JP2014504147A - HIV therapeutic antibody - Google Patents

Abstract

  The present invention is an isolated antibody that can not only bind to CXCR4, but also induce conformational changes of CXCR4 homodimer and inhibit HIV-1 primary isolate replication in PBMC Or a derivative or antigen-binding fragment thereof. More particularly, the present invention relates to 515H7 and 301aE5 monoclonal antibodies specific for CXCR4 protein and their use for the treatment of HIV infection. Also encompassed are pharmaceutical compositions comprising such antibodies and methods of selecting such antibodies.

Description

Background of the Invention

  The present invention relates to novel antibodies capable of specifically binding to chemokine receptors (CXCR), in particular murine monoclonal antibodies, chimeric antibodies and humanized antibodies, and amino acid and nucleic acid sequences encoding such antibodies. . From one aspect, the present invention relates to novel antibodies, functional fragments or derivatives that can specifically bind to CXCR4 and have strong activity against human immunodeficiency virus (HIV) infection. The present invention also comprises the use of such antibodies, functional fragments or derivatives as medicaments for the prophylactic and / or therapeutic treatment of HIV infection.

Chemokines are small secreted peptides that control leukocyte migration by chemical gradients of ligands known as chemokine gradients, particularly during immune responses (Zlotnick A. et al., 2000). Chemokines are divided into two major subfamilies, CC and CXC, based on the position of their NH ₂ terminal cysteine residues, which bind to G protein-coupled receptors, and the two major subfamilies of that receptor are It is called CCR and CXCR. To date, more than 50 human chemokines and 18 chemokine receptors have been found.

  Several members of the chemokine receptor family act as a co-receptor with the major receptor CD4, allowing various type 1 HIV strains to enter the cell, the major co-receptors being CCR5 and CXCR4. T-cell tropic X4 HIV-1 uses CD4 and CXCR4 for cell entry, whereas macrophage-tropic R5 HIV-1 uses CD4 and CCR5. Bidirectional strains can use both CXCR4 and CCR5 as co-receptors. Among other chemokine receptors, CCR3, CCR2, CCR8, CXCR6, CXCR7, CX3CR1 can function as co-receptors with a more limited subset of HIV strains.

  The natural ligand of CXCR4, SDF-1, and the natural ligands of CCR5, CCL3, CCL4, CCL4-L1 and CCL5 ligands, can inhibit cell fusion and infection by various HIV-1 strains. These findings facilitate the development of anti-HIV therapeutics that target chemokine receptors and, in patients infected with CCR5-directed HIV-1, maraviroc (CELSENTRI ™), a small molecule antagonist of CCR5 And approval of combinations with other anti-HIV-1 drugs. Nonetheless, maraviroc is not used in patients infected with bi-directional HIV-1 nor in patients infected with CXCR4-directed HIV-1 (VIDAL 2009). Thus, there is a clear medical need to extend this type of therapy to both X4-directed and dual-directed HIV-infected patients by identifying CXCR4 antagonists that can inhibit X4-directed HIV replication. There is.

  Chemokine receptor 4 (also known as Fusin, CD184, LESTR, or HUMSTR) exists as two isoforms comprising 352 or 360 amino acids. Residue Asn11 is glycosylated, residue Tyr21 is modified by the addition of sulfate groups, and Cys109 and 186 are joined by a disulfide bridge in the extracellular portion of the receptor (Juarez J. et al., 2004).

  This receptor can be found in various types of normal tissues, naive, non-memory T cells, regulatory T cells, B cells, neutrophils, endothelial cells, primary monocytes, dendritic cells, natural killer cells, CD34 + hematopoietic stem cells, Expressed in the heart, colon, liver, kidney, and brain, albeit at low levels. CXCR4 plays an important role in leukocyte trafficking, B cell lymphocyte generation, and bone marrow hematopoiesis.

  The unique ligand of the CXCR4 receptor described so far is stromal cell derived factor-1 (SDF-1) or CXCL12. SDF-1 is secreted in large amounts and to a lesser extent in the lymph nodes, bone marrow, liver, and lungs by the kidneys, brain, and skin. CXCR4 is also recognized by the antagonist chemokine, a viral macrophage inflammatory protein II (vMIP-II) encoded by a type III human herpesvirus.

  As previously described, the CXCR4 receptor is the major co-receptor for T cell directed HIV-1 isolate (X4 virus). Interfering with this receptor should be able to inhibit X4 virus replication very efficiently.

  One of the inventive aspects of the present invention is to produce mouse monoclonal antibodies (Mabs) that inhibit HIV replication. The present invention encompasses CXCR4 Mab 515H7 (or a fragment thereof) that can bind to CXCR4 homodimer and has strong activity against HIV infection. The invention also encompasses CXCR4 Mab 301aE5 (or a fragment thereof) that can bind to CXCR4 homodimer and has strong activity against HIV infection.

  Surprisingly, we not only can bind CXCR4, but also can induce a conformational change in CXCR4 homodimer and X4-HIV-1 primary isolate in PBMC Of monoclonal antibodies capable of inhibiting the replication of. More particularly, the antibodies of the present invention can also inhibit replication of X4 / R5-HIV-1 primary isolates in PBMC.

Preferably, the CXCR4 compound is
Genbank accession number NP_003458 SEQ ID NO: 27:
MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS,
A chemokine (C—X—C motif) receptor 4 isoform b [homo sapiens] having the sequence represented as:
Genbank accession number NP_001008540 SEQ ID NO: 28:
MSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS,
A chemokine (C—X—C motif) receptor 4 isoform a [Homo sapiens] having the sequence represented as:
A selective transcriptional splice variant having at least 95% identity with one of these b or a isoforms having SEQ ID NO: 27 or 28, or a natural variant thereof, and its natural ligand, stromal cell derived factor-1 ( Of two human CXCR4 isoforms that can be specifically recognized by SDF-1) and are preferably selected from the group consisting of fragments thereof having an amino acid length of at least 100, 150 and 200 one of.

CXCR2 is
Genbank accession number NP_001548 SEQ ID NO: 29:
MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNVSPACYEDMGNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFLLCWLPYNLVLLADTLMRTQVIQETCERRNHIDRALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVGSSSGHTSTTL,
Interleukin-8 receptor β [homo sapiens] having the sequence represented as
A selective transcriptional splice variant having at least 95% identity with this interleukin 8 receptor β having SEQ ID NO: 29, or a natural variant thereof, and can be specifically recognized by IL-8, and Preferably selected from the group consisting of fragments thereof having an amino acid length of at least 100, 150 and 200.

  The present invention also provides a method for selecting a compound having anti-HIV activity or that can be used for the manufacture of a composition for the treatment of HIV infection, comprising the following steps: Also includes how to:

  In a first aspect, the subject of the invention is a method for making and selecting an antibody according to the invention.

More particularly, the present invention is a method of selecting an anti-CXCR4 antibody, or one of its functional fragments or derivatives, capable of inhibiting HIV replication comprising:
i) screening the produced antibody and selecting an antibody capable of specifically binding to CXCR4;
ii) assaying the selected antibody of step i) and selecting an antibody capable of binding to peripheral blood mononuclear cells (PBMC);
iii) assaying the selected antibody of step ii) and selecting an antibody capable of binding to the CXCR4 homodimer, and then iv) assaying the selected antibody of step iii) and X4-directed HIV-in PBMC 1. A method comprising selecting an antibody capable of inhibiting replication of a primary isolate.

In another embodiment, the invention provides a method of selecting an anti-CXCR4 antibody, or one of its functional fragments or derivatives, capable of inhibiting HIV replication comprising:
i) screening the produced antibody and selecting an antibody capable of specifically binding to CXCR4;
ii) assaying the selected antibody of step i) and selecting an antibody capable of binding to peripheral blood mononuclear cells (PBMC);
iii) assaying the selected antibody of step ii) and selecting an antibody capable of binding to the CXCR4 homodimer, and then iv) assaying the selected antibody of step iii) and X4-directed HIV-in PBMC Selecting a antibody capable of inhibiting replication of the primary isolate of 1 and / or inhibiting replication of the X4 / R5-directed HIV-1 primary isolate in PBMC .

  The production of antibodies can be accomplished by methods known to those skilled in the art, such as, for example, fusion of myeloma cells with spleen cells from immunized mice or other species compatible with selected myeloma cells [Kohler & Milstein, 1975, Nature, 256: 495-497]. The immunized animal can include a transgenic mouse having a human immunoglobulin locus and then directly producing human antibodies. Another possible embodiment may consist of the use of phage display technology to screen the library.

  Screening steps i) and ii) can be achieved by any method or process known to those skilled in the art. Non-limiting examples include ELISA, BIAcore, immunohistochemistry, Western blot analysis, FACS analysis, and functional screening using CXCR4-expressing cell membrane extracts or purified CXCR4. A preferred method is by FACS analysis on CXCR4 transformants (step 1) and at least PBMC (step 2) to confirm that the antibodies produced can also recognize the native CXCR4 receptor conformation on the target cell surface. Consists of screening. This method is described more precisely in the following example.

  The screening step iii) can be realized by any method or step known to those skilled in the art. A preferred example includes, but is not limited to, Western blotting and / or immunoprecipitation using an antibody of interest against a membrane extract derived from CXCR4 transfected cells or PBMC.

  The screening step iv) can be realized by any method or process known to those skilled in the art. By way of non-limiting example, the preferred method described by Holl et al. (J. Immunol. 2004, 173, 6274-83) is used to isolate X4 primary HIV-1 isolates in PBMC and / or Or a method comprising screening an antibody for whether it can inhibit replication of X4 / R5 primary HIV-1 isolate.

  In a preferred embodiment of step iii) of the selection of the method of the invention, this step iii) comprises evaluating the antibody by BRET analysis against cells expressing CXCR4-RLuc / CXCR4-YFP, and at least 40% of the BRET signal Selecting antibodies that can inhibit, preferably 45%, 50%, 55%, most preferably 60%.

  The BRET method is a technique known as a representative example of protein dimer formation [Angers et al., (PNAS), 2000, 97: 3684-89].

  The BRET method used in step iii) of the method is well known to those skilled in the art and is described in detail in the examples below. More specifically, BRET (Bioluminescence Resonance Energy Transfer) is a mutant of a bioluminescent donor (Renilla luciferase (Rluc)) and a fluorescent acceptor GFP (green fluorescent protein) or YFP (yellow fluorescent protein). Is a non-radioactive energy transfer between and. In this case, EYFP (enhanced yellow fluorescent protein) was used. The effectiveness of migration depends on donor and acceptor orientation and distance. And energy transfer can only occur when the two molecules are in close proximity (1-10 nm). This property is used to create a protein-protein interaction assay. In fact, to examine the interaction between the two partners, the first one is gene fused with Renilla luciferase and the second with a yellow mutant of GFP. Fusion proteins are generally but not necessarily expressed in mammalian cells. In the presence of its membrane permeable substrate (coelenterazine), Rluc emits blue light. If the GFP mutant is closer than 10 nm from Rluc, energy transfer occurs and a yellow signal can be detected. The BRET signal is measured as the ratio between the light emitted by the receptor and the light emitted by the donor. Therefore, the BRET signal increases as the two fusion proteins are in close proximity, or the conformational change is in close proximity to Rluc and the GFP mutant.

  If the BRET analysis comprises a preferred embodiment, any method known to those skilled in the art can be used to measure the conformational change of the CXCR4 dimer. Non-limiting techniques include the following techniques: FRET (Fluorescence Resonance Energy Transfer), HTRF (Homogeneous Time Resolved Fluorescence), FLIM (Fluorescence Lifetime Imaging) or SW-FCCS (Single Wavelength Fluorescence Correlation Spectroscopy) ).

  Other conventional methods such as co-immunoprecipitation, alpha screen, chemical cross-linking, double hybrid, affinity chromatography, ELISA, or far western blot can also be used.

  In a particular aspect of the method according to the invention, step iii) is to evaluate the antibody by BRET analysis against cells expressing both CXCR4-RLuc / CXCR4-YFP and to inhibit at least 40% of the BRET signal. Consisting of selecting antibodies that can.

  In a second aspect, the subject of the present invention is an isolated antibody obtained by said method, or one of their functional fragments or derivatives. The antibody or one of their fragments or derivatives can specifically bind to human CXCR4 and the antibody can also induce conformational changes in CXCR4 homodimers.

For example CXCR4 Mab is HIV-1 can inhibit the invasion of PBMC experiments strain _{(X4HIV-1 NL4-3)} are known from the literature (Tanaka R. et al, such as Crohn A 120, J. Virol 2001, 75, 11534-11543). Furthermore, it is also known that CXCR4 Mab can inhibit entry of HIV-1 X4 primary isolates into cell lines expressing CXCR4. In contrast, no antibodies are disclosed that can inhibit such viruses in their natural environment, ie not only against experimental viruses or cell lines. However, again, it is novel and not an obvious aspect of the present invention that CXCR4 Mab can inhibit entry of HIV-1 X4 primary isolates into PBMC.

  The expressions “functional fragments and derivatives” and “antigen-binding fragments and derivatives” are similar and are defined in detail later in this specification.

  Here, the present invention does not relate to naturally occurring antibodies, i.e. they are not in their natural environment and can be isolated or obtained by purification from natural sources, or even genetically modified. Alternatively, it should be understood that they have been obtained by chemical synthesis and that they can contain unnatural amino acids as further described.

  More particularly, according to another aspect of the invention, an isolated antibody, or one of its functional fragments or derivatives, is described, said antibody comprising SEQ ID NO: 1 to 1 defined by the IMGT numbering system. It is characterized by comprising at least one complementarity determining region CDR selected from CDRs comprising amino acid sequences 6 and 30-33.

  According to a first aspect, the present invention relates to at least one CDR selected from the CDRs of the sequences SEQ ID NO: 1-6 defined according to the IMGT numbering system, or SEQ ID NO: 1 after the optimal alignment of the sequences. An isolated antibody, or a functional fragment or derivative thereof, comprising at least one CDR having at least 80%, preferably 85%, 90%, 95%, and 98% identity with the sequence of -6 About.

  According to a second aspect, the present invention provides at least one CDR selected from the CDRs of the sequences SEQ ID NO: 1, 2 and 30-33 as defined according to the IMGT numbering system, or the sequence of which is optimally aligned. Isolated later comprising at least one CDR having at least 80%, preferably 85%, 90%, 95%, and 98% identity with the sequence of SEQ ID NOs: 1, 2, and 30-33 It relates to an antibody, or a functional fragment or derivative thereof.

“Functional fragments” or “antigen-binding fragments” of an antibody include in particular the fragments Fv, scFv (sc = single chain), Fab, F (ab ′) ₂ , Fab ′, scFv-Fc or diabody. ) Or any fragment with an extended half-life. Such functional fragments are described in detail later in this specification.

  The “derivative compound” or “derivative” of an antibody means in particular a binding protein composed of a peptide scaffold and at least one CDR of the original antibody to retain the ability to recognize CXCR4. . Such derivative compounds well known to those skilled in the art are described in further detail later in this specification.

  More preferably, the present invention comprises antibodies according to the present invention, derivatives thereof or functional fragments thereof, in particular antibodies obtained by chimeric or humanized antibodies, genetic recombination, or chemical synthesis.

  According to a preferred embodiment, the antibody according to the invention, or a derivative compound or functional fragment thereof, is characterized in that it consists of a monoclonal antibody.

  “Monoclonal antibody” is understood to mean an antibody arising from a substantially homogeneous antibody population. More particularly, the individual antibodies of a population are identical except for a small number of potentially naturally occurring mutations found in a minimal proportion. In other words, a monoclonal antibody is a single cell clone (eg, a hybridoma, a eukaryotic host cell transfected with a DNA molecule encoding the homogeneous antibody, a prokaryote transfected with a DNA molecule encoding the homogeneous antibody, Consisting of homogenous antibodies resulting from the growth of a host cell, etc., and is generally characterized by one and only one class and subclass heavy chains and only one type of light chain. Monoclonal antibodies are highly specific and are directed against a single antigen. In addition, each monoclonal antibody is directed against a single epitope of the antigen, as opposed to making polyclonal antibodies that generally include different antibodies directed against different determinants or epitopes.

More particularly, according to a first preferred embodiment of the invention, the antibody, or derived compound or functional fragment thereof, is at least one CDR selected from CDR-L1, CDR-L2, and CDR-L3. Characterized in that it comprises a light chain comprising
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 1,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 2,
CDR-L3 comprises the amino acid sequence of SEQ ID NO: 3.

  According to another embodiment, the antibody of the invention, or one of the derived compounds or functional fragments thereof, is at least one of the three CDRs of the sequence of SEQ ID NO: 1, 2, or 3, or optimal Comprising a light chain comprising at least one sequence having at least 80%, preferably 85%, 90%, 95%, and 98% identity with the sequence of SEQ ID NO: 1, 2, or 3 after alignment. It is characterized by becoming.

  An antibody of the invention, or one of its functional fragments or derivatives, is also characterized in that it comprises a light chain comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1, L1 comprises the amino acid sequence of SEQ ID NO: 1, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 2, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 3.

  In another embodiment, the antibody of the invention, or one of its functional fragments or derivatives, is at least 80%, preferably 85% with the amino acid sequence of SEQ ID NO: 7, or the sequence of SEQ ID NO: 7 after optimal alignment. , 90%, 95%, and 98% identity, comprising a light chain of a sequence comprising at least one sequence.

According to a second preferred embodiment of the invention, the antibody, or derivative or functional fragment thereof, is a light comprising at least one CDR selected from CDR-L1, CDR-L2, and CDR-L3. Characterized in that it comprises a chain, where
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 1,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 2,
CDR-L3 comprises the amino acid sequence of SEQ ID NO: 30.

  According to another embodiment, the antibody of the invention, or one of the derived compounds or functional fragments thereof, is at least one of the three CDRs of the sequence SEQ ID NO: 1, 2, or 30, or optimal Comprising a light chain comprising at least one sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity with the sequence of SEQ ID NO: 1, 2, or 30 after alignment It is characterized by that.

  One of the antibodies of the invention, or a functional fragment or derivative thereof, is characterized in that it comprises a light chain comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 Comprises the amino acid sequence of SEQ ID NO: 1, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 2, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 30.

  In another embodiment, the antibody of the invention, or one of its functional fragments or derivatives, is at least 80%, preferably 85% with the amino acid sequence of SEQ ID NO: 34, or the sequence of SEQ ID NO: 34 after optimal alignment. , 90%, 95%, and 98% identity, comprising a light chain of a sequence comprising at least one sequence.

More particularly, the antibody of the invention, or one of their derived compounds or functional fragments, comprises a heavy chain comprising at least one CDR selected from CDR-H1, CDR-H2, and CDR-H3. Characterized in that it comprises
CDR-H1 comprises the amino acid sequence of SEQ ID NO: 4,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 5,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 6.

  According to another embodiment, the antibody of the invention, or one of the derived compounds or functional fragments thereof, is at least one of the three CDRs of the sequence SEQ ID NO: 4, 5 or 6, or optimal alignment Followed by a heavy chain comprising at least one sequence having at least 80%, preferably 85%, 90%, 95%, and 98% identity with the sequence of SEQ ID NO: 4, 5 or 6. It is characterized by.

  According to another particular embodiment, the antibody, or one of its derived compounds or functional fragments, comprises a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3. Wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 5 and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 6 .

  In another embodiment, the antibody of the invention, or one of its functional fragments or derivatives, is at least 80%, preferably 85% with the amino acid sequence of SEQ ID NO: 8, or the sequence of SEQ ID NO: 8 after optimal alignment. , 90%, 95%, and 98% identity, comprising a heavy chain of a sequence comprising at least one sequence.

More particularly, the antibody of the invention, or one of their derived compounds or functional fragments, comprises a heavy chain comprising at least one CDR selected from CDR-H1, CDR-H2, and CDR-H3. Characterized in that it comprises
CDR-H1 comprises the amino acid sequence of SEQ ID NO: 31,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 32,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 33.

  According to another embodiment, the antibody of the invention, or one of the derived compounds or functional fragments thereof, is at least one of the three CDRs of the sequence of SEQ ID NO: 31, 32, or 33, or optimal Comprising a heavy chain comprising at least one sequence having at least 80%, preferably 85%, 90%, 95%, and 98% identity with the sequence of SEQ ID NO: 31, 32, or 33 after alignment. It is characterized by becoming.

  According to another particular embodiment, the antibody, or one of its derived compounds or functional fragments, comprises a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3. Wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 31, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 32, and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 33. .

  In another embodiment, the antibody of the invention, or one of its functional fragments or derivatives, is at least 80%, preferably 85% with the amino acid sequence of SEQ ID NO: 35, or the sequence of SEQ ID NO: 35 after optimal alignment. , 90%, 95%, and 98% identity, comprising a heavy chain of a sequence comprising at least one sequence.

  One of the antibodies of the present invention, or a functional fragment or derivative thereof, is a light antibody comprising CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively. And a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

  Finally, an antibody of the invention, or one of its functional fragments or derivatives, also comprises a light chain comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 8. It can be characterized by

  One of the antibodies of the present invention, or a functional fragment or derivative thereof, is a light antibody comprising CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 30, respectively. And a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 31, 32, and 33, respectively.

  Finally, an antibody of the invention, or one of its functional fragments or derivatives, also comprises a light chain comprising the amino acid sequence of SEQ ID NO: 34 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 35. It can be characterized by comprising.

  As used herein, “polypeptide”, “polypeptide sequence”, “peptide”, and “antibody compound or protein associated with those sequences” are interchangeable.

  The present invention does not relate to naturally occurring antibodies, i.e., they are not taken from their natural environment, but have been isolated or obtained by purification from natural sources, or It should be understood that they have been obtained by chemical synthesis and therefore they can contain unnatural amino acids as described below.

  In a first embodiment, the complementarity determining region or CDR is defined by Kabat et al. (Kabat et al., “Sequences of proteins of immunological interest”, 5th edition, US Department of Health. and the hypervariable regions of immunoglobulin heavy and light chains as defined by Human Services, NIH, 1991 and subsequent editions). There are three heavy chain CDRs and three light chain CDRs. As used herein, “CDR” refers to one or more, or even all, of the region containing most of the amino acid residues responsible for the binding affinity of the antibody to the antigen or epitope recognized by the antibody, depending on the case. Used to indicate.

  In a second embodiment, the CDR regions or CDRs shall refer to the immunoglobulin heavy and light chain hypervariable regions as defined by IMGT.

  IMGT unique numbering has been defined for comparing variable domains of any antigen receptor, chain type, or species [Lefranc M. -P., Immunology Today 18, 509 (1997) / Lefranc M.-P., The Immunologist, 7, 132-136 (1999) / Lefranc, M.-P., Pommie, C, Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol, 27, 55-77 (2003)]. In IMGT unique numbering, for example, cysteine 23 (1st-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine 104 (2nd-CYS), phenylalanine, or tryptophan 118 (J-PHE or J-TRP) For example, conserved amino acids always have the same position. In IMGT unique numbering, a framework region (FR1-IMGT: 1-26, FR2-IMGT: 39-55, FR3-IMGT: 66-104 and FR4-IMGT: 118-128) and complementarity determining region Standardized definitions of: CDR1-IMGT: 27-38, CDR2-IMGT: 56-65 and CDR3-IMGT: 105-117 are obtained. Since the gap represents an unoccupied position, the CDR-IMGT length (for example, [8.8.13] or the like and indicated by a dot in parentheses) is important information. IMGT unique numbering is based on IMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)], and IMGT / 3Dstructure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., "T cell receptor and MHC structure data. Nucl. Acids. Res., 32, D208-D210 (2004)].

  There are three heavy chain CDRs and three light chain CDRs. Here, the CDRs optionally comprise one or several of these regions, or even these regions, which comprise most of the amino acid residues responsible for binding of the antibody to the antigen or epitope recognized by the antibody. Used to indicate all of

  For clarity, it should be understood that in the following description and more particularly in Tables 2 and 3, CDRs are defined by the IMGT numbering system and the Kabat numbering system.

  The IMGT numbering system defines CDRs according to the IMGT system defined above, and the Kabat numbering system defines CDRs according to the Kabat system defined above.

  More specifically, for an antibody called 515H7, CDR-L1 consists of SEQ ID NO: 1 in the IMGT numbering system and SEQ ID NO: 9 in the Kabat numbering system. Regarding CDR-L2, it consists of SEQ ID NO: 2 in the IMGT numbering system and SEQ ID NO: 10 in the Kabat numbering system. CDR-L3 consists of SEQ ID NO: 3 in each of these two numbering systems. For the heavy chain, CDR-H1 consists of SEQ ID NO: 4 in the IMGT numbering system and SEQ ID NO: 11 in the Kabat numbering system. CDR-H2 consists of SEQ ID NO: 5 in the IMGT numbering system and SEQ ID NO: 12 in the Kabat numbering system. Finally, CDR-H3 consists of SEQ ID NO: 6 in the IMGT numbering system and SEQ ID NO: 13 in the Kabat numbering system.

  And for an antibody called 301aE5, CDR-L1 consists of SEQ ID NO: 1 in the IMGT numbering system and SEQ ID NO: 9 in the Kabat numbering system. Regarding CDR-L2, it consists of SEQ ID NO: 2 in the IMGT numbering system and SEQ ID NO: 36 in the Kabat numbering system. CDR-L3 consists of SEQ ID NO: 30 in the IMGT numbering system and SEQ ID NO: 37 in the Kabat numbering system. For the heavy chain, CDR-H1 consists of SEQ ID NO: 31 in the IMGT numbering system and SEQ ID NO: 38 in the Kabat numbering system. CDR-H2 consists of SEQ ID NO: 32 in the IMGT numbering system and SEQ ID NO: 39 in the Kabat numbering system. Finally, CDR-H3 consists of SEQ ID NO: 33 in the IMGT numbering system and SEQ ID NO: 40 in the Kabat numbering system.

  In the sense of the present invention, “percent identity” between two nucleic acid sequences or amino acid sequences means the proportion of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment. However, this proportion is purely statistical and the differences between the two sequences are randomly distributed along their length. Comparison of two nucleic acid or amino acid sequences is conventionally performed by comparing the sequences after optimal alignment of them, and this comparison can be done by segment or by using an “alignment window”. In addition to manual comparison, the optimal alignment of sequences for comparison is the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2: 482], Neddleman and Wunsch (1970) [J. Mol. Biol. 48: 443] local homology algorithm, Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85: 2444] similarity search method, or computer software using these algorithms Software (by Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI GAP, BESTFIT, FASTA and TFASTA, or comparable software BLAST NR or BLAST P).

  The percent identity between two nucleic acid or amino acid sequences is determined by comparing two optimally aligned sequences, where the nucleic acid or amino acid sequence being compared is the optimal alignment between the two sequences. Can have additions or deletions to the reference sequence. The percent identity determines the number of positions where an amino acid nucleotide or residue is identical between the two sequences, preferably between the entire two sequences, and the number of identical positions is divided by the total number of positions in the alignment window, Calculated by multiplying the quotient by 100 to obtain the percent identity between the two sequences.

  For example, the BLAST program “BLAST2 sequences” (Tatusova et al., “Blast 2 sequences-a new tool for comparing” available at the site http://www.ncbi.nlm.nih.gov/gorf/bl2.html protein and nucleotide sequences ", FEMS Microbiol, 1999, Lett. 174: 247-250) as default parameters (especially as parameters," open gap penalty ": 5 and" extension gap penalty ": 2, the selection matrix is , Which is “BLOSUM 62” proposed by this program), and the percent identity between the two sequences is calculated directly by this program.

  Preferred examples of amino acid sequences that exhibit at least 80%, preferably 85%, 90%, 95%, and 98% identity with the reference amino acid sequence include reference sequences, certain modifications, particularly at least one amino acid Including deletions, additions or substitutions, truncations or extensions. In the case of substitution of one or more consecutive or non-consecutive amino acids, substitutions in which the substituted amino acid is replaced with an “equivalent” amino acid are preferred. As used herein, “equivalent amino acid” is intended to indicate an amino acid that can replace one of the structural amino acids without giving any modification to the biological activity of the corresponding antibody and the specific examples defined below.

  Equivalent amino acids are determined based on the structural homology with the amino acid to be substituted or the results of a comparative test of biological activity between the various antibodies that can be generated.

  As a non-limiting example, Table 1 below summarizes the substitutions that can be made without resulting in significant modification of the biological activity of the corresponding modified antibody. Note that reverse substitution is also possible under the same conditions.

  In certain embodiments, the present invention relates to murine antibodies, or derived compounds or functional fragments thereof.

  As seen above, the present invention also relates to compounds derived from the antibodies described herein.

  More particularly, the antibody of the invention, or a derivative or functional fragment thereof, is grafted with at least one CDR such that the derivative compound preserves all or part of the paratope recognition properties of the original antibody. It is characterized by comprising a binding protein comprising a peptide backbone.

  Also, one or more of the CDR sequences described in the present invention may be present in various immunoglobulin protein scaffolds. In this case, this protein sequence can again create a peptide backbone that favors folding of the grafted CDRs, allowing them to preserve their paratope antigen recognition properties.

In general, those skilled in the art know how to determine the type of protein scaffold for grafting at least one of the CDRs from the original antibody. More specifically, it is known that such selected skeletons must meet the following maximum number of criteria (Skerra A., J. Mol. Recogn., 2000, 13: 167- 187):
-Good phylogenetic preservation;
-The three-dimensional structure is known (eg by crystallography, NMR spectroscopy or any other method known to the person skilled in the art),
-Small size,
-Little or no post-transcriptional modification and / or-easy production, expression and purification.

  The origin of such a protein backbone is, but not limited to, fibronectin and preferentially fibronectin type III domain 10, lipocalin, anticalin (Skerra A., J. Biotechnol., 2001, 74 (4): 257-75), protein Z derived from domain A of protein A of Staphylococcus aureus, thioredoxin A or “ankyrin repeat” (Kohl et al., PNAS, 2003, vol. 100, No. 4, 1700 -1705), “armadillo repeats”, “leucine rich repeats” and “tetratricopeptide repeats”.

  For example, skeletons derived from toxins such as scorpions, insects, plants, molluscs and other toxins and protein inhibitors of neuronal NO synthase (PIN) should be mentioned.

  An example of such a hybrid construct includes, but is not limited to, an anti-CD4 antibody in one of the PIN loops, ie, a CDR-H1 (heavy chain) insertion of 13B8.2, thus obtained. The new binding protein preserves the same binding properties as the original antibody (Bes et al., Biochem. Biophys. Res. Commun., 2006, 343 (1), 334-344). By way of example only, grafting the anti-lysozyme VHH antibody CDR-H3 (heavy chain) to one of the loops of neocalcinostatin.

  Finally, as noted above, such a peptide backbone can comprise at least one of the CDRs derived from the original antibody. Preferably, but not necessarily, one skilled in the art can select at least one CDR from the heavy chain, which is known to be primarily responsible for the specificity of the antibody. The selection of one or more appropriate CDRs will be obvious to those skilled in the art, who can then select a suitable known technique (Bes et al., FEBS letters 508, 2001, 67- 74).

A particular aspect of the invention relates to a method for selecting a compound derived from an antibody according to the invention, wherein the inducing compound is capable of inhibiting HIV cell entry in vitro and / or in vivo, wherein the inducing compound comprises: Comprising a peptide backbone onto which at least one antibody CDR is grafted, the method comprising:
a) contacting in vitro a compound composed of a peptide backbone to which at least one antibody CDR is grafted with a biological sample containing HIV type 1 and PBMC, and b) the compound undergoes HIV-1 replication. If it can be inhibited, comprising selecting the compound;
At least one grafted CDR is at least 80%, preferably 85%, 90% with the sequence of SEQ ID NO: 1-6 and 30-33, or the sequence of SEQ ID NO: 1-6 and 30-33 after optimal alignment , 95%, and 98% sequence CDRs.

  According to a preferred mode, the method may comprise contacting in vitro in step a) a compound comprising a peptide backbone onto which at least two or three antibody CDRs are grafted.

  According to a further preferred mode of this method, the peptide backbone is selected from among the backbones or binding proteins whose structures are described above.

  Obviously, these examples are not limited in any way, and any other structure known or obvious to those skilled in the art should be considered to be covered by the protection conferred by this patent application.

  Thus, the present invention provides that the peptide backbone is a) a well-conserved protein, b) a protein with a robust structure, c) a protein with a well-known 3D molecular configuration, d) a small protein And / or e) an antibody selected from proteins comprising regions that can be modified by deletion and / or insertion without altering stability, or a derivative compound or function thereof Related to dynamic fragments.

  According to a preferred embodiment, the antibody of the present invention, or a derivative or functional fragment thereof, comprises a peptide backbone from i) a backbone derived from fibronectin, preferentially fibronectin type 3 domain 10, lipocalin, anticalin, Protein Z, thioredoxin A or "ankyrin repeat" derived from domain B of protein A of Staphylococcus aureus (Kohl et al., PNAS, 2003, vol. 100, No. 4, 1700-1705), " It is characterized in that it is selected from proteins having repetitive motifs such as “armadillo repeat”, “leucine rich repeat” and “tetratricopeptide repeat”, or iii) neuronal NO synthase (PIN) protein inhibitors.

  Another aspect of the invention relates to a functional fragment of said antibody.

More particularly, the present invention provides that the functional fragment has an extended half-life, such as fragments Fv, Fab, (Fab ′) ₂ , Fab ′, scFv, scFv-Fc, and diabodies, or PEGylated fragments. It is directed to an antibody characterized by being selected from any fragment, or a derivative compound or functional fragment thereof.

Such functional fragments of the antibodies according to the invention are for example the fragments Fv, scFv (sc = single chain), Fab, F (ab ′) ₂ , Fab ′, scFv-Fc or diabody, or polyethylene glycol ( By chemical modification such as addition of polyalkylene glycols such as (PEGylated) (PEGylated fragments are referred to as Fv-PEG, scFv-PEG, Fab-PEG, F (ab ′) ₂ -PEG and Fab′-PEG), or Consists of any fragment whose half-life has been extended by incorporation into liposomes, microspheres, or PLGA, which may in particular show the general activity, or even partial activity, of the antibody from which it is derived. And having at least one of the characteristic CDRs of the present invention.

  Preferably, the functional fragment comprises or comprises a partial sequence of the variable heavy or light chain of the antibody from which it is derived, the partial sequence having the same binding specificity as the antibody from which it is derived. And sufficient affinity, preferably sufficient to retain at least 1/100, more preferably at least 1/10 of the affinity of the antibody from which it is derived.

  Such a functional fragment may comprise at least 5 amino acids, preferably 6, 7, 8, 10, 15, 25, 50, or 100 consecutive amino acids of the sequence of the antibody from which it is derived.

  Preferably, these functional fragments are Fv, scFv, Fab, F (ab ′) 2, F (ab ′), scFv-Fc, or dia, which generally have similar binding specificity as the antibody from which they are derived. It can be of body type. According to the present invention, the fragment of the antibody of the present invention can be obtained from the aforementioned antibody by a method such as enzymatic digestion containing pepsin or papain and / or cleavage of disulfide bond by chemical reduction. The antibody fragments can also be obtained by recombinant gene technology, also known to those skilled in the art, or by peptide synthesis with an automated peptide synthesizer such as sold by, for example, Applied BioSystems.

  For further clarity, Table 2 below summarizes various amino acid sequences corresponding to the antibodies of the present invention.

  A particularly important additional aspect of the antibody subject of the present invention is that they do not exhibit effector functions such as antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

  More specifically, by way of example, one of the antibodies of the present invention, or a functional fragment or derivative thereof, is directed against FcγR (I, II, or III), or against C1q, or both. Has no affinity for.

  Structurally, this is appropriate for those skilled in the art that one of the antibodies of the invention, or a functional fragment or derivative thereof, lacks an Fc moiety, or that the Fc part may confer an effector function. Means no glycosylation.

  The result is that the antibodies of the invention are preferably selected from IgG4 or IgG2 isoforms, most preferably IgG4 isoforms.

Similarly, preferred fragments are Fv, scFv (sc is single chain), Fab, F (ab ′) ₂ , Fab ′, scFv-Fc fragment or diabody, or poly (ethylene) glycol (“PEGylated”). (Pegylated fragments are referred to as Fv-PEG, scFv-PEG, Fab-PEG, F (ab ′) ₂ -PEG, or Fab′-PEG) (“PEG” is poly (ethylene) glycol) and other poly (alkylenes) ) Fragments lacking ADCC, such as any fragment whose half-life has been extended by chemical modification such as the addition of glycols or by incorporation into liposomes.

  More particularly, a preferred functional fragment of the present invention derived from antibody 515H7 is scFv, hereinafter referred to as 515H7 scFv-Ck fragment, comprising the amino acid sequence of SEQ ID NO: 54.

  The nucleotide sequence corresponding to the scFv comprises the sequence of SEQ ID NO: 55.

  Another particular aspect of the present invention is a chimeric antibody, or derivative compound thereof, characterized in that the antibody also comprises light and heavy chain constant regions derived from antibodies that are heterologous to mice, in particular humans Or it relates to a functional fragment.

  Yet another aspect of the present invention is a humanized antibody, characterized in that the constant regions of the light and heavy chains derived from a human antibody are the λ or κ region and γ-2 or preferably γ-4 region, respectively. Or a derivative or functional fragment thereof.

  The antibodies of the present invention also comprise chimeric or humanized antibodies.

  A chimeric antibody is one that contains naturally occurring variable regions (light and heavy chains) derived from an antibody of a given species in combination with light and heavy chain constant regions of an antibody that is heterologous to the given species.

  Antibodies, or chimeric fragments thereof, can be prepared using recombinant gene technology. For example, the chimeric antibody is produced by cloning recombinant DNA comprising a promoter, a sequence encoding the variable region of a non-human, particularly murine monoclonal antibody of the present invention, and a sequence encoding the constant region of a human antibody. be able to. A chimeric antibody according to the present invention encoded by one such recombinant gene may be, for example, a mouse-human chimera, the specificity of which is determined by the variable region derived from murine DNA and its isoforms Is determined by the constant region derived from human DNA. See Verhoeyn et al. (BioEssays, 8:74, 1988) for methods of preparing chimeric antibodies.

  Table 3 below summarizes the amino acid sequences of various heavy and light chains of chimeric antibody 515H7 (referred to as c515H7 or C515H7) according to the present invention.

  The nucleotide sequences corresponding to the antibody c515H7 heavy chain SEQ ID NO: 56-58 and light chain SEQ ID NO: 59 correspond to the sequences of SEQ ID NO: 60-63 (heavy chain) and SEQ ID NO: 64 (light chain), respectively.

  In a preferred embodiment, these heavy chain sequences have lysine residues deleted from their C-terminus (found in pConPlus vector phylogeny from Lonza: pConPlusγ4ΔK, pConPlusγγ4PROΔK, and pConPlusγ2ΔK).

  Furthermore, the G4PRO heavy chain represents a human IgG4 isoform with a mutation in the hinge region to avoid the formation of half antibodies. This mutation is found in the parent strain pConPlusγ4PROΔK from Lonza [Angal S, King DJ, Bodmer MW, Turner A, Lawson AD, Roberts G, Pedley B, Adair JR. IgG4) eliminates heterogeneity of antibody (A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody). "Mol Immunol. (1993); 30 (1): 105-108].

  More particularly, the present invention relates to a chimeric antibody heavy chain characterized in that it comprises CDRs homologous to the corresponding CDRs of antibodies from different mammalian species, wherein the CDRs are according to IMGT. For example, it consists of CDR-H1, CDR-H2, and CDR-H3 comprising the sequences of SEQ ID NOs: 4, 5, and 6, respectively.

  More particularly, the invention relates to a chimeric antibody light chain, characterized in that it comprises a CDR homologous to the corresponding CDR of an antibody from various mammalian species, wherein the CDR is according to IMGT. For example, it consists of CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 1, 2, and 3, respectively.

  More particularly, the present invention relates to a chimeric antibody characterized in that it comprises heavy and light chains each having CDRs homologous to the corresponding CDRs of antibodies from various mammalian species, or a derivative compound thereof or For functional fragments, where the CDRs, according to IMGT, are the heavy chain CDR-H1, CDR-H2, and CDR-H3 comprising the sequences of SEQ ID NOs: 4, 5, and 6, respectively, It consists of the light chain CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of numbers 1, 2, and 3, respectively.

  In another embodiment, the invention relates to a chimeric antibody comprising a heavy chain variable region of the sequence consisting of SEQ ID NO: 8 and a light chain variable region of the sequence of SEQ ID NO: 7, or a derivative or functional fragment thereof. .

  In yet another embodiment, the invention provides a chimeric antibody comprising a heavy chain of a sequence selected from the group consisting of SEQ ID NO: 56, 57, or 58 and a light chain of the sequence of SEQ ID NO: 59, or Relates to derived compounds or functional fragments.

  In a preferred embodiment, the chimeric antibody c515H7 VH (G4wt) / VL-Ck, or a derivative or functional fragment thereof according to the present invention comprises a heavy chain variable region of sequence SEQ ID NO: 56 and a light chain of sequence SEQ ID NO: 59. And a variable region.

  In a preferred embodiment, the chimeric antibody c515H7 VH (G4PRO) / VL-Ck, or a derivative or functional fragment thereof according to the present invention comprises a heavy chain variable region of the sequence of SEQ ID NO: 57 and a light chain of the sequence of SEQ ID NO: 59. And a variable region.

  In a preferred embodiment, the chimeric antibody c515H7 VH (G2wt) / VL-Ck, or a derivative or functional fragment thereof according to the present invention comprises a heavy chain variable region of sequence SEQ ID NO: 58 and a light chain of sequence SEQ ID NO: 59. And a variable region.

  “Humanized antibody” refers to an antibody comprising a CDR region derived from an antibody of non-human origin, the other part of which is derived from one (or more) human antibody. In addition, some of the backbone segment residues (called FR) can be modified to preserve binding affinity (Jones et al., Nature, 321: 522-525, 1986, Verhoeyen et al., Science, 239: 1534-1536, 1988, Riechmann et al., Nature, 332: 323-327, 1988).

  The humanized antibody or fragment thereof of the present invention can be obtained by techniques known to those skilled in the art (for example, Singer et al., J. Immun., 150: 2844-2857, 1992, Mountain et al., Biotechnol. Genet. Eng. Rev. , 10: 1-142, 1992, and Bebbington et al., Bio / Technology, 10: 169-175, 1992). Such humanized antibodies are preferred for use in methods such as in vitro diagnostics or in vivo prophylactic and / or therapeutic treatments. Other humanization techniques are also known to those skilled in the art, such as EP 0451261, 0682040, 0939127, 0656647, or US Pat. The "PDR grafts" described by PDL in 530,101, 6,180,370, 5,585,089, and 5,693,761 There is a law. U.S. Pat. Nos. 5,639,641 or 5,639,641 or 6,054,297, 5,886,152, and 5,877, No. 293 can also be cited.

  Furthermore, the present invention also relates to a humanized antibody derived from the murine antibody described above.

  In a preferred manner, the light and heavy chain constant regions derived from human antibodies are the λ or κ and γ-2 or preferably γ-4 regions, respectively.

  More particularly, the invention includes i) a framework region that is homologous to the framework region of the corresponding human antibody heavy chain, and ii) a CDR that is homologous to the corresponding CDRs of antibodies from various mammalian species. Wherein the CDR is, according to IMGT, CDR-H1, CDR-H2, comprising the sequences of SEQ ID NOs: 4, 5, and 6, respectively, and It consists of CDR-H3.

  In another embodiment, the invention relates to a humanized antibody heavy chain comprising the variable region of the sequence consisting of SEQ ID NO: 64.

  In yet another embodiment, the invention relates to a humanized antibody heavy chain comprising the complete sequence selected from the group consisting of SEQ ID NOs: 67, 68, 69 and 95.

  More particularly, the invention includes i) a framework region that is homologous to the corresponding framework region of a human antibody light chain, and ii) a CDR that is homologous to the corresponding CDRs of antibodies from various mammalian species. Wherein the CDR is, according to IMGT, CDR-L1, CDR-L2, and CDR-L1, comprising the sequences of SEQ ID NOs: 1, 2, and 3, respectively, It consists of CDR-L3.

  In another embodiment, the invention relates to a humanized antibody light chain comprising a variable region of a sequence selected from the group consisting of SEQ ID NOs: 65, 66, 82, or 83.

  In yet another embodiment, the invention relates to a humanized antibody light chain comprising a complete sequence selected from the group consisting of SEQ ID NOs: 70, 71, 84, or 85.

  More particularly, the present invention relates to heavy chains each having i) a framework region that is homologous to the corresponding framework region of a human antibody, and ii) a CDR that is homologous to the corresponding CDRs of antibodies from various mammalian species. And the derived compound or functional fragment thereof, wherein the CDR comprises the sequences of SEQ ID NOs: 4, 5, and 6, respectively, according to IMGT CDR-H1, CDR-H2, and CDR-H3 of the heavy chain comprising, and CDR-L1, CDR-L2, and CDR-L3 of the light chain comprising the sequences of SEQ ID NOs: 1, 2, and 3, respectively. Consists of.

  In another embodiment, the invention comprises a heavy chain variable region of sequence consisting of SEQ ID NO: 64 and a light chain variable region of sequence selected from the group consisting of SEQ ID NO: 65, 66, 82, or 83. Or a derived compound or functional fragment thereof.

  In yet another embodiment, the invention provides a heavy chain of a sequence selected from the group consisting of SEQ ID NO: 67, 68, 69 or 95 and a sequence selected from the group consisting of SEQ ID NO: 70, 71, 84 or 85 And a derived compound or functional fragment thereof.

  In a preferred embodiment, the humanized antibody Hz515H7 VH1 D76N (G4wt) / VL2-Ck, or a derivative or functional fragment thereof according to the invention comprises a heavy chain of the sequence SEQ ID NO: 67 and a light chain of the sequence SEQ ID NO: 70. And comprising.

  In another preferred embodiment, the humanized antibody Hz515H7 VH1 D76N (G4PRO) / VL2-Ck, or a derivative or functional fragment thereof according to the present invention comprises a heavy chain of the sequence of SEQ ID NO: 68, and a sequence of SEQ ID NO: 70. A light chain.

  In another preferred embodiment, the humanized antibody Hz515H7 VH1 D76N (G2wt) / VL2-Ck, or a derivative or functional fragment thereof according to the present invention, comprises a heavy chain of the sequence of SEQ ID NO: 69 and a sequence of SEQ ID NO: 70. A light chain.

  In another preferred embodiment, the humanized antibody Hz515H7 VH1 D76N (G4wt) /VL2.1-Ck, or a derivative or functional fragment thereof, according to the present invention comprises the heavy chain of the sequence of SEQ ID NO: 67, the sequence of SEQ ID NO: 71, A light chain of the sequence.

  In another preferred embodiment, the humanized antibody Hz515H7 VH1 D76N (G4PRO) /VL2.1-Ck, or a derivative or functional fragment thereof according to the invention comprises the heavy chain of sequence SEQ ID NO: 68, A light chain of the sequence.

  In another preferred embodiment, the humanized antibody Hz515H7 VH1 D76N (G2wt) /VL2.1-Ck, or a derivative or functional fragment thereof according to the present invention comprises the heavy chain of sequence SEQ ID NO: 69, A light chain of the sequence.

  In another preferred embodiment, the humanized antibody Hz515H7 VH1 D76N (G4wt) /VL2.2-Ck, or a derivative or functional fragment thereof according to the present invention comprises the heavy chain of the sequence of SEQ ID NO: 67 and the sequence of SEQ ID NO: 84 A light chain of the sequence.

  In another preferred embodiment, the humanized antibody Hz515H7 VH1 D76N (G4PRO) /VL2.2-Ck, or a derivative or functional fragment thereof according to the present invention comprises the heavy chain of the sequence of SEQ ID NO: 68 and the sequence of SEQ ID NO: 84 A light chain of the sequence.

  In another preferred embodiment, the humanized antibody Hz515H7 VH1 D76N (G2wt) /VL2.2-Ck, or a derivative or functional fragment thereof according to the present invention comprises the heavy chain of the sequence of SEQ ID NO: 69, A light chain of the sequence.

  In another preferred embodiment, the humanized antibody Hz515H7 VH1 D76N (G4wt) /VL2.3-Ck, or a derivative or functional fragment thereof according to the invention comprises the sequence heavy chain of SEQ ID NO: 67 and the sequence of SEQ ID NO: 85 A light chain.

  In another preferred embodiment, the humanized antibody Hz515H7 VH1 D76N (G4PRO) /VL2.3-Ck, or a derivative or functional fragment thereof according to the present invention comprises the heavy chain of the sequence of SEQ ID NO: 68, the sequence of SEQ ID NO: 85 A light chain of the sequence.

  In another preferred embodiment, the humanized antibody Hz515H7 VH1 D76N (G2wt) /VL2.3-Ck, or a derivative or functional fragment thereof according to the present invention comprises the heavy chain of sequence SEQ ID NO: 69, A light chain of the sequence.

  Table 4 below summarizes the various heavy and light chain variable domains and full-length (or complete) amino acid sequences of the humanized antibody 515H7 according to the present invention, respectively.

  In a preferred embodiment, these heavy chain sequences have lysine residues deleted from their C-terminus (found in pConPlus vector phylogeny from Lonza: pConPlusγ4ΔK, pConPlusγγ4PROΔK, and pConPlusγ2ΔK).

  Furthermore, the G4PRO heavy chain represents a human IgG4 isoform with a mutation in the hinge region to avoid the formation of half antibodies. This mutation is found in the parent strain pConPlusγ4PROΔK from Lonza [Angal S, King DJ, Bodmer MW, Turner A, Lawson AD, Roberts G, Pedley B, Adair JR. IgG4) eliminates heterogeneity of antibody (A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody). "Mol Immunol., (1993); 30 (1): 105-108].

  Specifically, the present invention provides a humanized antibody termed hz515H7 IgG4 comprising the heavy chain of the human IgG4 isoform having the sequence represented by SEQ ID NO: 95.

  As an example, to avoid doubt, “VH1” is similar to the expression “VH variant 1”, “VH variant 1”, “VH Var 1”, or “VH var 1”.

  It should be understood that the VH / VL combinations illustrated above are not limiting. One skilled in the art will of course be able to rearrange all of the VH and VL disclosed herein without undue burden and without using inventive techniques.

The novel aspects of the invention include the following nucleic acids (including the degenerate genetic code):
a) a nucleic acid, DNA or RNA encoding an antibody according to the invention, or one of its functional fragments or derivatives,
b) a nucleic acid comprising a DNA sequence selected from the sequence group consisting of SEQ ID NOs: 14-19 and 41-45,
c) a nucleic acid comprising a DNA sequence selected from the sequence group consisting of SEQ ID NOs: 20, 21, 46, and 47;
d) a corresponding RNA nucleic acid of the nucleic acid as defined in b) or c),
e) hybridizing under high stringency conditions with a complementary nucleic acid of the nucleic acids defined in a), b) and c), and f) at least one of the CDRs of the sequences of SEQ ID NOs: 14-19 and 41-45. Relates to an isolated nucleic acid, characterized in that it is selected from nucleic acids of at least 18 nucleotides.

  Table 5 below summarizes the various nucleotide sequences for the antibodies of the present invention.

  As used herein interchangeably, “nucleic acid”, “nuclear sequence”, “nucleic acid sequence”, “polynucleotide”, “oligonucleotide”, “polynucleotide sequence”, and “nucleotide sequence” are non-natural nucleotides. Means the exact sequence of modified or unmodified nucleotides that define a fragment or region of nucleic acid that may or may not contain and is either double-stranded DNA, single-stranded DNA, or a transcript of these DNAs .

  It should also be included here that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, ie in the natural state. The sequences of the invention are isolated and / or purified, ie they are sampled directly or indirectly, eg by copying, and their environment is at least partially modified. An isolated nucleic acid obtained, for example, by recombinant gene technology with a host cell or obtained by chemical synthesis should also be mentioned here.

  “A nuclear sequence that exhibits at least 80%, preferably 85%, 90%, 95%, and 98% percent identity with a preferred sequence after optimal alignment” is particularly a deletion compared to a reference nuclear sequence. Means a nuclear sequence exhibiting certain modifications, such as truncations, extensions, chimeric fusions, and / or substitutions, especially regular ones. Preferably, these are sequences that encode the same amino acid sequence as the reference sequence, which is specifically degenerate from the genetic code, or preferably under highly stringent conditions, especially under the conditions defined below. Associated with a complementary sequence capable of hybridizing.

  For hybridization under high stringency conditions, conditions relating to temperature and ionic strength are selected such that hybridization can be maintained between two complementary DNA fragments. By way of example only, the high stringency conditions of the hybridization step for the purpose of defining the aforementioned polynucleotide fragments are advantageously as follows.

  DNA-DNA or DNA-RNA hybridization consists of (1) 5 × SSC (1 × SSC corresponds to a solution of 0.15 M NaCl + 0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulfate (SDS ) Prehybridization for 3 hours at 42 ° C. in phosphate buffer (20 mM, pH 7.5) containing 10 × Denhart solution, 5% dextran sulfate, and 1% salmon sperm DNA, (2) to probe length Main hybridization for 20 hours at the corresponding temperature (ie, 42 ° C. for probes> 100 nucleotides long) followed by two 20 minute washes in 2 × SSC + 2% SDS at 20 ° C., 0.1 × SSC + 0.1 Performed in two steps, one wash for 20 minutes at 20 ° C. in% SDS. The last wash is performed at 60 ° C. for 30 minutes in 0.1 × SSC + 0.1% SDS for probes> 100 nucleotides long. The high stringency hybridization conditions described above for size-defined polynucleotides are similar to Sambrook et al. ("Molecular cloning: a laboratory manual", Cold, for longer or shorter oligonucleotides. The procedures described in Spring Harbor Laboratory, 3rd edition, 2001) can be adapted by those skilled in the art.

The present invention also provides the following nucleic acids:
a) a nucleic acid, DNA or RNA encoding a humanized antibody heavy chain according to the invention, or a derivative or functional fragment thereof,
b) a nucleic acid, DNA or RNA encoding a humanized antibody light chain according to the invention, or a derivative or functional fragment thereof,
c) a nucleic acid, DNA or RNA encoding a humanized antibody according to the invention, or a derivative or functional fragment thereof,
d) a nucleic acid complementary to the nucleic acid defined in a), b) or c),
e) a nucleic acid of at least 18 nucleotides capable of hybridizing under high stringency conditions with at least the heavy chain comprising the nucleic acid sequence of SEQ ID NO: 72 or 75-77;
f) at least 18 nucleotides capable of hybridizing with at least the light chain comprising the nucleic acid sequence of SEQ ID NO: 73, 74, 86, 87, or 78, 79, 88, 89 under high stringency conditions. Nucleic acid,
An isolated nucleic acid molecule, characterized in that it is selected from:

  Table 6 below summarizes the various heavy and light chain variable domains and full-length (or complete) nucleotide sequences of humanized antibody 515H7 according to the present invention, respectively.

  Table 7 below summarizes the nucleotide sequences of various heavy and light chains of chimeric antibody 515H7 according to the present invention.

In other words, the present invention includes the following nucleic acids:
a) a nucleic acid, DNA or RNA encoding an antibody according to the invention, or one functional fragment or derivative thereof,
b) a nucleic acid comprising a DNA sequence selected from the CDR sequence group consisting of SEQ ID NOs: 14-19 and 41-45,
c) a nucleic acid comprising a DNA sequence selected from the heavy and light chain variable domain sequences consisting of SEQ ID NOs: 20, 21, 46, 47, 72, 73, 74, 86, and 87;
d) a nucleic acid comprising a DNA sequence selected from the heavy and light chain sequences consisting of SEQ ID NOs: 60-63, 75-79, 88, 89 and 94;
e) a nucleic acid comprising a DNA sequence consisting of SEQ ID NO: 55;
f) the corresponding RNA nucleic acid of the nucleic acid defined in b), c), d), or e),
g) complementary nucleic acids of the nucleic acids defined in a), b), c), d), and e), and h) CDRs of the sequences of SEQ ID NOs: 14-19 and 41-45 under high stringency conditions Handles an isolated nucleic acid characterized in that it is selected from nucleic acids of at least 18 nucleotides capable of hybridizing with at least one.

  The present invention also relates to vectors comprising the nucleic acids described herein.

  The present invention is particularly directed to cloning and / or expression vectors containing such nucleotide sequences.

  The vectors of the present invention preferably contain elements that allow the expression and / or secretion of the nucleotide sequence in a given host cell. Thus, the vector must contain a promoter, translation initiation and termination signals, and suitable transcriptional regulatory regions. The vector must be capable of being maintained in a stable manner in the host cell, and may optionally have specific signals that specify the secretion of the translated protein. These various elements are selected and optimized by one skilled in the art depending on the host cell used. For this purpose, these nucleotide sequences can be inserted into a vector that self-replicates in the selected host, or can be an integrated vector of the selected host.

  Such vectors are prepared by methods commonly used by those skilled in the art, and the resulting clones are introduced into a suitable host by standard methods such as lipofection, electroporation, heat shock, or chemical methods. Can do.

  These vectors are, for example, plasmid or viral origin vectors. They can be used to transform host cells in order to clone or express the nucleotide sequences of the invention.

  The present invention also comprises a host cell transformed with or comprising a vector described in the present invention.

  The host cell can be selected from prokaryotic or eukaryotic cell systems such as yeast cells or animal cells, especially mammalian cells, as well as bacterial cells. Insect or plant cells can also be used.

  The invention also relates to a non-human animal having a transformed cell according to the invention.

Another aspect of the present invention relates to a method for producing an antibody according to the present invention, or one functional fragment thereof, characterized in that it comprises the following steps:
a) a culture step of the host cell according to the invention in a culture medium and suitable culture conditions, and b) the recovery of the antibody, or one functional fragment thereof, thus produced from the culture medium or the culture cell. Process.

  The transformed cell according to the present invention is used in the method for preparing a recombinant polypeptide according to the present invention. The present invention also includes a method for preparing a polypeptide according to the present invention in a recombinant form, characterized by using the vector according to the present invention and / or a cell transformed with the vector. Preferably, the cells transformed with the vector according to the invention are cultured under conditions allowing the expression of said polypeptide and the recovery of said recombinant peptide.

  As already mentioned, the host cell can be selected from prokaryotic or eukaryotic systems. In particular, it is possible to identify the nucleotide sequences of the invention that aid secretion in such prokaryotic or eukaryotic systems. Thus, the vector according to the present invention having such a sequence can be advantageously used for the production of recombinant proteins to be secreted. In fact, purification of these noted recombinant proteins is facilitated by their presence in the cell culture supernatant rather than in the host cell.

  The polypeptides of the present invention can also be prepared by chemical synthesis. One such preparation method is also an object of the present invention. Those skilled in the art will be familiar with solid phase techniques by condensing fragments or by conventional synthesis in solution (in particular Steward et al., 1984, “Solid phase peptides synthesis”, Pierce Chem. Company, Rockford, 111 , See 2nd edition) or know chemical synthesis methods such as partial solid phase technology. Polypeptides obtained by chemical synthesis and which may contain the corresponding unnatural amino acid are also included in the present invention.

  Antibodies that may be obtained by the methods of the present invention, or derived compounds or functional fragments thereof are also included in the present invention.

  According to yet another aspect, the invention is further characterized in that it can specifically bind to human chemokine family receptors and / or can specifically inhibit X4-directed HIV replication. It relates to an antibody as described above.

  According to yet another aspect, the invention is characterized in that it can specifically bind to a human chemokine family receptor and / or can specifically inhibit X4 / R5-directed HIV replication. , And relates to an antibody as described above.

  According to a novel embodiment, the invention relates to, for example, CCR5, CD4, CXCR4 (other than the antibody of the invention, ie targeting another epitope) or CCR3, CCR2, CCR8, CXCR6, CXCR7, CX3CR1, etc. Relates to an antibody consisting of an antibody that is bispecific in the sense that it comprises a second motif capable of interacting with a receptor involved in cell entry of HIV, or a derivative or functional fragment thereof.

  Bispecific or bifunctional antibodies constitute a second generation monoclonal antibody in which two different variable regions are combined in the same molecule (Hollinger and Bohlen, 1999, Cancer and metastasis, rev. 18: 411- 419). Their utility has been demonstrated in both diagnostic and therapeutic domains with respect to their ability to target several molecules on the cell surface, and such antibodies have been demonstrated by chemical methods (Glennie MJ et al. , 1987, J. Immunol. 139, 2367-2375, Repp R. et al., 1995, J. Hemat., 377-382) or somatic cell method (Staerz UD and Bevan MJ, 1986, PNAS 83, 1453-1457). , Suresh MR et al., 1986, Method Enzymol, 121: 210-228) but preferentially imposes heterodimer formation on the desired antibody, thus facilitating purification of the antibody. Can be obtained by genetic engineering techniques (Merchand et al., 1998, Nature Biotech., 16: 677-681).

  These bispecific antibodies can be constructed not only as full IgG, bispecific Fab′2, Fab′PEG, diabody, or bispecific scFv, but also for each targeted antigen. It can also be constructed as a tetravalent bispecific antibody (Park et al, 2000, Mol Immunol., 37 (18): 1123-30) or a fragment thereof as described above in which one binding site is present.

  In addition to the economic advantages when considering the production and administration of a single bispecific antibody is cheaper than the production of two specific antibodies, the use of such a bispecific antibody is a treatment toxicity. It also has the advantage of reducing In fact, the use of bispecific antibodies can reduce the total amount of circulating antibody, resulting in reduced toxicity.

  In a preferred embodiment of the invention, the bispecific antibody is a bivalent or tetravalent antibody.

  Finally, the present invention relates to the aforementioned antibodies as drugs, or one of their functional fragments or derivatives.

  The present invention also relates to a pharmaceutical composition comprising as an active ingredient a compound comprising the antibody of the present invention, or one of its functional fragments or derivatives. Preferably, the antibody adds excipients and / or pharmaceutically acceptable carriers.

  The invention also relates to a composition as described above as a medicament.

In a particular aspect of the invention, the antibody, or one of its functional fragments or derivatives, replicates HIV-1 KON primary isolate in PBMC with an IC ₉₀ of at least 5 μg / ml, preferably at least 10 μg / ml. Inhibits.

  The invention also comprises the use of an antibody or composition according to the invention for the manufacture of a medicament and / or medicament for the prevention or treatment of HIV infection.

  More particularly, but not limited to, the HIV infection is an X4-directed HIV infection.

  In another embodiment, but not limited to, the HIV infection is an X4 / R5-directed HIV infection.

  The invention also relates to the use of an antibody according to the invention, or a functional fragment or derivative thereof, preferably humanized, and / or a composition for the manufacture of a medicament for inhibiting HIV replication. In general, the invention relates to the use of antibodies, or functional fragments or derivatives thereof, preferably humanized, and / or compositions for the manufacture of a medicament for the prevention or treatment of HIV disease.

  As used herein, a “pharmaceutical vehicle” does not cause a secondary reaction and, for example, facilitates administration of an active compound, prolongs its lifetime in an organism, and / or increases efficacy. Or a compound or combination of compounds (including pharmaceutical compositions) that increases its solubility in solution or increases its storage properties. Such pharmaceutical carriers are well known and will be adapted by those skilled in the art depending on the nature of the active compound chosen and the route of administration.

  Preferably, such compounds are administered by systemic routes, particularly intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intravaginal, or oral routes. More preferably, the composition composed of the antibody according to the present invention is administered in several doses separated by a certain time.

  Their route of administration, dosage regimen, and optimal dosage form will be used in establishing treatments that are appropriate for the patient, such as, for example, the patient's age or weight, the severity of the patient's health, tolerance to the patient's treatment, and the side effects experienced. Can be determined according to generally considered criteria.

  The present invention also relates to a composition further comprising an anti-HIV antibody or anti-HIV cell invasion antibody or anti-HIV replicating antibody other than an antibody against CXCR4 as a combination for use in a simultaneous, separate or extended manner.

  According to yet another embodiment, the present invention also specifically inhibits HIV entry and / or replication, such as anti-CCR5, anti-CD4 compounds, and anti-CXCR4 compounds other than those described in the present invention. Or a pharmaceutical composition as described above comprising at least one second anti-HIV compound selected from among compounds that can be made, or any other anti-HIV compound known to those skilled in the art.

  Another supplementary embodiment of the invention consists of a composition as described above further comprising an anti-HIV compound as a combination or composite for simultaneous, separate or extended use.

  “Simultaneous use” means that both compounds contained in a composition contained in a single dosage form are administered.

  “Individual use” means that both compounds of the composition contained in different dosage forms are administered simultaneously.

  “Extended use” means sequential administration of both compounds of the composition, each contained in different dosage forms.

  In general, the composition according to the invention significantly increases the effectiveness of HIV treatment. In other words, the therapeutic effect of the antibody of the present invention is enhanced unexpectedly by administration of an anti-HIV agent. Another major secondary advantage provided by the composition of the present invention relates to the possibility of using lower effective doses of the active ingredient, thus avoiding the risk of the appearance of side effects, particularly the action of anti-HIV agents. Or it becomes possible to reduce. Furthermore, this composition makes it possible to achieve the expected therapeutic effect more rapidly.

  “Therapeutic anti-HIV agent” means a substance that, when administered to a patient, treats or prevents HIV replication in the patient. Non-limiting examples of such agents include HIV protease inhibitors (PI), nucleoside / nucleotide HIV reverse transcriptase inhibitors (NRTI / NtRTI), non-nucleoside HIV reverse transcriptase inhibitors (NNRTI), HIV entry inhibitors And “antiretroviral agents” such as HIV integrase inhibitors.

  Such agents are listed, for example, on pages directed to compounds relating to VIDAL's “anti-HIV compounds”, and the anti-HIV compounds listed by the references in this document are limited herein. Preferred anti-HIV agents not mentioned.

  An HIV protease inhibitor refers to any substance that can inhibit HIV protease activity. Examples of such HIV protease inhibitors include, but are not limited to, saquinavir mesylate or SQV (Invirase ™), indinavir or IDV (Crixivan ™), ritonavir or RTV (Norvir ™) ), Nelfinavir or NFV (Viracept ™), amprenavir (Agenerase ™, Prozei ™), lopinavir / ritonavir or LPV / r (Kaletra ™, Aluvia ™), atazanavir or ATV ( Reyataz (TM), Zrivada (TM), fosamprenavir or FPV (Lexiva (TM), Telzir (TM)), tipranavir or TPV (Aptivus (TM)), darunavir or DRV (Prezista (TM)) It is done.

  An HIV nucleoside or nucleotide reverse transcriptase inhibitor (NRTI) refers to a substance that is a nucleoside or nucleotide analog that blocks reverse transcription of HIV RNA. Examples of NRTIs include, but are not limited to, zidovudine or AZT, ZDV (Retrovir / combivir / trixivir ™), didanosine or ddi (Videx ™), sarcitabine (HIVID ™), stavudine or d4T (Zerit ™), lamivudine or 3TC (Epivir / combivir / epzicom / trixivir ™), abacavir or ABC (Ziagen / trixivir / epzicom ™), tenofovir disoproxil fumarate or TDF (Viread / atripla / truvada (TM)), emtricitabine or FTC (Emtriva / atripla / truvada (TM)).

  A non-nucleoside HIV reverse transcriptase inhibitor (NNRTI) refers to a substance that is not a nucleoside or nucleotide analog that blocks reverse transcription of HIV RNA. Examples of NNRTI include, but are not limited to, nevirapine or NVP (Viramune ™), efavirenz or EFV (Sustiva / atripla ™, Stocrin ™), delavirdine or DLV (Rescriptor ™) And etravirin or ETR (Intelence ™).

  An HIV entry inhibitor refers to a substance that blocks HIV cell entry. Examples of HIV entry inhibitors include, but are not limited to, enfuvirtide or T20 (Fuzeon ™), maraviloc or MVC (Celsentri ™, Celzentry ™).

  An HIV integrase inhibitor refers to a substance that inhibits HIV integrase activity. Examples of integrase inhibitors include, but are not limited to, raltegravir or RAL (Isentress ™).

  Such agents also include, but are not limited to, VIDAL currently in clinical trials such as, but not limited to, bicribilok, PRO140, TNX-355, AMD070, rasibil, apricitabine, elvucitabine, flosalvudine, rilpivirine, erbitegravir The compounds belonging to the same kind of drugs described in 1.

  Such agents also include, but are not limited to, for example, maturation inhibitors (bevimat), glycoside analogs of β-galactosyl-ceramide, carbohydrate binders, RNase H inhibitors, HIV gene expression inhibitors, Compounds that belong to other possible classes of drugs, such as stimulators of HIV release from latent T cells (such as valproic acid).

  In a particularly preferred embodiment, the composition of the invention as a combination is characterized in that the anti-HIV agent is chemically conjugated to the antibody for simultaneous use.

  In order to facilitate the binding between the anti-HIV agent and the antibody according to the present invention, a spacer molecule such as poly (alkylene) glycol, polyethylene glycol, or amino acid can be introduced between the two compounds to be bound, Alternatively, in another embodiment, an active derivative of the anti-HIV agent into which a functional group capable of reacting with the antibody is introduced can be used. These coupling techniques are well known to those skilled in the art and will not be described in further detail here.

  Also preferably, the antibodies of the invention that form the complex are selected from functional fragments thereof, particularly fragments lacking their Fc component, such as scFv fragments.

  The invention also relates to a composition according to the invention as a combination, or an anti-CXCR4 Mab / anti-HIV drug complex, used as a drug.

  Preferably, excipients and / or pharmaceutical vehicles may be added to the composition or the complex as a combination.

  Thus, the present invention relates to the use of an antibody, or one of its functional fragments or derivatives, for the manufacture of a drug for the specific targeting of a biologically active compound for HIV replication.

  In another embodiment, the present invention is also a method for the prevention or treatment of HIV, wherein the antibody or one of the antigen-binding fragments or derivatives thereof and / or the composition according to the present invention is used in patients in need of prevention or treatment. A method comprising the steps of:

  More particularly, the method according to the invention also comprises a step consisting in administering to said patient an anti-CCR5 compound such as maraviroc.

  As previously indicated, CXCR4 Mabs 515H7 and 301aE5 have strong activity against HIV-1 replication in PBMC, and thus such antibodies are CXCR4 antagonist antiviruses for treating HIV-1 infection. It can be used in screening assays for agent identification. In the first step of these assays, cells expressing CXCR4 are incubated with Mab 515H7 and / or 301aE5, and then the molecule is evaluated for the potential to inhibit binding to Mab 515H7 and / or 301aE5. Can do. Cells used in this type of assay include transfected cell lines such as CHO-CXCR4, NIH3T3-CXCR4 or CXCR4 transfected human cell lines (eg, U373-MAGI-CXCR4), human cell lines expressing CXCR4 such as NALM6. Or it can be a primary cell such as PBMC. The method used to screen for antagonists of CXCR4 that inhibit binding of Mab 515H7 and / or 301aE5 to CXCR4-expressing cells is described by Zhao Q. et al. (AIDS Research And Human Retroviruses, 2003, 19, pp947-955). A protocol using a cell-based competitive enzyme-linked immunosorbent assay (ELISA) or a fluorescence activated cell sorting method (FACS) described by Juarez J. et al. (Leukemia 2003, 17, ppl294-1300) obtain.

Thus, in a particular aspect of the invention, a method for screening and / or identifying a molecule as a CXCR4 antagonist antiviral agent is contemplated, comprising the following steps:
a) selecting a cell that expresses CXCR4;
b) incubating the cells with an antibody of the invention, or one of its functional fragments or derivatives,
c) evaluating a test molecule for potential inhibition of binding between the antibody, or one of its functional fragments or derivatives, and CXCR4; and d) selecting a molecule capable of the inhibition. .

In another specific embodiment, the following step e) can be added:
e) Testing these molecules in an HIV-1 replication assay.

  Other features and advantages of the present invention will become more apparent from the description when read in conjunction with the examples and drawings (the legend is shown below).

1A and 1B show the gating strategy for CXCR4 expression in monocytes and lymphocytes. FIG. 1A: T-cell staining with CD3-PE antibody FIG. 1B: Monocyte staining with CD14-PE antibody Figures 2A and 2B show the binding of anti-CXCR4 Mab 515H7 and 301aE5 on monocytes and T lymphocytes. FIGS. 3A and 3B show modulation of CXCR4 receptor dimers by SDF-1 and anti-CXCR4 Mabs 515H7 and 301aE5, respectively, by a bioluminescence resonance energy transfer (BRET) approach in HEK293 cells. 4A and 4B and FIG. 5 show the ability of anti-CXCR4 Mabs 515H7 and 301aE5 to inhibit replication of HIV-1 isolate KON (X4 virus) in human PBMC. FIGS. 4A and 4B and FIG. 5 show the ability of anti-CXCR4 Mab 515H7 to inhibit replication of HIV-1 isolate KON (X4 virus) in human PBMC. Figures 6A, 6B and 6C show the inhibition of SDF-1 induced calcium release in CHO-CXCR4 cells by Mabs 515H7 (Figure 6A), 301aE5 (Figure 6B) and c515H7 (Figure 6C). Figures 7 and 8 show the ability of anti-CXCR4 Mabs 515H7, c515H7, and 301aE5 to inhibit replication of HIV-1 X4 virus primary isolates MN (Figure 7) and 92UG024 (Figure 8) in human PBMC. It is. Figures 7 and 8 show the ability of anti-CXCR4 Mabs 515H7, c515H7, and 301aE5 to inhibit replication of HIV-1 X4 virus primary isolates MN (Figure 7) and 92UG024 (Figure 8) in human PBMC. It is. FIG. 9 shows the ability of anti-CXCR4 Mabs 515H7, c515H7, and 301aE5 to inhibit replication of HIV-1 X4 virus primary isolates KON, MN, and 92UG024 in human PBMC. FIGS. 10 and 11 show the ability of anti-CXCR4 Mabs 515H7, c515H7, and 301aE5 to inhibit replication of HIV-1 X4 / R5 double virus primary isolate 89.6 in human PBMC. FIGS. 10 and 11 show the ability of anti-CXCR4 Mabs 515H7, c515H7, and 301aE5 to inhibit replication of HIV-1 X4 / R5 double virus primary isolate 89.6 in human PBMC. FIG. 12 shows the beneficial effect of the combination of Mab c515H7 and Maraviroc inhibiting the replication of HIV-1 primary isolate 89.6 (X4 / R5 double virus) in human PBMC. FIG. 13 shows the beneficial effect of the combination of Mab c515H7 and Maraviroc inhibiting the replication of HIV-1 primary isolate UG93067 (X4 / R5 double virus) in human PBMC. FIG. 14 shows the ability of anti-CXCR4 Mabs 515H7, c515H7, and 301aE5 to inhibit replication of HIV-1 X4 virus primary isolate KON in PBMC. FIG. 15 shows the binding specificity of c515H7 Mab by FACS analysis. FIG. 16 shows the effect of c515H7 Mab on CXCR4 homodimer via a bioluminescence resonance energy transfer (BRET) approach. FIG. 17 is an amino acid sequence alignment of the 515H7 heavy chain variable domain with human germline IGHV3-49 ^* 04 and IGHJ4 ^* 01. The 515H7 VH amino acid sequence is aligned with the selected human receptor framework sequence. The VH Var1 (VH1) sequence corresponds to a humanized variant of the 515H7 VH domain. A single back mutation at position 76 is shown in bold. FIG. 18 is an amino acid sequence alignment of 515H7 light chain with human germline IGKV4-1 ^* 01 and IGKJ1 ^* 01. The 515H7 VL amino acid sequence is aligned with the selected human receptor framework sequence. The VL Var2.1, Var2.2, and Var2.3 sequences correspond to the humanized variant of humanized 515H7 VL Var2 (mutated residues are shown in bold). Var2.1 and Var2.2 have four more humanized residues, and Var2.3 contains five more human residues. FIG. 19 shows cross-blocking of biotinylated murine antibody 515H7 by various variants of chimeric 515H7 and humanized 515H7. The activity of the humanized variant of 515H7 (hz515H7) to cross block the parent murine antibody 515H7 was assessed by flow cytometry using CXCR4 transfected NIH3T3 cells. The activity of the humanized variant was compared to the chimeric 515H7. The cross-blocking activity of mutant VH1 combined with chimeric VL (cVL) was very similar to that of chimera (A). When combined with mutant 2 of VL (B), the activity of VH mutant 1 (VH1, a mutant without back mutation) was not reduced. FIG. 20 shows inhibition of biotinylated SDF-1 binding by various variants of chimeric 515H7 and humanized 515H7. The ability of the humanized variant of 515H7 (hz515H7) to inhibit SDF-1 binding was assessed by flow cytometry using the cell line RAMOS. The inhibitory ability of the humanized mutant was compared with the chimeric 515H7. The humanized mutant hz515H7 VH1 D76N VL2 has the same ability to inhibit SDF-1 binding as the chimeric antibody. The humanized antibody fragment hz515 VH1 VL2 showed full activity in inhibiting SDF-1 binding to RAMOS cells. 21 shows humanized 515H7 Mabs that specifically bind to CXCR4 in NIH3T3-CXCR4 (hz515H7 VH1 D76N VL2, hz515H7 VH1 D76N VL2.1, hz515H7 VH1 D76N VL2.2, and hVV1H376). It is. Figure 22 shows a humanized 515H7 Mab (hz515H7 VH1 D76N VL2, hz515H7 VH1 D76N VL2.1, hz515H7 VH1 D76N VL2.2 and hz515H2 VH1 VL2.2 and hz515HLV1H2HH5V1H2HH5VH2HV5HH2H2 It is a figure which shows the effect of 3). FIG. 23 shows the ability of anti-CXCR4 Mab hz515H7 to inhibit X4 HIV-1 _IIIB- induced cytopathogenicity in MT-4 cells. FIG. 24 shows the ability of anti-CXCR4 Mab hz515H7 to inhibit replication of HIV-1 X4 virus primary isolate KON in human PBMC. FIG. 25 shows the beneficial effect of the combination of Mab hz515H7 and Maraviroc to inhibit replication of HIV-1 primary isolate 89.6 (double virus X4 / R5) in human PBMC. FIG. 26 shows the beneficial effect of the combination of Mab hz515H7 and Maravirok to inhibit replication of HIV-1 primary isolate UG93067 (double virus X4 / R5) in human PBMC. FIG. 27 shows the ability of anti-CXCR4 Mab hz515H7 IgG4 to inhibit replication of primary isolate KON of HIV-1 X4 virus in human PBMC. FIG. 28 shows the beneficial effect of the combination of Mab hz515H7 IgG4 and Maraviroc to inhibit replication of HIV-1 primary isolate 89.6 (double virus X4 / R5) in human PBMC.

Example 1: Production of monoclonal antibody (Mab) against human CXCR4 To produce a monoclonal antibody against CXCR4, Balb / c mice were sensitized with recombinant NIH3T3-CXCR4 cells and / or peptides corresponding to CXCR4 extracellular N-terminus and loop. I made it. Mice 6 to 16 weeks of age were sensitized subcutaneously (sc) once with complete Freund's adjuvant, and then challenged 2-6 times s. With Freund's incomplete adjuvant. c. I was sensitized. The immune response was measured by retroorbital bleeds. Serum was screened by ELISA (as described below) and mice with high anti-CXCR4 antibody titers were used for fusion. Mice were boosted intravenously 2 days before sacrifice and removal of the spleen.

ELISA
To select mice that produce anti-CXCR4 antibodies, sera from immunized mice were examined by ELISA. In short, microtiter plates were coated with purified [1-41] N-terminal peptide conjugated with BSA at an equivalent amount of 5 μg peptide / mL, 100 μL / well, incubated overnight at 4 ° C., and then in PBS. Blocked with 250 μL / well of 0.5% gelatin. Plasma dilutions from CXCR4 immunized mice were added to each well and incubated at 37 ° C. for 2 hours. These plates were washed with PBS and then incubated with a goat anti-mouse IgG antibody conjugated with HRP (Jackson Laboratories) for 1 hour at 37 ° C. After washing, the plate was developed with TMB substrate and the reaction was stopped after 5 minutes by adding 100 μL / well of 1 MH ₂ SO ₄ . Mice that produced the highest titer of anti-CXCR4 antibody were used for antibody production.

Generation of hybridoma producing Mab against CXCR4 Mouse splenocytes isolated from Balb / c mice that produced the highest titer anti-CXCR4 antibody were fused with mouse myeloma cell line Sp2 / O using PEG. Cells were seeded in microtiter plates at approximately 1 × 10 ⁵ / well and then incubated for 2 weeks in selective medium containing ultraculture medium + 2 mM L-glutamine + 1 mM sodium pyruvate + 1 × HAT. The wells were then screened for anti-CXCR4 monoclonal IgG antibody by ELISA. Subsequently, hybridomas secreting antibodies were subcloned at least twice by limiting dilution and cultured in vitro to produce antibodies for further analysis.

Example 2: Characterization of anti-CXCR4 Mab 515H7 and 301aE5 binding specificity (NIH3T3-CXCR4 transformants) by FACS analysis In this experiment, specific binding of anti-CXCR4 Mab 515H7 and 301aE5 to human CXCR4 (hCXCR4) was FACS Investigated by analysis. NIH3T3 and NIH3T3-hCXCR4 transfected cells were incubated with 10 μg / ml of monoclonal antibodies 515H7 and 301aE5. The cells were then washed with 1% BSA / PBS / 0.01% NaN ₃ . Next, Alexa-labeled secondary antibody was added to these cells and incubated at 4 ° C. for 20 minutes. Thereafter, the cells were washed again twice. After the second wash, FACS analysis was performed. The results of these binding tests are shown in Table 8 below showing [Average Fluorescence Intensity (MFI) Obtained by FACS]. Anti-CXCR4 Mabs 515H7 and 301aE5 specifically bound to the human CXCR4-NIH3T3 transfected cell line, but no recognition for parental NIH3T3 cells was seen.

Example 3: Characterization of anti-CXCR4 Mab 515H7 and 301aE5 binding to peripheral blood mononuclear cells (PBMC) by FACS analysis Blood was collected from healthy donors as a buffy coat. 100 μl of whole blood was incubated for 20 minutes at 4 ° C. with the indicated concentrations of anti-human CXCR4 antibodies (clone 515H7 and 301aE5). The blood was washed 3 times in PBS-BSA 1% -NaN ₃ 0.01% and incubated with 1: 500 dilution of goat anti-human Alexa 488 IgG (Invitrogen) for 20 minutes at 4 ° C. The cells were then washed, incubated with CD14-PE (Caltag) or CD3-PE (Caltag) for 10 minutes at 4 ° C. and washed 3 times. Red blood cells were lysed with High-Yield lysis solution (Caltag) for 10 minutes at room temperature. The cells were immediately analyzed using Facscalibur (Becton-Dickinson). CXCR4 expression on monocytes was performed on CD14 positive cells, and CXCR4 expression on T cells was performed on CD3 positive cells (FIG. 1). Results are expressed as antigen binding capacity (ABC).

  As shown in FIGS. 2A and 2B, anti-human CXCR4 clones 515H7 and 301aE5 stained both T lymphocytes (FIG. 2A) and monocytes (FIG. 2B), indicating that 515H7 and 301aE5 Mabs were monocytes. And the ability to recognize native CXCR4 expressed on the cell surface of T lymphocytes.

Example 4: Effect of 515H7 and 301aE5 Mabs on CXCR4 homodimer by bioluminescence resonance energy transfer (BRET) approach This functional assay is triggered when SDF-1 and / or 515H7 Mabs bind to CXCR4 receptor The conformational change that is made can be assessed at the CXCR4 homodimer level.

The expression vector of the interaction partner to be studied was constructed as a fusion protein with corresponding dyes (Renilla reniformis luciferase Rluc and yellow fluorescent protein YFP) by applying conventional molecular biology techniques. Two days before conducting the BRET test, HEK293 cells were transiently transfected with an expression vector encoding the corresponding BRET partner: [CXCR4 / Rluc + CXCR4 / YFP] and examined for CXCR4 homodimer formation. The next day, cells were dispensed into complete culture medium [DMEM supplemented with 10% FBS] on white 96 MW plates pre-coated with polylysine. First, in order to adhere the cells to the plate, the cells were cultured at 37 ° C. with 5% CO ₂ . The cells were then starved overnight with 200 μl DMEM / well. Just prior to the BRET test, DMEM was removed and the cells were quickly washed with PBS. Cells were then incubated in PBS in the presence or absence of antibody for 10 minutes at 37 ° C., followed by addition of 5 μM coelenterazine H with or without SDF-1 300 nM in a final volume of 50 μl. Following an additional 10 min incubation at 37 ° C., a Mitras LB940 multilabel reader (Berthold) (1 s / wavelength / well, 15 replicates at room temperature) was used to induce emission acquisition at 485 nm and 530 nm.

The BRET ratio was calculated as previously described (Angers et al., 2000): [(emission _{530 nm} ) − (emission _{485 nm} ) × Cf] / (emission _{485 nm} ), where Cf = same test conditions. Of cells expressing Rluc fusion protein alone at (emission _{530 nm} ) / (emission _{485 nm} ). To simplify this equation, the BRET ratio gives the 530/485 nm ratio obtained when two BRET partners are present when only the partner fused with Rluc is present in the assay under the same test conditions. It is shown that it corresponds to that corrected by the 530/485 nm ratio. For ease of reading, the results are expressed in milliBRET units (mBU), where mBU is the BRET ratio multiplied by 1000.

  SDF1 (300 nM) enhanced the BRET signal resulting from the spatial proximity of the adapter and acceptor proteins fused to the CXCR4 receptor by approximately 20%, which is probably CXCR4 / CXCR4 homodimer formation Or suggest a conformational change of the existing dimer (FIGS. 3A and B). 515H7 and 301aE5 Mabs were able to modulate the conformational changes induced by SDF-1 with respect to CXCR4 homodimers (69% inhibition of SDF-1-induced BRET enhancement for 515H7 and 301aE5, FIG. 3A and B). 515H7 and 301aE5 Mab could also themselves regulate the spatial proximity of CXCR4 / CXCR4, suggesting the effect of 515H7 and 301aE5 Mab on CXCR4 / CXCR4 homodimer conformation (Fig. 3A and 3B).

Example 5: Inhibition of HIV-1 primary isolate KON (X4 virus) replication in human PBMC by anti-CXCR4 Mabs 515H7 and 301aE5 PBMC from healthy donors seronegative for HIV-1 from buffy coat Or from cytapheresis by Ficoll-Hypaque gradient centrifugation. PBMC were added in RPMI 1640 cell culture medium containing 25 mM HEPES, 5 ml penicillin (10000 U / ml) -streptomycin (10000 μg / ml) 2 mM L-glutamine and supplemented with 10% heat-inactivated FCS in the presence of PHA. Activated and used as a cell target in a one-cycle neutralization assay. HIV-1 replication in primary human PBMC was performed by analyzing intracellular staining for viral p24 antigen by FACS analysis. In short, 25 μl / well of various dilutions of Mab 515H7, 301aE5 and 12G5 (R & D Systems) or culture medium (RPMI 1640, 10% FCS, 0.1% IL-2) as a control, 25 μl / well of HIV. -1 Incubated in duplicate with KON X4 primary isolate dilution for 1 hour at 37 ° C. PHA-activated human PBMC (25 μl / well) was added to the Mab / virus mixture in a 96-well plate (U-bottom, Costar 3599) at 20 × 10 ⁶ cells / ml, RPMI 1640, 10% FCS and 0.1% IL -2 at 37 ° C. for 24-36 hours. A control consisting of uninfected PBMC was introduced in medium without Mab. To detect HIV-infected PBMC, intracellular staining for viral p24 antigen was performed and analyzed by flow cytometry. Fluorescent anti-p24 Mab (Clone KC57-Coulter Beckman). After washing with PBS-3% FCS medium, PBMC was diluted with PBS before flow cytometric analysis. The percentage of p24 positive cells in the various samples was determined by gating 20,000 events on the live cell population. P24 expression of this subset of live cells was analyzed relative to background staining of uninfected cells. p24 antigen positive values were obtained after subtracting background events in mock infected cells. The percentage of neutralization was defined as the decrease in p24 positive cells compared to control infected wells without Mab. Neutralization titer was defined as the dilution of Mab that reduces the proportion of infected cells by 90%. Anti-CXCR4 Mabs 515H7 and 301aE5 were compared to the 12G5 Mab known as the reference anti-CXCR4 Mab for HIV application. As shown in FIGS. 4A and B and 5, anti-CXCR4 Mabs 515H7 and 301aE5 replicate IC-1 primary isolates in PBMC with IC ₉₀ 10 μg / ml (66 nM) and 150 μg / ml (1 μM), respectively. Although 12G5 Mab was unable to inhibit replication of HIV-1 KON primary isolates in PBMC (FIG. 4A).

Example 6: Recruitment of intracellular calcium stores mediated by CXCR4 receptor This functional assay demonstrates CXCR4 receptor signaling via stimulation of the phospholipase C pathway that induces calcium release from intracellular stores of the endoplasmic reticulum. Designed to measure.

  CHO-K1 cells that stably and constitutively express human CXCR4 receptor are expressed as naive CHO-K1 cells (ATCC CCL-61) in a mammalian expression vector containing the entire coding sequence of human CXCR4 receptor (RefSeq NM_003467). Obtained when transfected. Cells were grown in complete culture medium [DMEM-Ham F12 medium supplemented with 5% fetal calf serum (FCS) and 500 μg / ml geneticin]. Cells were seeded on black 96 MW plates at a density of 100,000 cells / well in an appropriate culture medium. Cells were starved overnight before testing. Cells are loaded with fluorescent calcium dye (Fluo-4 No Wash, Invitrogen US) in loading buffer [HBSS 1 ×, HEPES 20 mM, Probenicid acid 25 mM] at 37 ° C. for 30 minutes, then at 25 ° C. for 30 minutes. Loaded. SDF-1 stimulation was performed by direct injection into each well. For antagonism studies, 10 μl of Mab solution is added directly to loading buffer at least 10 minutes before SDF-1. Dynamic fluorescence measurements are performed on a multimode fluorescence microplate reader Mitras LB940 (Berthold) with the following settings: excitation at 485 nm, emission at 535 nm, excitation energy in 10000 arbitrary units. The fluorescence of each well is recorded for 0.1 seconds per second for 20 seconds before SDF-1 injection (basal signal). Thereafter, 20 μl of SDF-1 is injected and data recording is continued for 2 minutes. Each test condition is performed in duplicate. The value for each well is first corrected by subtracting the basal fluorescence and the fluorescence emitted by the control well without cells. Relative data is expressed as the percentage of maximum stimulation obtained with SDF-1 (100 nM).

  SDF1 (100 nM) induced rapid and strong release of intracellular calcium in recombinant CHO / CXCR4, but no fluorescent signal was detected in naive CHO-K1 cells. Maximum intensity reached more than 140% of basal fluorescence and was seen in approximately 40 seconds upon stimulation with SDF-1 (FIGS. 6A, 6B, and 6C). Mabs 515H7 (133 nM) (FIG. 6A) and c515H7 (133 nM) (FIG. 6C) resulted in strong inhibition of the calcium signal induced by SDF-1 (100 nM). Mab 301aE5 (133 nM) (FIG. 6B) resulted in partial inhibition of the calcium signal induced by SDF-1 (100 nM).

Example 7: Inhibition of replication of HIV-1 primary isolates KON, MN, and 92UG024 (X4 virus) in human PBMC by anti-CXCR4 Mabs 515H7, c515H7 and 301aE5
One cycle neutralization assay This assay is performed for 36 hours with primary isolates KON, MN, and 92UG024 correspondingly concentrated and diluted to allow detection of 2% of infected CD4 T lymphocytes 2 days after infection. Do.

Various dilutions of Mab 515H7, c515H7 and 301aE5 25 μl were incubated with 25 μl virus for 1 hour at 37 ° C. 20 × 10 ⁶ cells / ml human PBMC (25 μl) are added to the Mab / virus mixture in a 96-well plate (U bottom, Costar 3599), RPMI 1640 10% FCS and 20 U / ml IL-2 (R & D Systems, Minneapolis , MN) for 36 hours.

  After 2 days of culture, HIV-infected lymphocytes were detected by intracellular staining for virus p24 Ag. Fluorescent anti-p24 cells are fixed and permeabilized using both Cytofix / Cytoperm and Perm / Wash kits (BD Biosciences) according to manufacturer and added for 15 minutes at 4 ° C. used at 1/160 dilution in Perm / Wash solution Stained with Mab (FITC- or PE-anti-p24, clone KC57, Beckman Coulter / Immunotech, Hialeah, FL). After washing with PBS containing 3% FBS, PBMC was diluted in 300 μl of PBS, and flow cytometry analysis (LSRII, BD Biosciences) was performed using DIVA software (BD Biosciences). The percentage of p24 positive cells in the various samples was determined by gating 20,000 events on the live cell population identified by the forward and side scatter parameters. These live cell subsets were analyzed using a live / dead solution kit (Invitrogen). p24 Ag positive values were obtained after subtracting background events in mock infected cells.

  Percent neutralization was defined as the decrease in p24 positive cells compared to control infected wells without Mab. The neutralization titer was defined as the concentration of antibody that reduced the percentage of infected cells by 90% (inserted between serial dilutions made in triplicate).

  As shown in FIGS. 7, 8 and 9, anti-CXCR4 Mabs 515H7, c515H7, and 301aE5 can inhibit replication of HIV-1 X4 MN, KON, and 92UG024 primary isolates in PBMC. The results of IC (in μg / ml) are summarized in Table 9.

Example 8: Inhibition of replication of HIV-1 primary isolate 89.6 (X4 / R5 double virus) in human PBMC by anti-CXCR4 Mabs 515H7, c515H7 and 301aE5
One-Cycle Neutralization Assay This assay is performed for 36 hours with primary isolate 89.6 correspondingly concentrated and diluted to allow detection of 2% infected CD4 T lymphocytes 2 days after infection.

Various dilutions of Mab 515H7, c515H7 and 301aE5 25 μl were incubated with 25 μl virus for 1 hour at 37 ° C. 20 × 10 ⁶ cells / ml human PBMC (25 μl) are added to the Mab / virus mixture in a 96-well plate (U bottom, Costar 3599), RPMI 1640 10% FCS and 20 U / ml IL-2 (R & D Systems, Minneapolis , MN) for 36 hours.

  After 2 days of culture, HIV-infected lymphocytes were detected by intracellular staining for virus p24 Ag. Fluorescent anti-p24 cells are fixed and permeabilized using both Cytofix / Cytoperm and Perm / Wash kits (BD Biosciences) according to manufacturer and added for 15 minutes at 4 ° C. used at 1/160 dilution in Perm / Wash solution Stained with Mab (FITC- or PE-anti-p24, clone KC57, Beckman Coulter / Immunotech, Hialeah, FL). After washing with PBS containing 3% FBS, PBMC was diluted in 300 μl of PBS, and flow cytometry analysis (LSRII, BD Biosciences) was performed using DIVA software (BD Biosciences). The percentage of p24 positive cells in the various samples was determined by gating 20,000 events on the live cell population identified by the forward and side scatter parameters. These live cell subsets were analyzed using a live / dead solution kit (Invitrogen). p24 Ag positive values were obtained after subtracting background events in mock infected cells.

  Percent neutralization was defined as the decrease in p24 positive cells compared to control infected wells without Mab. The neutralization titer was defined as the concentration of antibody that reduced the percentage of infected cells by 90% (inserted between serial dilutions made in triplicate).

  As shown in FIGS. 10 and 11, anti-CXCR4 Mabs 515H7, c515H7, and 301aE5 can inhibit replication of the HIV-1 89.6 primary isolate in PBMC. IC (μg / ml) results are summarized in Table 10.

Example 9: Inhibition of replication of HIV-1 primary isolate 89.6 and UG93067 (X4 / R5 double virus) in human PBMC by a combination of anti-CXCR4 Mab c515H7 and anti-CCR5 molecule maraviroc
Neutralization assay, analysis of multiple HIV primary isolate replicates in primary PBMC:
This assay, combining serial dilutions of c515H7 Mab or Maraviroc or a combination of both with serial dilutions of virus, analyzes multiple infections against PBMC (peripheral blood mononuclear cells). In short, four 25 μl aliquots of serial dilutions (2 ×) of c515H7 Mab or Maraviroc or a combination of both are each placed in a pre-hydrated 96 well filter plate (pore size 1.25 μm, Durapor Dv, Millipore, Molsheim, France). And incubated with 25 μl of serial dilutions of the virus. Viral control titration (25 μl RPMI instead of diluted c515H7 Mab or Maraviroc) was performed on the same plate as the titration in the presence of dilutions of c515H7 Mab or Maraviroc or a combination of both. After 1 hour at 37 ° C., 25 μl of PHA-stimulated PBMC at a concentration of 4 × 10 ⁶ cells / ml (PHA-activated PBMC pool from 5 healthy donors) was added to a final culture volume of 75 μl of RPMI, 10% fetal bovine Serum (FCS) and 20 IU of interleukin-2 (IL-2) / ml (R & D System) were added. After 24 hours at 37 ° C., 100 μl of the same culture medium was added. On the fourth day, two washes were performed by filtration (200 μl RPMI each) to remove c515H7 Mab and Marabilok, and 200 μl fresh culture medium was added. On day 7, the presence of p24 in the culture supernatant was measured by ELISA and compared to the negative control (culture infected with virus diluent and maintained in the presence of 10 ⁻⁶ M zidovudine [AZT]), Positive wells were determined. Using 4 replicate wells, the virus titer (50% tissue culture infectious dose [TCID ₅₀ ]) in the absence (V ₀ ) and presence (V _n ) of each dilution of c515H7 Mab or Maraviroc or a combination of both. It was determined. Neutralization titer was defined as the dilution of c515H7 Mab or Maraviroc or a combination of both resulting in a 90% reduction in virus titer (V _n / V ₀ = 0.1).

As shown in FIG. 12, replication of the bi-directional X4R5 virus 89.6 was inhibited by c515H7 Mab with an IC _{90 of} 2 μg / ml (FIG. 12). In Maraviroc 50 [mu] g / ml, did not reach _{IC 90} inhibitory activity (Figure 12). Furthermore, when 2 μg / ml of marabiroc was added to antibody 515H7, the inhibitory activity of c515H7 Mab was enhanced with IC _{90 of} 0.2 μg / ml (FIG. 12).

  The beneficial effect of the combination of c515H7 Mab and Maraviroc was evaluated using these two molecules at different dilutions and another bi-directional virus UG93067. As shown in FIG. 13, the inhibitory activities of Mab c515H7 and Maraviroc were comparable. These results suggest that the ability of the virus UG93067 was comparable to use either the CCR5 receptor or the CXCR4 receptor. With UG93067 virus, better activity can be demonstrated, and only the combination of these X4 (c515H7 Mab) and R5 (marabiroc) inhibitors (10 μg / ml each) can reduce viral titer by 90% (FIG. 13).

Example 10: Production of anti-CXCR4 chimeric Mab c515H7 A chimeric format for the murine 515H7 Mab was designed. This corresponds to the genetic fusion of the murine antibody light and heavy chain variable domains of interest with human Cκ and IgG1 / IgG2 / IgG4 constant domains. This recombinant Mab was created during transient transfection by using the HEK293 / EBNA system with the pCEP4 expression vector (InVitrogen, US).

  Individual amino acid and nucleotide sequences are described herein above. Furthermore, since the sequences of the IgG2 and 4 isoforms (preferred isoforms) are disclosed in Table 3 above, again the heavy chain of the IgG1 isoform, ie the amino acid sequence of SEQ ID NO: 80 and the nucleotide of SEQ ID NO: 81 A sequence of c515H7 VH (G1 wt) corresponding to the sequence can be mentioned.

  The entire nucleotide sequence corresponding to the 515H7 Mab light and heavy chain variable domains was synthesized by global gene synthesis (Genecust, Luxembourg). They are subcloned into the pCEP4 vector (InVitrogen, US) with the entire coding sequence of the constant domain of either the light chain [Cκ] or heavy chain [CH1-Hinge-CH2-CH3] of human IgG1 / IgG2 / IgG4 immunoglobulin did. All cloning steps were performed according to conventional molecular biology techniques described in the Laboratory manual (Sambrook and Russel, 2001) or according to the supplier's instructions. Each gene construct was fully validated by nucleotide sequencing using a Big Dye terminator cycle sequencing kit (Applied Biosystems, US) and analyzed using a 3100 Genetic Analyzer (Applied Biosystems, US).

Suspension-compatible HEK293 EBNA cells (InVitrogen, US) were grown in a conventional manner in 50 ml of serum-free medium Excell 293 (SAFC Biosciences) supplemented with 6 mM glutamine on an orbital shaker (rotation speed 110 rpm) in a 250 ml flask. It was. Transient transfections consisted of linear 25 kDa polyethyleneimine (PEI) (Polysciences) prepared at a final concentration of 1 mg / ml in water, mixed and plasmid DNA (heavy chain: light chain plasmid ratio 1: 1, final concentration of 1.25 μg / ml). ml) and 2.10 ⁶ cells / ml. Four hours after transfection, the culture was diluted with 1 volume of fresh culture medium to a final cell density of 10 ⁶ cells / ml. The culture process was measured based on cell viability and Mab production. In general, the culture was maintained for 4-5 days. Mabs were purified using a conventional chromatographic approach with protein A resin (GE Healthcare, US). Mabs were produced at a level suitable for functional evaluation. Productivity levels generally range from 6-15 mg / l of purified Mab.

Example 11: Characterization of anti-CXCR4 chimeric Mab c515H7 binding specificity by FACS analysis In this study, the specific binding of anti-CXCR4 chimeric Mab c515H7 to human CXCR4 was examined by FACS analysis.

  NIH3T3-hCXCR4 transfected cells were incubated with monoclonal antibody c515H7 in a dose range of 0 μg / ml to 10 μg / ml. The cells were then washed with 1% BSA / PBS / 0.01% NaN3. A secondary antibody labeled with Alexa was then added to these cells and incubated at 4 ° C. for 20 minutes. Thereafter, these cells were washed again twice. After the second wash, FACS analysis was performed. The results of this binding test are shown in FIG. 15, which shows that the anti-CXCR4 chimeric Mab C515H7 specifically binds to the human CXCR4-NIH3T3 transfected cell line. No binding with NIH3T3wt cells was detected (data not shown).

Example 12: Effect of c515H7 Mab on CXCR4 homodimer by bioluminescence resonance energy transfer (BRET) approach This functional assay is triggered when SDF-1 and / or c515H7 Mab binds to CXCR4 receptor Conformational changes can be assessed at the CXCR4 homodimer level.

The expression vector of the interaction partner to be studied was constructed as a fusion protein with corresponding dyes (Renilla reniformis luciferase Rluc and yellow fluorescent protein YFP) by applying conventional molecular biology techniques. Two days before conducting the BRET test, HEK293 cells were transiently transfected with an expression vector encoding the corresponding BRET partner: [CXCR4 / Rluc + CXCR4 / YFP] and examined for CXCR4 homodimer formation. The next day, cells were dispensed into complete culture medium [DMEM supplemented with 10% FBS] on white 96 MW plates pre-coated with polylysine. First, in order to adhere the cells to the plate, the cells were cultured at 37 ° C. with 5% CO ₂ . The cells were then starved overnight with 200 μl DMEM / well. Just prior to the BRET test, DMEM was removed and the cells were quickly washed with PBS. Cells were then incubated in PBS in the presence or absence of antibody at 37 ° C. for 10 minutes, followed by addition of 5 μM coelenterazine H with or without SDF-1 100 nM in a final volume of 50 μl. Following an additional 10 min incubation at 37 ° C., a Mitras LB940 multilabel reader (Berthold) (1 s / wavelength / well, 15 replicates at room temperature) was used to induce emission acquisition at 485 nm and 530 nm.

The BRET ratio was calculated as previously described (Angers et al. 2000): [(emission _{530 nm} ) − (emission _{485 nm} ) × Cf] / (emission _{485 nm} ), where Cf = under the same test conditions. (Emission _{530 nm} ) / (Emission _{485 nm} ) of cells expressing Rluc fusion protein alone. To simplify this equation, the BRET ratio gives the 530/485 nm ratio obtained when two BRET partners are present when only the partner fused with Rluc is present in the assay under the same test conditions. It is shown that it corresponds to that corrected by the 530/485 nm ratio. For ease of reading, the results are expressed in milliBRET units (mBU), where mBU is the BRET ratio multiplied by 1000.

  SDF1 (100 nM) enhanced the BRET signal resulting from the spatial proximity of the donor protein fused to the CXCR4 receptor and the acceptor protein by about 10%, which is probably CXCR4 / CXCR4 homodimer It suggests somatic formation or a conformational change of the existing dimer (Figure 16). Mab c515H7 was able to modulate the conformational changes induced by SDF-1 with respect to CXCR4 homodimers (96% inhibition of BRET enhancement induced by SDF-1, FIG. 16). Mab c515H7 could also itself modulate the spatial proximity of CXCR4 / CXCR4, suggesting the effect of this Mab on CXCR4 / CXCR4 homodimer conformation (FIG. 16).

Example 13: In vitro evaluation of Mab 515H7 anti-HIV-1 activity using GFP-transduced human osteosarcoma (GHOST) cells expressing CD4 and CXCR4 or CCR5 To determine the specificity of CXCR4 515H7 Mab They evaluated the anti-HIV-1 activity of this Mab using GHOST cells expressing CD4 and CXCR4 or CCR5.

This assay is performed for 48 hours using either X4 HIV-1 LAI virus (with Ghost cells expressing CXCR4) or R5 HIV-1 BaL virus (with Ghost cells expressing CCR5). The 500μl of Ghost cells was 24 hours plated in culture medium Dulbecco's supplemented with 10% FCS (2.5 10 ⁵ cells / ml). After various dilutions of Mab 515H7 were incubated for 1 hour at 37 ° C., diluted HIV-1 LAI virus (1/10) and HIV-1 BaL virus (1/7) were added to these cells for 48 hours. Cells were trypsinized and washed once with PBS. To fix the cells and inactivate the virus, 300 μL of 1.5% paraformaldehyde was added to the cell pellet for 2 hours at + 4 ° C. in the dark. GFP positive cells were analyzed by flow cytometry and the inhibition of HIV-1 infection was calculated.

  The percent inhibition of infected cells was defined relative to control infected wells without Mab. The results for ICs (μg / ml) are summarized in Table 11. Anti-CXCR4 Mab 515H7 was able to inhibit the infection of HIV-1 X4 Lai virus in CXCR4 expressing Ghost cells, but not at all to inhibit the infection of HIV-1 R5 BaL virus in CCR5 expressing Ghost cells. It was active.

Example 14: Humanization of 515H7 anti-CXCR4 murine antibody and generation of the h515H7 fragment
General Procedure 515H7 anti-CXCR4 antibody was humanized by applying the global rules of the CDR grafting method. Immunogenicity analysis and CDR and framework (FR) region definitions were performed by applying the IMGT unique numbering scheme and the IMGT library and tools (Lefranc, 1997-www.imgt.org).

  The binding of the humanized variant of 515H7 was determined in the NIH3T3 cell line stably transfected with human CXCR4. Binding activity was assessed by a competition assay using biotinylated mouse antibody. In a second attempt, humanized antibodies were evaluated for their ability to inhibit the binding of biotinylated SDF-1 to RAMOS cells. RAMOS cells were selected because of high CXCR4 expression and low CXCR7 and SDF-1 expression.

  These assays were used to characterize recombinant humanized forms of anti-CXCR4 antibodies. The variable domain was formatted with a human IgG1 / k constant domain and cloned into the mammalian expression vector pCEP. Recombinant IgG1 / κ-derived antibody was transiently expressed in HEK293 cells. The expression culture supernatant was filtered and the antibody was purified using protein A sepharose. PBS buffer was added again to the purified antibody, and the antibody concentration was measured by ELISA.

  Recombinant antibody fragments were generated by PCR using oligonucleotides specific for the variable domain of the humanized antibody and these were subcloned into the E. coli system. The antibody fragment was purified by immobilized metal ion affinity chromatography (IMAC).

Various sequence alignments of the humanized heavy and light chain variable domains of the 515H7 variable domain are shown in FIGS. 17 and 18.

  In a first series of tests, the three first humanized variants were analyzed for anti-CXCR4 binding activity. VH variant 1 (VH1) was combined with murine VL to assess the ability of these constructs to inhibit the binding of biotinylated murine 515H7 parent antibody. The amino acid sequence of the variable domain of VH1 comprises SEQ ID NO: 90, and the nucleotide sequence comprises SEQ ID NO: 91. The amino acid sequence of full length VH1 comprises SEQ ID NO: 92 and the nucleotide sequence comprises SEQ ID NO: 93. This construct showed the same ability to compete with the murine antibody as the chimeric antibody (FIG. 19A). This suggests that most human VH variants have the same binding ability as chimeras. Therefore, VH1 was combined with VL variant 2 (FIG. 19B).

  In a further study, it was determined whether humanized variants of antibody 515H7 inhibit SDF-1 binding to CXCR4-expressing cells (FIG. 20). The ability of hz515H7 to humanize mutants was evaluated by detecting biotinylated SDF-1 in flow cytometry. The humanized antibody hz515H7 VH1 D76N VL2 has the same ability to inhibit SDF-1 binding as the chimeric c515H7.

  An antibody fragment of the humanized variant hz515H7 VH1 VL2 was also tested and found to be able to completely inhibit SDF-1 binding (FIG. 20).

Example 15: Characterization of anti-CXCR4 humanized Mab 515H7 binding specificity by FACS analysis In this study, the specific binding of anti-CXCR4 humanized Mab 515H7 to human CXCR4 was examined by FACS analysis.

  NIH3T3, NIH3T3-hCXCR4 transfectants were transferred to 100 μl Facs buffer in the dark for 20 minutes at 4 ° C. for 0-10 μg / mL humanized Mab 515H7 (hz515H7 VH1 D76N VL2, hz515H7 VH1 D76N 5H7 VH1 D76N VL2.2, hz515H7 VH1 D76N VL2.3). After three washes in Facs buffer, the cells were incubated with the secondary antibody goat anti-human Alexa 488 (diluted 1/500) for 20 minutes in the dark at 4 ° C. After washing 3 times in Facs buffer, propidium iodide was added to each well and only viable cells were analyzed by Facs. At least 5000 viable cells were evaluated and an average fluorescence intensity was estimated for each condition.

  The results of these binding tests are shown in FIG. 21, which shows [Mean fluorescence intensity (MFI) obtained by FACS]. Anti-CXCR4 humanized Mab hz515H7 specifically bound to the human CXCR4-NIH3T3 transfected cell line (MFI = 2.2 in NIH3T3 parental cells).

Example 16: Effect of hz515H7 Mab on CXCR4 homodimer by bioluminescence resonance energy transfer (BRET) approach This functional assay was performed using SDF-1 and / or hz515H7 VH1 D76N VL2, hz515H7 VH1 D76N VL2.1, hz515H7 VH1. D76N VL2.2, hz515H7 VH1 The conformational change induced when D76N VL2.3 binds to the CXCR4 receptor can be assessed at the CXCR4 homodimer level.

The expression vector of the interaction partner to be studied was constructed as a fusion protein with corresponding dyes (Renilla reniformis luciferase Rluc and yellow fluorescent protein YFP) by applying conventional molecular biology techniques. Two days before conducting the BRET test, HEK293 cells were transiently transfected with an expression vector encoding the corresponding BRET partner: [CXCR4 / Rluc + CXCR4 / YFP] and examined for CXCR4 homodimer formation. The next day, cells were dispensed into complete culture medium [DMEM supplemented with 10% FBS] on white 96 MW plates pre-coated with polylysine. First, in order to adhere the cells to the plate, the cells were cultured at 37 ° C. with 5% CO ₂ . The cells were then starved overnight with 200 μl DMEM / well. Just prior to the BRET test, DMEM was removed and the cells were quickly washed with PBS. Cells were then incubated in PBS in the presence or absence of antibody at 37 ° C. for 10 minutes, followed by addition of 5 μM coelenterazine H with or without SDF-1 100 nM in a final volume of 50 μl. Following an additional 10 min incubation at 37 ° C., a Mitras LB940 multilabel reader (Berthold) (1 s / wavelength / well, 15 replicates at room temperature) was used to induce emission acquisition at 485 nm and 530 nm.

The BRET ratio was calculated as previously described (Angers et al., 2000): [(emission _{530 nm} ) − (emission _{485 nm} ) × Cf] / (emission _{485 nm} ), where Cf = same test conditions. Of cells expressing Rluc fusion protein alone at (emission _{530 nm} ) / (emission _{485 nm} ). To simplify this equation, the BRET ratio gives the 530/485 nm ratio obtained when two BRET partners are present when only the partner fused with Rluc is present in the assay under the same test conditions. It is shown that it corresponds to that corrected by the 530/485 nm ratio. For ease of reading, the results are expressed in milliBRET units (mBU), where mBU is the BRET ratio multiplied by 1000.

  SDF1 (100 nM) enhanced the BRET signal resulting from the spatial proximity of the donor protein fused to the CXCR4 receptor and the acceptor protein, which is probably CXCR4 / CXCR4 homozygous. Suggests conformer change or conformation change of existing dimer (Figure 22).

  515H7 humanized Mab can modulate the conformational changes induced by SDF-1 with respect to CXCR4 homodimer, and the inhibition rate of BRET enhancement induced by SDF-1 is about hz515H7 VH1D76N-VL2 Mab. 88%, 65% for hz515H7 VH1D76N-VL2.1 Mab, 33% for hz515H7 VH1D76N-VL2.2 Mab and 21% for hz515H7 VH1D76N-VL2.3 Mab (FIG. 22).

Example 17: Inhibition of replication of HIV-1 _IIIB (X4 virus) in MT-4 cells by anti-CXCR4 Mab hz515H7 In this assay, the activity of hz515H7 Mab against HIV-1 _IIIB was determined by the virus in MT-4 cells. Based on inhibition of induced cytopathogenicity. Cells were infected with HIV-1 _IIIB isolate 5 times the 50% tissue culture infectious dose (TCID ₅₀ ) of viral load that reduced the number of viable cells by 90% within 5 days. After adsorbing at 37 ° C. for 30 minutes, infected cells were cultured in RPMI 1640 medium supplemented with 20% heat-inactivated fetal calf serum (FCS), 100 IU / ml penicillin, 100 μg / ml streptomycin, 2 mM glutamine. Medium 2 10 ⁵ cells / ml and seeded in 96 well flat bottom tissue culture plates (COSTAR 3596) (100 μl / well) with 100 μl of various concentrations of hz515H7 Mab. On day 5, cell viability was measured by colorimetric reaction MTT. The percentage of protection of infected cells treated with hz515H7 Mab was calculated by the following formula:

As shown in FIG. 23, hz515H7 Mab exhibits significant anti-HIV-1 activity because it can inhibit the cytopathogenicity induced by HIV-1 _IIIB − in MT-4 cells.

Example 18: Inhibition of HIV-1 primary isolate KON (X4 virus) replication in human PBMC by anti-CXCR4 Mab hz515H7
One-Cycle Neutralization Assay This assay was performed for 36 hours with primary isolate KON correspondingly concentrated and diluted to allow detection of 2% infected CD4 T lymphocytes 2 days after infection.

25 μl of Mab 515H7 at various dilutions were incubated with 37 μl of virus for 1 hour at 37 ° C. 20 × 10 ⁶ cells / ml human PBMC (25 μl) are added to the Mab / virus mixture in a 96-well plate (U bottom, Costar 3599) and RPMI 1640, 10% FCS and 20 U / ml IL-2 (R & D Systems, Minneapolis, Minn.) For 36 hours.

  After 2 days of culture, HIV-infected lymphocytes were detected by intracellular staining for virus p24 Ag. Fluorescent anti-p24 cells are fixed and permeabilized using both Cytofix / Cytoperm and Perm / Wash kits (BD Biosciences) according to manufacturer and added for 15 minutes at 4 ° C. used at 1/160 dilution in Perm / Wash solution Stained with Mab (FITC- or PE-anti-p24, clone KC57, Beckman Coulter / Immunotech, Hialeah, FL). After washing with PBS containing 3% FBS, PBMC was diluted in 300 μl of PBS, and flow cytometry analysis (LSRII, BD Biosciences) was performed using DIVA software (BD Biosciences). The percentage of p24 positive cells in the various samples was determined by gating 20,000 events on the live cell population identified by the forward and side scatter parameters. These live cell subsets were analyzed using a live / dead solution kit (Invitrogen). p24 Ag positive values were obtained after subtracting background events in mock infected cells.

  Percent neutralization was defined as the decrease in p24 positive cells compared to control infected wells without Mab. Neutralization titer was defined as the concentration of antibody that reduces the proportion of infected cells (inserted between serial dilutions made in triplicate).

  As shown in FIG. 24, anti-CXCR4 Mab 515H7 can inhibit replication of HIV-1 X4 KON primary isolates in PBMC.

Example 19: Inhibition of replication of HIV-1 primary isolate 89.6 and UG93067 (X4 / R5 double virus) in human PBMC by a combination of anti-CXCR4 Mab hz515H7 and anti-CCR5 molecule maraviroc
Neutralization assay, analysis of multiple HIV primary isolate replicates in primary PBMC:
This assay, combining serial dilutions of hz515H7 Mab or Maraviroc or a combination of both with serial dilutions of virus, analyzes multiple infections against PBMC (peripheral blood mononuclear cells). In short, four 25 μl aliquots of serial dilutions (2 ×) of hz515H7 Mab or Maraviroc or a combination of both were each placed in a pre-hydrated 96 well filter plate (pore size 1.25 μm, Durapor Dv, Millipore, Molsheim, France). And incubated with 25 μl of serial dilutions of the virus. Control titration of the virus (25 μl RPMI instead of diluted hz515H7 Mab or Maraviroc) was performed on the same plate as the titration in the presence of dilutions of hz515H7 Mab or Maraviroc or a combination of both. After 1 hour at 37 ° C., 25 μl of PHA-stimulated PBMC at a concentration of 4 × 10 ⁶ cells / ml (PHA-activated PBMC pool from 5 healthy donors) was added to a final culture volume of 75 μl of RPMI, 10% fetal bovine Serum (FCS) and 20 IU of interleukin-2 (IL-2) / ml (R & D System) were added. After 24 hours at 37 ° C., 100 μl of the same culture medium was added. On the fourth day, two washes (200 μl RPMI each) were performed by filtration to remove hz515H7 Mab and Maraviroc, and 200 μl fresh culture medium was added. On day 7, the presence of p24 in the culture supernatant was measured by ELISA and compared to the negative control (culture infected with virus diluent and maintained in the presence of 10 ⁻⁶ M zidovudine [AZT]), Positive wells were determined. Using 4 replicate wells, virus titer (50% tissue culture infectious dose [TCID ₅₀ ]) in the absence (V ₀ ) and presence (V _n ) of each dilution of 515H7 Mab or Maraviroc or a combination of both It was determined. Neutralization titer was defined as the dilution of 515H7 Mab or Maraviroc or a combination of both resulting in a 90% reduction in virus titer (V _n / V ₀ = 0.1).

  The potential for synergy between the hz515H7 Mab and Maraviroc was assessed using the bi-directional virus 89.6 and UG93067, using combinations of these two molecules at various dilutions. As shown in FIGS. 25 and 26, the inhibitory activities of Mab hz515H7 and Marabilok were comparable. The combination of these X4 (hz515H7 Mab) and R5 (maraviroc) inhibitors allowed a 90% reduction in 89.6 and UG93067 double virus X4 / R5 virus titers in PBMC (FIGS. 25 and 26, respectively). ).

Example 20: Inhibition of replication of HIV-1 primary isolate KON (X4 virus) in human PBMC by anti-CXCR4 Mab hz515H7 IgG4
One-Cycle Neutralization Assay This assay is performed for 36 hours with primary isolate KON correspondingly concentrated and diluted to allow detection of 2% infected CD4 T lymphocytes 2 days after infection.

Various dilutions of Mab hz515H7 IgG4 25 μl were incubated with 25 μl of virus for 1 hour at 37 ° C. 20 × 10 ⁶ cells / ml human PBMC (25 μl) are added to the Mab / virus mixture in a 96-well plate (U bottom, Costar 3599), RPMI 1640 10% FCS and 20 U / ml IL-2 (R & D Systems, Minneapolis , MN) for 36 hours.

  After 2 days of culture, HIV-infected lymphocytes were detected by intracellular staining for virus p24 Ag. Fluorescent anti-p24 cells are fixed and permeabilized using both Cytofix / Cytoperm and Perm / Wash kits (BD Biosciences) according to manufacturer and added for 15 minutes at 4 ° C. used at 1/160 dilution in Perm / Wash solution Stained with Mab (FITC- or PE-anti-p24, clone KC57, Beckman Coulter / Immunotech, Hialeah, FL). After washing with PBS containing 3% FBS, PBMC was diluted in 300 μl of PBS, and then flow cytometric analysis (LSRII, BD Biosciences) was performed using DIVA software (BD Biosciences). The percentage of p24 positive cells in the various samples was determined by gating 20,000 events on the live cell population identified by the forward and side scatter parameters. These live cell subsets were analyzed using a live / dead solution kit (Invitrogen). p24 Ag positive values were obtained after subtracting background events in mock infected cells.

  Percent neutralization was defined as the decrease in p24 positive cells compared to control infected wells without Mab. Neutralization titer was defined as the concentration of antibody that reduces the proportion of infected cells (inserted between serial dilutions made in triplicate).

  As shown in FIG. 27, anti-CXCR4 Mab hz515H7 IgG4 can inhibit replication of HIV-1 X4KON primary isolate in PBMC.

Example 21: Inhibition of replication of HIV-1 primary isolate 89.6 (X4 / R5 double virus) in human PBMC by a combination of anti-CXCR4 Mab hz515H7 IgG4 and anti-CCR5 molecule maraviroc
Neutralization assay, analysis of multiple HIV primary isolate replicates in primary PBMC:
This assay, combining serial dilutions of hz515H7 IgG4 Mab or Maraviroc or a combination of both with serial dilutions of virus, analyzes multiple infections against PBMC (peripheral blood mononuclear cells). In short, 96 well filter plates (pore size 1.25 μm, Durapor Dv, Millipore, Molsheim, France) each containing four 25 μl aliquots of serial dilutions (2 ×) of hz515H7 IgG4 Mab or Maraviroc or a combination of both. And incubated with 25 μl of serial dilutions of virus. Viral control titration (25 μl RPMI instead of diluted hz515H7 IgG4 Mab or Maraviroc) was performed on the same plate as the titration in the presence of dilutions of hz515H7 IgG4 Mab or Maraviroc or a combination of both. After 1 hour at 37 ° C., 25 μl of PHA-stimulated PBMC at a concentration of 4 × 10 ⁶ cells / ml (PHA-activated PBMC pool from 5 healthy donors) was added to a final culture volume of 75 μl of RPMI, 10% fetal bovine Serum (FCS) and 20 IU of interleukin-2 (IL-2) / ml (R & D System) were added. After 24 hours at 37 ° C., 100 μl of the same culture medium was added. On the 4th day, two washes (200 μl RPMI each) were performed by filtration to remove hz515H7 IgG4 Mab and Maraviroc, and 200 μl fresh culture medium was added. On day 7, the presence of p24 in the culture supernatant was measured by ELISA and compared to the negative control (culture infected with virus diluent and maintained in the presence of 10 ⁻⁶ M zidovudine [AZT]), Positive wells were determined. Using 4 replicate wells, virus titer (50% tissue culture infectious dose [TCID ₅₀ ]) in the absence (V ₀ ) and presence (V _n ) of each dilution of hz515H7 IgG4 Mab or Maraviroc or a combination of both )It was determined. Neutralization titer was defined as the dilution of hz515H7 IgG4 Mab or Maraviroc or a combination of both resulting in a 90% reduction in virus titer (V _n / V ₀ = 0.1).

  The potential for synergy between hz515H7 IgG4 Mab and Maraviroc was assessed using a bi-directional virus 89.6, using a combination of various dilutions of these two molecules. As shown in FIG. 28, the combination of these X4 (hz515H7 IgG4 Mab) and R5 (marabiroc) inhibitors allows a 90% reduction in the viral titer of 89.6 double virus X4 / R5 in PBMC. (FIG. 28).

Claims (32)

  An isolated antibody comprising at least one complementarity determining region CDR selected from CDRs comprising the amino acid sequences of SEQ ID NOs: 1-6 and 30-33 defined by the IMGT numbering system, or an antigen thereof One of the binding fragments or derivatives.   A light chain comprising CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively, and a CDR comprising amino acid sequences of SEQ ID NOs: 4, 5 and 6 respectively. The antibody of claim 1, or one of its antigen-binding fragments or derivatives, comprising a heavy chain comprising -H1, CDR-H2 and CDR-H3.   The antibody according to claim 2, or one of its antigen-binding fragments or derivatives, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 8. .   The antibody according to claim 2, which is a chimeric antibody and comprises a heavy chain of a sequence selected from the group consisting of SEQ ID NO: 56, 57 or 58, and a light chain of the sequence of SEQ ID NO: 59, or the antibody One of antigen-binding fragments or derivatives.   A humanized antibody, comprising a heavy chain variable region of the sequence consisting of SEQ ID NO: 64 and a light chain variable region of a sequence selected from the group consisting of SEQ ID NO: 65, 66, 82 or 83. 3. The antibody according to 2, or one of its antigen-binding fragments or derivatives.   A humanized antibody comprising a heavy chain of a sequence selected from the group consisting of SEQ ID NO: 67, 68 or 69 and a light chain of a sequence selected from the group consisting of SEQ ID NO: 70, 71, 84 or 85 3. The antibody of claim 2, or one of its antigen-binding fragments or derivatives, comprising a humanized antibody comprising: or a derivative compound or functional fragment thereof. The functional fragment consists of the fragment Fv, scFv, Fab, F (ab ′) ₂ , Fab ′, scFv-Fc, diabody, or any fragment whose half-life is extended by chemical modification. Or an antigen-binding fragment or derivative thereof.   8. The antibody of claim 7, or one of its antigen-binding fragments or derivatives, which is an scFv comprising the amino acid sequence of SEQ ID NO: 54.   A light chain comprising CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 1, 2 and 30, respectively, and a CDR comprising amino acid sequences of SEQ ID NOs: 31, 32 and 33, respectively. The antibody of claim 1, or one of its antigen-binding fragments or derivatives, comprising a heavy chain comprising -H1, CDR-H2 and CDR-H3.   The antibody according to claim 9, or one of the antigen-binding fragments or derivatives thereof, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 34 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 35. An isolated nucleic acid selected from the following nucleic acids:
a) a nucleic acid, DNA, or RNA encoding the antibody of any one of claims 1 to 10, or an antigen-binding fragment or derivative thereof,
b) a nucleic acid comprising a DNA sequence selected from the CDR sequence group consisting of SEQ ID NOs: 14-19 and 41-45,
c) a nucleic acid comprising a DNA sequence selected from the heavy and light variable domain sequence groups consisting of SEQ ID NOs: 20, 21, 46, 47, 72, 73, 74, 86, and 87;
d) a nucleic acid comprising a DNA sequence selected from the heavy and light chain sequences consisting of SEQ ID NOs: 60-63, 75-79, 88, and 89;
e) a nucleic acid comprising a DNA sequence consisting of SEQ ID NO: 55;
f) the corresponding RNA nucleic acid of the nucleic acid defined in b), c), d) or e),
g) complementary nucleic acids of the nucleic acids defined in a), b), c), d) and e) and h) at least the CDRs of the sequences of SEQ ID NOs: 14-19 and 41-45 under high stringency conditions A nucleic acid of at least 18 nucleotides capable of hybridizing to one.   A vector comprising the nucleic acid of claim 11.   A host cell comprising the vector of claim 12.   A transgenic non-human animal comprising at least one cell transformed with the vector of claim 12. A method for producing the antibody according to any one of claims 1 to 10, or one of its antigen-binding fragments or derivatives, comprising: a) the cell culture medium according to claim 13 and suitable culture conditions. And b) one recovery step of the antibody, or an antigen-binding fragment or derivative thereof, thus produced from the culture medium or the cultured cells.
Comprising a method.   An antibody, or a functional fragment or derivative thereof, obtainable or obtained by the method of claim 15.   The antibody according to any one of claims 1 to 10 and 16, as a drug. 18. An antibody according to any one of claims 1-10 or 16-17, which inhibits replication of HIV-1 KON primary isolate in PBMC with an IC ₉₀ of at least 5 μg / ml, preferably at least 10 μg / ml. Or one of its functional fragments or derivatives.   A composition comprising as an active ingredient a compound comprising the antibody according to any one of claims 1 to 10 and 16 to 18, or an antigen-binding fragment or derivative thereof.   20. A composition according to claim 19 as a medicament.   21. A composition according to claim 19 or 20 for the prevention or treatment of HIV infection.   24. The composition of claim 21, wherein the HIV infection is an X4-directed HIV infection.   24. The composition of claim 21, wherein the HIV infection is an X4 / R5-directed HIV infection.   24. At least one second anti-HIV compound selected from among compounds capable of specifically inhibiting HIV entry and / or replication, according to any one of claims 19-23. Composition.   At least a second anti-HIV compound is an HIV protease inhibitor (PI), a nucleoside / nucleotide HIV reverse transcriptase inhibitor (NRTI / NtRTI), a non-nucleoside HIV reverse transcriptase inhibitor (NNRTI), an HIV entry inhibitor, HIV 25. The composition of claim 24, selected from the group consisting of antiretroviral drugs such as integrase inhibitors.   26. The composition of claim 24 or 25, wherein at least the second anti-HIV compound is an anti-CCR5 compound.   27. The composition of claim 26, wherein the anti-CCR5 compound is marabilok. A method for screening and / or identifying a molecule as a CXCR4 antagonist antiviral agent comprising:
a) selecting a cell that expresses CXCR4;
b) incubating the cells with the antibody of any one of claims 1-10 or 16-18, or one of its antigen-binding fragments or derivatives,
c) evaluating a test molecule for potential inhibition of binding between the antibody, or one of its antigen-binding fragments or derivatives, and CXCR4; and d) selecting a molecule capable of said inhibition. Comprising a method.   An antibody according to any one of claims 1 to 10 and 16, or one of its antigen-binding fragments or derivatives, and / or a claim 19 to 27 for preparing a medicament for inhibiting HIV replication. Use of the composition according to any one of the above.   20. The antibody according to any one of claims 1 to 10 and 16, or one of its antigen-binding fragments or derivatives, and / or claim 19 for the preparation of a medicament for the prevention or treatment of HIV disease. Use of the composition according to any one of -27.   A method for the prevention or treatment of HIV, the antibody according to any one of claims 1 to 10 and 16, or one of its antigen-binding fragments or derivatives, and / or any one of claims 19 to 27. Said method comprising the step of administering the composition of claim 1 to a patient in need thereof.   32. The method of claim 31, further comprising the step of administering an anti-CCR5 compound to the patient.