RU2393873C2 - Antibody or fragment thereof, having neutralising effect on hiv, but not on il2 - Google Patents

Abstract

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

Description

The invention relates to an antibody or fragment thereof having a neutralizing activity against HIV and suitable for the treatment and / or prevention of HIV infection. The invention also relates to a pharmaceutical composition comprising said antibody, to a diagnostic composition and a method for providing passive immunotherapy.

Human Immunodeficiency Virus (HIV) is a member of the lentivirus family of animal retroviruses and is the causative agent of Acquired Immunodeficiency Syndrome (AIDS). To date, two closely related types of HIV have been identified and characterized at the molecular level - type 1 (HIV-1) and type 2 (HIV-2).

The use of antiviral anti-HIV agents, such as inhibitors of RNA-dependent DNA polymerase (reverse transcriptase), has significantly improved the condition of patients suffering from AIDS. However, in most cases, the therapeutic efficacy of these drugs in relation to AIDS is partial or temporary, and, in addition, these drugs exhibit toxicity or inhibit the growth of hematopoietic cells and, therefore, inhibit the restoration of the immune system, which has acquired insufficiency.

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

Mucous surfaces are the main site of HIV-1 penetration (NICOLOSI, et al. 1994, J. Acquir. Immun. Defic. Syndr., 7: 296). HIV-1 transmission can occur through mucosal contact with HIV-1-infected fluids such as sperm, 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 mucosal surface, a component of the vaccine developed against HIV should cause the mucosal immune system to interfere with the early stages of the transmission of the virus through the mucosal surface and interact with potential receptors.

It is recognized that antibodies that neutralize AIDS viruses can certainly play an important role in antiviral protection.

However, although it was possible to obtain neutralizing antibodies against a specific strain of the virus grown in the laboratory, it has not yet been possible to achieve similar success in creating antibodies that could neutralize a wide range of viral strains and which could exhibit effective in vivo activity.

The ectodomain of the HIV gp41 protein, being the most conserved region in the envelope of the virus, is an extremely immunogenic glycoprotein.

It is known from US 6,455,265 that the conserved region and the region of antigenic determinants of the HIV gp41 sheath retroviral protein can cause harmful autoimmune phenomena due to analogies of the three-dimensional structure and / or cross-reactivity with respect to certain regions of the protein of the human immune system, and, in particular, IL-2 (interleukin-2).

Gp41 recombinant loop proteins are known from WO 2005/010033, containing a linker fragment in the connecting loop between the gp41 N and C helices and lacking or exhibiting reduced autoimmune side effects. As a consequence, there is a need for an antibody that can neutralize HIV infection, and in particular HIV-1 infection, without an autoimmune side effect.

There is a need for an antibody that can prevent and / or reduce HIV infection through the mucous surface.

There is also a need for an antibody for the manufacture of a medicament for use in passive immunotherapy.

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

The invention aims to satisfy the above needs in whole or in part.

The inventors have obtained an effective monoclonal antibody that can satisfy the aforementioned urgent needs.

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

In particular, a newly identified Fab IgA antibody may recognize a recombinant gp41 loop protein or wild-type gp41 protein, but may not recognize a peptide called P1 that is located in a recombinant gp41 loop protein or wild-type gp41 protein.

The PI peptide (SEQ ID NO 12) corresponds, for example, to amino acid sequence 649 to 683 of the wild-type gp41 protein amino acid sequence shown in SEQ ID N0 11 and obtained from HIV-1 strain HxB2.

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

Thus, according to one aspect, the present invention relates to a monoclonal antibody or fragment thereof comprising in the variable region of the H chain at least one complementarity determining region (CDR) selected from CDR1, CDR2 and CDR3, respectively having amino acid the sequence shown in SEQ ID NO I, SEQ ID NO 2 and SEQ ID NO 3, or functional analogs thereof.

According to another aspect, the present invention relates to a monoclonal antibody or fragment thereof recognizing a recombinant gp41 loop protein, as defined below, and not recognizing a peptide having the amino acid sequence shown in SEQ ID NO 12. As an example, a recombinant gp41 loop protein, which can recognized by the antibody of the invention, a gp41 recombinant loop protein having the amino acid sequence shown in SEQ ID NO 13 can be cited.

Accordingly, the antibody of the invention can also recognize the wild-type sequence of the gp41 protein, which can exist naturally in various strains of HIV.

According to another aspect, the present invention relates to a monoclonal antibody or fragment thereof comprising an L chain variable region (light chain) comprising at least one complementarity determining region (CDR) selected from CDR1, CDR2, and CDR3 having respectively, the amino acid sequence shown in SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, or functional analogues thereof.

The term "antibody fragment", as used in the present description, refers to an antibody that has an amino-terminal and / or carboxy-terminal deletion, but in which the remaining amino acid sequence is identical to the corresponding positions in the natural sequence, and its biological properties are preserved or not deteriorated . This antibody fragment may contain further modifications, such as insertion, deletion and / or substitution of amino acid residues and / or fusion with other peptides or proteins to produce chimeric proteins. The term "antibody fragment" may also encompass various parts of an antibody, i.e. constant, variable, heavy and light chains.

Within the meaning of the invention, the expression “functional analogue” with respect to a peptide is intended to mean a peptide that has an amino acid sequence homologous to or identical to another amino acid sequence, and which has and retains or has similar biological properties compared to the indicated other peptide sequence. Typically, peptide analogues contain conservative amino acid substitutions (and / or insertions and / or deletions) compared to the natural sequence.

According to another aspect, the present invention also relates to an H chain variable region comprising at least one CDR selected from CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence shown in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, or their functional analogs.

According to another aspect, the present invention relates to an H chain variable region that recognizes a recombinant gp41 loop protein as defined below, for example, having the amino acid sequence shown in SEQ ID NO 13 and not recognizing a peptide with the amino acid sequence shown in SEQ ID NO 12.

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

According to another aspect, the present invention also relates to a nucleic acid sequence encoding an antibody of the invention or a fragment thereof, as well as to an expression vector and a host cell containing said nucleic acid sequence.

According to another aspect, the present invention relates to a pharmaceutical composition comprising, as an active agent, an effective amount of an agent selected from an antibody of the invention or a fragment thereof, a nucleic acid sequence of the invention, a vector of the invention or a host cell of the invention and a suitable carrier.

According to another aspect, the present invention also relates to a diagnostic composition and method for in vitro detection of an HIV strain in a sample.

According to another aspect, the present invention also relates to a method for passive immunotherapy of an individual susceptible to HIV infection, comprising administering a therapeutically effective amount of at least an antibody of the invention or a fragment thereof.

ANTIBODIES

An antibody of the invention relates to an intact immunoglobulin or fragment thereof, and in particular to its antigen binding site.

Immunoglobulin (Ig) is a tetrameric molecule consisting of two identical pairs of polypeptide chains, each pair having one “light” (L) (about 25 kDa) and one “heavy” (H) chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region (V) of from about 100 to 110 or more amino acids, primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region (C), primarily responsible for effector function. Human light chains are classified as κ and λ light chains. Permanent heavy chain regions are classified as µ, δ, γ, α or ε, and the type of antibodies is determined as IgM, IgD, IgG, IgA and IgE, respectively. The variable regions of each light / heavy chain pair form an antibody binding site, so that an intact immunoglobulin typically has at least two binding sites.

In a specific embodiment of the invention, the monoclonal antibody of the invention or a fragment thereof may be IgA, and more specifically, secretory IgA (S-IgA).

According to another embodiment, the monoclonal antibody of the invention or a fragment thereof may be a human antibody.

Immunoglobulin chains are characterized by the same general structure from relatively conservative framework regions (FR - framework regions) connected by free hypervariable regions, also called complementarity determining regions, or CDRs. The CDRs of the two chains of each pair are aligned using framework regions, which allows the binding of a specific epitope (antigen-binding region of the antibody). Both light and heavy chains contain, from the N-terminus to the C-terminus, domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

According to one embodiment, the antibody of the invention may comprise in the variable region of the H chain at least one complementarity determining region (CDR) selected from CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence shown in SEQ ID NO I, SEQ ID NO 2 and SEQ ID NO 3, or their functional analogs.

According to a specific embodiment, the H region variable region of an antibody of the invention or a fragment thereof may contain, as CDR regions, complementarity determining regions, CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence shown in SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, or functional analogues thereof.

In another embodiment, the antibody of the invention is also an H chain variable region, as previously defined. In particular, the H region variable region of the invention may comprise at least one CDR selected from CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence shown in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, or their functional counterparts.

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 shown in SEQ ID NO 1 and SEQ ID NO 2 and SEQ ID NO 3, or their functional analogues.

An antibody of the invention or an H chain variable region of the invention may not recognize a peptide called P1 and having the amino acid sequence shown in SEQ ID NO 12.

In another embodiment of the present invention, the antibody of the invention or a fragment thereof may also comprise an L chain variable region comprising at least one complementarity determining region selected from CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence shown in SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, or functional analogues thereof.

Thus, according to another embodiment, the present invention also relates to a variable region of an L chain containing at least one CDR selected from CDR1, CDR2 and CDR3 having, respectively, the amino acid sequence shown in SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, or functional analogues thereof.

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 shown in SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, or functional analogs thereof .

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

According to another embodiment, the antibody of the invention may be a recombinant anti-HIV antibody or fragment thereof. A recombinant antibody may comprise an H chain variable region of the invention.

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

Thus, the complementarity determining regions of the H chain variable region of the invention, or the entire H chain variable region, can be used to construct a chimeric, or recombinant, antibody having a framework different from an IgA isotype, such as, for example, an IgG framework isotype. This design can be obtained using any molecular biology tools known in the art, such as those described in the "Molecular Cloning - A Laboratory Manual" (2 ^nd ed.), Sambrook et al., 1989, Coldspring Harbor Laboratory, Coldspring Harbor Press , NY (Sambrook). A chimeric or recombinant antibody may be a whole antibody or fragment thereof.

Accordingly, a functional analog of the complementarity determining region, the H chain variable region, or the L chain variable region of the invention, when inserted instead of the original sequence in the antibody of the invention, retains the ability of said antibody or does not impair its ability to recognize the recombinant gp41 loop protein, as defined lower, or wild-type gp41 protein. As an example, the recombinant loop protein gp41 may have the amino acid sequence shown in SEQ ID NO 13, and the wild-type sequence of gp41 protein may have, for example, the amino acid sequence shown in SEQ ID NO 11 or its functional analogs. Additionally, its ability to not recognize a peptide with the sequence represented by SEQ ID NO 12 and its ability to neutralize HIV and / or inhibit HIV infection are also retained.

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

Thus, according to one embodiment, the invention also relates to an antigen binding site of an antibody of the invention or a fragment thereof comprising an H chain variable region as previously described. This antigen binding site may optionally comprise a variable region of the L chain as previously described.

Antigen binding sites can be obtained using any known DNA recombination technique or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding sites may include, but are not limited to, Fab (a monovalent fragment consisting of VL, VH, CL and CH1 domains), F (ab ') ₂ (a bivalent fragment containing two Fab fragments connected by a disulfide bridge in the hinge region), F _v (a fragment consisting of VL and VH domains from one arm of an antibody), F _d (a fragment consisting of VH and CH1 domains), a dAb fragment (consisting of a VH domain), scF _v (consisting of an antibody in which VL and VH sections are grouped in pairs to form monovalent molecules via a synthetic linker), chimeric antibody, dimers antibody fragments; and fragments of complementarity determining regions (CDRs).

The ability of an antibody of the invention or its fragment to neutralize HIV can be assessed by analysis of the inhibition of transcytosis and analysis of blocking infection of CD4 ⁺ T cells, as described below and illustrated by examples.

Recombinant Loop Protein GP41

An antibody of the invention can recognize gp41 recombinant loop proteins that are derived from the wild type gp41 HIV glycoprotein amino acid sequences. As an example of the amino acid sequence of the wild-type gp41 protein that can be used in the practice of the present invention, mention may be made of the amino acid sequence represented as SEQ ID NO 11 obtained from HIV-1 strain HxB2.

Recombinant gp41 loop proteins that may be suitable for the practice of the present invention can be obtained by introducing certain mutations (deletion, substitution and / or insertion) into the antigenic determinant region in order to reduce homology with human interleukin-2 (IL-2), to eliminate or reduce the risk of triggering an autoimmune reaction. Such recombinant gp41 loop proteins, in particular, are described in WO 2005/010033, which is incorporated herein by reference.

As used herein, “mutation” means any modification of a region (possibly reduced to a single amino acid residue) of a polypeptide made by physical means, chemical means (covalent or non-covalent modification) and / or biological means (mutations by substitution, deletion and / or insertion of one or more amino acids), leading to a modification of the functional potential of the amino acid (s) entering (them) into the specified region, called the "mutated region". As an example, it is possible to carry out mutations leading to the loss, acquisition and / or modulation of the properties of disulfide bridges, hydrogen bonds, electrostatic interactions and / or hydrophobic interactions, to modify the ability of a protein to form a heterocomplex, or, alternatively, in the case of oligomeric proteins, to modify the state oligomerization or oligomer stability.

Modification of the regions of antigenic determinants leads to the introduction of a hydrophilic and non-immunogenic or weakly immunogenic flexible linker to or substitution of a part of the loop, and possibly with the introduction of mutation (s).

The recombinant loop protein gp41 may also include at least one mutation in its region of antigenic determinant, which leads to a cross-reaction in vitro, type B and / or type T, with the host protein and, in particular, with IL-2.

Some of the mutations critical to the manifestation of this change in antigenicity are disclosed in US Pat. No. 6,455,265 and WO 2005/010033, the contents of which are incorporated herein by reference in their entirety.

Recombinant gp41 loop proteins useful in the invention may also include modifications, such as shortening a portion of the amino acid sequence at the N- or C-terminal regions or adding a peptide sequence to produce a chimeric protein such as His-tag (histidine tag) or HA-tag (hemagglutinin label) as described in WO 2005/010033.

To produce recombinant gp41 loop proteins for use in the present invention, any known peptide synthesis methods or genetic engineering techniques, such as those described in the “Molecular Cloning - A Laboratory Manual (2 ^nd ed.)," Can be used. Sambrook et al., 1989, Coldspring Harbor Laboratory, Coldspring Harbor Press, NY (Sambrook).

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

Additionally, the antibody of the invention or a fragment thereof not only recognizes a gp41 recombinant loop protein having, for example, the amino acid sequence shown in SEQ ID NO 13, but also recognizes wild-type gp41 proteins, however, without recognizing a peptide having the sequence presented on SEQ ID NO 12.

As an example of a wild-type gp41 protein that can be recognized by the antibody of the invention, mention may be made of a gp41 protein having the amino acid sequence shown in SEQ ID NO 11, which is derived from the HIV strain HxB2 and which corresponds to amino acid sequence 540 through 683.

PEPTID P1

The antibody of the invention does not recognize a peptide called P1 and having the peptide sequence shown in SEQ ID NO 12, or an analog thereof.

Peptide P1 corresponds to the amino acid sequence present in gp41, an HIV envelope protein that is exposed on the surface of viral particles before viruses interact with the target cells. As an example, the strain HIV-1HxB2 in this sequence contains amino acids 649 to 683 from the wild-type gp41 protein.

In an aqueous solution, this peptide can assume a concentration-dependent structured oligomeric state, namely a dimeric or tetrameric state (ALFSEN & BOMSEL, 2002, J. Biol. Chem., 277: 25649).

Thus, the antibody or H region variable region of the invention, or a fragment thereof, does not recognize the P1 peptide in monomeric or oligomeric form.

ANTIBODY NEUTRALIZING ACTIVITY

The neutralizing activity of an antibody of the invention, or fragment thereof, against HIV can be assessed by inhibiting HIV transcytosis through epithelial cells and / or inhibiting HIV infection of CD4 ⁺ T cells.

According to one embodiment, the neutralizable HIV is, in particular, an HIV-1 strain.

Evaluation of the ability of the monoclonal antibody of the invention to inhibit HIV transcytosis through epithelial cells can be performed on any polarized cells. In one embodiment, the polarized cells may be epithelial cells, such as, for example, an intestinal cell HT-29 cell line or an HEC-1 endometrial cell line, or cells from a human mucosa biopsy (BOMSEL et al., Immunity, 1998, 3: 277). As a rule, cell lines are grown as a dense polarized monolayer on a permeable filter substrate (having, for example, a pore size of 0.45 μm), forming a contact surface between two separate chambers, of which the upper one washes the apical surface of the epithelial monolayer and the lower one washes the basolateral surface , or biopsy samples are placed inside using cameras.

Transcytosis can be initiated by interaction with HIV-infected cells, such as, for example, HIV-1 infected peripheral blood mononuclear cells (PBMC peripheral blood mononuclear cells), in the apical chamber.

The antibody to be tested 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 of the invention to 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.

Evaluation of viral transcytosis and possibly its inhibition can be performed by detecting a nucleic acid or HIV protein in a basolateral medium using any technique known in the art, such as PCR (polymerase chain reaction), RT-PCR (polymerase reverse transcriptase polymerase chain reaction) or ELISA (enzyme-linked immunosorbent assay - enzyme linked immunosorbent assay).

In one embodiment, the HIV peptide that can be used to evaluate viral transcytosis can be a p24 peptide that can be detected using an ELISA assay using, for example, the reagent kit provided by PASTEUR-SANOFI (FRANCE).

In a specific embodiment, when a concentration of about 0.5 ng / ml to about 5 ng / ml is added to about 10 ⁶ HIV-1 infected PBMC cells and incubated for about 1 hour at about 17 ° C or at about 4 ° C before by introducing infected cells into the apical chamber, the monoclonal antibody of the invention or a fragment thereof can inhibit viral transcytosis initiated by the addition of 10 ⁶ HIV-1 infected PBMC cells into the apical chamber by at least about 50%, in particular at least about 75 % and more specific at least about 95%

compared with transcytosis of the virus, performed without antibodies.

In another embodiment, the antibody of the invention or a fragment thereof can be introduced into the apical chamber before the addition of HIV-infected PBMC cells. In such an embodiment, the inhibition of transcytosis can be at least about 50% compared to transcytosis of a virus performed without an antibody.

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

In a specific embodiment, Fab and Fd of clone 69, which are also objects of the present invention, are capable of specifically binding to gp41 expressed on the surface of HIV-1 (JRCSF clone R5) infected PBMC cells, as measured by flow cytometry in accordance with a common method, known in the art.

In another embodiment, the monoclonal antibodies of the invention or fragments thereof may exhibit the ability to inhibit HIV infection of CD4 ⁴⁺ T cells.

In a specific embodiment, after incubating the virus at a concentration of about 1 ng / ml for about 30 minutes at a temperature of about 37 ° C. with the antibody of the invention or a fragment thereof, HIV infection of the CD4 ⁺ T cells can be inhibited by at least about 75%, and more specifically, at least about 95%, compared with HIV infection of CD4 ⁺ T cells performed without an antibody or with a non-specific antibody that does not recognize the virus.

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

NUCLEIC ACIDS, VECTORS AND HOST-CELLS

An antibody of the invention has been identified by screening a Fab IgA phage display library prepared as described in the experimental part.

The methods of obtaining and working with a combinatorial display library have been exhaustively described in the literature and are included in the general knowledge of a specialist in this field.

The Fab combinatorial phage display library was screened in a two-step manner.

In a first step, the Fab phage display library was screened for peptide P1 having the sequence shown as SEQ ID NO 12 immobilized on a solid support (such as plates used in an ELISA) to deplete the library antibodies recognizing this peptide.

This first step can be carried out with an antigen concentration of about 100 μM, at which the peptide P1 acquires a dimer / tetrameric oligomerized state.

In a second step, the Fab phage display library was screened with the recombinant gp41 loop protein by repeatedly panning on an antigen immobilized on a solid support; this method is known as micropanning and is described by AZZAY & HIGHSMITH (Clinical Biochemistry, 2002, 35: 425-445).

The recombinant loop protein gp41, which can be used to screen the Fab phage display library of the invention, may have, for example, the amino acid sequence shown in SEQ ID NO 13 or a functional analog thereof.

The initial amount may be, for example, from about 2 to about 1 μg, then the amount can be divided into two in each panning cycle.

The determination of the interaction between the antigen, namely, the recombinant loop protein gp41, and the antigen binding domain exposed on the outside of the bacteriophages can be evaluated using any methods known in the art. For example, you can use the ELISA method, where the recombinant loop protein gp41 cover the wells of the tablet, and then make the phage library.

Genes encoding an antigen binding site that are unique to each phage can then be isolated from the phage nucleic acid of the selected phage, sequenced and used to construct the genes of an entire antibody molecule or its analogue, such as a Fab fragment, F (ab ') ₂ or scFv, as described above.

The sequence of genes encoding the antigen binding site isolated from the selected phage can be sequenced according to any technique known to a person skilled in the art.

For example, sequencing can be performed using an automated DNA sequencer with a set of reagents (Applied Biosystems) containing fluorescently labeled dideoxynucleotides acting as terminators for the cyclic Taq sequencing reaction. Double-stranded DNA can be obtained, for example, from bacteria, and sequencing can be performed using a set of primers that specifically bind to the vector used to clone Fab H and L chains before and after each Fab chain. For example, using pComb3X vector, primers having the sequences shown in Table II can be used.

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

In particular, the nucleic acid sequences of the invention may be the H chain variable region sequence shown in SEQ ID NO 9 and the L chain variable region sequence shown in SEQ ID NO 10.

Of course, due to the degeneracy of the genetic code, we can assume variations in the nucleic acid sequence of the variable regions of the heavy and light chains shown, respectively, on SEQ ID NO 9 and SEQ ID NO 10, which will, as a result, give nucleic acid sequences that can be capable of producing antibodies or their functional analogs containing the amino acid sequence of the variable region of the heavy chain shown in SEQ ID NO 7 and the amino acid sequence of the variable the light chain region shown in SEQ ID NO 8.

According to one embodiment, the invention also relates to a nucleic acid sequence encoding an antibody of the invention or a fragment thereof. This nucleic acid may be RNA, cDNA, and the like. The nucleic acid sequence of the invention can be fused to other nucleic acid sequences to construct recombinant proteins using any molecular biology techniques known in the art. For example, the nucleic acid sequence of the invention can be fused to a nucleic acid encoding a His tag or HA tag, which can then be used, for example, to purify antibodies of the invention.

According to another embodiment, the present invention also relates to an expression vector comprising a nucleic acid sequence encoding a monoclonal antibody in accordance with the invention or a fragment thereof.

Such an expression vector can be used to express a monoclonal antibody of the invention, or a fragment thereof, in cells of various types. Such a vector may be a plasmid or a viral vector. Such a vector may have the ability to autonomously replicate in the host cell, or it can be integrated into the genome of the host cell.

As an example of vectors that can serve to carry out the invention, pComb3X or pASK88 can be mentioned.

The present invention also provides for the expression of an antibody of the invention, or a fragment thereof, using various host cells, such as bacteria, mammalian cells (CHO or HEK293), insect cells (such as Sf9 cells) or plant cells (tobacco, tomatoes).

After expression, the antibody of the invention can be isolated and purified using any techniques known in the art. For example, an antibody can be expressed with a His tag or HA tag and can subsequently be purified on a nickel column or using a specific antibody, as is usually done in the art.

Thus, the present invention also relates to a host cell transformed with a nucleic acid sequence encoding a monoclonal antibody in accordance with the invention or a fragment thereof. A host cell can be obtained in accordance with any transformation technique known in the art, such as electroporation, calcium phosphate treatment techniques, lipofection, infection with, for example, a recombinant virus. The present invention relates to a host cell transformed with a nucleic acid sequence encoding a monoclonal antibody according to the invention or a fragment thereof, for example, by electroporation in the case of bacteria or lipofection in the case of mammalian cells (CHO - Chinese hamster ovary - (Chinese ovary cells) hamster).

PHARMACEUTICAL COMPOSITION

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

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

The term "effective amount" means the minimum amount necessary for the manifestation of the expected effect, i.e. neutralize HIV and / or prevent HIV infection. A therapeutically or prophylactically effective amount of an antibody of the invention or a fragment thereof for a particular patient can be defined as the amount of antibody administered to an individual to provide a therapeutic or prophylactic effect (i.e., reducing or preventing infection) while minimizing side effects. An effective amount can be measured by decreasing the amount of HIV antigens in the serum of an individual.

By way of example, a non-limiting range of a therapeutically or prophylactically effective amount of an antibody of the invention or fragment thereof is about 0.1-100 mg / kg, more specifically, about 0.5-50 mg / kg, more specifically, about 1-20 mg / kg and, even more specifically, about 1-10 mg / kg.

According to another embodiment, the monoclonal antibody of the invention, or fragments thereof, or the pharmaceutical composition of the invention can be used in passive immunotherapy by administering to a person at risk of being infected or having suspected contact with HIV, or who has been in contact with HIV, a therapeutically effective amount of at least least an antibody according to the invention, or a fragment thereof, or a pharmaceutical composition according to the invention. Passive immunotherapy of the invention can be practiced with individuals exhibiting AIDS symptoms or related disease states caused by HIV infections, or individuals at risk for HIV infections.

According to one embodiment, the passive immunotherapy of the invention can also be used prophylactically, i.e. before alleged exposure to HIV.

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

Thus, the pharmaceutical composition of the invention can be formulated in various formulations, such as injectable or infusion sterile solutions, dispersions or suspensions, tablets, pills, powders, liposomes, suppositories and creams.

A suitable carrier for use in the manufacture of the pharmaceutical composition of the invention must be adapted in accordance with the dosage form that is intended to be manufactured. Such suitable carriers include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerin and the like, as well as combinations thereof. Pharmaceutically acceptable compounds, such as wetting or emulsifying agents, preservatives or buffers, may also be included in the pharmaceutical compositions of the invention.

The pharmaceutical composition of the invention may also include, in addition to the active agents of the invention, other anti-HIV active agents, such as antiretroviral drugs. According to another embodiment, such additional antiretroviral drugs can be administered in combination with the pharmaceutical composition of the invention simultaneously, separately or sequentially in time.

In another embodiment, the antibody of the invention or a fragment thereof may also be labeled for therapeutic purposes, in which case the label may be a drug conjugate or toxin, for example, a radioisotope or radionuclide ( ¹³¹ I, ⁹⁹ Tc, ¹¹¹ In or the like) , pertussis toxin, taxol, cytokalazin B, doxorubicin and the like.

DIAGNOSTIC COMPOSITION AND APPLICATION

The monoclonal antibody of the invention or a fragment thereof can also be used as a diagnostic agent in an in vitro method for detecting an HIV strain, and in particular an HIV-1 strain, in a sample.

In one of its embodiments, the method according to the invention includes at least the stages of:

A. Bringing the sample into contact with at least the antibody of the invention or a fragment thereof under conditions suitable for forming a complex between said antibody or fragment thereof and gp41 protein or its functional analogue.

B. Detection of the presence of the specified complex.

As an example of a gp41 protein that can complex with an antibody of the invention, wild-type gp41 proteins can be mentioned, such as, for example, a gp41 protein having the amino acid sequence shown in SEQ ID NO 11 or a functional analogue thereof, or modified gp41 proteins, such as a recombinant gp41 loop protein having, for example, the amino acid sequence shown in SEQ ID NO 13, or a functional analog thereof.

Detection of the complex can be carried out using any method of immune analysis known in this field.

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

Additionally, to facilitate detection, the monoclonal antibody of the invention, or a fragment thereof, can be labeled with a detectable marker. As an example of a labeling agent, mention may be made of fluorescent compounds such as fluorescein or rhodamine; enzymes such as horseradish peroxidase, beta-galactosidase or luciferase; biotin allowing detection through indirect measurement of avidin or streptavidin binding; or radionuclides ³ H, ¹⁴ C or ¹²⁵ I.

According to another embodiment, the present invention also relates to a diagnostic composition comprising an antibody of the invention or a fragment thereof.

To make the invention more clear, the following examples are provided.

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

DESCRIPTION OF DRAWINGS

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

Figure 2 shows a schematic representation of the plasmid pComb3X, where the variable and constant regions of the heavy and light chains from the Fab IgA phage library were inserted, respectively, between restriction sites by Sac 1 and Xba I enzymes, and between Xho 1 and Spe 1.

Figure 3 shows the inhibition of HIV-1 transcytosis through endometrial cells of the HEC-1 line by selected IgA Fab clones. A negative control was obtained by performing an experiment without an antibody (standard) or with a product obtained by screening a phage library obtained using the empty TOR10 plasmid. Positive control was performed using 2F5 IgA antibodies.

Figure 4 presents the results of a dot blot analysis performed with Fab 69 clone and IgA alpha 2F5 antibody on a wild-type gp41 protein from HIV HxB2 strain (amino acids 546 to 682) using a peptide with a leucine fastener, BSA (bovine serum albumin - bovine serum albumin) and lysozyme. After washing the nitrocellulose membranes, the antibody / protein complexes were incubated with mouse anti-His antibody for Fab clone 69 and mouse anti-human antibody for 2F5 antibody. Both complexes were detected with a goat anti-mouse antibody conjugated to horseradish peroxidase, followed by electro-generated chemoluminescence and autoradiography.

Figure 5 presents the amino acid sequences corresponding to the complementarity determining sequences of the variable region of the H chain of an antibody according to the invention (CDR1 as SEQ ID NO 1, CDR2 as SEQ ID NO 2, CDR3 as SEQ ID NO 3), and regions for determining complementarity, variable regions of the L chain of an antibody of the invention, wherein CDR1, CDR2 and CDR3 are shown in SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6.

6A and 6B show the amino acid and nucleic acid sequences 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) of clone Fab 69.

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

Fig. 8 shows the amino acid sequences corresponding to the wild-type gp41 protein, SEQ ID NO 11 (HIV-1 strain HxB2), PI peptide SEQ ID NO 12.

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

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

Table II presents the sequences of the primers used to identify and sequence the Fab IgA nucleic acid sequences from the phage display library.

Example 1

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

Cervical secretions of 56 HEPS individuals from Cambodia were examined for the presence of HIV S-IgA envelope glycoprotein. Discharges were collected after two days of sexual abstinence using 3 ml of sterile PBS (phosphate buffered saline - phosphate buffered saline). Samples were centrifuged and frozen and stored at -80 ° C. Sperm contamination of the sample was measured using a seminal fluid detection analysis method (SEMA; HUMANGEN FERTILITY DIAGNOSTICS, Charlottesville, VA) according to the manufacturer's instructions, but optimized for increasing incubation time and using O-phenylenediamine dihydrochloride as a substrate. Contaminated samples were removed from the study.

The presence of specific IgA antibodies against HIV was tested using Western blot using a commercially available reagent kit (New Lav Blot1, Sanofi-Pasteur, France) in accordance with the manufacturer's instructions.

In 22 of the tested samples, a large amount of anti-HIV contained the secretory form of IgA (S-IgA) against enveloped gp41 (Figure 1).

Example 2

Construction of a combinatorial phage display library expressing Fab from IgA

Mucosal B cells from 22 identified HEPS individuals from Example 1 were used to construct a combinatorial phage display library expressing Fab from IgA.

Whole RNA was obtained from an enriched population of B cells from cervical-vaginal secretions from 22 HEPS individuals using standard techniques. Whole RNA (60 μg) was purified from cervical B cells by a quick, one-step RNA isolation procedure based on guanidine isothiocyanate / phenol chloroform (CHOMCZYNSKI & SACCHI, Anal. Biochem., 1987, 162: 156-9). This RNA was completely transformed into complementary DNA (cDNA) using reverse transcription polymerase chain reaction (RT-PCR). The resulting cDNA library was amplified using the specific mucosal Fab IgA primers shown in Table I.

The amplified DNA was then digested with Sac I and Xba 1 for the light chain (L) and Xho 1 and Spe 1 for the heavy chain (H), and the resulting digestion products were inserted into the pComb3X vector (BARBAS et al., Proc Natl Acad Sci USA, 1991, 88: 7978-82), digested with the same enzymes. The digested vector and digested PCR product were ligated using DNA ligase from T4 phage (4 inserts plus 1 vector plus T4DNA ligase, overnight at 14 ° C) (Figure 2). The resulting recombinant plasmids were then used to transform E. coli bacteria (pilus +, male) TG1 by electroporation.

The library of complementary DNA antibodies in culture after bacterial transformation was then expressed on the bacteriophage by superinfection using the helper phage VCS-M13 (10 ¹² pfu - (plague forming units - plaque forming units)) (Stratagene, La Jolla, CA).

Phage preparations were collected. The phages were precipitated by the addition of 20% (w / v) polyethylene glycol 8000 and 2.5 M NaCl, followed by incubation on ice for one hour. After centrifugation, the phage pellet was resuspended in phosphate buffered saline (PBS) and microcentrifuged for several minutes to separate cell debris.

A restriction analysis of the colonies from the titer plates showed that approximately 35% of the phages contained Fab IgA and that library-containing titers were typically close to 10 ⁷ CFU / ml (colony units per ml) and 10 ⁷ different clones.

Example 3

Screening Fab IgA phage library on recombinant loop protein gp41 (SEQ ID NO 13)

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

Before screening a Fab IgA phage display library with recombinant loop protein gp41 (SEQ ID NO 13), the first panning cycle was performed with the PI peptide (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 an oligomeric state. Such a protocol allows the separation of any Fab IgA capable of binding the P1 peptide in an oligomeric state.

The initial amount of the used recombinant loop protein gp41 was 1 μg, and the amount was divided into two in each cycle so that ultimately the amount in the fourth cycle was 125 ng.

Wells of 96-well plates (Exiqon peptide Immobilirez, 10202-111-10) were coated with 1 μg of gp41 recombinant loop protein, then blocked with BSA for 2 hours at 37 ° C. Concentrated phages obtained from the library were then added to the wells in an amount of 10 ¹² -10 ¹³ plaque-forming units and left for 2 hours at 37 ° C. Unbound phages were then removed by intensive washing, for the first and second selection cycles, 10 times with PBS containing 0.1% Tween-20, then 10 times with PBS to remove detergent. For subsequent selection cycles, washing was carried out 20 times with PBS containing 0.1% Tween-20, then 20 times with PBS. Specific elution of phages carrying epitopes of surface Fabs binding the gp41 recombinant loop protein was carried out by treatment with 0.1 M glycine-HCl, with a pH adjusted to 2.2, for 10 min. The eluted fraction was immediately neutralized and used for infection with 2 ml of fresh E. coli TG1 bacterial culture (OD ₆₀₀ (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 10 ¹² plaque forming units / ml of the VCS-M13 helper phage were added to produce recombinant phages overnight . Eluted phages were amplified between each panning cycle.

After panning, individual clones from four rounds were grown and Fab was monitored by PCR using specific primers shown in Table II that hybridize with the vector before and after the Fab light and heavy chains. Clones containing Fab IgA were converted to a soluble Fab form by transforming them with E. coli cells of the non-suppressor strain carrying the amber codon and sequencing to determine the variable regions of the heavy (H) and light (L) chains using the IMGT / V- program QUEST (Fig. 7).

Sequencing of immunoglobulin genes

Sequencing was performed using an automated DNA sequencer with a set of reagents (Applied Biosystems) containing fluorescently labeled dideoxynucleotides acting as terminators for the cyclic Taq sequencing reaction. Double-stranded DNA was obtained from bacteria and sequencing was performed using a set of primers (see Table II) that bind specifically to the pComb3X vector before and after each Fab chain.

6A and 6B show the result of sequencing the variable regions of the H and L chains of clone 69 (SEQ ID NO 9 and SEQ ID NO 10).

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

The vector used for cloning carries an amber codon between the tag sequences (His tag and HA tag) and the phage protein pIII, which makes it possible to either obtain an antibody fused to the phage coat protein using an E. coli suppressor strain such as TG1 or obtain soluble antibodies by expression of a vector in a non-suppressor strain, such as TOP-10. Given this advantage, phage DNAs were used to transform E. coli TOP-10 (a non-suppressor strain carrying the amber codon) to express soluble Fab fragments without a pIII fusion protein. Fab expression was induced using 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

For large-scale expression of the monoclonal antibody of the invention, the intact operon was transferred from pComb3X to pASK.88 vector (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).

The Fab monoclonal antibody DNA of the invention was amplified using specific primers introducing restriction sites necessary for sub-cloning into the pASK88 vector. Amplification products were purified by agarose gel electrophoresis and digested with restriction enzymes PstI and NcoI, in the case of the heavy chain, and SacI and HindIII, in the case of the light chain. Fd genes (VH-CH1) and light chain genes (VL-CL) were inserted separately and ligated into pASK88 digested with the same restriction enzymes using the standard protocol. Ligation products were transformed into JM83 thermocompetent bacterial cells (provided by Dr. Skerra) and plated on Petri dishes to separate into individual clones. Plasmid DNA for several clones was analyzed by restriction analysis and, for some of them, double-stranded DNA sequencing using specific primers (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).

The pASK88 expression vector was designed for convenient cloning of immunoglobulin variable region genes, as well as for periplasmic secretion of the corresponding Fabs fragments in Escherichia coli. Using this plasmid, expression was monitored using the tetracycline promoter.

Large amounts of the monoclonal antibody of the invention were prepared using this vector. Preparative expression was performed on a 1 L scale using E. coli K-12 JM83 as expressing host cells. Cells were grown to the middle of the logarithmic phase and Fab expression was then induced with 0.2 mg / L anhydrous tetracycline for 4 hours or overnight.

Purification of the monoclonal antibody of the invention was carried out on Ni-NTA Spin Columns (Qiagen) columns by reaction with a His tag. The periplasmic fraction sterilized by filtration was applied to a column and a gradient of 250-500 mM imidazole in chromatography buffer was used to collect samples.

Example 4

ELISA analysis performed with soluble IgA Fab clones from the phage display library.

An ELISA was performed with soluble IgA Fab obtained from TOP-10F 'bacteria (pComb3x system) and with soluble IgA Fab obtained from K-12 JM83 bacteria (pASK88 system).

Wells of the plates (Exiqon peptide Immobilirez, 10202-111-10 or NUNC, 439454) were coated with 100 ng of the recombinant loop protein gp41 (SEQ ID NO 13) and blocked with BSA for 2 hours at 37 ° C. Then purified IgA was added at a concentration of 2 ng / ml and incubated for 2 hours at 37 ° C. Detection was performed using a mouse anti-HA antibody (clone 12CA5, Roche) or anti-His (PentaHis, Qiagen 34660) antibodies, followed by use of a goat anti-mouse antibody (Caltag Laboratories, H1003) labeled with horseradish peroxidase. The enzymatic reaction was manifested by the addition of TMB (3.3 ', 5.5'-tetramethylbenzidine, Kikergaard & Perry Laboratories Inc.) as a substrate, and the absorbance was measured at 450 nm after adding 1M phosphoric acid using an ELISA plate spectrophotometer. The positive control was a 2F5 antibody, and the negative control was a clone obtained after panning that did not recognize the recombinant gp41 loop protein during ELISA.

Example 5

Transcytosis inhibition

HIV-1 transcytosis through epithelial cells and neutralization of transcytosis by antibodies was performed on the HEC-1 cell line of intestinal cells grown as a dense polarized monolayer for 7 days on a permeable filter substrate (pore size 0.45 μm), forming a contact surface between two separate cameras, of which the upper one washes the apical (luminal) surface of the epithelial monolayer, and the lower one washes the basolateral (serous) surface.

PBMCs were prepared and prepared as described by LAGAYE et al. (J. Virol, 2001, 75: 4780). Then, PBMC cells were activated with phytohemagglutinin (PhA) for 48 hours and inoculated with 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 viral transcytosis, 2 · 10 ⁶ HIV-1 ⁺ PBMCs were added to the apical chamber. Contact between HIV-1 ⁺ PBMC and a monolayer of epithelial cells led to the rapid budding of HIV-1 virions, followed by their transcytosis from the apical to the basolateral pole of epithelial cells. After 2 hours, inhibition of antibody transcytosis was determined by detecting the p24 HIV protein in basolateral medium using an ELISA (Coulter, France or PASTEUR SANOFI, FRANCE). The p24 level in the absence of antibody or in the presence of a nonspecific Fab to the recombinant gp41 loop protein or in the presence of a control IgA 2F5 was measured, respectively, as a negative and positive control with a value of 100, 98 and 35%, respectively. The significance for the negative control was taken as 100% transcytosis and was used to present the results.

The experiments were performed in triplicate independent repetition.

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

Example 6

Dot blotting

BSA, peptide with leucine fastener, lysozyme and gp41 wild-type protein from the HIV strain HxB2 (amino acids 546 to 682, obtained from ABI) were spotted onto nitrocellulose membranes, respectively, each sample at a concentration of 100 ng / ml. After that, Fab clone 69, at a concentration of 5 ng / ml, or 2F5 alpha-IgA antibody, at a concentration of 2 μg / ml, diluted 1: 2500, was incubated on nitrocellulose membranes.

After washing the nitrocellulose membranes (blocking reagent for Western blotting, Roche: 1% for blocking and washing, 0.1% for binding the antibody 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 a goat anti-mouse antibody labeled with horseradish peroxidase (Caltag Laboratories, H1003.1: 4000 dilution), followed by transfer to an ECL X-ray film and autoradiography (ECL, Amersham, according to the manufacturer's instructions).

Figure 4 presents the result of an experiment repeated twice.

Claims (17)

1. Anti-HIV monoclonal antibody or its functional fragment containing in the variable region of the heavy H- (heavy) chain complementarity determining regions, CDR1, CDR2 and CDR3 having respectively the amino acid sequence shown in SEQ ID NO I, SEQ ID NO 2 , SEQ ID NO 3, and in the variable region of the L- (light) chain CDR1, CDR2, and CDR3 having respectively the amino acid sequence shown in SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6. 2. The monoclonal antibody according to claim 1, characterized in that it does not recognize the peptide of the amino acid sequence shown in SEQ ID NO 12. 3. The monoclonal antibody or its functional fragment according to claim 1, recognizing the recombinant gp41 loop protein having the amino acid sequence shown in SEQ ID NO 13 and not recognizing the peptide of the amino acid sequence shown in SEQ ID NO 12. 4. The monoclonal antibody or its functional fragment according to claim 1, characterized in that it is an IgA. 5. The monoclonal antibody or its functional fragment according to claim 1, characterized in that it is a human antibody. 6. The monoclonal antibody or its functional fragment according to claim 1, characterized in that the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO 7, and the variable region of the light chain has the amino acid sequence shown in SEQ ID NO 8. 7. The monoclonal antibody or its functional fragment according to claim 1, characterized in that it has the ability to neutralize the human immunodeficiency virus (HIV). 8. The monoclonal antibody or its functional fragment according to claim 7, characterized in that the neutralizable HIV is a strain of HIV-1. 9. The fragment of the heavy chain of the antibody according to claim 1, which is 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 an antibody according to claim 1, which is 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 according to claim 9. 12. An expression vector encoding an antibody light chain fragment of claim 10. 13. A host cell transformed with the expression vector according to claim 11 and producing a fragment of the antibody heavy chain, which is its variable region, according to claim 9. 14. A host cell transformed with an expression vector according to claim 12 and producing a fragment of the antibody light chain, which is its variable region, according to claim 10. 15. The use of an antibody, or a functional fragment thereof according to claim 1, or an antibody heavy chain fragment according to claim 9, or an antibody light chain fragment according to claim 10, an expression vector according to claim 11 or 12, or a host cell according to claim 13 or 14 for the manufacture of a medicament for use in the prevention and / or treatment of HIV infection. 16. A method for detecting an HIV strain in an in vitro sample, comprising at least a step:
A) bringing the sample into contact with at least the antibody or its functional fragment according to claim 1 under conditions suitable for the formation of a complex between the specified antibody or its functional fragment and gp41 protein or its functional analogue, and B) detecting the presence of the specified complex. 17. A method for conducting passive immunotherapy of an individual susceptible to HIV infection, comprising administering to said individual a therapeutically effective amount of at least an antibody or a functional fragment thereof according to claim 1.

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