDR selected from the CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence represented by SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, or their functional equivalents.

According to another embodiment, the variable region of the H-chain according to the invention may contain as CDR regions CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence represented by SEQ ID NO 1 and SEQ ID NO 2 and SEQ ID NO 3, or their functional equivalents.

The antibody according to the invention or the variable region of the H-chain according to the invention may not recognize the peptide, called P1 and having the amino acid sequence represented by SEQ ID NO 12.

In another embodiment of the present invention, the antibody according to the invention or its fragment may also contain variable region of the L-chain, containing at least one region defining complementarity selected from the CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence represented by SEQ ID NO 4, SEQ ID O 5 and SEQ ID NO 6, or their functional equivalents.

Thus, according to another embodiment, the present invention also refers to the variable region of the L-chain, containing at least one CDR selected from the CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence represented by SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, or their functional equivalents.

According to another embodiment, the variable region of the L-chain according to the invention contains as CDR regions CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence represented by SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, or their functional equivalents.

According to one embodiment, the antibody according to the invention contains the variable region of the H-chain according to the invention and the variable region of the L-chain according to the invention, having, respectively, the amino acid sequence represented by SEQ ID NO 7 and SEQ ID NO 8, or its functional analogs. In a specific embodiment, the antibody according to the invention is a Fab, specified as a clone 69 in the experimental part and containing the variable regions of H(heavy(heavy)and L-chains.

According to another embodiment, the antibody according to the invention can be a recombinant anti-HIV antibody or its fragment. Recombinant antibody can contain a variable region of the H-chain according to the invention.

According to another the embodiment, recombinant antibody according to the invention may further comprise a variable region of the L-chain according to the invention.

Thus, the area determining complementarity, variable regions of H chain according to the invention, or variable region of H chain as a whole, can be used to construct chimeric or recombinant antibodies that have a frame that is different from the IgA isotype, such as, for example, the skeleton of the IgG isotype. This construction can be obtained by any means of molecular biology known in this field, such as described in "Molecular Cloning - A Laboratory Manual" (2nded.), Sambrook et al., 1989, Coldspring Harbor Laboratory, Coldspring Harbor Press, N.Y. (Sambrook). Chimeric or recombinant, the antibody may be a whole antibody or fragment.

Accordingly, the functional analogue of the region that defines complementarity, variable regions of H chain or variable region of the L-chain according to the invention, being inserted instead of the original sequence in the antibody according to the invention retains the ability of the indicated antibodies or does not impair his ability to recognize recombinant loop protein gp41, as defined below, or protein gp41 wild type. As an example, recombinant loop protein gp41 can have the amino acid sequence represented by SEQ ID NO 13, and sequentially the th wild-type protein gp41 may have, for example, the amino acid sequence represented by SEQ ID NO 11, or its functional analogs. Additionally, also saved his ability to recognize peptide with the sequence represented as SEQ ID NO 12, and its ability to neutralize HIV and/or inhibit HIV infection.

The present invention also relates to antibodies containing Fab-fragment, derived from a human antibody according to this invention, and a human Fc region originating from another Ig subtype, such as IgG and the like.

Thus, according to one embodiment, the invention also relates to antigennegative section of the antibodies according to the invention or its fragment containing the variable region of the H chain, as described previously. This antigennegative site may contain variable region of the L-chain, as described previously.

Antigennegative plots can be obtained using any known methods of DNA recombination or by enzymatic or chemical cleavage of intact antibodies. Antigennegative areas may include, inter alia, Fab (monovalent fragment consisting of the VL, VH, CL and CN domains), F(ab')2(bivalent fragment comprising two Fab fragment, connected by a disulfide bridge at the hinge region), Fv(fragment consisting of the VL and VH from one shoulder antibodies) Fd(fragment consisting of the VH and CN domains), a dAb fragment (consisting of a VH domain), scFv(consisting of an antibody in which VL and VH plots are grouped in pairs for education monovalent molecules through synthetic linker), a chimeric antibody, a dimer fragments of the antibodies and fragments of the scopes that define complementarity, (CDR).

The ability of the antibodies according to the invention or its fragment to neutralize HIV can be assessed through analysis of inhibition of transcytosis are activated and analysis of blocking infection of CD4+T cells, as described below and illustrated with examples.

Recombinant loop protein GP41

The antibody according to the invention can recognize recombinant loop proteins gp41 derived amino acid sequence of the glycoprotein of HIV gp41 wild type. As an example, the amino acid sequence of the wild type protein gp41, which can be used when implementing the present invention, may be mentioned amino acid sequence presented as SEQ ID NO 11, obtained from HIV-1 strain Nhw.

Recombinant loop gp41 proteins that may be suitable for implementing the present invention can be obtained by introducing in the field of antigenic determinants of some mutations (deletion, substitution and/or insertion)in order to reduce the homology with the human interleukin-2 (IL-2), to eliminate or reduce the risk of starting an autoimmune reaction. Such recombinant loop gp41 proteins, in particular, as described in the application WO 2005/010033, which is incorporated into this description by reference.

In the present description, "mutation" refers to any modification of the site (possibly reduced to a single amino acid residue) polypeptide, performed by physical means, chemical means (covalent or non-covalent modification) and/or biological means (mutation by substitution, deletion and/or insertion of one or more amino acids), leading to the modification of the functional potential of amino acids (amino acids), member(s) in the specified area, called "the mutated region. As an example of possible mutations leading to loss, acquisition and/or modulating the properties of disulfide bonds, hydrogen bonds, electrostatic interactions and/or hydrophobic interactions, the modification of the ability of the protein to form a heterocomplex, or, alternatively, in the case of oligomeric proteins, the modification of the state of oligomerization or stability of the oligomer.

Modification areas antigenic determinants leads to the introduction or replacement of part of the hydrophilic loop and not immunogenic or weakly immunogenic flexible linker, and possibly with the introduction of the mutation(s).

Recombinant loop protein gp41 may include, in addition, at least one mutation in its field antigenic determinants, which leads to cross-reactions in vitro, In type and/or T-type, the host protein and, in particular, with IL-2.

Some of the mutations that are critical for the manifestation of this change in antigenicity, are disclosed in the publications US 6,455,265 and WO 2005/010033, the contents of which are fully incorporated into the present description by reference.

Recombinant loop gp41 proteins that are suitable according to the invention may also include modifications, such as shortening the part of the amino acid sequence at the N - or C-terminal regions or adding a peptide sequence to obtain a chimeric protein, such as His-tag (his-tag label or ON-label (hemagglutinin label), as described in the publication WO 2005/010033.

To obtain recombinant loop gp41 proteins for use in the present invention can use any known methods of peptide synthesis or genetic engineering techniques, such as described in "Molecular Cloning - A Laboratory Manual (molecular cloning - a laboratory manual)" (2nded.), Sambrook et al., 1989, Coldspring Harbor Laboratory, Coldspring Harbor Press, N.Y. (Sambrook).

According to a specific embodiment, antibodies of the invention can recognize the recombinant protein loop with gp41 amino acid sequence, presented at SEQ ID NO 13, or its analogs.

Additionally, the antibody of the invention or its fragment not only recognize recombinant loop protein gp41, which has, for example, amino acid sequence represented by SEQ ID NO 13, but also recognizes proteins gp41 wild type, however, is not recognizing this peptide having a sequence represented by SEQ ID NO 12.

As an example, protein gp41 wild type, which can be recognized by an antibody according to the invention, mention can be made of protein gp41, having the amino acid sequence represented by SEQ ID NO 11, which is derived from the HIV strain Nhw and which corresponds to the amino acid sequence of from 540 to 683.

PEPTIDE P1

The antibody according to the invention does not recognize the peptide, called P1 and having the peptide sequence represented by SEQ ID NO 12 or its equivalent.

Peptide P1 corresponds to the amino acid sequence present in gp41, the protein shell of HIV, which is exposed on the surface of viral particles before the viruses interact with target cells. As an example, the strain HIV-nhv in this sequence contains amino acids 649 at 683 of protein gp41 wild-type.

In aqueous solution this peptide can adopt depending on the concentration structured oligomeric state, and the property named is, dimer or tetramer state (ALFSEN & BOMSEL, 2002, J. Biol. Chem., 277:25649).

Thus, the antibody or variable region of the H-chain according to the invention, or fragment, do not recognize the peptide P1 in Monomeric or oligomeric form.

The NEUTRALIZING ACTIVITY of ANTIBODIES

The neutralizing activity of the antibodies according to the invention or its fragment, HIV can be assessed by inhibition of HIV transcytosis are activated through the epithelial cells and/or inhibition of HIV infection of CD4+T cell.

According to one embodiment, neytralizuya HIV represents, in particular, HIV-1 strain.

The ability of the monoclonal antibodies according to the invention to inhibit HIV transcytosis are activated through the epithelial cells can be run on any of polarized cells. In one embodiment, polarized cells can be an epithelial cell, such as, for example, cell line HT-29 intestinal cells or endometrial cell line BORE-1, or cells from a biopsy of the mucosa of the person (BOMSEL et al., Immunity, 1998, 3:277). Typically, a cell line grown as a dense polarized monolayer on permeable filter substrate (having, for example, pore size 0.45 μm), forming a contact surface between two separate compartments, of which the upper bathes the apical surface of epithelia inogo monolayer, and the bottom washes basolateral surface, or biopsy samples placed inside with the use of cameras.

The transcytosis are activated, you can initiate interaction with HIV-infected cells, such as, for example, HIV-1 infected peripheral blood mononuclear cells (RVMS peripheral blood mononuclear cells), in the apical chamber.

The antibody under test can be placed in the apical chamber before or after the introduction of HIV-infected cells, or it can be pre-incubated with cells infected with HIV-1 or HIV-1.

The antibody according to the invention, which should be tested, can be applied in various concentrations, for example, from about 0.01 to about 10 ng/ml, in particular from about 0.1 to about 5 ng/ml, or most preferably, from about 0.5 to about 1 ng/ml.

Assessment of virus transcytosis are activated and, possibly, its inhibition can be performed by identifying nucleic acid or protein of the HIV virus in basolateral environment using any method known in this field, such as PCR (polymerase chain reaction - polymerase chain reaction), RT-PCR (polymerase chain reaction with reverse transcription - reverse transcriptase polymerase chain reaction) or ELISA (enzyme-linked immunosorbent assay - enzyme linked immunosorbent assay).

In one embodiment, HIV-peptide, which can be used to assess viral transcytosis are activated may be a peptide P24, which can be detected using the ELISA, using, for example, a set of reagents supplied PASTEUR-SANOFI (FRANCE).

In a particular embodiment of adding to about 106HIV-1 infected RVMS cells at a concentration of from about 0.5 ng/ml to about 5 ng/ml and incubation for about 1 hour at about 17°C. or at about 4°C before introduction of infected cells in the apical chamber, monoclonal antibody according to the invention or its fragment can inhibit transcytosis are activated virus initiated by adding 106HIV-1 infected RVMS cells in the apical chamber, at least about 50%, in particular at least about 75%, more particularly at least about 95%

compared with virus transcytosis are activated, performed without antibody.

In another embodiment, the antibody of the invention or its fragment can be made in the apical chamber before adding HIV-infected RVMS cells. In this embodiment, inhibition of transcytosis are activated may be at least about 50% compared to virus transcytosis are activated, performed without antibody.

As an example, Fab and Fd clone 69, which are also objects of the present invention, can inhibit HIV transcytosis are activated, respectively, at least about 70% and about 80%.

In particular OPL is not the same, Fab and Fd clone 69, which are also objects of the present invention is able to specifically bind and gp41 expressed on the surface of HIV-1 (JRCSF clone R5) infected RVMS cells, as measured using flow cytometry in accordance with a commonly used method known in this field.

In another embodiment, the monoclonal antibodies according to the invention or their fragments can exhibit the ability to inhibit HIV infection of CD44+T cell.

In a particular embodiment, after incubation of the virus at a concentration of about 1 ng/ml within about 30 minutes at a temperature of approximately 37°C with the antibody according to the invention or its fragment infection of CD4+T cells by the HIV virus can be ingibirovalo at least about 75%, and more specifically at least about 95%, compared with HIV-infected CD4+T cells, performed without antibody or with a nonspecific antibody that does not recognize the virus.

As an example, Fab clone 69, which is an object of the present invention, can inhibit HIV infection of CD4+T cells of at least 95%.

NUCLEIC ACIDS, VECTORS AND cells of the HOST

The antibody according to the invention were identified by screening a phage-display library Fab IgA, obtained as described in the experimental the second part.

Methods of obtaining and working with combinatorial display library were exhaustively described in the literature and included in the total knowledge of a specialist in this field.

Combinatorial phage-display Fab library was subjected to two-stage screening method.

In the first stage, the phage-display Fab library was subjected to screening the peptide P1 having the sequence represented as SEQ ID NO 12, immobilized on a solid medium (such as tablets, used in the method of enzyme-linked immunosorbent assay (ELISA)), in order to Deplete the library of antibodies that recognize the peptide.

This first stage can be carried out with the concentration of the antigen of approximately 100 μm, in which the peptide P1 acquires dimanno/tetramer oligomerization state.

In the second stage, the phage-display Fab library was subjected to screening recombinant loop protein gp41 by a multiple of panning on the antigen immobilized on a solid carrier; this method is known as microbending and described in AZZAY & HIGHSMITH (Clinical Biochemistry, 2002, 35:425-445).

Recombinant loop protein gp41, which can be used for screening a phage-display library Fab according to the invention can have, for example, amino acid sequence represented by SEQ ID NO 13, or its functional equivalent.

Recognize the number may be, for example, from about 2 to about 1 μg, then the number can be divided into two in each cycle of panning.

Determination of the interaction between the antigen, namely, recombinant loop protein gp41, and antigennegative domain exposed on the outer side of bacteriophages can be assessed by any methods known in this field. For example, you can use the ELISA method, where recombinant loop protein gp41 cover the wells, and then make a phage library.

Genes encoding antigennegative site that are unique for each phage, you can then select from ragovoy nucleic acid selected phage, sequenced and used to design gene of the whole molecule antibody or its equivalent, such as a Fab fragment, F(ab')2or scFv as described above.

The sequence of the genes encoding antigennegative site isolated from selected phage can be sequenced according to any method known to a person skilled in the field.

For example, sequencing can be performed using automated DNA sequencing machine with a set of reagents (Applied Biosystems)containing fluorescently labeled dideoxynucleotide, act as terminators for cyclic reaction Taq sequencing. Double-stranded DNA can be obtained, for example, from bacteria, and with conromania you can do, using a set of primers that specifically associated with the vector used for cloning of H - and L-chains Fab, before and after each Fab-chain. For example, applying Comb3 vector, you can use the primers having the sequences shown in Table II.

Thus, according to another embodiment, the present invention also relates to nucleic acid molecules, such as cDNA (complementary DNA), RNA and the like, encoding amino acid sequences of variable regions of heavy and light chains according to the invention, where these sequences are presented, respectively, in SEQ ID NO 7 and SEQ ID NO 8.

In particular, the nucleic acid sequences according to the invention can be represented as a sequence of variable regions of H chain represented by SEQ ID NO 9, and the sequence of the variable region of the L-chain represented by SEQ ID NO 10.

Indeed, due to the degeneracy of the genetic code, it can be assumed variations in the nucleic acid sequences of variable regions of heavy and light chains are shown, respectively, in SEQ ID NO 9 and SEQ ID NO 10, which will, in result, to give the sequence of nucleic acids that may be able to provide the production of antibodies or their functional analogues containing the amino acid sequence is lnost variable regions of the heavy chain, shown in SEQ ID NO 7, and the amino acid sequence of the variable region of the light chain shown in SEQ ID NO 8.

According to one embodiment, the invention also relates to nucleic acid sequences that encodes the antibody of the invention or its fragment. This nucleic acid can be an RNA, cDNA and the like. The sequence of the nucleic acids according to the invention can be merged with other sequences of nucleic acids to construct recombinant proteins using any of the techniques of molecular biology known in this field. For example, the sequence of nucleic acids according to the invention can be drained with a nucleic acid that encodes a His-tag or tag that can be used subsequently, for example, for the purification of antibodies according to the invention.

According to another embodiment, the present invention also relates to an expression vector containing a nucleic acid sequence that encodes a monoclonal antibody in accordance with the invention or its fragment.

This expression vector can be used for the expression of monoclonal antibodies in accordance with the invention or its fragment in cells of different types. Such a vector can be a plasmid or viral vector. Such a vector can the possession is ü capacity for Autonomous replication in a cell-master, or it can be integrated into the genome of the host cell.

As examples of vectors that can be used for carrying out the invention, it is possible to mention pComb3 or pASK88.

The present invention also provides for expression of the antibodies according to the invention or its fragment, using different host cells such as bacteria, mammalian cells (Cho or NECK), insect cells (such as Sf9 cells) or cells of plants (tobacco, tomatoes).

After the expression of the antibody according to the invention can be extracted and cleaned using any of the methods known in this field. For example, the antibody can Express with His-label or ON-label and can subsequently be cleaned on a Nickel column or using specific antibodies, as it is usually performed in the field.

Thus, the present invention also relates to the cell host transformed using a nucleic acid sequence that encodes a monoclonal antibody in accordance with the invention or its fragment. Cage-the owner can be obtained in accordance with any method of transformation known in this field, such as electroporation, methods of processing calcium phosphate, lipofection, infection with, for example, recombinant virus. The present invention relates to a cell-master cord is armirovannoj sequence of nucleic acid, encodes a monoclonal antibody in accordance with the invention or its fragment, for example, using electroporation in the case of bacteria or lipofectin in the case of mammalian cells (Cho - Chinese hamster ovary (cells) Chinese hamster ovary).

The PHARMACEUTICAL COMPOSITION

According to one embodiment, the invention relates to pharmaceutical compositions containing as active agent an effective amount of at least one agent selected from the antibodies according to the invention or its fragment, in particular the clone 69, variable regions of H chain according to the invention, the recombinant anti-HIV antibody according to the invention or its fragment, a nucleic acid according to the invention, the expression vector according to the invention or a host cell according to the invention and a suitable carrier.

According to another embodiment, the above-described active agent can be used for the manufacture of a medicinal product intended for use in the prophylaxis and/or treatment of HIV infection, and in particular, HIV-1 infection.

The term "effective amount" means the minimum number required for the manifestation of the desired effect, i.e. neutralization of HIV and/or prevention of HIV infection. Therapeutically or prophylactically effective amount of the antibody according to izopet the tion or its fragment for a particular patient can be determined, as the number of antibodies, administered to an individual to provide a therapeutic or prophylactic effect (i.e. reduction or prevention of infection) while minimizing side effects. An effective amount can be measured by the decrease in the number of HIV antigens in the serum of the individual.

As an example, non-limiting range for a therapeutically or prophylactically effective amount of the antibody according to the invention or its fragment is about 0.1-100 mg/kg, more specifically, about 0.5-50 mg/kg, more specifically, about 1-20 mg/kg and, more specifically, about 1-10 mg/kg

According to another embodiment, the monoclonal antibody according to the invention, or fragments, or a pharmaceutical composition according to the invention can be used in passive immunotherapy by introducing the individual with the risk of being infected or having alleged contact with HIV, or who have been in contact with HIV, a therapeutically effective amount of at least one antibody according to the invention or its fragment or pharmaceutical composition according to the invention. Passive immunotherapy according to the invention can be practiced in relation to individuals exhibiting symptoms of AIDS or related painful condition caused by HIV infection, or individuals, are at risk In The H infections.

According to one embodiment, passive immunotherapy according to the invention can also be used prophylactically, i.e. before the planned contact with HIV.

The pharmaceutical composition according to the invention can be administered orally, parenterally (intravenously or intranasally, or the like), topically, rectally, vaginally, or the like.

Thus, the pharmaceutical composition according to the invention can be produced in the form of various finished dosage forms, such as injection or infusion of sterile solutions, dispersions or suspensions, tablets, pills, powders, liposomes, suppositories and creams.

Suitable media for use in the manufacture of pharmaceutical compositions according to the invention must be adapted in accordance with the dosage form, which is supposed to produce. Such suitable carriers include, but are not limited to specified, water, saline, phosphate buffered saline, dextrose, glycerol and the like, and combinations thereof. Pharmaceutically acceptable compounds such as moisturizing or emulsifying agents, preservatives or buffers, can also be included in pharmaceutical compositions according to the invention.

The pharmaceutical composition according to the invention may also include, in addition to the active agents is about invention, other active agents against HIV, such as antiretroviral drugs. According to another embodiment, such additional antiretroviral drugs can be introduced in combination with the pharmaceutical composition according to the invention simultaneously, separately or sequentially in time.

In another embodiment, the antibody of the invention or its fragment can also be marked for therapeutic purposes, and in this case, the label may be a conjugate of a drug or toxin, for example, a radioisotope or radionuclide (131I99TC111In or the like), pertussis toxin, Taxol, cytochalasin, doxorubicin and the like.

DIAGNOSTIC COMPOSITION AND APPLICATION

Monoclonal antibody according to the invention or its fragment can also be used as a diagnostic agent in the method of detection in vitro of HIV strains, and in particular, HIV-1 strain in the sample.

In one of its variants embodiment, the method according to the invention comprises at least the stages of:

A. bringing the sample into contact with at least one antibody according to the invention or fragment under conditions suitable for the formation of a complex between the specified antibody, or its fragment, and protein gp41 or its functional equivalent.

B. Detection of the presence of the specified sets the KSA.

As an example, protein gp41, which can form a complex with the antibody according to the invention, mention can be made of proteins gp41 wild type, such as, for example, protein gp41, having the amino acid sequence represented by SEQ ID NO 11 or its functional equivalent, or modified gp41 proteins, such as recombinant loop protein gp41, which has, for example, amino acid sequence represented by SEQ ID NO 13, or its functional equivalent.

The detection complex can be done using any method of immunological analysis, well known in this field.

Such assay methods include, but are not limited to, radioimmunoassay analysis, analysis by Western blot, immunofluorescence assay, enzyme immunoassay (such as ELISA), chemiluminescent analysis, immunohistochemical analysis, and the like.

Additionally, to facilitate detection, monoclonal antibody according to the invention, or fragment, can be marked using the detected marker. As an example agent for tagging mention can be made of fluorescent compounds such as fluorescein or rhodamine; enzymes such as horseradish peroxidase, beta-galactosidase or luciferase; Biotin, allowing detection by indirect measurement of the binding avidin or streptavidin; or radionuclides3H 14With or125I.

According to other variant embodiments, the present invention also relates to a diagnostic composition comprising the antibody according to the invention or its fragment.

To make the invention more understandable, given the following examples.

These examples are intended for illustration only and should not be construed as limiting in any way the scope of the invention.

Description of the DRAWINGS

Figure 1 shows the immunoblot derived protein gp41 of HIV-1 are in contact with the cervical-vaginal secretion persistently seronegative against IgG individuals high-risk groups (Highly Exposed Persistently IgG Seronegative - HEPS)containing anti-HIV-1 IgA. As a positive control was performed immunoblot gp41-peptide serum, obtained from HIV seropositive individuals. As a negative control was carried out the same experiment with the serum obtained from HIV-seronegative individuals.

Figure 2 schematically shows the image plasmids pComb3, where the variable and constant region of the heavy and light chains from ragovoy library Fab IgA were inserted, respectively, between the sites of the restriction enzymes Sac 1 and Xba I, and between the Xho 1 and Spe 1.

3 shows the inhibition of transcytosis are activated HIV-1 through endometrial cells and line BORE-1 selected clones IgA Fab. Negative controls were obtained by running the experiment without antibodies (standard) or with the product obtained by screening ragovoy library, obtained using an empty plasmid TOP10. Positive control was performed using the 2F5 antibody IgA.

Figure 4 presents the results of dot-blot analysis, performed with the Fab clone 69 and alpha antibody 2F5 IgA to protein gp41 wild-type strain of HIV Nhw (amino acids 546 on 682) using peptide with latinboy clasp, BSA (bovine serum albumin - bovine serum albumin and lysozyme. After washing nitrocellulose membranes, antibody/protein complexes were incubated with mouse anti-His antibody to Fab clone 69 and mouse anti-human antibody for the 2F5 antibody. Both complexes were detected using anti-mouse antibodies goat conjugated to horseradish peroxidase, followed by use electrogenerating chemoluminescence and autoradiography.

Figure 5 presents the amino acid sequence corresponding complementarity determining sequences of the variable regions of H chain antibodies according to the invention (as CDR1 SEQ ID NO 1, CDR2 as SEQ ID NO 2, CDR3 as SEQ ID NO 3), and the field defining complementarity, the variable region of the L-chain antibodies according to the invention, and CDR1, CDR2 and CDR3 represented by SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6.

the and Figa and 6B presents the amino acid sequence and nucleic acid sequence of the variable region of the heavy chain (SEQ ID NO 7 and SEQ ID NO 9) and the variable region of the light chain (SEQ ID NO 8 and SEQ ID NO 10) clone Fab 69.

Figure 7 presents the amino acid sequence of the framework region (FR) and the region complementarity determining (CDR) light and heavy chains of the Fab IgA clone 69, obtained using IMGT program, available free of charge from the Internet (created M.P.LEFRANC, France). IMGT/V-QUEST program (http://imtg.cines.fr).

On Fig presents the amino acid sequence corresponding to a protein gp41 wild-type SEQ ID NO 11 (HIV-1 strain Nhv), the peptide PI SEQ ID NO 12.

Figure 9 presents the amino acid sequence of the recombinant protein loop gp41, SEQ ID NO 13.

Table I presents the sequence of the primers used for amplification of DNA obtained from HEPS individuals.

Table II presents the sequence of the primers used for the identification and sequencing of nucleic acids sequences Fab IgA from phage-display libraries.

Example 1

Identification of anti-HIV-1 IgA in cervical-vaginal secretions steadily IgG seronegative individuals high-risk groups (HEPS individuals).

Cervical allocation 56 HEPS individuals from Cambodia investigated for the presence of glycoprotein shell HIV S-IgA. Highlight was collected after two days of abstinence, using 3 ml of sterile PBS (phosphate buffered saline - phosphate buffered saline). Samples centrifuges who believed and were frozen and stored at -80°C. Contamination of the sperm sample was measured using the method of analysis for the detection of seminal fluid (SEMA; HUMANGEN FERTILITY DIAGNOSTICS, Charlottesville, VA) according to the manufacturer's instructions, but are optimized in terms of increasing the time of incubation and the use of O-phenylenediamine dihydrochloride as substrate. Contaminated samples were withdrawn from the study.

The presence of specific IgA antibodies to HIV were tested using Western blot using commercially available assay kit (New Lav Blot1, Sanofi-Pasteur, France) according to the manufacturer's instructions.

In 22 of the tested samples contained in a large number of anti-HIV secretory form of IgA (S-IgA) against gp41 envelope (Figure 1).

Example 2

Designing combinatorial phage-display libraries expressing Fab from IgA

In cells of the mucous membrane of 22 identified HEPS individuals from Example 1 was used for constructing combinatorial ragovoy display library expressing Fab from IgA.

Whole RNA was obtained from enriched populations of b cells from the cervical-vaginal secretions from 22 HEPS individuals using standard techniques. Whole RNA (60 μg) was purified from In cervical cells through rapid one-step method of RNA extraction on the basis of guanidine isothiocyanate/phenolchloroform (CHOMCZYNSKI & SACCH, Anal. Biochem., 1987, 162:156-9). This RNA is entirely transformed into complementary DNA (cDNA) using polymerase chain reaction with reverse transcription (RT-PCR). The obtained cDNA library is amplified using specific primers Fab IgA mucosal given in Table I.

Then, the amplified DNA was digested using Sac I and b 1 for the light chain (L) and Xho 1 and Spe 1 for the heavy chain (H), and the resulting cleavage products were inserted into the vector pComb3 (BARBAS et al., Proc Natl Acad Sci USA, 1991, 88:7978-82), cleaved with the same enzymes. Split vector and split the PCR product ligated using DNA ligase of phage T4 (4 inserts plus 1 vector plus TANK ligase, overnight at 14°C) (Figure 2). The obtained recombinant plasmid is then used to transform bacteria E. coli (pilus+, male) TG1 by electroporation.

Library of complementary DNA antibodies in the culture of the transformed bacteria are then expressed on the phage by superantisypware using helper phage VCS-M13 (1012pfu (plague forming units - plaque-forming units)) (Stratagene, La Jolla, CA).

Collected phage preparations. The phage precipitated with addition of 20% (wt./about.) polyethylene glycol 8000 and 2.5 M NaCl, followed by incubation on ice for one hour. After centrifugation the precipitate phage which was suspendible in phosphate buffered saline (PBS) and were microcentrifuged for several minutes to separate cell debris.

Restriction analysis of the colonies from the title of the tablets showed that approximately 35% of the phages contain IgA Fab and that contains a library of titles, as a rule, were close to the content of 107CFU/ml (colinesterase units per ml) and 107different clones.

Example 3

Screening Fab IgA ragovoy library of recombinant loop protein gp41 (SEQ ID NO 13)

Fab IgA phage display library was subjected to screening by microbending, in which the concentration of the antigen was a parameter variable in each cycle. The antigen used for screening was a recombinant protein loop gp41 (SEQ ID NO 13).

Before screening Fab IgA ragovoy display library with recombinant loop protein gp41 (SEQ ID NO 13) the first cycle of panning performed with peptide PI (SEQ ID NO 12) (ALFSEN & BOMSEL, 2002, J. Biol. Chem., 277:25649-59; obtained from Eurogentec, Belgium) in an amount of 20 μg (100 μm), in which the peptide is in oligomeric state. This Protocol allows to separate any Fab IgA, capable of binding the peptide P1 in oligomeric state.

The original number used recombinant loop protein gp41 was 1 µg, and the number was divided by two in each cycle so that eventually the number in the fourth cycle was 125 ng.

Wells of 96-well plates (Exiqon peptide Immobilirez, 10202-111-10) were coated with 1 mg of recombi the based loop protein gp41, then blocked using BSA for 2 hours at 37°C. Concentrated phages obtained from the library, then added to the wells in an amount of 1012-1013plaque-forming units and left for 2 hours at 37°C. Unbound phage were then removed by intensive washing, for the first and second cycles of selection, 10 times with PBS containing 0.1% tween-20, then 10 times with PBS to remove the detergent. For subsequent cycles of selection lavage were 20 times with PBS containing 0.1% tween-20, then 20 times with PBS. Specific elution of phages bearing surface epitopes Fabs that bind recombinant loop protein gp41, was carried out by processing using 0.1 M glycine-HCl, pH adjusted to 2.2, within 10 minutes Elyuirovaniya fraction was immediately neutralized and used to infect 2 ml of fresh E. coli TG1 bacterial culture (OD600(optical density at 600 nm) =1). Bacteria were incubated at 30°C for 30 minutes, the culture volume was increased, and the culture was incubated in a shaker at 37°C for 1 hour and then was added 1012plaque-forming units/ml of VCS-M13 helper phage to produce recombinant phages during the night. Erwerbende phages amplified between each cycle of panning.

After penning individual clones from four rounds to grow the Ali and controlled the presence of Fab using PCR using specific primers, in Table II, which hybridize with the vector before and after the Fab light and heavy chains. Clones containing Fab IgA, translated in soluble form by Fab transform their cells of E. coli despressing strain carrying the amber codon, and sequenced to determine the sequences of the variable regions of the heavy (H) and light (L) chains, using the IMGT/V-QUEST (Fig.7).

Sequencing of immunoglobulin genes

Sequencing was performed using automated DNA sequencing machine with a set of reagents (Applied Biosystems)containing fluorescently labeled dideoxynucleotide, act as terminators for cyclic reaction Taq sequencing. Double-stranded DNA was obtained from bacteria and sequencing was performed using the primer set (see Table II)that are associated specifically with pComb3 vector before and after each Fab-chain.

On Figa and 6B shows the result of the sequencing of the variable regions of the H and L chain clone 69 (SEQ ID NO 9 and SEQ ID NO 10).

Figure 7 presents the results of identification of amino acid sequences corresponding to the areas of complementarity determining, using the IMGT program.

The vector used for cloning, is amber codon between sequences tag (His-tag and TO-tag) and the phage protein pIII, which provides the opportunity to either receive the antibody, merged with rahovym protein shell, using E. coli suppressor strain such as TG1, or to obtain a soluble antibody expression vector in acupressure strain, such as top-10. Considering this advantage, phage DNA was used to transform top 10 E. coli (non-suppressor strain that carries the amber codon)to Express the soluble Fab fragments without R fused protein. The Fab expression was induced using 1 mm isopropyl-β-D-thiogalactopyranoside (IPTG).

For large-scale expression of monoclonal antibodies according to the invention is intact operon moved from Comb3 in vector pASK.88 (SKERRA A., Gene, 1994, 151(1-2): p 131-5; SKERRA, A., Gene, 1994, 141(1): p. 79-84; SKERRA, A., et al., Biotechnology (NY), 1991. 9(3), p.273-8; SKERRA, A. & A. PLUCKTHUN, Protein Eng, 1991,4(8): p.971-9).

Fab DNA monoclonal antibodies according to the invention is amplified using specific primers, introducing the restriction sites required for sub-cloning in pASK88 vector. Amplification products were purified agarose gel electrophoresis and digested with restricted > PST and NcoI, in the case of heavy chains, and SacI and HindIII, in the case of the light chain. Genes Fd (VH-CH1) and the genes for the light chain (VL-CL) was inserted separately and ligated in pASK88, split using the same restricted using a standard Protocol. The ligation products were transformed in termokonstantnye bacterial cells JM83 (courtesy of Dr. Skera) and were sown on Petri dishes for separation into individual clones. Plasmid DNA for several clones were analyzed by restriction analysis and, for some of them, by sequencing double-stranded DNA using specific primers (SKERRA A., Gene, 1994, 151(1-2): R-5; SKERRA, A., Gene, 1994, 141(1): p.79-84; SKERRA, A., et al., Biotechnology (NY), 1991. 9(3), p.273-8; SKERRA, A. & A. PLUCKTHUN, Protein Eng, 1991,4(8): p.971-9).

Expression vector pASK88 developed for convenient cloning of genes of immunoglobulin variable regions, as well as for periplasmic secretion of the corresponding Fabs fragments in Escherichia coli. When using this plasmid expression was controlled by tetracycline promoter.

Large quantities of monoclonal antibodies according to the invention was obtained with the use of this vector. Preparative expression was performed at a scale of 1 l using E. coli K-12 JM83 as expressing host cells. Cells were grown to mid-logarithmic phase and then induced Fab expression using 0.2 mg/l of anhydrous tetracycline for 4 h or overnight.

Purification of monoclonal antibodies according to the invention was carried out on columns Ni-NTA Spin Columns (Qiagen) interaction with His-tag. Periplasmic faction, sterilized by filtration, was applied to a column and used a gradient from 250 to 500 mm imidazole in buffer for chromatography sample collection.

Example 4

ELISA analysis, vypolnennym soluble IgA Fab clones from ragovoy display library.

Performed ELISA analysis with soluble IgA Fab obtained from TOP-10F' bacteria (pComb3 system), and soluble IgA Fab obtained from K-12 JM83 bacteria (pASK88 system).

The wells of plates (Exiqon peptide Immobilirez, 10202-111-10 or NUNC, 439454) were coated with 100 ng of recombinant protein loop gp41 (SEQ ID NO 13) and blocked with BSA for 2 hours at 37°C. Then added purified IgA at a concentration of 2 ng/ml, and incubated for 2 hours at 37°C. Detection was performed using mouse anti-antibody (clone SA, Roche) or anti-His (PentaHis, Qiagen 34660) antibodies, followed by anti-mouse antibodies goat (Caltag Laboratories, H1003), peroxidase labeled horseradish. The enzymatic reaction was shown by the addition of TMB (3,3',5,5'-tetramethylbenzidine, Kikergaard & Perry Laboratories Inc.) as the substrate, and the absorption was measured at 450 nm after addition of 1M phosphoric acid using a spectrophotometer to read the tablets ELISA. The positive control consisted of 2F5 antibody and the negative control consisted received after penning clone, which does not recognize the recombinant protein loop gp41 during ELISA.

Example 5

Inhibition of transcytosis are activated

HIV-1 transcytosis are activated through the epithelial cells and neutralization of transcytosis are activated antibodies were performed on cell lines was CARRIED-1 intestinal cells grown in the form of a dense polarized monolayer for 7 bottom of the permeable filter substrate (pore size 0.45 µm), forming a contact surface between two separate compartments, of which the upper apical washes (luminally) surface epithelial monolayer, and the bottom washes basolateral (serosal) surface.

RVMS were obtained and prepared as described in LAGAYE et al. (J. Virol, 2001, 75:4780). Then RWMS-cells activated by phytohemagglutinin (PhA) for 48 h and were inoculable HIV-1 JRCSF clone R5 or YU2 and used on day 7 after infection. Purified S-IgA (5 ng/ml) was added to the apical chamber and incubated for 10 min at 37°C.

To initiate the virus transcytosis are activated, 2·106HIV-1+RWMS was added to the apical chamber. Contact between HIV-1+RUMS and a monolayer of epithelial cells resulted in the rapid separation of HIV-1 virions and their subsequent transcytosis are activated from the apical to basolateral pole of epithelial cells. After 2 hours the inhibition of transcytosis are activated antibody was determined by detection of HIV protein P24 in basolateral environment using ELISA (Coulter, France or SANOFI PASTEUR, FRANCE). The level of P24 in the absence of antibody or in the presence of non-specific recombinant Fab hinge protein gp41 or in the presence of a control IgA 2F5 were measured, respectively, as negative and positive control value, respectively, equal to 100, 98 and 35%. The value for the negative control received C is 100% transcytosis are activated and used for presentation of results.

The experiments were performed in three independent replicate samples.

The results allow to identify the 3 clones that were able to inhibit HIV transcytosis are activated by more than 50%, among which was the clone 69 (Figure 3).

Example 6

Dot-blotting

BSA peptide with latinboy clasp, lysozyme and gp41 protein from wild-type HIV strain Nhw (amino acids 546 on 682 obtained from ABI) were scatter plotted on a nitrocellulose membrane, respectively, each sample at a concentration of 100 ng/ml thereafter, Fab clone 69, at a concentration of 5 ng/ml, or antibody 2F5 alpha-IgA, at a concentration of 2 μg/ml, dilution 1:2500, incubated on nitrocellulose membranes.

After washing nitrocellulose membranes (blocking reagent for Western blotting, Roche: 1% for blocking and washing, 0.1% for binding of the antibodies according to the manufacturer's instructions), the immune complexes were incubated with mouse anti-His antibody (Qiagen, 0.2 mg/ml, 1:5000 dilution) for Fab clone 69 and with mouse anti-human antibody for 2F5 alpha-IgA. Both complexes were detected using anti-mouse antibodies goat horseradish peroxidase (Caltag Laboratories, H1003,1:4000 dilution) followed by transfer to x-ray film ECL and autoradiography (ECL, Amersham, according to manufacturer's instructions).

Figure 4 presents the result of the experiment, repeated dward is.

1. Anti-HIV monoclonal antibody or functional fragment containing the variable regions of the heavy N-(heavy) chain region, defining complementarity, CDR1, CDR2 and CDR3, respectively having the amino acid sequence represented by SEQ ID nos I, SEQ ID NO 2, SEQ ID NO 3, and variable regions L(light) chain CDR1, CDR2 and CDR3, respectively having the amino acid sequence represented by SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6.

2. Monoclonal antibody according to claim 1, characterized in that it does not recognize the peptide amino acid sequence represented by SEQ ID NO 12.

3. Monoclonal antibody or functional fragment according to claim 1, recognizes recombinant loop protein gp41, having the amino acid sequence represented by SEQ ID NO 13, and does not recognize the peptide amino acid sequence represented by SEQ ID NO 12.

4. Monoclonal antibody or functional fragment according to claim 1, characterized in that it is an IgA.

5. Monoclonal antibody or functional fragment according to claim 1, characterized in that it is a human antibody.

6. Monoclonal antibody or functional fragment according to claim 1, characterized in that the variable region of the heavy chain and has inoculate sequence, presented at SEQ ID NO 7, and the variable region of the light chain has the amino acid sequence represented by SEQ ID NO 8.

7. Monoclonal antibody or functional fragment according to claim 1, characterized in that it has the ability to neutralize human immunodeficiency virus (HIV).

8. Monoclonal antibody or functional fragment according to claim 7, characterized in that neytralizuya HIV is an HIV-1 strain.

9. The fragment of the heavy chain of the antibody according to claim 1, which represents its variable region encoded by a nucleic acid molecule having the sequence of SEQ ID NO 9.

10. The fragment of the light chain of the antibody according to claim 1, which represents its variable region encoded by a nucleic acid molecule having the sequence of SEQ ID NO 10.

11. Expression vector encoding a fragment of the heavy chain of the antibody of claim 9.

12. Expression vector encoding a fragment of the light chain of the antibody of claim 10.

13. A host cell transformed by the expression vector according to claim 11 and producing a fragment of heavy chain antibodies, representing its variable region according to claim 9.

14. A host cell transformed by the expression vector according to item 12 and producing a fragment of the light chain of the antibody, representing its variable region, on item 10.

15. The use of antibodies, is whether its functional fragment according to claim 1, or fragment of the heavy chain of the antibody according to claim 9, or a fragment of the light chain of the antibody of claim 10, the expression vector according to claim 11 or 12, or a host cell according to item 13 or 14 for the manufacture of medicinal products intended for use in the prophylaxis and/or treatment of HIV infection.

16. The method of detection of the strain of HIV in a sample in vitro, comprising at least stage:
A) bringing the sample into contact with at least one antibody or functional fragment according to claim 1 under conditions suitable for the formation of a complex between the specified antibody or the functional fragment and protein gp41 or its functional equivalent, and B) detecting the presence of the complex.

17. Way for passive immunotherapy of an individual susceptible to HIV infection, including the introduction of a specified individual a therapeutically effective amount of at least one antibody or functional fragment according to claim 1.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and aims at HIV-infection treatment. That is ensured by introduction of a T-cell depletor which effectively disintegrates all T-cells of the patient. Said depletor is a CD52 monoclonal antibody Alemtuzumab.

EFFECT: invention allows suppressing the immune system by the controlled method.

8 cl, 2 ex, 2 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention relates to biochemistry and specifically to a modified polypeptide of a HIV-1 gp41 envelope glycoprotein, a polynucleotide which codes the modified polypeptide and an expression vector which contains a coding modified polypeptide of the HIV-1 gp41 envelope glycoprotein. The modified polypeptide of the HIV-1 gp41 envelope glycoprotein contains an amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 14, where the sequence between positions 603 and 615 or 598 and 622 SEQ ID NO: 1 or the sequence between positions 530 and 542 or 525 and 549 SEQ ID NO: 14 is replaced by a linker fragment which is an oligopeptide SEQ ID NO: 2.

EFFECT: improved solubility of a modified polypeptide of the HIV-1 gp41 envelope glycoprotein without changing its immunogenic reactivity.

10 cl, 8 dwg, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to new compounds of formula , to their salts, where R1 and R2 each independently is hydrogen or C1-10alkyl which can be optionally substituted with substitutes selected from a group comprising a hydroxyl group, NR4R5, pyrrolidinyl, piperidinyl, morpholinyl; R3 is a radical of formula , where n equals 1; R3a is nitro; X is -NR7 - or -O-; R4 and R5 each independently is C1-6alkyl; R7 is hydrogen, C1-6alkyl, optionally substituted with pyrrolidinyl. The invention also pertains to use of the compounds, to a pharmaceutical composition, to a method of preparing the pharmaceutical composition, as well as to a method of obtaining the chemical compound in any of paragraphs 1-3.

EFFECT: obtaining new biologically active compounds with antiviral activity.

7 cl, 4 ex, 2 tbl

FIELD: chemistry.

SUBSTANCE: present invention relates to novel compounds of formula , where X is -O-; values of Ar, R1-R5, R11 are given in the formula of invention. The said compounds have inhibitory effect on HIV reverse transcriptase. The invention also relates to a pharmaceutical composition containing the invented compounds or their pharmaceutically acceptable salts.

EFFECT: obtaining new compounds and a pharmaceutical composition containing said compounds.

8 cl, 61 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: there are described salts of 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB). Particularly, there is disclosed production process, pharmaceutical estimation and bioavailability estimation in vivo of N-methyl-D-glucamine salt and alkali salt. The pharmaceutical compositions containing these salt forms are used in methods of treating HIV infection and related diseases. There is also described method for preparing DBS salts.

EFFECT: improved clinical effectiveness.

12 cl, 5 ex, 5 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing HIV protease inhibitor atazanavir sulphate in form of Form A crystals, which involves reacting a solution of a free base of atazanavir in an organic solvent in which atazanavir sulphate is virtually insoluble, at temperature ranging from 35°C to 55°C with a first portion of concentrated sulphuric acid in an amount sufficient for reaction with less than approximately 15 wt % free base of atazanavir, addition of nucleating centres of Form A atazanavir sulphate crystals, addition of an additional amount of concentrated sulphuric acid in several steps, where the acid is added at increasing rate to form atazanavir sulphate crystals and drying the atazanavir sulphate to form Form A crystals. A method of producing atazanavir sulphate in form of Form C crystals is also proposed.

EFFECT: improved method.

20 cl, 11 dwg, 6 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to pharmaceutical composition, suitable for peroral introduction in form of solid medicinal form, which contains efficient amount of main salt of formula compound and composition with controlled rate of release, which contains solubilising agent, gel-forming agent and eater-soluble filler.

EFFECT: compositions are suitable for application in inhibiting of HIV integrase, treatment and prevention of HIV infection, and in treatment, prevention and retardation of AIDS development.

15 cl, 4 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula I with anti-HIV activity , where R1 represents C1-6(Ar1)alkyl or C1-6(Ar1)oxyalkyl; R2 represents hydrogen or OR14; R3 represents hydrogen, halogen, hydroxyl, cyano, C1-6alkyl, C5-7cycloalkenyl, C1-6halogenalkyl, C1-6alkoxy, C1-6alkylthio, N(R8)(R9), NHAr2, N(R6)COR7, OCON(R8)(R9), OCH2CON(R9)(R9), CO2R6, CON(R8)(R9), SOR7, S(=N)R7, SO2R7, SO2N(R6)(R6), PO(OR6)2, C2-4(R12)alkynyl, R13, Ar2 or Ar3; R4 represents hydrogen, halogen, C1-6alkyl or C1-6alkoxy; R5 represents hydrogen, halogen, C1-6alkyl or C1-6alkoxy; R6 represents hydrogen or C1-6alkyl; R7 represents C1-7alkyl; R8 represents hydrogen or C1-6alkyl; R9 represents hydrogen, C1-6alkyl, C1-6hydroxyalkyl or C1-6(C1-6dialkylamino)alkyl; or N(R8)(R9) taken together represent azetidinyl, pyrrolydinyl, (R10)-piperidinyl, N-(R11)-piperazinyl, morpholinyl or dioxothiazinyl; R10 represents hydrogen; R11 represents hydrogen, C1-6alkyl, COR6 or CO2R6 ; R12 represents hydrogen, hydroxyl, N(R6)(R6), OSO2R7 or dioxothiazinyl; R13 represents dioxothiazinyl; R4 represents hydrogen or C1-6alkyl; Ar1 represents ,,,,,,,,; or Ar2 represents tetrazolyl, triazolyl, thiadiazolyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, furanyl, thienyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyridinyl, quinolinyl or indolyl, and is substituted with 0-2 substitutes selected from a group consisting of halogen, benzyl, C1-6alkyl, C1-6alkoxy, N((R8)(R9), CON(R8)(R9) and CO2R8; Ar3 represents phenyl substituted with 0-2 substitutes selected from a group consisting of halogen, cyano, hydroxy, C1-6alkyl, C1-6alkoxy, (C1-6alkoxy)methyl, C1-6halogenalkoxy, N(R8)(R9), CON(R6)(R6) and CH2N(R8)(R9), or represents dioxolanylphenyl; and X-Y-Z represents C(R14)2OC(R14)2C(R14)2, C(R14)2OC(R14)2C(R14)2C(R14)2; or pharmaceutically acceptable salt thereof. The invention also relates to a pharmaceutical composition.

EFFECT: bicyclic heterocycles are disclosed, as well as their use HIV integrase inhibitors.

21 cl, 38 dwg, 8 tbl, 282 ex

FIELD: medicine.

SUBSTANCE: present invention concerns lysine compounds of formula (I) or its pharmaceutically acceptable salts, a based pharmaceutical compositions and application for treatment or prevention of HIV-infection. The compounds of formula (I) where n is equal to 3 or 4, where X and Y identical or different are chosen from the group consisting of H, F, Cl, Br, I and -NR4R5, where R6 is chosen from the group consisting of unbranched alkyl group, containing 1 to 6 carbon atoms, and branched alkyl group containing 3 to 6 carbon atoms, where R3 is chosen from the group consisting of the group of formula R3A-CO-, and R3a is chosen from the group consisting of unbranched or branched alkyl group containing 1 to 6 carbon atoms, alkyloxygroup containing 1 to 6 carbon atoms, and 4-morpholinyl, where R4 and R5 are identical and represent H, where R2 is chosen from the group consisting of diphenylmethyl group, naphthyl-1-CH2-group, and naphthyl-2-CH2-group, where X' and Y' are identical and represent H, and where R1 is chosen from the group consisting of from (HO)2P(O) and (MO)2P(O), where M represents alkaline metal.

EFFECT: lysine compounds representing effective inhibitors of aspartyl-protease.

15 cl, 3 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely medical products for HIV/AIDS infection therapy. There is offered application of Glimurid as a drug for HIV-infection treatment.

EFFECT: application of said drug ensures to increase the clinical effectiveness due to viral load reduction and immunocorrection.

1 ex, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and concerns a composition for treatment or prevention of infection and/or infectious disease of respiratory tracts and the method for application of said composition for specified purposes which involves oral introduction to a mammal of the composition containing a galactose-containing indigestible oligosaccharide and at least 5 wt % of digested galactose saccharide.

EFFECT: invention provides that the active substance is safe and can be included in the dietary intake.

20 cl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention presents the method and composition for treatment and-or prevention of infection, and said method includes oral introduction of the composition to a mammal, and said composition involves galactose-containing indigestible oligosaccharide and immunoglobulin of milk or colostrum of hyperimmunised cows.

EFFECT: development of the effective method for the compositions for treatment and-or prevention of infection.

15 cl, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method is suggested for production of virus on lung cells of cotton rats, and also line of lung cells of cotton rats ATCC PTA-3930 for growth, reproduction and cultivation of viruses.

EFFECT: improved efficiency of method.

8 cl, 1 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, particularly therapy, and can be applied in chronic disease treatment. Method involves causative agent extraction or determination of antibodies to causative agents or extraction of genetic components from blood, body fluid or smear. Treatment is performed by vaccine drugs specific to one or several identified causative agents, additionally immunomodulators are administered.

EFFECT: activated repair processes, arrested development of disease state of organs and systems due to elimination of identified pathogen from organism and to immune correction.

3 cl, 8 ex

FIELD: biotechnology, preparative biochemistry.

SUBSTANCE: method involves hyperimmunization of animal-producers with inactivated, purified and concentrated rabies virus with aluminum phosphate and/or sodium nucleinate as an adjuvant. Virus obtained in culture transplantable cells VERO and in primary culture of Syrian hamster kidney cells is concentrated by adsorption of aluminum phosphate gel or centrifugation, or ultrafiltration methods and purified passing through porous silica, or by ion-exchange chromatography. For immunization antigen with the immunogenicity index 9.5 IU/ml, not less, is used and with the content of protein less 200 mcg/ml, bovine serum albumin less 0.5 mcg/ml Syrian hamster kidney cells and VERO cells less 0.5 mcg/ml, cellular DNA 5.0-10.0 ng/ml, aluminum ions concentration in antigen 0.2-1.25 mcg/ml, and sodium nucleinate 0.002-0.5 mg/ml. Scheme of grund-immunization - 1-3 injections of antigen in the dose 3-15 ml with interval for 14-30 days, the main immunization cycle - on 45-60 day involving 10-15 injections with interval for 5-15 days and in the antigen dose 5-70 ml. Then producers are immunized once per a month with the dose 20-70 ml, and once per 6 month the shortened cycle of immunization is carried out involving 2-4 injections of antigen with interval for 5-15 days in the dose 20-70 ml. Method provides preparing heterologous antirabies serum of high specific activity.

EFFECT: improved preparing method, improved and valuable properties of serum.

12 cl, 1 tbl, 4 ex

FIELD: biotechnology, protein engineering.

SUBSTANCE: claimed library represents E.coli TGI cells wherein each cell contains fragmid DNA providing biosynthesis of filamentous bacteriophages exposing unique human single-stranded antibody on surface thereof. Also disclosed is recombinant fragmid pHEN-2A8 DNA containing artificial gene of human single-stranded antibody under control of lactose operon promoter providing synthesis of human single-stranded antibody in composition of chimerical protein with membrane pIII protein of M13 bacteriophage in E.coli cells. Also disclosed is method for production of artificial human single-stranded 2A8 antibody by using such fragmid DNA.

EFFECT: fragmid library useful in medicine.

3 cl, 7 dwg, 10 ex

FIELD: veterinary medicine.

SUBSTANCE: means has alcohol extract of herb and inflorescences of Echinacea purpurea, Tussilago farfara, Thymus serpyllum herb and Glycyrrhiza glabra roots taken in equal quantities. Method involves introducing intramuscular recovalescent serum injection containing specific antibodies to coronary rotaviruses in 1:128 and 1:64 proportions respectively. Alcohol extract of herb and inflorescences of Echinacea purpurea, Tussilago farfara, Thymus serpyllum herb and Glycyrrhiza glabra roots taken in equal quantities as 7-8% water solution is additionally administered for 7-10 days with 24 h long intervals at a dose of 1.0-1.5 ml/kg of animal weight as prophylaxis and at a dose of 2.5-3.0 ml/kg as treatment with 12 h long intervals until clinical recovery takes place.

EFFECT: simplified and high accuracy diagnosis method.

2 cl, 4 tbl

FIELD: veterinary science.

SUBSTANCE: the suggested remedy contains alcoholic extract of the mixture of the equal parts of Echinacea and coltsfoot herb and inflorescences, flowers of small-leaved linden and licorice roots. Method for preventing and treating respiratory diseases in calves includes subcutaneous injection of hyperimmune serum that contains antibodies to the viruses of parainfluenza-3 and infectious rhinotracheitis in titers being 1:1280 and 1:256, not less, moreover, additionally for inner intake it is necessary to apply alcoholic extract of equal quantities of Echinacea and coltsfoot herb and inflorescences, flowers of small-leaved linden and licorice roots as a 7-8%-aqueous solution at prophylactic purpose at the dosage of about 1.5-2.5 ml/kg animal body weight for 15 d at interval of 12-24 h, at curative purpose - at the dosage of about 3.5-4.5 ml/kg animal body weight at 12-h-long interval till clinical recovery.

EFFECT: higher efficiency.

2 cl, 3 ex, 3 tbl

FIELD: veterinary, immunology.

SUBSTANCE: healthy calves are immunized three time with 10-day interval by hypodermic injection of immune serum from donor animal with hemagglutinin titer to infective rhinotracheitis virus of 1:256; to parainfluenza-3 virus of 1:1280; and to viral diarrhoea virus of 1:1024. After the first injection additionally probiotic preparation lactobifadol in amount of 16x108 microbial bifido- cells and 2x107 lactobacteria for 10 days in three courses.

EFFECT: nocifensor activation; decreased calf morbidity rate.

3 ex, 5 tbl

FIELD: veterinary science.

SUBSTANCE: on proving the diagnosis one should introduce immune serum of animals-donors for sick calves till clinical recovery at the titers of hemagglutinins to IRT virus being 1:256, to PG-3 virus being 1:1280 and to VD-BC virus being 1:1024, and, additionally, it is necessary to apply a probiotic preparation lactobifadol at 32x108 microbial cells bifido- and 4x107 lactobacteria. The innovation increases the quantity of recovered animals, shortens the terms of therapy and the number of relapses.

EFFECT: higher efficiency of therapy.

2 ex, 3 tbl

FIELD: clinical immunology.

SUBSTANCE: preparation represents 9.0-11.0% solution of horse blood serum immunoglobulins with pH 7.0-7.5 and content of γ-immunoglobulin fraction at least 90.0% of the total protein. Nonspecific fractions in preparation are albumin, α- and β-globulin fractions in amount not larger than 10.0%, and residual ethyl alcohol in amount not larger than 4.0%. Preparation is characterized by virus-neutralizing antibodies against virus Marburg. Titer of virus-neutralizing antibodies is at least 1:2048 and can be used for emergency prevention of Marburg fever in humans.

EFFECT: extended immunoglobulin application area.

1 tbl

Up!