CN103180342A

CN103180342A - Antibodies for the treatment of HIV

Info

Publication number: CN103180342A
Application number: CN2011800520640A
Authority: CN
Inventors: C·克林格-汉默
Original assignee: Pierre Fabre Medicament SA
Current assignee: Pierre Fabre Medicament SA
Priority date: 2010-10-27
Filing date: 2011-10-27
Publication date: 2013-06-26
Also published as: EP2632953A1; RU2013122770A; AU2011322508A1; JP2014504147A; ZA201302639B; KR20140009174A; MX2013004710A; WO2012055980A1; CA2814908A1

Abstract

The present invention relates to isolated antibodies, or the derivatives or antigen binding fragments of same, capable of binding to CXCR4 but also of inducing conformational change of the CXCR4 homodimers and able to inhibit HIV-1 primary isolate replication in PBMC. More particularly, the present invention relates to the 515H7 and 301 aE5 monoclonal antibodies, specific to the CXCR4 protein, as well as their use for the treatment of HIV infection. Pharmaceutical compositions comprising such antibodies and a process for the selection of such antibodies are also covered.

Description

The antibody that is used for the treatment of HIV

Technical field

The present invention relates to can specific binding in the new antibodies of Chemokine Receptors (CXCR), particularly chimeric and humanized mouse monoclonal antibody, and the amino acid of this antibody of encoding and nucleotide sequence.From on the one hand, the present invention relates to can specific binding in CXCR4 and new antibodies, functional fragment or derivative with strong activity that anti-human immunodeficiency virus (HIV) is infected.The present invention comprises that also this antibody, functional fragment or derivative are as the purposes of the medicine of preventing and/or treating property treatment HIV infection.

Background technology

Chemokine is little, secretion peptide, and it is controlling (particularly during immune response) white corpuscle along the migration of (being called as the chemokine gradient) of part chemical gradient people such as (, 2000) Zlotnick A..Based on its NH ₂The position of-end cysteine residues, and and the combination of g protein coupled receptor (two main subfamily is named as CCR and CXCR), chemokine is divided into two main subfamilies, CC and CXC.Up to the present, 50 human chemokines and 18 Chemokine Receptors have been found to surpass.

Some members of Chemokine Receptors family play a role as the co-receptor of principal recipient CD4, make the not homophyletic of HIV1 type can enter cell, and main co-receptor is CCR5 and CXCR4.T cytotropism X4HIV-1 uses CD4 and CXCR4 to enter cell, and scavenger cell tropism R5HIV-1 uses CD4 and CCR5.Amphicheirality's strain can use CXCR4 and CCR5 as co-receptor.CCR3 in other Chemokine Receptors, CCR2, CCR8, CXCR6, CXCR7, CX3CR1 can play a role as the co-receptor that the HIV strain more limits subgroup.

The native ligand of SDF-1(CXCR4) and the CCL3 of CCR5, CCL4, CCL4-L1 and CCL5 part can suppress the not cytogamy and the infection that cause of homophyletic of HIV-1.These discoveries have promoted the development of the anti-HIV treatment of target Chemokine Receptors, cause CCR5 small molecular antagonists Malawi's promise (maraviroc)

Get permission to be used for other anti-HIV-1 agent combination the patient that CCR5 tropism HIV-1 infects.Yet Malawi's promise can not be used for the patient who is infected by amphicheirality HIV-1 or the patient (VIDAL2009) who is infected by CXCR4 tropism HIV-1.Therefore, can suppress this type for the treatment of of expansion in the patient that X4 tropism and amphicheirality HIV infect of CXCR4 antagonist that X4 tropism HIV copies clear and definite medical need is arranged for being tested and appraised.

Chemokine Receptors 4(is also referred to as fusin, CD184, LESTR or HUMSTR) exist to comprise 352 or 360 amino acid whose two kinds of isotype forms.Residue A sn11 is glycosylated, and residue Tyr21 is added into sulfate group and modifies, and Cys109 and 186 in the extracellular of acceptor part with disulfide linkage bridge combine (people such as Juarez J., 2004).

Dissimilar healthy tissues, initial

, non-memory property T cell, regulatory T cells, B cell, neutrophil leucocyte, endotheliocyte, primary monocyte, dendritic cell, natural killer cell, CD34+ hemopoietic stem cell all express this acceptor, and in heart, colon, liver, kidney and brain with low expression level.CXCR4 plays a crucial role in white corpuscle transportation, the generation of B cell lymphocyte and myelocyte generate.

Up to the present unique part of described CXCR4 acceptor is mesenchymal cell derived factor-1 (SDF-1) or CXCL12.SDF-1 is secretion in a large number in lymphoglandula, marrow, liver, lung, and kidney, brain and skin secretion degree are less.CXCR4 is also by the Antagonism chemokine, the virus macrophage inflammatory protein II(vMIP-II that human herpes simplex vicus III type is coded), identify.

As previously mentioned, the CXCR4 acceptor is the main co-receptor of T cytotropism HIV-1 strain isolated (X4 virus).Disturb this acceptor to suppress the X4 virus replication in effectively mode.

Summary of the invention

An invention aspect of the present invention is to generate to suppress the mouse monoclonal antibody (Mab) that HIV copies.The present invention includes CXCR4Mab515H7(or its fragment that can be incorporated into the CXCR4 homodimer and have the strong activity of anti-HIV infection).Invention also comprises CXCR4Mab301aE5(or its fragment that can be incorporated into the CXCR4 homodimer and have the strong activity of anti-HIV infection).

It is shocking, the contriver has managed to generate monoclonal antibody, and this antibody can be incorporated into CXCR4, and can induce the conformational change of CXCR4 homodimer, and can suppress Primary isolate X4-HIV-1 copying in PBMC.More particularly, antibody of the present invention can also suppress Primary isolate X4/R5-HIV-1 copying in PBMC.

Preferably, the CXCR4 compound is to be selected from one of two following people CXCR4 isotypes:

-chemokine (C-X-C motif) acceptor 4 isotype b[people (Homo sapiens)], have the described sequence SEQ ID of Genbank accession number NP_003458 No.27:

MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGN?GLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLC?KAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPA?LLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCI?IISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENT?VHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRG?GHSSVSTESESSSFHSS；

-chemokine (C-X-C motif) acceptor 4 isotype a[people], have the described sequence SEQ ID of Genbank accession number NP_001008540 No.28:

MSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGI?VGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGN?FLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVW?IPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSC?YCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFE?NTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGK?RGGHSSVSTESESSSFHSS；

-optionally transcribe splice variant or its natural variant, its with these b with SEQ ID No.27 or 28 or a isotype in one have at least 95% identity; And

-its fragment, it can be by its native ligand mesenchymal cell derived factor-1 (SDF-1) institute specific recognition, and has preferably at least 100,150 and 200 amino acid whose length.

CXCR2 is selected from as follows:

-interleukin-8 receptor β [people] has the described sequence SEQ ID of Genbank accession number NP_001548 No.29:

MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIY?ALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVN?GWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFIC?LSIWGLSLLLALPVLLFRRTVYSSNVSPACYEDMGNNTANWRMLLRILPQSFGFI?VPLLIMLFCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFLLCWLPYNLVLLADT?LMRTQVIQETCERRNHIDRALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIH?GLISKDSLPKDSRPSFVGSSSGHTSTTL；

-optionally transcribe splice variant or its natural variant, have at least 95% identity with this interleukin-8 receptor β with SEQ ID No.29; And

-its fragment can be by IL-8 institute specific recognition, and has preferably at least 100,150 and 200 amino acid whose length.

Invention also comprises for the method for the compound of the compound of selecting to have HIV (human immunodeficiency virus)-resistant activity or the composition that can infect for the preparation for the treatment of HIV, is characterised in that described method comprises step:

In first aspect, theme of the present invention is the method for generation and selective basis antibody of the present invention.

More particularly, the present invention relates to comprise the following steps: for selecting to suppress anti-CXCR4 antibody that HIV copies or the method for one of its functional fragment or derivative

I) antibody of screening generation and selection energy specific combination are in the antibody of CXCR4;

Ii) checking procedure i) selected antibody, and select can be in conjunction with the antibody of peripheral blood lymphocytes (PBMC),

Iii) ii) selected antibody of checking procedure, and select to be incorporated into the antibody of CXCR4 homodimer, and subsequently

Iv) iii) selected antibody of checking procedure, and select to suppress the antibody that Primary isolate X4-tropism HIV-1 copies in PBMC.

In another embodiment, invention relates to for selecting to suppress anti-CXCR4 antibody that HIV copies or the method for one of its functional fragment or derivative, comprises the following steps:

Iv) iii) selected antibody of checking procedure, and select to suppress Primary isolate X4-tropism HIV-1 and copy in PBMC and/or can suppress the antibody that Primary isolate X4/R5-tropism HIV-1 copies in PBMC.

The generation of antibody can realize by any means known to those skilled in the art, as for example, with the myeloma cell with merge mutually [Kohler﹠amp from immune mouse or with the splenocyte of other species of selected myeloma cell's compatibility; Milstein, 1975, Nature, 256:495-497].Immune animal can comprise the transgenic mice with human immunoglobulin gene seat direct production human antibodies after it.Another possible embodiment can comprise with display technique of bacteriophage and screens the library.

Screen step I) and ii) can realize by any means known to those skilled in the art or process.As limiting examples, can mention western blot analysis, facs analysis and the functional screening of the CXCR4 of ELISA, BIAcore, immunohistochemical methods, use CXCR4 express cell film extract or purifying.Preferred method comprise by facs analysis on the CXCR4 transfectant (step 1) and at least on PBMC (step 2) screen to guarantee that the antibody of producing also can identify the natural CXCR4 receptor conformation of target cell surface.This method is more accurately described in the following example.

The screening step I ii) can realize by any means known to those skilled in the art or process.As non-limiting but preferred example, what can mention is Western blot and/or the immunoprecipitation technology of using target antibody to carry out on from the film extract of CXCR4 transfectional cell or PBMC.

The screening step I v) can realize by any means known to those skilled in the art or process.As non-limiting but preferred example, what can mention is the method that comprises antibody screening, by the described rules of people (J.Immunol.2004 such as use Holl, 173,6274-83) screen the antibody that the capable primary HIV-1 of X4 of inhibition and/or the primary HIV-1 strain isolated of X4/R5 copy in PBMC.

In the screening step I preferred implementation ii) of the inventive method, described step I ii) comprises analyzing by BRET assesses antibody expressing on the cell of CXCR4-RLuc/CXCR4-YFP, and selects to suppress at least 40%, preferred 45%, 50%, 55% and the antibody of 60%BRET signal most preferably.

The BRET technology is the technology that represents [people such as Angers, PNAS, 2000,97:3684-89] that is called as protein dimer.

The step I of method ii) in BRET technology used, be well known to those skilled in the art, and have a detailed description in the following example.More particularly, BRET(bioluminescence resonance energy shifts) be at bioluminescence donor (renilla luciferase (Rluc)) and fluorescent receptor GFP(green fluorescent protein) or the YFP(yellow fluorescence protein)) the non-radiation type energy transfer that occurs between mutant.Use EYFP(enhancement type yellow fluorescence protein in present case).Transfer efficiency depends on direction and the distance between donor and acceptor.So, only have the energy transfer could occur when (1-10nm) when two molecules.This character is used to generate the protein-protein interaction analysis.In fact, in order to study the interaction between two mating partners (partner), generally first is blended in renilla luciferase and second yellow mutant that is blended in GFP.Fusion rotein general (but not necessarily) is expressed in mammalian cell.When existing, Rluc launches blue light at its membrane permeability substrate (coelenterazine (coelenterazine)).If GFP mutant distance R luc is not enough 10nm, energy can occur shift and extra yellow signal can be detected.The BRET signal is measured as the ratio of the light that light that acceptor launches and donor launch.Therefore, along with two fusion roteins are furthered or conformational change makes Rluc and GFP mutant more close the time, the BRET signal will strengthen.

If comprise that in a preferred embodiment BRET analyzes, can use any means known to those skilled in the art to measure the dimeric conformational change of CXCR4.Can mention following technology, unrestricted: the FRET(FRET (fluorescence resonance energy transfer)), the HTRF(homogeneous phase time discrimination fluorescence), FLIM(fluorescence lifetime imaging microscopy mirror) or the single wavelength fluorescent crosscorrelation of SW-FCCS(spectrum).

Also can use other classical technology, as coimmunoprecipitation, α screening, chemically crosslinked, double cross, affinity chromatography, ELISA or Far Western blot.

In aspect the described method of invention concrete, step I ii) comprises analyzing by BRET assesses antibody expressing on both cells of CXCR4-RLuc/CXCR4-YFP, and selection can suppress the antibody of 40%BRET signal at least.

In second aspect, theme of the present invention is separation antibody or one of its functional fragment or the derivative that obtains by described method.Described antibody or one of its described fragment or derivative can specific binding people CXCR4, and described antibody also can be induced CXCR4 homodimer conformational change.

CXCR4Mab and so on (for example cloning A120) can suppress HIV-1 experiment strain (X4HIV-1 as can be known from document _NL4-3) enter the people such as PBMC(Tanaka R., J.Virol.2001,75,11534-11543).And, know that also CXCR4Mab can suppress the HIV-1X4 Primary isolate and enter the clone of expressing CXCR4.Conversely, never announced the antibody of this viroid (namely not just in zoo virus or clone) that can be suppressed in its physical environment.In any case novelty of the present invention and non-obvious aspect are that CXCR4Mab can suppress the HIV-1X4 Primary isolate and enters PBMC.

Expressing " functional fragment and derivative " is similar with " Fab and derivative ", and general's specific definition in the present note subsequently.

Be understood that herein the present invention does not relate to the antibody of natural form, antibody is not in its natural surroundings that is to say, but can be by being isolated or obtaining from the natural origin purifying, perhaps pass through in addition genetic recombination or obtain by chemosynthesis, and therefore antibody capable comprises after this described alpha-non-natural amino acid.

More particularly; according to invention on the other hand; the antibody of claimed separation or one of its functional fragment or derivative; described antibody is characterised in that: they comprise at least one complementary determining region CDR, and this CDR is selected from the CDR that comprises as by IMGT numbering system defined aminoacid sequence SEQ ID No.1 to 6 and 30 to 33.

According to first aspect, the antibody that the present invention relates to separate or its functional fragment or derivative, at least one CDR that comprises the CDR that is selected from sequence SEQ ID No.1 to 6 defined according to the IMGT numbering system, perhaps its sequence and sequence SEQ ID No.1 to 6 carry out having after the best is compared at least 80%, at least one CDR of preferred 85%, 90%, 95% and 98% identity.

According to second aspect, the antibody that the present invention relates to separate or its functional fragment or derivative, comprise at least one CDR that is selected from sequence SEQ ID No.1 defined according to the IMGT numbering system, 2 and 30 to 33 CDR, perhaps its sequence and sequence SEQ ID No.1,2 and 30 to 33 carry out having after the best is compared at least 80%, at least one CDR of preferred 85%, 90%, 95% and 98% identity.

" functional fragment " or " Fab " of antibody mean, and particularly, antibody fragment is as fragment Fv, scFv(sc=strand), Fab, F (ab ') ₂, any fragment of being extended of Fab ', scFv-Fc or bispecific antibody or its transformation period.This functional fragment will be described in detail in this explanation subsequently.

" derivative compound " or " derivative " of antibody mean, and particularly, are the activity that keeps its identification CXCR4, the conjugated protein that is made of peptide support and at least one CDR of original antibody.This derivative compound is well known to those skilled in the art, will be in this explanation subsequently more detailed description.

More preferably, according to the present invention, the present invention includes particularly chimeric or humanized antibody, its derivative compound or its functional fragment that obtain by genetic recombination or chemosynthesis.

According to preferred embodiment, according to antibody of the present invention or its derivative compound or functional fragment, be characterised in that it is comprised of monoclonal antibody.

Should be appreciated that " monoclonal antibody " means to come from almost homologous antibody group's antibody.More refer in particular to, the individual antibody in the group (except some may the sudden change with the natural generation of minimum proportion discovery) be identical.In other words, monoclonal antibody is made of the homologous antibody that comes from individual cells clone (having eukaryotic host cell, the transfection of the DNA molecular of coding homologous antibody that the prokaryotic host cell etc. of the DNA molecular of coding homologous antibody is arranged such as hybridoma, transfection) growth, and generally take a class and only a class and subclass heavy chain, and only the light chain of a type as feature.Monoclonal antibody be high special and for single antigen.In addition, compare with the polyclonal antibody prepared product that generally includes for the various antibody of various determinants or epi-position, each monoclonal antibody is for single epitope.

More particularly, according to the first preferred embodiment of the present invention, antibody or its derivative compound or functional fragment are characterised in that it comprises the light chain that contains at least one CDR that is selected from CDR-L1, CDR-L2 and CDR-L3, wherein:

-CDR-L1 comprise aminoacid sequence SEQ ID No.1,

-CDR-L2 comprise aminoacid sequence SEQ ID No.2,

-CDR-L3 comprises aminoacid sequence SEQ ID No.3.

According to another embodiment, antibody or one of its derivative compound or functional fragment of invention, be characterised in that it comprise contain in sequence SEQ ID No.1, three CDR of 2 or 3 at least one or carry out having after the best is compared at least 80% with sequence SEQ ID No.1,2 or 3, the light chain of at least one sequence of preferred 85%, 90%, 95% and 98% identity.

Antibody or one of its functional fragment or derivative of invention, feature is that also it comprises the light chain that contains CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 comprises aminoacid sequence SEQ ID No.1, and CDR-L2 comprises that aminoacid sequence SEQ ID No.2 and CDR-L3 comprise aminoacid sequence SEQ ID No.3.

In another embodiment, antibody of the present invention or one of its functional fragment or derivative, be characterised in that it comprise contain aminoacid sequence SEQ ID No.7 or carry out having after the best is compared at least 80% with sequence SEQ ID No.7, the sequence of light chain of at least one sequence of preferred 85%, 90%, 95% and 98% identity.

According to second preferred implementation of invention, antibody or its derivative compound or functional fragment are characterised in that it comprises the light chain that contains at least one CDR that is selected from CDR-L1, CDR-L2 and CDR-L3, wherein:

-CDR-L1 comprises aminoacid sequence SEQ ID No.1,

-CDR-L2 comprises aminoacid sequence SEQ ID No.2,

-CDR-L3 comprises aminoacid sequence SEQ ID No.30.

According to another embodiment, antibody or one of its derivative compound or functional fragment of invention, be characterised in that they comprise contain in sequence SEQ ID No.1, three CDR of 2 or 30 at least one or carry out having after the best is compared at least 80% with sequence SEQ ID No.1,2 or 30, the light chain of at least one sequence of preferred 85%, 90%, 95% and 98% identity.

Antibody of the present invention or one of its functional fragment or derivative, feature is that also it comprises the light chain that contains CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 comprises aminoacid sequence SEQ ID No.1, and CDR-L2 comprises that aminoacid sequence SEQ ID No.2 and CDR-L3 comprise aminoacid sequence SEQ ID No.30.

In another embodiment, antibody or one of its functional fragment or derivative of invention, be characterised in that it comprise contain aminoacid sequence SEQ ID No.34 or carry out having after the best is compared at least 80% with sequence SEQ ID No.34, the sequence of light chain of at least one sequence of preferred 85%, 90%, 95% and 98% identity.

More particularly, antibody of the present invention or one of its derivative compound or functional fragment are characterised in that it comprises the heavy chain that contains at least one CDR that is selected from CDR-H1, CDR-H2 and CDR-H3, wherein:

-CDR-H1 comprises aminoacid sequence SEQ ID No.4,

-CDR-H2 comprises aminoacid sequence SEQ ID No.5,

-CDR-H3 comprises aminoacid sequence SEQ ID No.6.

According to another embodiment, antibody of the present invention or one of its derivative compound or functional fragment, be characterised in that it comprise contain in sequence SEQ ID No.4, three CDR of 5 or 6 at least one or carry out having after the best is compared at least 80% with sequence SEQ ID No.4,5 or 6, the heavy chain of at least one sequence of preferred 85%, 90%, 95% and 98% identity.

The embodiment concrete according to another, antibody or one of its derivative compound or functional fragment, be characterised in that it comprises the heavy chain that contains CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 comprises aminoacid sequence SEQ ID No.4, and CDR-H2 comprises that aminoacid sequence SEQ ID No.5 and CDR-H3 comprise aminoacid sequence SEQ ID No.6.

In another embodiment, antibody of the present invention or one of its functional fragment or derivative, be characterised in that it comprise contain aminoacid sequence SEQ ID No.8 or carry out having after the best is compared at least 80% with sequence SEQ ID No.8, the sequence of heavy chain of at least one sequence of preferred 85%, 90%, 95% and 98% identity.

More particularly, one of antibody of the present invention, its derivative compound or functional fragment are characterised in that it comprises the heavy chain that contains at least one CDR that is selected from CDR-H1, CDR-H2 and CDR-H3, wherein:

-CDR-H1 comprises aminoacid sequence SEQ ID No.31,

-CDR-H2 comprises aminoacid sequence SEQ ID No.32,

-CDR-H3 comprises aminoacid sequence SEQ ID No.33.

According to another embodiment, antibody of the present invention or one of its derivative compound or functional fragment, be characterised in that it comprise at least one that contains sequence SEQ ID No.31,32 or 33 3 CDR or carry out having after the best is compared at least 80% with sequence SEQ ID No.31,32 or 33, the heavy chain of at least one sequence of preferred 85%, 90%, 95% and 98% identity.

According to another embodiment, one of antibody, its derivative compound or functional fragment, be characterised in that it comprises the heavy chain that contains CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 comprises aminoacid sequence SEQ ID No.31, and CDR-H2 comprises that aminoacid sequence SEQ ID No.32 and CDR-H3 comprise aminoacid sequence SEQ ID No.33.

In another embodiment, antibody of the present invention or one of its functional fragment or derivative, be characterised in that it comprise contain aminoacid sequence SEQ ID No.35 or carry out having after the best is compared at least 80% with sequence SEQ ID No.35, the sequence of heavy chain of at least one sequence of preferred 85%, 90%, 95% and 98% identity.

Antibody of the present invention or one of its functional fragment or derivative, be characterised in that it comprises a light chain and a heavy chain, wherein this light chain comprises and contains respectively aminoacid sequence SEQ ID No.1,2 and 3 CDR-L1, CDR-L2 and CDR-L3, and this heavy chain comprises and contains respectively aminoacid sequence SEQ ID No.4,5 and 6 CDR-H1, CDR-H2 and CDR-H3.

At last, antibody of the present invention or one of its functional fragment or derivative, feature is that also it comprises the light chain that contains aminoacid sequence SEQ ID No.7 and the heavy chain that contains aminoacid sequence SEQ ID No.8.

Antibody of the present invention or one of its functional fragment or derivative, be characterised in that it comprises a light chain and a heavy chain, this light chain comprises and contains respectively aminoacid sequence SEQ ID No.1,2 and 30 CDR-L1, CDR-L2 and CDR-L3, and this heavy chain comprises and contains respectively aminoacid sequence SEQ ID No.31,32 and 33 CDR-H1, CDR-H2 and CDR-H3.

At last, antibody of the present invention or one of its functional fragment or derivative, feature is that also it comprises the light chain that contains aminoacid sequence SEQ ID No.34 and the heavy chain that contains aminoacid sequence SEQ ID No.35.

In this manual, term " polypeptide ", " peptide sequence ", " peptide " and " being attached to the protein of antibody compound or its sequence " are interchangeable.

It must be understood that at this, the present invention does not relate to the antibody of natural form, be that antibody is not taken from its natural surroundings, but can be by being isolated or obtaining from the natural origin purifying, perhaps obtain by genetic recombination or chemosynthesis, and therefore antibody capable carries following described alpha-non-natural amino acid.

In first embodiment, complementary determining region or CDR, mean the heavy chain of the defined immunoglobulin (Ig)s of people such as Kabat and the hypervariable region of the light chain (people such as Kabat, Sequences of proteins of immunological interest, the 5th edition, U.S.Department of Health and Human Services, NIH, 1991, and later version).Three heavy chain CDR and three light chain CDR are arranged., depend on situation herein, term " CDR " and " CDRs " are used to indicate one or more or even all comprise the zone of most of amino-acid residues of the antibodies affinity of the antigen of being responsible for its identification or epi-position.

In the second embodiment, by CDR zone or CDR(s), mean the hypervariable region of heavy chain immunoglobulin as defined in IMGT and light chain.

The IMGT unique number is given for variable domains [Lefranc M.-P., Immunology Today18,509 (1997)/Lefranc M.-P. of more any antigen receptor, chain type or species, The Immunologist, 7,132-136 (1999)/Lefranc, M.-P., Pommi é, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev.Comp.Immunol., 27,55-77 (2003)].In the IMGT unique number, conserved amino acid always has same site, for example Cys2 3(the one CYS), tryptophane 41(conservative-TRP), hydrophobic amino acid 89, halfcystine 104(the 2nd CYS), phenylalanine or tryptophane 118(J-PHE or J-TRP).The IMGT unique number provides (FR1-IMGT:1 to 26 of frame area, FR2-IMGT:39 to 55, FR3-IMGT:66 to 104 and FR4-IMGT:118 to 128) and the stdn description of complementary determining region (CDR1-IMGT:27 to 38, CDR2-IMGT:56 to 65 and CDR3-IMGT:105 to 117).Because the room represents vacant position, CDR-IMGT length (be presented between bracket and with point and separate, for example [8.8.13]) becomes important information.The IMGT unique number is used to 2D figure and presents, be appointed as 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 [Kaas in the 3D structure of IMGT/3D structure-DB, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data.Nucl.Acids.Res., 32, D208-D210 (2004)].

There are three heavy chain CDR and three light chain CDR.According to circumstances, term CDR purpose is to indicate one of these zones or several or even these zones whole as used herein, and it comprises is responsible for antibody for most of amino-acid residues of the affinity combination of the antigen of its identification or epi-position.

For more clear, should be appreciated that in following explanation and be more in particular in table 2 and 3, with IMGT numbering system and Kabat numbering system definition CDR.

According to the system of IMGT as defined above, with IMGT numbering system definition CDR, and according to the system of Kabat as defined above, with Kabat numbering system definition CDR.

More particularly, about being called as the antibody of 515H7, CDR-L1 is made of the SEQ ID No.1 in the IMGT numbering system and the SEQ ID No.9 in the Kabat numbering system.About CDR-L2, it is made of the SEQ ID No.2 in the IMGT numbering system and the SEQ ID No.10 in the Kabat numbering system.CDR-L3 is made of each the SEQ ID No.3 in two kinds of numbering systems.For heavy chain, CDR-H1 is made of the SEQ ID No.4 in the IMGT numbering system and the SEQ ID No.11 in the Kabat numbering system.CDR-H2 is made of the SEQ ID No.5 in the IMGT numbering system and the SEQ ID No.12 in the Kabat numbering system.At last, CDR-H3 comprises the SEQ ID No.6 in the IMGT numbering system, and it is made of the SEQ ID No.13 in the Kabat numbering system.

Then, about being called as the antibody of 301aE5, CDR-L1 is made of the SEQ ID No.1 in the IMGT numbering system and the SEQ ID No.9 in the Kabat numbering system.About CDR-L2, it is made of the SEQ ID No.2 in the IMGT numbering system and the SEQ ID No.36 in the Kabat numbering system.CDR-L3 is made of the SEQ ID No.30 in the IMGT numbering system and the SEQ ID No.37 in the Kabat numbering system.For heavy chain, CDR-H1 is made of the SEQ ID No.31 in the IMGT numbering system and the SEQ ID No.38 in the Kabat numbering system.CDR-H2 is made of the SEQ ID No.32 in the IMGT numbering system and the SEQ ID No.39 in the Kabat numbering system.At last, CDR-H3 comprises the SEQ ID No.33 in the IMGT numbering system, and it is made of the SEQ ID No.40 in the Kabat numbering system.

In implication of the present invention, " per-cent identity " between two nucleic acid or aminoacid sequence means the per-cent of identical Nucleotide between two sequences to be compared or amino-acid residue, it obtains after the best comparison, this per-cent is purely statistical, and the difference between two sequences is along its length stochastic distribution.Article two, being undertaken by comparative sequences after best comparison to it more traditionally of nucleic acid or aminoacid sequence describedly relatively can be carried out by sections (segment) or by use " contrast window ".except manual comparison, local homology's algorithm (1981) [Ad.App.Math.2:482] by Smith and Waterman, local homology's algorithm (1970) [J.Mol.Biol.48:443] by Neddleman and Wunsch, by the similarity searching method (1988) [Proc.Natl.Acad.Sci.USA85:2444] of Pearson and Lipman or by using the computer software (GAP in Wisconsin genetics software package of these algorithms, BESTFIT, FASTA and TFASTA, genetics calculating group, 575Science Dr., Madison, WI, or by comparison software BLAST NR or BLAST P) can carry out the best comparison of sequence to be compared.

Determine per-cent identity between two nucleic acid or aminoacid sequence by the sequences of relatively two best comparisons, wherein nucleic acid to be compared or aminoacid sequence, compared to the reference sequences that is used for best comparison between two sequences, can have and add or deletion.Number by the site of (between preferred two complete sequence) same amino acid Nucleotide or residue between definite two sequences calculates per-cent identity, and identical bits is counted divided by total number of sites of comparison window and result be multiply by 100 to obtain the per-cent identity between two sequences.

For example, can use with default parameter (particularly parameter " is opened gap penalty ": 5, and " extension gap penalty ": 2; Selected matrix is " BLOSUM62 " matrix of proposing of program for example) available blast program on webpage http://www.ncbi.nlm.nih.gov/gorf/bl2.html, " BLAST2 sequence " (people such as Tatusova, " Blast2sequences-a new tool for comparing protein and nucleotide sequences ", FEMS Microbiol., 1999, Lett.174:247 – 250); Directly calculate per-cent identity between two sequences to be compared with program.

Have at least 80% for demonstrating with reference amino acid sequence, the aminoacid sequence of preferred 85%, 90%, 95% and 98% identity, preferred example comprises those that comprise reference sequences, some modification (particularly delete, insert or replace at least one amino acid, brachymemma or prolongation).In the substituted situation of one or more continuous or discrete amino acid, the amino acid that preferably replaces in replacement is replaced by " equivalence " amino acid.At this, express " amino acid of equal value " and mean to replace a structure amino acid and the arbitrary amino acid that do not change the corresponding antibodies biologic activity, and the arbitrary amino acid of those specific embodiments that define below.

The amino acid whose structural homology that can replace according to itself and they or determine amino acid of equal value according to the result that biological activity between the various antibody that may generate is relatively checked.

As limiting examples, below table 1 summed up may the replacing of antibody biological activity noticeable change that may carry out and not cause corresponding modification; Nature can be done opposite replacement under identical conditions.

Table 1

Initial residue	Replace
		Ala(A)	Val,Gly,Pro
Arg(R)	Lys,His
		Asn(N)	Gln

Asp(D)	Glu
		Cys(C)	Ser
Gln(Q)	Asn
		Glu(G)	Asp
Gly(G)	Ala
		His(H)	Arg
Ile(I)	Leu
		Leu(L)	Ile,Val,Met
Lys(K)	Arg
		Met(M)	Leu
Phe(F)	Tyr
		Pro(P)	Ala
Ser(S)	Thr,Cys
		Thr(T)	Ser
Trp(W)	Tyr
		Tyr(Y)	Phe,Trp
Val(V)	Leu,Ala

In embodiment, the present invention relates to murine antibody or its derivative compound or functional fragment.

As above finding, the invention still further relates to any compound derived from antibody of the present invention.

More particularly, antibody of the present invention or its derivative compound or functional fragment, be characterised in that, on it, grafting has the conjugated protein of the peptide support of at least one CDR to consist of to described derivative compound by containing, and keeps in this way all or part of paratope recognition property of initial antibodies.

One or more sequences in CDR sequence of the present invention also can appear on various immunoglobulin (Ig) supports.In the case, protein sequence can rebulid the folding peptide backbone of CDR that is conducive to grafting, makes CDR can keep its paratope antigen recognition attribute.

Usually, those skilled in the art will know that type how to determine protein scaffolds, at least one CDR from initial antibody of grafting on protein scaffolds.More particularly, known this type of support that will select must satisfy maximum number following standard (Skerra A., J.Mol.Recogn., 2000,13:167-187):

-phylogenetic conservative property is good;

-known three-dimensional structure (as is known to the person skilled in the art, for example, by crystallography, NMR spectrum or any other technology);

-size is little;

-rare or there is no a post transcriptional modificaiton; And/or

-easily production, expression and purifying.

the origin of this type of protein scaffolds can be but be not limited to be selected from following structure: fibronectin and optimum fiber connect albumen III type structural domain 10, lipophorin, anticalin(is without corresponding translation) (Skerra A., J.Biotechnol., 2001, 74 (4): 257 – 75), protein Z from streptococcus aureus (Staphylococcus aureus) albumin A structural domain B, Trx A or band repeat motif as " ankyrin the repetition " (people such as Kohl, PNAS, 2003, vol.100, No.4, 1700-1705), " tatou repetition ", the protein of " being rich in leucine repeats " and " three tetradecapeptides repeat ".

Should further be mentioned that the support derived from the protein supressor (PIN) of toxin (as such as the toxin from scorpion, insect, plant, mollusk etc.) and neuronal nitric oxide synthase.

The example of this type of heterozygosis construct (having no intention to limit) is to insert the CDR-H1(heavy chain of anti-CD 4 antibodies in the ring of PIN) (being 13B8.2), new conjugated protein thereby the binding property that obtains to keep same with the initial antibody (people such as Bes, Biochem.Biophys.Res.Commun., 2006,343 (1), 334-344).On illustrative basis merely, also mentioned the CDR-H3(heavy chain of the anti-N,O-Diacetylmuramidase VHH of grafting antibody on a ring of neocarzinostatin) (people such as Nicaise, Protein Science, 2004,13 (7): 1882-1891).

At last, as mentioned above, this type of peptide support can comprise that at least one is from the CDR of initial antibody.Preferably, but not necessarily, those skilled in the art can be selected from least one from the CDR of heavy chain, and known heavy chain is the principal element of being responsible for antibodies specific.It is apparent selecting to those skilled in the art one or more relevant CDR, thus they will select suitable known technology (people such as Bes, FEBS letters508,2001,67-74).

Concrete aspect of the present invention relates to for selecting derived from the method according to the compound of antibody of the present invention, described derivative compound can be external and/or body in suppress the HIV cell and enter, and described derivative compound has comprised on it grafting peptide support of at least one antibody CDR, the method is characterised in that it comprises the following steps:

A) will prop up the compound that is configured to and the biological specimen that comprises HIV1 type and PBMC by peptide and contact placement external, wherein on the peptide support grafting at least one antibody CDR); And

B) if described compound can Inhibit the replication of HIV-1, select described compound,

And, be characterised in that the CDR of described at least one grafting is selected from following CDR: sequence SEQ ID No.1 to 6 and 30 to 33 or carry out best comparison after and sequence SEQ ID No.1 to 6 and 30 to 33 has at least 80%, the sequence of preferred 85%, 90%, 95% and 98% identity.

According to preference pattern, method can comprise step a) will contain on it grafting at least the compound of the peptide support of two or three antibody CDR place in external contact.

According to the even preferred pattern of this method, the peptide support is selected from its structure at support or the conjugated protein of mistake as mentioned above.

Obviously, these embodiment have no intention restriction, and should think it is that known or apparent other structure arbitrarily is covered by in the protection domain that present patent application gives for those skilled in the art.

The present invention thereby relate to antibody or its derivative compound or functional fragment, be characterised in that the peptide support is selected from protein, this protein is very conservative in a) phylogeny, b) strong system structure is arranged, c) the 3D molecular organization that has people to know, d) the little and/or e of size) contain can be by deletion and/or insert modify but do not change the zone of Properties in Stability.

according to preferred embodiment, antibody of the present invention, or its derivative compound or functional fragment, be characterised in that described peptide support is selected from i) from fibronectin, optimum fiber connects albumen 3 type structural domains 10, lipophorin, anticalin, protein Z from staphylococcus aureus protein A structural domain B, Trx A or band repeat motif as " ankyrin the repetition " (people such as Kohl, PNAS, 2003, vol.100, No.4, 1700-1705), " tatou repetition ", the support of the protein of " being rich in leucine repeats " and " three tetradecapeptides repeat ", or iii) neuronal nitric oxide synthase protein supressor (PIN).

The functional fragment that relates on the other hand above-mentioned antibody of invention.

More particularly, targeting antibodies of the present invention or its derivative compound or functional fragment are characterised in that described functional fragment is to be selected from fragment Fv, Fab, (Fab ') ₂, any fragment such as the Pegylation fragment that are extended of Fab ', scFv, scFv-Fc and bispecific antibody or its transformation period.

According to this type of functional fragment of antibody of the present invention by for example fragment Fv, scFv(sc=strand), Fab, (Fab ') ₂, Fab ', scFv-Fc or bispecific antibody or by chemically modified, as adding polyalkylene glycol such as polyoxyethylene glycol (Pegylation), (the Pegylation fragment is called as Fv-PEG, scFv-PEG, Fab-PEG, F (ab ') ₂-PEG and Fab '-PEG), or form by incorporating any fragment that improves its transformation period in liposome, microballoon or PLGA into, described fragment have at least one feature CDR(of the present invention particularly its can show general fashion, even part its from the activity of antibody).

Preferably, described functional fragment contains or comprises variable heavy chain or the light chain partial sequence of source antibody, described partial sequence be enough to keep with its from antibody identical binding specificity with and from antibody, preferably equal at least 1/100, more preferably at least 1/10, enough affinities.

This type of functional fragment contain its from five amino acid at least, preferred 6,7,8,10,15,25,50 or 100 continuous amino acid of antibody sequence.

Preferably, these functional fragments are Fv, scFv, Fab, F (ab ') ₂, F (ab '), scFv-Fc or bispecific antibody type, its usually have with its from the identical binding specificity of antibody.According to the present invention, antibody fragment of the present invention can be by obtaining from above-mentioned antibody as enzymic digestion (comprising stomach en-or papoid) and/or the method for cutting disulfide linkage by chemical reduction.The genetic recombination technology that antibody fragment can also also be known by those skilled in the art or by synthesizing to obtain by carrying out peptide such as automatic peptide synthesizer (as those of the sale such as Applied BioSystems).

For more clear, below table 2 summed up various aminoacid sequences corresponding to antibody of the present invention.

Table 2(is the Mu.=mouse wherein)

The aspect of another particularly important of antibody object of the present invention is, they do not bring into play effector function, as antibody dependent cellular cellulotoxic effect (ADCC) and/or CDC (CDC).

More particularly, as an example, antibody of the present invention or one of its functional fragment or derivative be not for Fc γ R(I, II or III) or for C1q or for both affinity.

This means to those skilled in the art, structurally, antibody of the present invention or one of its functional fragment or derivative do not have Fc part or its Fc partly there is no to exercise the normal glycosylation of effector function.

This consequence is that antibody of the present invention is preferably selected from IgG4 or IgG2 isotype, more preferably IgG4.

Similarly, preferred fragment is the fragment that there is no ADCC, and for example Fv, scFv(sc are strand), Fab, (Fab ') ₂, Fab ', scFv-Fc fragment or bispecific antibody or (the Pegylation fragment is called as Fv-PEG, scFv-PEG, Fab-PEG, F (ab ') as adding polyalkylene glycol such as polyoxyethylene glycol (" Pegylation ") by chemically modified ₂-PEG or Fab '-PEG) (" PEG " is polyoxyethylene glycol) or improved any fragment of its transformation period by incorporating liposome into.

More particularly, the preferred function fragment of the present invention that comes from antibody 515H7 is scFv, referred to here as the 515H7scFv-Ck fragment, comprises aminoacid sequence SEQ ID No.54.

Nucleotide sequence corresponding to described scFv comprises sequence SEQ ID No.55.

Another concrete aspect of the present invention relates to chimeric antibody or its derivative compound or functional fragment, is characterised in that described antibody also comprises light chain and the CH that comes from the antibody of mouse, particularly human heterogenous species.

Yet another concrete aspect of the present invention relates to humanized antibody or its derivative compound or functional fragment, is characterised in that constant region from the light chain of human antibodies and heavy chain is respectively λ or κ district and γ 2 or preferred γ 4th district.

Antibody of the present invention also comprises chimeric or humanized antibody.

Chimeric antibody is a kind of antibody, and it comprises the natural variable region (light chain and heavy chain) that comes from given species antibody and allos in the combination of the constant region of the light chain of antibody of the species of described given species and heavy chain.

Can be by using genetic recombination technology Dispersal risk or its Chimeric fragment.For example, can by the clone contain promotor and code book invention non-human (particularly mouse) variable region of mab sequence, and the recombinant DNA of the sequence of encoding human antibody-like constant region produce chimeric antibody.Chimeric antibody by this type of recombination coding according to the present invention can be mouse-people's mosaic for example, determines the specificity of this antibody from the variable region of mouse DNA, and determines its isotype from the constant region of people DNA.The method for the preparation of chimeric antibody with reference to the people such as Verhoeyn (BioEssays, 8:74,1988).

Summed up chimeric antibody 515H7(according to the present invention in herein following table 3 and be called as c515H7 or C515H7) various heavy chains and the aminoacid sequence of light chain.

Wherein c=is chimeric for table 3()

Corresponding to the nucleotide sequence of described antibody c515H7 heavy chain SEQ ID No.56 to 58 and light chain SEQ ID No.59 respectively with sequence SEQ ID No.60 to 63(heavy chain) and SEQ ID No.64(light chain) corresponding.

In a preferred embodiment, from its C end lysine residue deletion sequence of heavy chain (as seen in the initial pConPlus carrier series of Lonza: pConPlus γ 4 Δ K, pConPlus γ 4PRO Δ K﹠amp; PConPlus γ 2 Δ K).

And the G4PRO heavy chain is corresponding to IgG 4 isotypes, and it carries a sudden change to avoid the formation of incomplete antibody at hinge area.Find to have this sudden change [Angal S in from the parent pConPlus γ 4PRO Δ K of Lonza, King DJ, Bodmer MW, Turner A, Lawson AD, Roberts G, Pedley B, Adair JR.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 the chimeric antibody heavy chain, be characterised in that the CDR that it comprises CDR homology corresponding to the antibody that comes from different mammalian species, wherein said CDR, according to IMGT, by contain respectively sequence SEQ ID No.4,5 and 6 CDR-H1, CDR-H2 and CDR-H3 consist of.

More particularly, the present invention relates to the chimeric antibody light chain, be characterised in that the CDR that it comprises CDR homology corresponding to the antibody that comes from different mammalian species, wherein said CDR, according to IMGT, by contain respectively sequence SEQ ID No.1,2 and 3 CDR-L1, CDR-L2 and CDR-L3 consist of.

More particularly, the present invention relates to chimeric antibody or its derivative compound or functional fragment, be characterised in that it comprises heavy chain and light chain, every chain contains the CDR of CDR homology corresponding to the antibody that comes from different mammalian species, wherein said CDR, according to IMGT, consisted of by CDR-H1, the CDR-H2 and the CDR-H3 that contain respectively sequence SEQ ID No.4,5 and 6 heavy chain and CDR-L1, the CDR-L2 and the CDR-L3 that contain respectively sequence SEQ ID No.1,2 and 3 light chain.

In another embodiment, the present invention relates to chimeric antibody or its derivative compound or functional fragment, comprise the weight chain variabl area sequence that consisted of by SEQ ID No.8 and the variable region of light chain of sequence SEQ ID No.7.

Also in another embodiment, the present invention relates to chimeric antibody or its derivative compound or functional fragment, comprise being selected from SEQ ID No.56,57 or 58 sequence of heavy chain and the light chain of sequence SEQ ID No.59.

In a preferred embodiment, chimeric antibody c515H7VH (G4wt)/VL-Ck or its derivative compound or functional fragment according to the present invention, comprise the variable region of heavy chain of sequence SEQ ID No.56 and the variable region of light chain of sequence SEQ ID No.59.

In a preferred embodiment, chimeric antibody c515H7VH (G4PRO)/VL-Ck or its derivative compound or functional fragment according to the present invention, comprise the variable region of heavy chain of sequence SEQ ID No.57 and the variable region of light chain of sequence SEQ ID No.59.

In a preferred embodiment, chimeric antibody c515H7VH (G2wt)/VL-Ck or its derivative compound or functional fragment according to the present invention, comprise the variable region of heavy chain of sequence SEQ ID No.58 and the variable region of light chain of sequence SEQ ID No.59.

" humanized antibody " means to contain the antibody in the CDR zone of the antibody that comes from non-human source, and the other parts of antibody molecule derive from the one (or several) human antibodies.In addition, some skeleton section residue (being called FR) can be modified to keep binding affinity (people such as Jones, Nature, 321:522-525,1986; The people such as Verhoeyen, Science, 239:1534-1536,1988; The people such as Riechmann, Nature, 332:323-327,1988).

Humanized antibody of the present invention or its fragment can with technology well known by persons skilled in the art (as, such as in the people such as document Singer, J.Immun., 150:2844-2857,1992; The people such as Mountain, Biotechnol.Genet.Eng.Rev., 10:1-142,1992; With the people such as Bebbington, Bio/Technology, 10:169-175, those described in 1992) preparation.Preferred this type of humanized antibody is used for comprising the method for preventing and/or treating property treatment in in-vitro diagnosis or body.Other humanization technology that those skilled in the art also know as, for example, PDL is at patent EP0 451 216, EP0 682 040, EP0 939 127, EP0 566 647 or US5, and 530,101, US6,180,370, US5,585,089 and US5, " CDR grafting " technology described in 693,761.Can also quote United States Patent (USP) 5,639,641 or 6,054,297,5,886,152 and 5,877,293.

In addition, invention also relates to the humanized antibody that is produced by above-mentioned murine antibody.

In a preferred manner, be respectively λ or κ and γ 2 or preferred γ 4 zones from the light chain of human antibodies and CH.

More particularly, the present invention relates to the humanized antibody heavy chain, be characterised in that it comprises i) with the framework region of human antibodies heavy chain respective frame district homology, reach the ii) CDR of CDR homology corresponding to the antibody that comes from different mammalian species, wherein said CDR, according to IMGT, by comprise respectively sequence SEQ ID No.4,5 and 6 CDR-H1, CDR-H2 and CDR-H3 consist of.

In another embodiment, the present invention relates to the humanized antibody heavy chain, it comprises the variable region of the sequence that is made of SEQ ID No.64.

Also have in another embodiment, the present invention relates to the humanized antibody heavy chain, it comprises and is selected from SEQ ID No.67,68,69 and 95 complete sequence.

More particularly, invention relates to the humanized antibody light chain, be characterised in that it comprises i) with the framework region of human antibodies light chain respective frame district homology, reach the ii) CDR of CDR homology corresponding to the antibody that comes from different mammalian species, wherein said CDR, according to IMGT, by comprise respectively sequence SEQ ID No.1,2 and 3 CDR-L1, CDR-L2 and CDR-L3 consist of.

In another embodiment, the present invention relates to the humanized antibody light chain, it comprises the variable region that is selected from SEQ ID No.65,66,82 or 83 sequence.

Also have in another embodiment, the present invention relates to the humanized antibody light chain, it comprises and is selected from SEQ ID No.70,71,84 or 85 complete sequence.

More particularly, the present invention relates to humanized antibody or its derivative compound or functional fragment, be characterised in that it comprises heavy chain and light chain, every chain has i) with the framework region of human antibodies respective frame district homology, reach the ii) CDR of CDR homology corresponding to the antibody that comes from different mammalian species, wherein said CDR, according to IMGT, consisted of by CDR-H1, the CDR-H2 and the CDR-H3 that comprise respectively sequence SEQ ID No.4,5 and 6 heavy chain and CDR-L1, the CDR-L2 and the CDR-L3 that comprise respectively sequence SEQ ID No.1,2 and 3 light chain.

In another embodiment, the present invention relates to humanized antibody or its derivative compound or functional fragment, it comprises the weight chain variabl area sequence that is made of SEQ ID No.64 and is selected from the variable region of light chain of SEQ ID No.65,66,82 or 83 sequence.

Also have in another embodiment, the present invention relates to humanized antibody or its derivative compound or functional fragment, it comprises the heavy chain that is selected from SEQ ID No.67,68,69 or 95 sequence and is selected from the light chain of SEQ ID No.70,71,84 or 85 sequence.

In a preferred embodiment, according to the present invention, humanized antibody Hz515H7VH1D76N (G4wt)/VL2-Ck or its derivative compound or functional fragment comprise the heavy chain of sequence SEQ ID No.67 and the light chain of sequence SEQ ID No.70.

In another preferred embodiment, according to the present invention, humanized antibody Hz515H7VH1D76N (G4PRO)/VL2-Ck or its derivative compound or functional fragment comprise the heavy chain of sequence SEQ ID No.68 and the light chain of sequence SEQ ID No.70.

In another preferred embodiment, according to the present invention, humanized antibody Hz515H7VH1D76N (G2wt)/VL2-Ck or its derivative compound or functional fragment comprise the heavy chain of sequence SEQ ID No.69 and the light chain of sequence SEQ ID No.70.

In another preferred embodiment, according to the present invention, humanized antibody Hz515H7VH1D76N (G4wt)/VL2.1-Ck or its derivative compound or functional fragment comprise the heavy chain of sequence SEQ ID No.67 and the light chain of sequence SEQ ID No.71.

In another preferred embodiment, according to the present invention, humanized antibody Hz515H7VH1D76N (G4PRO)/VL2.1-Ck or its derivative compound or functional fragment comprise the heavy chain of sequence SEQ ID No.68 and the light chain of sequence SEQ ID No.71.

In another preferred embodiment, according to the present invention, humanized antibody Hz515H7VH1D76N (G2wt)/VL2.1-Ck or its derivative compound or functional fragment comprise the heavy chain of sequence SEQ ID No.69 and the light chain of sequence SEQ ID No.71.

In another preferred embodiment, according to the present invention, humanized antibody Hz515H7VH1D76N (G4wt)/VL2.2-Ck or its derivative compound or functional fragment comprise the heavy chain of sequence SEQ ID No.67 and the light chain of sequence SEQ ID No.84.

In another preferred embodiment, according to the present invention, humanized antibody Hz515H7VH1D76N (G4PRO)/VL2.2-Ck or its derivative compound or functional fragment comprise the heavy chain of sequence SEQ ID No.68 and the light chain of sequence SEQ ID No.84.

In another preferred embodiment, according to the present invention, humanized antibody Hz515H7VH1D76N (G2wt)/VL2.2-Ck or its derivative compound or functional fragment comprise the heavy chain of sequence SEQ ID No.69 and the light chain of sequence SEQ ID No.84.

In another preferred embodiment, according to the present invention, humanized antibody Hz515H7VH1D76N (G4wt)/VL2.3-Ck or its derivative compound or functional fragment comprise the heavy chain of sequence SEQ ID No.67 and the light chain of sequence SEQ ID No.85.

In another preferred embodiment, according to the present invention, humanized antibody Hz515H7VH1D76N (G4PRO)/VL2.3-Ck or its derivative compound or functional fragment comprise the heavy chain of sequence SEQ ID No.68 and the light chain of sequence SEQ ID No.85.

In another preferred embodiment, according to the present invention, humanized antibody Hz515H7VH1D76N (G2wt)/VL2.3-Ck or its derivative compound or functional fragment comprise the heavy chain of sequence SEQ ID No.69 and the light chain of sequence SEQ ID No.85.

Below table 4 has been summed up describedly according to invention herein, is respectively the aminoacid sequence of humanized antibody 515H7 different heavy chains and light chain variable structural domain and total length (or complete).

Table 4(is the Hz=humanization wherein)

In a preferred embodiment, delete sequence of heavy chain (seen in from the original pConPlus carrier series of Lonza: pConPlus γ 4 Δ K, pConPlus γ 4PRO Δ K﹠amp from its C end lysine residue; PConPlus γ 2 Δ K).

And the G4PRO heavy chain is corresponding to IgG 4 isotypes, and it carries a sudden change to avoid forming incomplete antibody at hinge area.[Angal S is found in this sudden change in from the parent pConPlus γ 4PRO Δ K of Lonza, King DJ, Bodmer MW, Turner A, Lawson AD, Roberts G, Pedley B, Adair JR.A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody.Mol Immunol. (1993); 30 (1): 105-108].

Especially, the humanized antibody of called after hz515H7IgG4 provided herein, it comprises the heavy chain of human IgG 4 isotypes, and described heavy chain has the sequence of SEQ ID NO:95 representative.

As an example, for avoiding query, express " VH1 " and be similar to expression " VH variant 1 ", " VH variant 1 ", " VH Var1 " or " VH var1 ").

It must be understood that above-mentioned illustration VH/VL combination is not restrictive.Certainly, those skilled in the art can reset all VH and the VL that announces in this explanation, and need not undue burden and need not to use creative skill.

A new aspect of the present invention relates to the nucleic acid of separation, is characterised in that it is selected from following nucleic acid (comprising any degeneracy genetic code):

A) nucleic acid (DNA or RNA), it is encoded according to antibody of the present invention or one of its functional fragment or derivative;

B) nucleic acid, it comprises the DNA sequence dna that selects free SEQ ID No.14 to 19 and 41 to 45 sequences that form;

C) nucleic acid, it comprises the DNA sequence dna that selects free SEQ ID No.20,21,46 and 47 sequences that form;

D) as b) or c) in the corresponding RNA nucleic acid of nucleic acid of definition;

E) as a), b) and c) in the complementary nucleic acid of nucleic acid of definition; And

F) nucleic acid of at least 18 Nucleotide, it can be hybridized with at least one CDR of sequence SEQ ID No.14 to 19 and 41 to 45 under high rigor condition.

Below table 5 summed up various nucleotide sequences about antibody of the present invention.

Table 5(is the Mu.=mouse wherein)

The term of Alternate " nucleic acid ", " core sequence ", " nucleotide sequence ", " polynucleotide " in the present note, " oligonucleotide ", " polynucleotide sequence " and " nucleotide sequence ", mean the accurate sequence of Nucleotide, no matter whether modify, fragment or the zone of its definition nucleic acid, comprise or do not contain non-natural nucleotide, and being the transcription product of double-stranded DNA, single stranded DNA or described DNA.

Comprise also that herein the present invention does not relate to the nucleotide sequence in its natural dyeing body environment (namely being in native state).Sequence of the present invention has been separated and/or purifying, i.e. directly or indirectly sampling of their (for example with copies), and its environment is at least part of the modification.Also should mention by genetic recombination at this and learn the isolating nucleic acid that the mode of host cell (for example with) obtains or obtain by chemosynthesis.

" show at least 80% after comparing with preferred sequence the best, the nucleotide sequence of preferred 85%, 90%, 95% and 98% per-cent identity " means nucleotide sequence (with respect to reference nucleic acid sequence) and shows especially some modification of fixed point, for example particularly deletion, brachymemma, prolongation, chimeric fusion and/or replacement.Preferably, these sequence encodings aminoacid sequence (this relate to the degeneracy of genetic code) identical with reference sequences or preferred under highly rigorous condition might with the complementary sequence of reference sequences specific hybridization, those that particularly define below.

Hybridize under highly rigorous condition and mean, two complementary DNA sheets are intersegmental keeps hybridization to the condition of selecting to relate to temperature and ionic strength under this mode allowing.On simple illustrative basis, the rigorous condition of height of above-mentioned hybridization step for limiting the polynucleotide passage purpose is favourable, and is as follows.

Carry out DNA-DNA or DNA-RNA hybridization in two steps: (1) is at 42 ° of C, at phosphate buffered saline buffer (20mM, pH7.5 comprises 5X SSC(1X SSC corresponding to the solution of 0.15M NaCl+0.015M Trisodium Citrate), 50% methane amide, 7% sodium lauryl sulphate (SDS), 10X Denhardt ' s, 5% T 500 and 1% salmon sperm DNA) in prehybridization three hours; (2) in the temperature that depends on probe length (namely, for being 42 ° of C greater than the long probe of 100 Nucleotide) under carry out initial hybridization 20 hours, then 20 ° of C, carry out in 2X SSC+2%SDS twice 20 minutes the washing, 20 ° of C, carry out in 0.1X SSC+0.1%SDS one time 20 minutes the washing.For greater than the long probe of 100 Nucleotide, at 60 ° of C, carry out last washing 30 minutes in 0.1X SSC+0.1%SDS.For longer or shorter oligonucleotide, those skilled in the art can be according to the described program of the people such as Sambrook (Molecular cloning:a laboratory manual, Cold Spring Harbor Laboratory; The third edition, 2001) adjust the rigorous hybridization conditions of above-mentioned height for the polynucleotide of specific dimensions.

The present invention also comprises the nucleic acid molecule of separation, is characterised in that it is selected from following nucleic acid:

A) nucleic acid (DNA or RNA), according to the present invention, coding humanization heavy chain of antibody or its derivative compound or functional fragment;

B) nucleic acid (DNA or RNA), according to the present invention, coding humanization light chain of antibody or its derivative compound or functional fragment;

C) nucleic acid (DNA or RNA), according to the present invention, coding humanized antibody or its derivative compound or functional fragment;

D) be complementary to as a), b) or the nucleic acid of the nucleic acid that c) limits;

E) nucleic acid of at least 18 Nucleotide can be hybridized with at least one heavy chain that comprises nucleic acid sequence SEQ ID No.72 or 75 to 77 under highly rigorous condition;

F) nucleic acid of at least 18 Nucleotide, can be under highly rigorous condition with comprise nucleic acid sequence SEQ ID No.73,74,86,87 or 78,79,88,89 at least one light chain hybridization.

Thereafter table 6 has been summed up respectively the nucleotide sequence according to different heavy chains and light chain variable structural domain and the total length (or complete) of humanized antibody 515H7 of the present invention.

Table 6(is the Hz=humanization wherein)

Table 7 has herein been summed up according to the different heavy chains of chimeric antibody 515H7 of the present invention and the nucleotide sequence of light chain.

Wherein c=is chimeric for table 7()

In other words, the nucleic acid that the present invention relates to separate is characterised in that it is selected from following nucleic acid:

A) nucleic acid (DNA or RNA), coding is according to antibody of the present invention or one of its functional fragment or derivative;

B) nucleic acid comprises the DNA sequence dna that selects free sequence SEQ ID No.14 to 19 and 41 to the 45 CDR sequences that consist of;

C) nucleic acid comprises and selects free SEQ ID No.20,21,46,47,72,73,74,86 and 87 heavy chains that consist of and the DNA sequence dna of light chain variable structural domain sequence;

D) nucleic acid comprises and selects free SEQ ID No.60 to 63,75 to 79,88,89 and 94 heavy chains that consist of and the DNA sequence dna of sequence of light chain;

E) nucleic acid comprises the DNA sequence dna that is made of SEQ ID No.55;

F) as b), c), d) or e) in the corresponding RNA nucleic acid of the nucleic acid that limits;

G) as a), b), c), d) and e) in the complementary nucleic acid of the nucleic acid that limits; And

H) nucleic acid of at least 18 Nucleotide can be hybridized with at least one CDR of sequence SEQ ID No.14 to 19 and 41 to 45 under high rigor condition.

The invention still further relates to the carrier that comprises nucleic acid of the present invention.

The present invention especially target comprises clone and/or the expression vector of this type of nucleotide sequence.

Carrier of the present invention preferably is contained in the element that allows nucleotide sequence to express and/or secrete in given host cell.Thereby carrier must comprise promotor, translation initiation and termination signal, also have suitable transcription regulatory region.It must be kept in stable mode in host cell, and randomly has the special signal that instructs the translated protein secretion.According to host cell used, those skilled in the art can these different elements of choice and optimization.For this purpose, nucleotide sequence can be inserted into the autonomously replicationg vector in selected host, or selected host's conformability carrier.

Examples of such carriers is with the normally used method preparation of those skilled in the art, and the clone who produces can be introduced into the standard method as lipofection, electroporation, heat shock or chemical process suitable host.

Carrier is the carrier of (for example) plasmid or viral source.They are used to transformed host cell with the clone or express nucleotide sequence of the present invention.

The present invention also comprises and transforms host cell carrier of the present invention is arranged or comprise carrier of the present invention.

Host cell can be selected from protokaryon or eukaryotic system, as bacterial cell for example, also has yeast cell or zooblast, particularly mammalian cell.Can also use insect or vegetable cell.

Except the mankind, the invention still further relates to the animal that has according to transformant of the present invention.

Another aspect of the present invention relates to for the production of the method according to one of antibody of the present invention or its functional fragment, it is characterized in that described method comprises the following steps:

A) cultivate according to host cell of the present invention in matrix and under suitable culture condition; And

B) so from culture medium or from described culturing cell one of the described antibody of remanufacture or its functional fragment.

Can be used for preparation method according to recombinant polypeptide of the present invention according to transformant of the present invention.The present invention also comprises preparation according to the method for the polypeptide of recombinant forms of the present invention, is characterised in that described method uses according to carrier of the present invention and/or transform the cell of the described carrier of with good grounds invention.Preferably, the cell of the described carrier of the with good grounds invention of conversion is expressed under the condition that is reclaimed with described recombinant peptide at the aforementioned polypeptide of permission and is cultivated.

As already mentioned, host cell can be selected from protokaryon or eukaryotic system.Particularly, may identify be conducive to the nucleotide sequence of the present invention secreted in this protokaryon or eukaryotic system.Carry therefore being advantageously used according to the described carrier of invention of such sequence and produce recombinant protein to be secreted.In fact, protein is in cell culture supernatant but not the fact that exists in host cell will be conducive to the purifying of these target recombinant protein matter.

Polypeptide of the present invention can also prepare by chemosynthesis.A kind of this type of preparation method is also the object of invention.Those skilled in the art will know that the method for chemosynthesis, as by concentrating fragment or (see especially the people such as Steward by solid phase technique synthetic in traditional solution, 1984, Solid phase peptides synthesis, Pierce Chem.Company, Rockford, 111, second edition) or the part solid phase technique.Invention also comprises the polypeptide that obtains and can comprise corresponding alpha-non-natural amino acid by chemosynthesis.

The present invention also comprises can obtainable antibody or its derivative compound or functional fragment by the method for invention.

According on the other hand, the present invention relates to antibody as above, be characterised in that in addition antibody capable specific combination human chemokine family receptors and/or can copy by special inhibition X4-tropism HIV.

According on the other hand, the present invention relates to antibody as above, be characterised in that in addition the antibody capable specific combination in the human chemokine family receptors and/or can copy by special inhibition X4/R5-tropism HIV.

According to new embodiment, the present invention relates to antibody or its derivative compound or functional fragment, its antibody by dual specific consists of, the meaning be antibody comprise can to the HIV cell enter relevant any acceptor (as, for example, CCR5, CD4, CXCR4(except antibody of the present invention, i.e. another epi-position of target) or CCR3, CCR2, CCR8, CXCR6, CXCR7, CX3CR1) interactional the second motif.

Dual specific or bifunctional antibody have formed s-generation monoclonal antibody, have wherein made up two kinds of different variable regions (Hollinger and Bohlen, 1999, Cancer and metastasis, rev.18:411-419) in a part.About the ability of the some molecules in its targeted cells surface, all illustrated the effectiveness of antibody in diagnosis and treatment field; This antibody-like can chemical process (people such as Glennie MJ, 1987, J.Immunol.139,2367 – 2375; The people such as Repp R., 1995, J.Hemat., 377-382) or somatic method (Staerz U.D. and Bevan M.J., 1986, PNAS83,1453-1457; The people such as Suresh M.R., 1986, Method Enzymol., 121:210-228) and preferably (this technology can force the heterodimerization of gained antibody and therefore be conducive to the purifying (Merchand etc. of gained antibody with gene engineering, 1998, Nature Biotech., 16:677-681)) obtain.

These bi-specific antibodies can be configured to whole IgG, dual specific Fab ' 2, Fab ' PEG, bispecific antibody or dual specific scFv, (wherein the antigen for each target exists two binding sites (people such as Park also can to become tetravalence bi-specific antibody as above, 2000, Mol.Immunol., 37 (18): 1123-30) or its fragment.

Except guaranteeing bi-specific antibody production and use than producing the more cheap economic interests of two specific antibodies, the use of this type of bi-specific antibody has the advantage that reduces treatment toxicity.In fact, the use of bi-specific antibody makes the total amount and the consequent possible toxicity that reduce circulating antibody become possibility.

In the preferred embodiment of the present invention, bi-specific antibody is divalence or tetravalent antibody.

At last, the present invention relates to above-mentioned antibody or one of its functional fragment or derivative as medicine.

The invention still further relates to pharmaceutical composition, it comprises that the compound that is made of antibody of the present invention or one of its functional fragment or derivative is as active ingredient.Preferably, described antibody supplement has vehicle and/or pharmaceutically acceptable carrier.

Invention also relates to the composition as mentioned above as medicine.

Of the present invention one concrete aspect, antibody or one of its functional fragment or derivative suppress HIV-1KON Primary isolate copying in PBMC, have IC ₉₀Be at least 5 μ g/ml, preferred at least 10 μ g/ml.

The present invention also comprises according to inventing described antibody or composition for the preparation of prevention or the medicine for the treatment of HIV infection and/or the purposes of medicine.

More particularly, as limiting examples, described HIV infects the infection for X4-tropism HIV.

In another embodiment, as limiting examples, described HIV infects the infection for X4/R5-tropism HIV.

The invention still further relates to describedly, preferably humanized according to invention, antibody or its functional fragment or derivative and/or composition are for the preparation of the purposes that suppresses the medicine that HIV copies.Usually, the present invention relates to (preferably humanized) antibody or its functional fragment or derivative and/or composition for the preparation of the purposes of the medicine of HIV disease prevention or treatment.

In this manual, " pharmaceutical carrier " means to participate in compound or the compound combination of pharmaceutical composition, this material does not cause second order reaction, and for example this material be conducive to active compound use, increase its life-span and/or effect in organism, improve its in solution solubleness or improve its storage.This type of pharmaceutical carrier is well-known, and according to character and the route of administration of selected active compound, those skilled in the art can adjust it.

Preferably, this compound will be with system mode, particularly use with intravenously, intramuscular, intracutaneous, intraperitoneal, subcutaneous, intravaginal or oral route.More preferably, used with the multiple doses at same interval in certain hour by the composition that forms according to the described antibody of invention.

The standard that usually will consider when the planning of its route of administration, dosage and best Galenic formula form (galenic forms) can be suitable for patient's treatment according to foundation (as, for example patient's age or body weight, his seriousness, his treatment tolerance and side effect of experiencing of overall state) determine.

The invention still further relates to composition, its as combination product with simultaneously, separately or the mode of extending use, comprise that in addition anti-HIV antibody or anti-HIV cell enter antibody or anti-HIV copies antibody, other antibody for CXCR4.

According to another embodiment, the invention still further relates to pharmaceutical composition as above, it comprises at least the second anti-HIV-1 compounds, this compound be selected from can be special the inhibition HIV compound that enters and/or copy (anti-CCR5, anti-CD4 compound and anti-CXCR4 compounds beyond those that describe as the present invention, or other anti-HIV-1 compounds arbitrarily well known by persons skilled in the art).

Another embodiment that is complementary in addition invention is made of composition as above, said composition as simultaneously, separately or extend combination or the coupled product that uses, formed by anti-HIV-1 compounds.

" use simultaneously " two kinds of compounds that mean to use the composition that is included in single dose form.

" separately use " two kinds of compounds that mean to use simultaneously the composition that is included in the various dose form.

" extend and use " compound that means two kinds of compositions of continuous administration, every kind is included in the various dose form.

Usually, greatly improved the validity of HIV treatment according to the described composition of invention.In other words, strengthened the result for the treatment of of invention antibody by using anti-HIV reagent in beyond thought mode.Another main later stage advantage that composition of the present invention produces is, might use the active ingredient of lower effective dose, thereby might avoid or reduce the risk that side effect (the particularly effect of anti-HIV reagent) occurs.And this composition makes the result for the treatment of that realizes quickly expectation become possibility.

" the anti-HIV reagent of therapeutic " means a kind of material, and when it was applied to the patient, its was treated or has prevented the interior HIV of patient to copy.The limiting examples of this type of reagent comprises " antiretroviral drugs such as hiv protease inhibitor (PI), nucleoside/nucleotide hiv reverse transcriptase inhibitor (NRTI/NtRTI) non-nucleoside hiv reverse transcriptase inhibitor (NNRTI), HIV entry inhibitor, hiv integrase inhibitor ".

In (for example) VIDAL, quoted this type of reagent on the page about the compound place of " anti-HIV-1 compounds "; Anti-HIV-1 compounds with reference to this reference citation is incorporated herein by non-limiting preferred anti-HIV reagent.

The hiv protease inhibitor means to suppress the arbitrary substance of hiv protease activity.The example of this type of hiv protease inhibitor includes, but not limited to saquinavir mesilate or SQV

Indinavir or IDV Ritonavir or RTV

Viracept see nelfinaivr or NFV

Amprenavir

Rltonavir/ritonavir or LPV/r

Reyataz R or ATV

Fosamprenavir or FPV

Tipranavir or TPV

Prezista or DRV

HIV nucleosides or nucleotide reverse transcriptase inhibitors (NRTI) mean a kind of material, and it is nucleosides or the nucleotide analog of blocking-up HIV RNA reverse transcription.The example of NRTI includes, but not limited to zidovudine or AZT, ZDV Didanosine or ddi

Zalcitabine

Stavudine or d4T

Lamivudine or 3TC

Abacavir or ABC

Tenofovir disoproxil (Tenofovir disoproxil fumarate) or TDF

Emtricitabine or FTC

Non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTI) means a kind of material, and it can be blocked HIV RNA reverse transcription but not be nucleosides or nucleotide analog.The example of NNRTI includes, but not limited to nevirapine or NVP

Efavirenz or EFV

Delavirdine or DLV

And etravirine (Etravirine) or ETR

The HIV entry inhibitor means to block a kind of material that the HIV cell enters.The example of HIV entry inhibitor includes, but not limited to En Fuwei or T20 Malawi's promise or MVC

Hiv integrase inhibitor means to suppress a kind of material of hiv integrase activity.The example of integrase inhibitor includes, but not limited to Merck or RAL

This type of reagent is also the compound that (for example) belongs to the same class medicine described in VIDAL, now the same class medicine in clinical case for example (but being not limited to) Vicriviroc(without corresponding translation), PRO140, TNX-355, AMD070, Racivir(be without corresponding translation), A Lita shore (Apricitabine), Elvucitabine (Elvucitabine), Fu Luowei pyridine (Flosalvudine), a profit dimension woods (Rilpivirine), Lei Getewei (Elvitegravir).

This type of reagent is also the compound that (for example) belongs to other potential class medicine, the stimulator (valproic acid ...) that the HIV of inhibitor as ripe in (but being not limited to) (Bevirimat), beta galactose ceramide glucoside analogue, carbohydrate binding reagents, RNA enzyme H inhibitor, HIV gene expression inhibitor, latent T cell discharges.

In particularly preferred embodiments, be characterised in that as the described invention composition of combination product, described anti-HIV reagent Chemical bond in described antibody in order to use simultaneously.

In order to be conducive to described anti-HIV reagent and according to the combination of invention between described antibody, can will in conjunction with two compounds between introduce the spacer molecule, as polyalkylene glycol polyoxyethylene glycol or amino acid; Perhaps, in another embodiment, can use the reactive derivative (wherein introduced can with the function of described antibody response) of described anti-HIV reagent.These combination technologies are known by this area researchist, and will more not discuss in detail in this manual.

Further preferably, the described antibody of invention that forms described conjugate is selected from its functional fragment, particularly loses the fragment (as the scFV fragment) of its Fc component.

According to invention, the invention still further relates to the composition as combination product as medicine, or relate to anti-CXCR4Mab/ inverase conjugate.

Preferably, described composition or described conjugate as combination product will be supplemented with vehicle and/or pharmaceutical carrier.

Thereby, the present invention relates to antibody or one of its functional fragment or derivative for the preparation of the selectively targeted purposes that HIV is copied the medicine with bioactive compound.

In another embodiment, the invention still further relates to the method for HIV prevention or treatment, wherein said method comprises that the patient by the Xiang Youqi needs uses the step that forms according to antibody of the present invention or one of Fab or derivative and/or composition.

More particularly, the method according to this invention also comprises by use the step that anti-CCR5 compound such as Malawi's promise form to described patient.

As previously described, CXCR4Mab515H7 and 301aE5 have the strong activity that anti-HIV-1 is copied in PBMC, so this type of antibody capable is used to screen the CXCR4 antagonist Anti-virus agent of analyzing to identify that treatment HIV-1 infects.In the first step of these analyses, the cell of expression CXCR4 and Mab515H7 and/or 301aE5 are hatched, and assess subsequently the potentiality of molecules in inhibiting Mab515H7 and/or 301aE5 combination.The cell that uses in this type analysis can be clone such as the human cell line of CHO-CXCR4, NIH3T3-CXCR4 or CXCR4 transfection such as human cell line such as NALM6 or primary cell such as the PBMC of U373-MAGI-CXCR4, expression CXCR4 of transfection.The method that suppresses Mab515H7 and/or 301a5 CXCR4 antagonist of combination on the CXCR4 express cell in order to screening can be (the AIDS Research And Human Retroviruses of the competitiveness enzyme-linked immune absorption analysis (ELISA) based on cell that the people such as Zhao Q describes, 2003,19, pp947-955) or use as the method (Leukemia2003 of the fluorescence-activated cell sorting (FACS) of the people such as Juarez J. description, 17, pp1294-1300).

Therefore, invention one concrete aspect, relate to for screening and/or identify as the method for the molecule of CXCR4 antagonist Anti-virus agent, comprise step:

A) select to express the cell of CXCR4,

B) antibody of described cell and invention or one of its functional fragment or derivative are hatched, and

C) the assessment check molecule potential inhibition of being combined with CXCR4 for antibody or one of its functional fragment or derivative, and

D) select to produce the molecule of described inhibition.

In another embodiment, can add the following step e):

E) copy these molecules of check in analysis at HIV-1.

Further feature and the advantage of invention further present with embodiment and Tu in specification sheets, and caption shows below.

Description of drawings

Figure 1A and 1B show (gating) strategy of establishing that on monocyte and lymphocyte, CXCR4 expresses.

Figure 1A: T cell CD3-PE antibody staining.

Figure 1B: monocyte CD14-PE antibody staining.

Fig. 2 A and 2B show anti-CXCR4Mab515H7 and the combination of 301aE5 on monocyte and T lymphocyte.

Fig. 3 A and 3B shift (BRET) method via the bioluminescence resonance energy in the HEK293 cell, show respectively by SDF-1 with by anti-CXCR4Mab515H7 and 301aE5 to regulate the CXCR4 receptor dimer.

Fig. 4 A and 4B and Fig. 5 have shown that anti-CXCR4Mab515H7 and 301aE5 suppress HIV-1 strain isolated KON(X4 virus) ability that copies in the human PBMC.

Fig. 6 A, 6B and 6C have shown in the CHO-CXCR4 cell with Mab515H7(Fig. 6 A), 301aE5(Fig. 6 B) and c515H7(Fig. 6 C) suppress the calcium that SDF-1 induces and discharge.

Fig. 7 and 8 has shown that anti-CXCR4Mab515H7, c515H7 and 301aE5 suppress HIV-1X4 virus Primary isolate MN(Fig. 7) and 92UG024(Fig. 8) ability that copies in the human PBMC.

Fig. 9 has shown the ability that anti-CXCR4Mab515H7, c515H7 and 301aE5 inhibition HIV-1X4 virus Primary isolate KON, MN and 92UGO24 copy in the human PBMC.

Figure 10 and 11 has shown that anti-CXCR4Mab515H7, c515H7 and 301aE5 suppress the ability that amphicheirality HIV-1X4/R5 virus Primary isolate 89.6 copies in the human PBMC.

Figure 12 has shown that Mab c515H7 and Malawi promise combination suppress HIV-1 Primary isolate 89.6(amphicheirality X4/R5 virus) beneficial effect that copies in the human PBMC.

Figure 13 has shown that Mab c515H7 and Malawi promise combination suppress HIV-1 Primary isolate UG93067(amphicheirality X4/R5 virus) beneficial effect that copies in the human PBMC.

Figure 14 has shown that anti-CXCR4Mab515H7, c515H7 and 301aE5 suppress the ability that HIV-1X4 virus Primary isolate KON copies in the human PBMC.

Figure 15 has illustrated the binding specificity of c515H7Mab by facs analysis.

Figure 16 shifts (BRET) method by the bioluminescence resonance energy and has illustrated the effect of c515H7Mab for the CXCR4 homodimer.

Figure 17: the aminoacid sequence of 515H7 weight chain variable structural domain and people's reproductive tract IGHV3-49*04 and IGHJ4*01 is compared.The 515H7VH aminoacid sequence compares with the acceptor Frame sequence of choosing.VH Var1(VH1) sequence is corresponding to the humanization variant of 515H7VH structural domain.Single reverse mutation at 76 shows with runic.

Figure 18: the aminoacid sequence of 515H7 light chain and people's reproductive tract IGKV4-1*01 and IGKJ1*01 is compared.The 515H7VL aminoacid sequence compares with the acceptor Frame sequence of choosing.The sudden change residue that VL Var2.1, Var2.2 and Var2.3 sequence show with runic corresponding to humanization 515H7VL Var2() enforcement humanization variant.Var2.1 and Var2.2 carry other 4 humanization residues, and Var2.3 comprises other 5 mankind's residues.

Figure 19: with the different variants intersection blocking-up biotinylation murine antibody 515H7 of chimeric 515H7 and humanization 515H7.Use the activity of NIH3T3 cell assessment 515H7 humanization variant (hz515H7) the intersection blocking-up parent murine antibody 515H7 of CXCR4 transfection with flow cytometry.The activity of humanization variant is compared with chimeric 515H7.Variant VH1 and chimeric VL(cVL) combination intersect the active and chimeric activity very similar (A) of blocking-up.When with variant 2 combination of VL, determine VH variant 1(VH1, without the variant of reverse mutation) activity do not reduce (B).

Figure 20: the different variants with chimeric 515H7 and humanization 515H7 suppress biotinylation SDF-1 combination.Use clone RAMOS assessment 515H7 humanization variant (hz515H7) to suppress the ability of SDF-1 combination with flow cytometry.The inhibition ability of humanization variant is compared with chimeric 515H7.Humanization variant hz515H7VH1D76N VL2 has the ability that similarly suppresses the SDF-1 combination with chimeric antibody.Humanized antibody fragment hz515VH1VL2 has complete activity in inhibition SDF-1 is incorporated into the RAMOS cell.

Figure 21 has shown humanization 515H7Mab(hz515H7VH1D76N VL2, hz515H7VH1D76N VL2.1, hz515H7VH1D76N VL2.2 and hz515H7VH1D76N VL2.3) CXCR4 of specific combination on NIH3T3-CXCR4.

Figure 22 shifts (BRET) method by the bioluminescence resonance energy, shown humanization 515H7Mab(hz515H7VH1D76N VL2, hz515H7VH1D76N VL2.1, hz515H7VH1D76N VL2.2 and hz515H7VH1D76N VL2.3) to the effect of CXCR4 homodimer.

Figure 23 has shown that anti-CXCR4Mab hz515H7 suppresses X4HIV-1 in the MT-4 cell _IIIBThe cytopathogenic ability of inducing.

Figure 24 has shown that anti-CXCR4Mab hz515H7 suppresses the ability that HIV-1X4 virus Primary isolate KON copies in the human PBMC.

Figure 25 has shown that Mab hz515H7 and Malawi promise combination suppress HIV-1 Primary isolate 89.6(amphicheirality X4/R5 virus) beneficial effect that copies in the human PBMC.

Figure 26 has shown that Mab hz515H7 and Malawi promise combination suppress HIV-1 Primary isolate UG93067(amphicheirality X4/R5 virus) beneficial effect that copies in the human PBMC.

Figure 27 has shown that anti-CXCR Mab hz515H7IgG4 suppresses the ability that HIV-1X4 virus Primary isolate KON copies in the human PBMC.

Figure 28 has shown that Mab hz515H7IgG4 and Malawi promise combination suppress HIV-1 Primary isolate 89.6(amphicheirality X4/R5 virus) beneficial effect that copies in the human PBMC.

Embodiment

Embodiment 1: the monoclonal antibody (Mab) that generates anti-human CXCR4

In order to generate the monoclonal antibody of CXCR4, with the peptide immunity Balb/c mouse of recombinating the NIH3T3-CXCR4 cell and/or holding and encircling corresponding to the outer N of CXCR4 born of the same parents.With the antigen in complete Freund's adjuvant to mouse subcutaneous (s.c.) immunity in age in 6-16 week once, then with the antigen subcutaneous inoculation in incomplete Freund's adjuvant 2 to 6 times.To get blood monitoring immune response after eye socket.As described below with ELISA() screening serum, and the mouse with the anti-CXCR4 antibody of higher titre is used to merge.Then mouse extracts spleen a few days ago strengthening with the antigen intravenous injection of being condemned to death.

-ELISA

For selecting to produce the mouse of anti-CXCR4 antibody, with the serum of ELISA check from immune mouse.Simply say, with the coated microwell plate of the purifying that is conjugated to BSA [1-41] N of 5 μ g nef polypeptide/mL end peptide, 100 μ L/ holes are 4 ° of C overnight incubation, then with 0.5% gelatin sealing in the PBS in 250 μ L/ holes.Add the diluted plasma thing of CXCR4 immune mouse and hatched 2 hours at 37 ° of C in every hole.Also hatched 1 hour at 37 ° of C with the sheep anti-mouse igg antibody (Jackson Laboratories) that is conjugated to HRP subsequently with the PBS wash plate.After washing, present plate with tmb substrate, pass through to add 100 μ L/ hole 1M H after 5 minutes ₂SO ₄Termination reaction.The mouse that generates the anti-CXCR4 antibody of the highest titre is used to antibody and generates.

The generation of the hybridoma of the Mab of the anti-CXCR4 of-production

The isolated mouse boosting cell of Balb/c mouse that produces the anti-CXCR4 antibody of the highest titre merges to mouse myeloma cell line Sp2/O with PEG.With about 1x10 ⁵/ hole is laid on microwell plate with cell, then hatches for two weeks in the selection culture medium that comprises ultra culture medium (ultra culture medium)+2mM L-glutaminate+1mM Sodium.alpha.-ketopropionate+1 * HAT.Then with the anti-CXCR4 mono-clonal IgG antibody in the ELISA screen holes.Then by the hybridoma of restricted dilution subclone at least twice secretory antibody, vitro culture is used for further analyzing to generate antibody.

Embodiment 2: (NIH3T3-CXCR4 transfectant) binding specificity of identifying anti-CXCR4Mab515H7 and 301aE5 by facs analysis

This experiment in, check anti-CXCR4Mab515H7 and 301aE5 and people CXCR4(hCXCR4 by facs analysis) specific binding.

Hatch NIH3T3 and NIH3T3-hCXCR4 transfectional cell with 10 μ g/ml monoclonal antibody 515H7 and 301aE5.Then with the 1%BSA/PBS/0.01%NaN3 washed cell.Then, add two of Alexa-mark to resist and it was hatched 20 minutes at 4 ° of C in cell.And then twice of washed cell.After washing for the second time, carry out facs analysis.These binding results are provided in the following Table 8, and [average fluorescent strength (MFI) that obtains with FACS] demonstrates anti-CXCR4Mab515H7 and 301aE5 specific binding people CXCR4-NIH3T3 transfectional cell series, and for parent NIH3T3 cell without identification.

Table 8

Embodiment 3: identify the combination of anti-CXCR4Mab515H7 and 301aE5 and peripheral blood lymphocytes (PBMC) by facs analysis

Collect the blood of buffy coat from healthy donors.100 μ l whole bloods were hatched 20 minutes at 4 ° of C with the anti-human CXCR4 antibody (clone 515H7 and 301aE5) with the concentration indicated.Washed blood is three times in PBS-BSA1%-NaN30.01%, and with the anti-human Alexa488IgG(Invitrogen of goat of 1:500 dilution) hatched 20 minutes at 4 ° of C.Then washed cell and and CD14-PE(Caltag) or CD3-PE(Caltag) hatch 10 minutes at 4 ° of C, and wash three times.Under room temperature with high yield cracked solution (Caltag) splitting erythrocyte 10 minutes.Use immediately Facscalibur(Becton-Dickinson) analysis of cells.Express and carry out the CXCR4 expression (Fig. 1) of T cell in carrying out monocytic CXCR4 on the CD14 positive cell on the cell of the CD3 positive.Result is expressed as antigen binding capacity (ABC).

As shown in Fig. 2 A and 2B, T lymphocyte (Fig. 2 A) and the monocyte (Fig. 2 B) of anti-human CXCR4 clone 515H7 and 301aE5 dyeing have illustrated that 515H7 and 301aE5Mab can the identification form karyocytes and the CXCR4 of the natural form of T lymphocyte cell surface expression.

Embodiment 4: shift (BRET) method by the bioluminescence resonance energy, 515H7 and 301aE5Mab are for the effect of CXCR4 homodimer

The conformational change that this functional analysis allows on CXCR4 homodimer level assessment SDF-1 and/or 515H7Mab to cause in conjunction with the CXCR4 acceptor.

By using traditional Protocols in Molecular Biology, will be with corresponding dyestuff (renilla luciferase, Rluc and yellow fluorescence protein, fused protein YFP) for the expression vector establishment of investigation interaction mating partner.Carry out BRET experiment a few days ago, with the corresponding BRET mating partner of encoding: the expression vector transient transfection HEK293 cell of [CXCR4/Rluc+CXCR4/YFP] is with research CXCR4 homodimer.After one day, cell is scattered in the perfect medium matter [adding the DMEM of 10%FBS] of the pre-coated white 96MW plate of poly-lysine.First at 37 ° of C with 5%CO ₂Culturing cell so that cell attachment to plate.Then with 200 μ l DMEM/ holes, cell hunger is spent the night.Just before BRET experiment, remove DMEM and with the quick washed cell of PBS.Then 5 μ M coelenterazine (coelenterazine) H(that add final volume 50 μ l have or without 300nMSDF-1) before, incubated cell is 10 minutes in the PBS that contains or do not contain antibody, under 37 ° of C.After 37 ° of C are hatched again 10 minutes, use the light emission at the initial 485nm of Mithras LB940 multiple labeling reading apparatus (Berthold) and 530nm place to obtain (1s/ wavelength/hole, under room temperature, repetition is 15 times).

Carry out as previously mentioned calculating people such as (, 2000) Angers of BRET ratio: [(emission _530nm)-(emission _485nm) * Cf]/(emission _485nm), herein for the cell of only expressing the Rluc fused protein under same experiment condition, the Cf=(emission _530nm)/(emission _485nm).Simplify this equation, show BRET than the 530/485nm that is equivalent to obtain when two BRET mating partners exist than (the 530/485nm ratio that obtains when existing with the mating partner that only is blended in Rluc in analysis under same experiment condition is proofreaied and correct).For the purpose of readability, result is expressed as milliBRET unit (mBU); MBU multiply by 1000 corresponding to the BRET ratio.

Adaptor protein causes SDF-1(300nM with the spatial proximity that is blended in the receptor protein of CXCR4 acceptor) improved approximately 20% BRET signal, may show the formation of CXCR4/CXCR4 homodimer or the dimeric conformational change (Fig. 3 A and B) that exists in advance.515H7 and 301aE5Mab can regulate the conformational change (for 515H7 and 301aE5, suppressed the BRET that 69% SDF-1 induces and raise, Fig. 3 A and B) of the CXCR4 homodimer that SDF-1 induces.515H7 and 301aE5Mab can also self-regulation CXCR4/CXCR4 spatial proximity, show that 515H7 and 301aE5Mab are for the impact (Fig. 3 A and 3B) of CXCR4/CXCR4 homodimer conformation.

Embodiment 5: suppress HIV-1 Primary isolate KON(X4 virus with anti-CXCR4Mab515H7 and 301aE5) copying in the human PBMC

From the PBMC of the seronegative normal donor of HIV-1, separate from buffy coat or pass through cytapheresis with the Ficoll-Hypaque gradient centrifugation.PBMC activates in containing the RPMI1640 cell culture substrate of PHA (contain 25mM HEPES, 5ml penicillin (10000U/ml)-Streptomycin sulphate (10000 μ g/ml), 2mM L-glutaminate, be added with 10% hot deactivation FCS), and as the cellular targets in the monocycle neutralization test.By the cell inner dyeing of FACS experimental analysis virus p24 antigen, the HIV-1 that detects in primary human PBMC copies.Simply say different dilution Mab515H7,301aE5 and the 12G5(R﹠amp in 25 μ l every holes; D system) or culture medium (RPMI1640,10%FCS, 0.1%IL-2) in contrast, hatched 1 hour at the HIV-1KON X4 Primary isolate dilution in 37 ° of C and 25 μ l every holes, duplicate.The human PBMC of PHA-activation (25 μ l/ hole) is with 20x10 ⁶Cells/well joins 96 orifice plates and (at the bottom of U-, in the Mab/ virus mixture in Costar3599), and cultivated 24 to 36 hours in RPMI164010%FCS and 0.1%IL-2, under 37 ° of C.Introducing is by the not contrast that consists of of infection of PBMCs in the culture medium that does not contain Mab.The PBMC that infects for detecting HIV carries out cell inner dyeing and the analysis of viral p24 antigen with flow cytometry.Use Cytofix/Cytoperm test kit (Becton Dickinson) fixed cell and make its infiltration according to the method for manufacturer, and the anti-p24Mab(clone of the fluorescence KC57-Coulter Beckman that uses with 1/160 dilution) in the dark, hatch under 4 ° of C and dyeed in 10 minutes.After washing, dilute PBMC before flow cytometry in PBS in PBS-3%FCS matrix.By establish on the viable cell group door 20,000 events determine different samples in p24 positive cell percentage ratio.Analyze the viable cell subgroup with respect to the p24 expression of non-infected cells background dyeing.Obtain p24 antigen positive value after deducting simulated infection cell background event.In be defined as infecting with the contrast that does not contain Mab the minimizing that the p24 positive cell is compared in the hole with percentage ratio.In and titre be defined as allowing cells infected percentage ratio that the 90% Mab extent of dilution that reduces is arranged.Anti-CXCR4Mab515H7 and 301aE5 are compared with the 12G5Mab of the known anti-CXCR4Mab reference that copies as HIV.As shown in Fig. 4 A and B and 5, anti-CXCR4Mab515H7 and 301aE5 respectively can be with 10 μ g/ml(66nM) and 150 μ g/ml(1 μ M) IC ₉₀Suppress HIV-1KON Primary isolate copying in PBMC, and 12G5Mab can not suppress HIV-1KON Primary isolate copying in PBMC (Fig. 4 A).

Flowing of the receptor-mediated intracellular calcium store of embodiment 6:CXCR4

This functional analysis is designed to monitor the CXCR4 receptor signal via stimulating the phospho-esterase c path, discharges this signal induction calcium stores in the endoplasmic reticulum cell.

By to carry the initial CHO-K1 cell of mammalian expression vector transfection (ATCC CCL-61) of the whole encoding sequence of people CXCR4 acceptor (RefSeq NM_003467), obtained the CHO-K1 cell of stable and constitutive expression people CXCR4 acceptor.Propagated cell in perfect medium matter [being added with DMEM-Ham ' the s F12 of 5% foetal calf serum (FCS) and 500 μ g/ml Geneticins].Density cell in appropriate incubation matrix with 100,000 cells/well is laid on black 96MW plate.Before testing, cell hunger is spent the night.In sample-loading buffer [HBSS1x, HEPES20mM, probenecid acid 25mM], loaded cells 30 minutes with fluorescence calcium dyestuff (Fluo-4No Wash, Invitrogen US) at 37 ° of C, then loaded cells 30 minutes at 25 ° of C.Completing SDF-1 by each cell of direct injection stimulates.For antagonistic experiment, directly 10 μ l Mab solution were added to sample-loading buffer at least 10 minutes before SDF-1.At multiplex mode fluorescence microwell plate reading apparatus Mithras LB940(Berthold) upper complete the kinetics fluorometric assay with following setting: excite at the 485nm place, launch the excitation energy of 10000 arbitrary units at the 535nm place.In per second with the fluorescence that recorded every hole in 0.1 second and before the SDF-1 injection record time (basis signal) of 20 seconds.Then inject 20 μ l SDF-1, and record subsequently the data of 2 minutes durations.Each experiment situation is carried out in duplicate.At first the value by fluorescence correction every hole of deducting basic fluorescence and thering is no that the control wells of cell sends.Relative data is expressed as SDF-1(100nM) percentage ratio of the maximal stimulation of gained.

SDF-1(100nM) induced the quick and strong release of intracellular Ca2+ in recombinant C HO/CXCR4, and fluorescent signal do not detected in initial CHO-K1 cell.Maximum strength reaches over basic fluorescence and observes (Fig. 6 A, 6B and 6C) more than 140% and in the time of about 40 seconds when stimulating with SDF-1.Mab515H7(133nM) (Fig. 6 A) and c515H7(133nM) (Fig. 6 C) produced for SDF-1(100nM) strongly inhibited of the calcium signal of inducing.Mab301aE5(133nM) (Fig. 6 B) produced for SDF-1(100nM) part of the calcium signal of inducing suppresses.

Embodiment 7: suppress HIV-1 Primary isolate KON, MN and 92UG024(X4 virus with anti-CXCR4Mab515H7, c515H7 and 301aE5) copying in the human PBMC

The monocycle neutralization analysis

This analyzes and uses corresponding Primary isolate KON, MN and the 92UG024 that concentrates and dilute to carry out in 36 hours, to guarantee the detecting 2% CD4T lymphocyte that infects after infecting 2 days.

Mab515H7, c515H7 and the 301aE5 of the different dilutions of 25 microlitres were hatched 1 hour with 25 μ l viruses under 37 ° of C.Human PBMC (25 μ l) is with 20x10 ⁶Cell/ml join 96 orifice plates (at the bottom of U, the Mab/ virus mixture in Costar3599) and at RPMI164010%FCS and 20U/ml IL-2(R﹠amp; D system, Minneapolis, MN) the middle cultivation 36 hours.

After cultivating 2 days, detect the lymphocyte of HIV infection with the cell inner dyeing of viral p24 antigen.Use Cytofix/Cytoperm and Perm/Wash test kit (BD Biosciences) fixed cell and make its infiltration according to manufacturer's suggestion, and to be added in the anti-p24Mab(FITC-of fluorescence or the anti-p24 of PE-that in Perm/Wash solution, 1/160 dilution is used, clone KC57; Beckman Coulter/Immunotech, Hialeah, FL) 4 ° of C dyeing 15 minutes.After washing in containing the PBS of 3%FBS, in flow cytometry (LSRII; BD Biosciences) with before DIVA software (BD Biosciences) analysis, dilute PBMC in 300 μ l PBS.By establishing the percentage ratio that 20,000 events of door are determined p24 positive cell in different samples in the viable cell group who identifies with forward direction and lateral scattering parameter.Analyze the viable cell subgroup with work/dead solution reagent box (Invitrogen).Obtain p24 antigen positive value after background event in deducting the cell of simulated infection.

In be defined as with percentage ratio and infect without the contrast of Mab the minimizing that the p24 positive cell is compared in the hole.In and titre be defined as making the percentage ratio of cells infected to reduce by 90% antibody concentration (interpolation between the serial dilution that carries out in triplicate).

As shown in Fig. 7,8 and 9, anti-CXCR4Mab515H7, c515H7 and 301aE5 can suppress HIV-1X4MN, KON and 92UG024 Primary isolate copying in PBMC.Sum up the result of (representing with μ g/ml) IC in table 9.

Table 9

Embodiment 8: suppress HIV-1 Primary isolate 89.6(amphicheirality X4/R5 virus with anti-CXCR4Mab515H7, c515H7 and 301aE5) copying in the human PBMC

The monocycle neutralization analysis

This analyzes and uses the corresponding Primary isolate 89.6 that concentrates and dilute to carry out in 36 hours, to guarantee the detecting 2% CD4T lymphocyte that infects after infecting 2 days.

Mab515H7, c515H7 and the 301aE5 of the different dilutions of 25 microlitres were hatched 1 hour with 25 μ l viruses under 37 ° of C.Human PBMC (25 μ l) is with 20x10 ⁶Cell/ml join 96 orifice plates (at the bottom of U, the Mab/ virus mixture in Costar3599) and at RPMI164010%FCS and 20U/ml IL-2(R﹠amp; D Systems, Minneapolis, MN) the middle cultivation 36 hours.

After cultivating 2 days, detect the lymphocyte of HIV infection with the cell inner dyeing of viral p24 antigen.Use Cytofix/Cytoperm and Perm/Wash test kit (BD Biosciences) fixed cell and make its infiltration according to manufacturer's suggestion, and to be added in the anti-p24Mab(FITC-of fluorescence or the anti-p24 of PE-that in Perm/Wash solution, 1/160 dilution is used, clone KC57; Beckman Coulter/Immunotech, Hialeah, FL) 4 ° of C dyeing 15 minutes.After washing in containing the PBS of 3%FBS, in flow cytometry (LSRII; BD Biosciences) with before DIVA software (BD Biosciences) analysis, dilute PBMC in 300 μ l PBS.By establishing the percentage ratio that 20,000 events of door are determined the p24 positive cell in different samples in the viable cell group who identifies with forward direction and lateral scattering parameter.Analyze the viable cell subgroup with work/dead solution reagent box (Invitrogen).Obtain p24 antigen positive value after background event in deducting the cell of simulated infection.

In be defined as with percentage ratio and infect the hole without the contrast of Mab and compare, the minimizing of p24 positive cell.In and titre be defined as making the percentage ratio of cells infected to reduce by 90% antibody concentration (interpolation between the serial dilution that carries out in triplicate).

As shown in FIG. 10 and 11, anti-CXCR4Mab515H7, c515H7 and 301aE5 can suppress HIV-189.6 Primary isolate copying in PBMC.Sum up the result of (representing with μ g/ml) IC in table 10.

Table 10

Embodiment 9: with anti-CXCR4Mab c515H7 and anti-CCR5 molecule Malawi's promise combination inhibition HIV-1 Primary isolate 89.6 and UG93067(amphicheirality X4/R5 virus) copying in the human PBMC

Neutralization analysis is analyzed the many wheels of HIV Primary isolate on primary PBMC and is copied

This analyzes, and (with the virus combination of c515H7Mab or Malawi's promise or both combinations and the serial dilution of serial dilution) analyzed the PBMC(peripheral blood lymphocytes) on many wheels infection.Simply say, at prehydration 96 hole screen plates (1.25 μ m apertures, Durapor DV; Millipore, Molsheim, France) in, the c515H7Mab of the serial dilution (twice) of 4 part of 25 μ l equal portions or Malawi's promise or each of both combinations and the virus of 25 μ l serial dilutions are hatched.Carry out the contrast titration (25 μ l RPMI replace c515H7Mab or Malawi's promises of dilution) of virus on the same model of the c515H7Mab that contains dilution or Malawi's promise or the titration that both make up.After 1 hour, add 4x10 under 37 ° of C ⁶The PBMC(that 25 μ l PHA of cell/ml concentration stimulate is from the set of the PHA activating PBMC of five healthy donors) with interleukin-22 (the IL-2) (R﹠amp of the RPMI, 10% foetal calf serum (FCS) and the every milliliter of 20IU that realize the whole volume of culture of 75 μ l; D system).After 24 hours, add the 100 same culture mediumes of μ l under 37 ° of C.Carry out twice washing (each 200 μ l RPMI) with removal c515H7Mab and Malawi's promise at the 4th day with filtration, and add 200 μ l fresh culture matrix.At the 7th day, existing and (infect and maintain 10 with virus dilution with negative control with p24 in ELISA mensuration culture supernatant ^-6Comparing with the definite positive hole culture in M zidovudine [AZT]).Determine that with quadruplicate hole there are not (V in the c515H7Mab of each dilution or Malawi's promise or both combinations ₀) and the virus titer (50% [TCID of TCID when having (Vn) ₅₀]).In and titre be defined as causing virus titer to reduce 90%(Vn/V ₀The dilution of c515H7Mab=0.1) or Malawi's promise or both combinations.

As shown in figure 12, c515H7Mab is with the IC of 2 μ g/ml ₉₀Suppress amphicheirality X4R5 virus 89.6 and copy (Figure 12).The promise of 50 μ g/ml Malawis does not reach IC ₉₀Suppress active (Figure 12).And, add in the antibody 515H7 promise of 2 μ g/ml Malawis to strengthen the inhibition of c515H7Mab active, its IC ₉₀Be 0.2 μ g/ml(Figure 12).

Use two molecules of different dilutions and the beneficial effect that another amphicheirality virus UG93067 has assessed c515H7Mab and Malawi's promise combination.As shown in figure 13, the inhibition activity of Mab c515H7 and Malawi's promise is similar.These results show that it is comparable that viral UG93067 uses the ability of CCR5 or CXCR4 acceptor.Use UG93067 virus to confirm better active, only have the combination (each 10 μ g/ml) of these X4 inhibitor (c515H7Mab) and R5 inhibitor (Malawi's promise) to make virus titer reduce 90%(Figure 13).

Embodiment 10: the production of the chimeric Mab c515H7 of anti-CXCR4

Design the mouse 515H7Mab of chimeric form: it is blended in people C κ and IgG1/IgG2/IgG4 constant region corresponding to light chain and the weight chain variable structural domain of target mouse antibodies in heredity.By using HEK293/EBNA system and pCEP4 expression vector (InVitrogen, US) transient transfection to come Restruction Mab.

Amino acid and nucleotide sequence separately described in above-mentioned specification sheets.And, as above the open IgG2 of table 3 and 4 isotype sequences (IgG4 is preferred isotype), mentioned also that at this IgG1 isotype sequence of heavy chain is c515H7VH(G1wt) (it is corresponding to aminoacid sequence SEQ ID No.80 and nucleotide sequence SEQ ID No.81).

With the synthetic whole nucleotide sequence corresponding to 515H7Mab light chain and weight chain variable structural domain of overall gene synthetic (Genecust, Luxembourg).It is subcloned in the pCEP4 carrier (InVitrogen, the U.S.) of whole encoding sequence of carrier IgG1/IgG2/IgG4 light chain immunoglobulin [C κ] or heavy chain [CH1-hinge-CH2-CH3] constant region.Indicate to carry out all clone's steps according to the described traditional Protocols in Molecular Biology of laboratory manual (Sambrook and Russel, 2001) or according to supplier.Each genetic constructs is fully verified with nucleotide sequencing through Big Dye end cycle sequencing test kit (Applied Biosystems, the U.S.), and is used 3100 genetic analyzers (Applied Biosystems, the U.S.) analysis.

Upper at orbital shaker (110rpm rotating speed), as to add 6mM glutamine 50ml serum-free culture matrix Excell293(SAFC Biosciences) the HEK293EBNA cell (InVitrogen, the U.S.) that in, conventional growth adaptation suspends in the 250ml shaking flask.The linear 25kDa polymine (PEI) of the final concentration 1mg/ml that use prepares in water (Polysciences) mixes with plasmid DNA (final concentration is 1.25 μ g/ml, and heavy chain is 1:1 with light chain plasmid ratio), with 2.10 ⁶Cell/ml carries out transient transfection.After transfection 4 hours, with a volume fresh culture matter dilution culture to realize that whole cell density is as 10 ⁶Cell/ml.Based on cell viability and Mab production monitoring culturing process.In general, maintain is 4 to 5 days.At the traditional purification by chromatography Mab of the upper use of a-protein resin (GE Healthcare, the U.S.).Produce Mab with the level that is suitable for functional assessment.Production level is generally purifying Mab between 6 to 15mg/l.

Embodiment 11: the binding specificity of identifying the chimeric Mab c515H7 of anti-CXCR4 with facs analysis

In this experiment, check that with facs analysis the chimeric Mab c515H7 of anti-CXCR4 is to the specific combination of people CXCR4.

NIH3T3-hCXCR4 transfectional cell and dosage range are hatched from the monoclonal antibody c515H7 of 0 μ g/ml to 10 μ g/ml.Then with the 1%BSA/PBS/0.01%NaN3 washed cell.Next, add two of Alexa-mark to resist and it was hatched 20 minutes at 4 ° of C in cell.And then twice of washed cell.After washing for the second time, carry out facs analysis.Figure 15 provides this binding result, shows that the chimeric Mab c515H7 of anti-CXCR4 specific binding is in people CXCR4-NIH3T3 transfectional cell series.Do not detect in the NIH3T3 wild-type cell in conjunction with (data do not show).

Embodiment 12: shift (BRET) method by the bioluminescence resonance energy and detect c515H7Mab for the effect of CXCR4 homodimer

This functional analysis allows on CXCR4 homodimer level assessment SDF-1 and/or c515H7Mab to be incorporated into the conformational change of CXCR4 receptor-inducible.

By using traditional Protocols in Molecular Biology, will be with corresponding dyestuff (renilla luciferase, Rluc and yellow fluorescence protein, fused protein YFP) for the expression vector establishment of investigation interaction mating partner.Carrying out BRET experiment a few days ago, with the corresponding BRET mating partner of encoding: the expression vector transient transfection HEK293 cell of [CXCR4/Rluc+CXCR4/YFP] is with research CXCR4 homodimer.After one day, cell dispersion is in perfect medium matter [being added with the DMEM of 10%FBS] on the pre-coated white 96MW plate of poly-lysine.At first under 37 ° of C, with 5%CO ₂Culturing cell so that cell attachment to plate.Then with 200 μ l DMEM/ holes, cell hunger is spent the night.Just before BRET experiment, remove DMEM and with the quick washed cell of PBS.Then under 37 ° of C, be with or without the PBS of antibody in incubated cell 10 minutes, then add the 5 μ M coelenterazine H(of final volume 50 μ l to contain or do not contain 100nM SDF-1).Hatch again 10 minutes under 37 ° of C after, use the light emission of the initial 485nm of Mithras LB940 multiple labeling reading apparatus (Berthold) and 530nm to obtain (1 second/wavelength/hole, at room temperature repeat 15 times).

Carry out as previously mentioned calculating people such as (, 2000) Angers of BRET ratio: [(emission _530nm)-(emission _485nm) * Cf]/(emission _485nm), herein for the cell of only expressing the Rluc fused protein under same experiment condition, the Cf=(emission _530nm)/(emission _485nm).Simplify this equation, show BRET than the 530/485nm that is equivalent to obtain when two BRET mating partners exist than (the 530/485nm ratio that obtains when existing with the mating partner that only is blended in Rluc in analysis under same experiment condition is proofreaied and correct).For ease of readability, result is expressed as milliBRET unit (mBU); MBU multiply by 1000 corresponding to the BRET ratio.

Donor causes SDF1(100nM with the spatial proximity that is blended in the receptor protein of CXCR4 acceptor) improved approximately 10% BRET signal, may show the dimeric conformational change (Figure 16) that the CXCR4/CXCR4 homodimer forms or is pre-existing in.Mab c515H7 can regulate the CXCR4 homodimer that SDF-1 induces conformational change (suppress that SDF-1 induces that BRET increases 96%, Figure 16).Mab c515H7 also can self-regulation CXCR4/CXCR4 spatial proximity, shows that this Mab is for the impact (Figure 16) of CXCR4/CXCR4 homodimer conformation.

Embodiment 13: use the external assessment Mab515H7 of GFP transduction human osteosarcoma cell (GHOST) Anti-HIV-1 Active of expressing CD4 and CXCR4 or CCR5.

In order to determine the specificity of CXCR4515H7Mab, we use the GHOST cell of expressing CD4 and CXCR4 or CCR5 to assess the Anti-HIV-1 Active of this Mab.

In 48 hours, use X4HIV-1LAI virus (with the Ghost cell of expressing CXCR4) or R5HIV-1BaL virus (with the Ghost cell of expressing CCR5) to carry out this analysis.In the Dulbecco culture medium that is added with 10%FCS, paving is by 500 μ l Ghost cells (2.510 ⁵Cell/ml) 24 hours.Hatch Mab515H71 hours of different dilutions under 37 ° of C, and with viral (1/7) 48 hour of the HIV-1LAI virus (1/10) that adds dilution in backward cell and HIV-1BaL.Cell stands tryptic digestion and washs with 1 * PBS.In the dark ,+added 300 μ L1.5% paraformaldehyde 2 hours to the cell precipitation thing under 4 ° of C, with fixed cell and inactivation of viruses.With flow cytometry GFP positive cell and calculate the inhibition that HIV-1 infects.

The inhibition percentage ratio of cells infected is defined as and infects the hole without the contrast of Mab and compare.Summed up IC result (take μ g/ml as unit) in table 11, anti-CXCR4Mab515H7 can suppress the infection of HIV-1X4Lai virus in the Ghost cell of expressing CXCR4, yet there is no activity fully in suppressing the infection of HIV-1R5BaL virus in the Ghost cell that CCR5 expresses.

Table 11

The humanization of the anti-CXCR4 mouse antibodies of embodiment 14:515H7 and the generation of described h515H7 fragment

General procedure

Carry out the humanization of the anti-CXCR4 antibody of 515H7 by the whole rule of using the CDR grafting.Carry out CDR and regional immune genetic analysis and the definition of framework (FR) by using IMGT unique number scheme and IMGT library and instrument (Lefranc, 1997 – www.imgt.org).

Determine the combination of 515H7 humanization variant on the NIH3T3 clone of stable transfection people CXCR4.To assess in conjunction with active with the analysis of biotinylation mouse antibodies competition.In attempting for the second time, the ability of assessment humanization antibody suppression biotinylation SDF-1 and RAMOS Cell binding.Select the RAMOS cell, because its high expression level CXCR4 and low CXCR7 and the SDF-1 of expressing.

Analyze to identify the recombinant humanized version of anti-CXCR4 antibody with these.Variable domains and human IgG1/k constant domain formats and is cloned into mammalian expression vector pCEP.Transient expression restructuring IgG1/ κ-derivative antibody in the HEK293 cell.Filter and express culture supernatant and use a-protein agarose antibody purification.Again cushion the antibody of purifying and determine antibody concentration with ELISA in PBS.

The PCR that is specific to the oligonucleotide of humanized antibody variable domains by use generates recombinant antibody fragment and these fragments of subclone in the intestinal bacteria system.Carry out the purifying of antibody fragment with immobilized metal ion affinity chromatography (IMAC).

The humanization of-515H7 variable domains

The different sequence alignments of heavy chain and light chain variable structural domain have been set forth in Figure 17 and 18.

In the experiment of First Series, analyzed three at first the anti-CXCR4 of humanization variant in conjunction with activity.And assessed the ability that these constructs suppress the combinations of biotinylation mouse 515H7 parental antibody VH variant 1(VH1) and mouse VL combination.The aminoacid sequence of VH1 variable domains comprises SEQ ID No.90 and nucleotide sequence comprises SEQ ID No.91.Total length VH1 aminoacid sequence comprises SEQ ID No.92 and nucleotide sequence comprises SEQ ID No.93.This construct shows ability (Figure 19 A) similar with chimeric antibody and the mouse antibodies competition.This shows that most mankind VH variants have the binding ability identical with chimeric antibody.Therefore, VH1 combine with VL variant 2 (Figure 19 B).

In further experiment, determine whether the humanization variant of antibody 515H7 suppresses the combination of SDF-1 and CXCR4 express cell (Figure 20).Inhibition ability by the assessment of detection of biological elementization SDF-1 in flow cytometer hz515H7 humanization variant.Humanized antibody hz515H7VH1D76N VL2 has the ability that similarly suppresses the SDF-1 combination with chimeric c515H7.

Also checked the antibody fragment of humanization variant hz515H7VH1VL2, and determined that described antibody fragment can suppress the combination (Figure 20) of SDF-1 fully.

Embodiment 15: identify anti-CXCR4 humanization Mab515H7 binding specificity with facs analysis

In this experiment, check that with facs analysis anti-CXCR4 humanization Mab515H7 is for the specific binding of people CXCR4.

In the dark, in 4 ° of C, 100 μ l Facs damping fluids with NIH3T3 and 0 to the 10 μ g/mL humanization Mab515H7(hz515H7VH1D76N VL2 of NIH3T3, hCXCR4 transfection, hz515H7VH1D76N VL2.1, hz515H7VH1D76N VL2.2, hz515H7VH1D76N VL2.3) hatched 20 minutes.After Facs damping fluid washing three times, in the dark, at 4 ° of C, cell and two anti-(goat-anti people Alexa488,1/500 dilutions) were hatched 20 minutes.Wash three times in the Facs damping fluid after, add propidium iodide in every hole and only analyze viable cell with Facs.Estimate at least 5000 viable cell to assess the fluorescence intensity mean value under each condition.

Show [with the average fluorescent strength (MFI) of FACS acquisition] in Figure 21, the result of these bindings is provided, anti-CXCR4 humanization Mab hz515H7 specific binding is in people CXCR4-NIH3T3 transfectional cell series (MFI=2.2 of NIH3T3 parental cell).

Embodiment 16: shift (BRET) method with the bioluminescence resonance energy and detect hz515H7Mab for the effect of CXCR4 homodimer

This functional analysis allows on CXCR4 homodimer level assessment SDF-1 and/or hz515H7VH1D76N VL2, hz515H7VH1D76N VL2.1, hz515H7VH1D76N VL2.2, hz515H7VH1D76N VL2.3 to be incorporated into the conformational change of CXCR4 receptor-inducible.

Donor causes SDF1(100nM with the spatial proximity that is blended in the receptor protein of CXCR4 acceptor) improved approximately 12% BRET signal, may show the dimeric conformational change (Figure 22) that the CXCR4/CXCR4 homodimer forms or is pre-existing in.

515H7 humanization Mab can regulate the conformational change of the CXCR4 homodimer that SDF-1 induces, the inhibition percentage ratio that the BRET that induces for hz515H7VH1D76N-VL2Mab SDF-1 increases is about 88%, hz515H7VH1D76N-VL2.1Mab be 65%, hz515H7VH1D76N-VL2.2Mab be 33% and hz515H7VH1D76N-VL2.3Mab is 21%(Figure 22).

Embodiment 17: by anti-CXCR4Mab hz515H7 to HIV-1 _IIIBThe inhibition that (X4 virus) copies in the MT-4 cell

In this was analyzed, hz515H7Mab was to anti-HIV-1 _IIIBActivity be based on the cytopathogenic inhibition of virus induction in the MT-4 cell.Infect metering 50(TCID with tissue culture ₅₀) HIV-1 of 5 times _IIIBStrain isolated cells infected, this virus dosage reduced by 90% viable count in 5 days.After 30 minutes, the cell that infects is adjusted to 210 37 ° of C absorption in the RPMI1640 matrix of the foetal calf serum that is supplemented with 20% heat inactivation (FCS), 100IU/ml penicillin, 100 μ g/ml Streptomycin sulphates, 2mM glutamine ⁵Cell/ml is seeded in (COSTAR3596) in the 96 flat tissue culturing plates in hole (100 μ l/ hole), and this flat board has the hz515H7Mab of 100 μ l different concns.At the 5th day, measure cytoactive with colorimetric reaction MTT.Calculate the per-cent of the cells infected protection of processing with hz515H7Mab by following formula:

As shown in figure 23, hz515H7Mab shows significant Anti-HIV-1 Active, because it can suppress HIV-1 in the MT-4 cell _IIIBThe cytopathogenic effect that – induces.

Embodiment 18: anti-CXCR4Mab hz515H7 is to HIV-1 Primary isolate KON(X4 virus) inhibition that copies in the human PBMC

The monocycle neutralization analysis

This analyzes and uses the corresponding Primary isolate KON that concentrates and dilute to carry out in 36 hours, to guarantee the detecting 2% CD4T lymphocyte that infects after infecting 2 days.

The Mab hz515H7 of the different dilutions of 25 microlitres was hatched 1 hour with 25 μ l viruses under 37 ° of C.(at the bottom of U, add 20x10 in the Mab/ virus mixture in Costar3599) to 96 orifice plates ⁶The human PBMC of individual cell/ml (25 μ l), and at RPMI164010%FCS and 20U/ml IL-2 (R﹠amp; D Systems, Minneapolis, MN) the middle cultivation 36 hours.

Cultivate after 2 days, detect the lymphocyte of HIV infection by the cell inner dyeing of viral p24Ag.Use Cytofix/Cytoperm and Perm/Wash test kit (BD Biosciences) fixed cell and make its infiltration according to manufacturer's suggestion, and to be added in the anti-p24Mab(FITC-of fluorescence or the anti-p24 of PE-that in Perm/Wash solution, 1/160 dilution is used, clone KC57; Beckman Coulter/Immunotech, Hialeah, FL) 4 ° of C dyeing 15 minutes.After washing in containing the PBS of 3%FBS, in flow cytometry (LSRII; BD Biosciences) with before DIVA software (BD Biosciences) analysis, dilute PBMC in 300 μ l PBS.By establishing the percentage ratio that 20,000 events of door are determined the p24 positive cell in different samples in the viable cell group who identifies with forward direction and lateral scattering parameter.Analyze the viable cell subgroup with work/dead solution reagent box (Invitrogen).Obtain p24 antigen positive value after background event in deducting the cell of simulated infection.

The per-cent of neutralization is defined as and infects the hole with the contrast that does not have Mab and compare, the reduction of p24 positive cell.In and titre be defined as the antibody concentration (interpolation between the serial dilution that carries out in triplicate) that the per-cent that allows cells infected reduces.

As shown in figure 24, anti-CXCR4Mab hz515H7 can suppress HIV-1X4KON Primary isolate copying in PBMC.

Embodiment 19: the combination of anti-CXCR4Mab hz515H7 and the promise of anti-CCR5 molecule Malawi is to HIV-1 Primary isolate 89.6 and UG93067(amphicheirality X4/R5 virus) inhibition that copies in the human PBMC

This analyzes, and (with the virus combination of hz515H7Mab or Malawi's promise or both combinations and the serial dilution of serial dilution) analyzed the PBMC(peripheral blood lymphocytes) on many wheels infection.Simply say, at prehydration 96 hole screen plates (1.25 μ m apertures, Durapor DV; Millipore, Molsheim, France) in, the hz515H7Mab of the serial dilution (twice) of 4 part of 25 μ l equal portions or Malawi's promise or each of both combinations and the virus of 25 μ l serial dilutions are hatched.Carry out the contrast titration (25 μ l RPMI replace hz515H7Mab or Malawi's promises of dilution) of virus on the same model of the hz515H7Mab that contains dilution or Malawi's promise or the titration that both make up.After 1 hour, add 4x10 under 37 ° of C ⁶The PBMC(that 25 μ l PHA of cell/ml concentration stimulate is from the set of the PHA activating PBMC of five healthy donors) with interleukin-22 (the IL-2) (R﹠amp of the RPMI, 10% foetal calf serum (FCS) and the every milliliter of 20IU that realize the whole volume of culture of 75 μ l; D System).After 24 hours, add the 100 same culture mediumes of μ l under 37 ° of C.Carry out twice washing (each 200 μ l RPMI) with removal hz515H7Mab and Malawi's promise at the 4th day with filtration, and add 200 μ l fresh culture matrix.At the 7th day, existing and (infect and maintain 10 with virus dilution with negative control with p24 in ELISA mensuration culture supernatant ^-6Comparing with the definite positive hole culture in M zidovudine [AZT]).Determine that with quadruplicate hole there are not (V in the 515H7Mab of each dilution or Malawi's promise or both combinations ₀) and the virus titer (50% [TCID of TCID when having (Vn) ₅₀]).In and titre be defined as causing virus titer to reduce 90%(Vn/V ₀The dilution of 515H7Mab=0.1) or Malawi's promise or both combinations.

Use amphicheirality's virus 89.6 and UG93067, use two molecules different dilution combined evaluation the possible synergy between hz515H7Mab and Malawi's promise.As shown in Figure 25 and 26, Mab hz515H7 is similar with the inhibition activity of Malawi's promise.These X4 (hz515H7Mab) and the promise of R5(Malawi) combination of inhibitor allowed in PBMC 89.6 and UG93067 amphicheirality X4/R5 virus titer reduced 90%(and be respectively Figure 25 and 26).

Embodiment 20: anti-CXCR4Mab hz515H7IgG4 is to HIV-1 Primary isolate KON(X4 virus) inhibition that copies in people PRMC

The monocycle neutralization analysis

This analyze to use the corresponding concentrated Primary isolate KON with dilution to carry out at 36 hours, with the CD4T lymphocyte that allows to detect afterwards 2% infection in 2 days infecting.

The Mab hz515H7IgG4 of the different dilutions of 25 microlitres was hatched 1 hour with 25 μ l viruses under 37 ° of C.(at the bottom of U, add 20x10 in the Mab/ virus mixture in Costar3599) to 96 orifice plates ⁶The human PBMC of individual cell/ml (25 μ l), and at RPMI164010%FCS and 20U/ml IL-2 (R﹠amp; D Systems, Minneapolis, MN) the middle cultivation 36 hours.

As shown in figure 27, anti-CXCR4Mab hz515H7IgG4 can suppress HIV-1X4KON Primary isolate copying in PBMC.

Embodiment 21: the combination of anti-CXCR4Mab hz515H7IgG4 and the promise of anti-CCR5 molecule Malawi is to HIV-1 Primary isolate 89.6(amphicheirality X4/R5 virus) inhibition that copies in the human PBMC

This analyzes, and (with the virus combination of hz515H7IgG4Mab or Malawi's promise or both combinations and the serial dilution of serial dilution) analyzed the PBMC(peripheral blood lymphocytes) on many wheels infection.Simply say, at prehydration 96 hole screen plates (1.25 μ m apertures, Durapor Dv; Millipore, Molsheim, France) in, the hz515H7IgG4Mab of the serial dilution (twice) of 4 part of 25 μ l equal portions or Malawi's promise or each of both combinations and the virus of 25 μ l serial dilutions are hatched.Carry out the contrast titration (25 μ l RPMI replace hz515H7IgG4Mab or Malawi's promises of dilution) of virus on the same model of the hz515H7IgG4Mab that contains dilution or Malawi's promise or the titration that both make up.After 1 hour, add 4x10 under 37 ° of C ⁶The PBMC(that 25 μ l PHA of cell/ml concentration stimulate is from the set of the PHA activating PBMC of five healthy donors) with interleukin-22 (the IL-2) (R﹠amp of the RPMI, 10% foetal calf serum (FCS) and the every milliliter of 20IU that realize the whole volume of culture of 75 μ l; D System).After 24 hours, add the 100 same culture mediumes of μ l under 37 ° of C.Carry out twice washing (each 200 μ l RPMI) with removal hz515H7IgG4Mab and Malawi's promise at the 4th day with filtration, and add 200 μ l fresh culture matrix.At the 7th day, existing and (infect and maintain 10 with virus dilution with negative control with p24 in ELISA mensuration culture supernatant ^-6Comparing with the definite positive hole culture in M zidovudine [AZT]).Determine that with quadruplicate hole there are not (V in the hz515H7IgG4Mab of each dilution or Malawi's promise or both combinations ₀) and the virus titer (50% [TCID of TCID when having (Vn) ₅₀]).In and titre be defined as causing virus titer to reduce 90%(Vn/V ₀The dilution of hz515H7IgG4Mab=0.1) or Malawi's promise or both combinations.

Use amphicheirality's virus 89.6, use two molecules different dilution combined evaluation the possible synergy between hz515H7IgG4Mab and Malawi's promise.As shown in figure 28, these X4 (hz515H7IgG4Mab) and the promise of R5(Malawi) combination of inhibitor allowed that in PBMC, 89.6 amphicheirality X4/R5 virus titers have reduced 90%(Figure 28).

Claims

1. the antibody of a separation or one of its Fab or derivative, wherein, it comprises at least one complementary determining region CDR, and described complementary determining region CDR is selected from the CDR that comprises IMGT numbering system defined aminoacid sequence SEQ ID No.1 to 6 and 30 to 33.

2. antibody according to claim 1 or one of its Fab or derivative, wherein it comprises a light chain and a heavy chain, described light chain comprises and comprises respectively aminoacid sequence SEQ ID No.1,2 and 3 CDR-L1, CDR-L2 and CDR-L3; Described heavy chain comprises and comprises respectively aminoacid sequence SEQ ID No.4,5 and 6 CDR-H1, CDR-H2 and CDR-H3.

3. antibody according to claim 2 or one of its Fab or derivative, wherein it heavy chain that comprises the light chain of aminoacid sequence SEQ ID No.7 and comprise aminoacid sequence SEQ ID No.8.

4. antibody according to claim 2 or one of its Fab or derivative, wherein it is chimeric antibody, and it comprises the heavy chain that is selected from SEQ ID No.56,57 or 58 sequence, and the light chain of sequence SEQ ID No.59.

5. antibody according to claim 2 or one of its Fab or derivative, wherein, it is humanized antibody, and it comprises the variable region of heavy chain of the sequence that is comprised of SEQ ID No.64, and is selected from the variable region of light chain of SEQ ID No.65,66,82 or 83 sequence.

6. antibody according to claim 2 or one of its Fab or derivative, wherein, it is humanized antibody, and it comprises humanized antibody or its derivative compound or functional fragment, it comprises the heavy chain that is selected from SEQ ID No.67,68 or 69 sequence, and is selected from the light chain of SEQ ID No.70,71,84 or 85 sequence.

7. antibody according to claim 2 or one of its Fab or derivative, wherein, described functional fragment is by fragment Fv, scFv, Fab, F (ab ') ₂, Fab ', scFv-Fc, bi-specific antibody or extended any fragment of transformation period by chemically modified and form.

8. antibody according to claim 7 or one of its Fab or derivative, wherein, it is the scFv that comprises aminoacid sequence SEQ ID No.54.

9. antibody according to claim 1 or one of its Fab or derivative, wherein, it comprises a light chain and a heavy chain, and described light chain comprises and comprises respectively aminoacid sequence SEQ ID No.1,2 and 30 CDR-L1, CDR-L2 and CDR-L3; Described heavy chain comprises and comprises respectively aminoacid sequence SEQ ID No.31,32 and 33 CDR-H1, CDR-H2 and CDR-H3.

10. antibody according to claim 9 or one of its Fab or derivative, wherein, the heavy chain that it comprises the light chain of aminoacid sequence SEQ ID No.34 and comprises aminoacid sequence SEQ ID No.35.

11. the nucleic acid of a separation, wherein, it is selected from following nucleic acid:

A) coding antibody as described in any one in claim 1 to 10 or its Fab or derivative one of nucleic acid, DNA or RNA;

B) comprise the nucleic acid of DNA sequence dna, this DNA sequence dna selects free SEQ ID No.14 to 19 and 41 to the 45 CDR sequences that form;

C) comprise the nucleic acid of DNA sequence dna, this DNA sequence dna selects free SEQ ID No.20,21,46,47,72,73,74,86 and 87 heavy chain and the light chain variable structural domain sequences that form;

D) comprise the nucleic acid of DNA sequence dna, this DNA sequence dna selects free SEQ ID No.60 to 63,75 to 79,88 and 89 heavy chain and the sequence of light chain that form;

E) comprise the nucleic acid of the DNA sequence dna that is formed by SEQ ID No.55;

F) as b), c), d) or e) defined in the corresponding RNA nucleic acid of nucleic acid;

G) as a), b), c), d) and e) defined in the complementary nucleic acid of nucleic acid; And

H) nucleic acid of at least 18 Nucleotide, it can be under high rigor condition and at least one hybridization of the CDR of SEQ ID No.14 to 19 and 41 to 45 sequences.

12. a carrier, it comprises nucleic acid as desired in claim 11.

13. a host cell, it comprises carrier as desired in claim 12.

14. the transgenic animal except the people, it comprises the have the right cell of requirement 12 desired carriers of at least one conversion.

15. the method for the production of antibody as desired in any one of claim 1 to 10 or one of its Fab or derivative, wherein said method comprises following phases:

A) cultivate cell as desired in claim 13 in matrix and under suitable culture condition; And

B) reclaim consequent described antibody or one of its Fab or derivative from culture medium or described culturing cell.

16. obtainable by the desired method of claim 15 or obtain antibody or one of its functional fragment or derivative.

17. according to claim 1 to 10 and 16 described antibody as medicine.

18. according to claim 1 to 10 or 16 to 17 described antibody or one of its functional fragment or derivative, wherein, it suppresses HIV-1KON Primary isolate copying in PBMC, its IC ₉₀Be at least 5 μ g/ml, preferably at least 10 μ g/ml.

19. a composition, it comprises that the compound that forms by the desired antibody of any one or one of its Fab or derivative as claim 1 to 10 and 16 to 18 is as activeconstituents.

20. the composition as desired in claim 19 as medicine.

21. according to claim 19 with 20 desired compositions are used for prevention or are used for the treatment of HIV infecting.

22. it is that X4 tropism HIV infects that desired composition according to claim 21, wherein said HIV infect.

23. it is that X4/R5 tropism HIV infects that desired composition according to claim 21, wherein said HIV infect.

24. according to claim 19 to 23 described compositions, wherein, it comprises that being selected from the energy specificity suppresses compound at least the second anti-HIV-1 compounds that HIV enters and/or copies.

25. composition according to claim 24, wherein, described at least the second anti-HIV-1 compounds is selected from antiretroviral drugs such as hiv protease inhibitor (PI), nucleoside/nucleotide hiv reverse transcriptase inhibitor (NRTI/NtRTI), non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTI), HIV entry inhibitor, hiv integrase inhibitor.

26. according to claim 24 or 25 described compositions, wherein said at least the second anti-HIV-1 compounds are anti-CCR5 compounds.

27. composition according to claim 26, wherein said anti-CCR5 compound is Malawi's promise.

28. be used for screening and/or identify as the method for the molecule of CXCR4 antagonist Anti-virus agent, comprising step:

A) select to express the cell of CXCR4,

B) hatch described cell and claim 1 to 10 or 16 to 18 described antibody or one of its Fab or derivative, and

C) ability of combination between the molecules in inhibiting antibody of assessment check or one of its Fab or derivative and CXCR4, and

D) select to produce the molecule of described inhibition.

29. according to claim 1 to 10 and the described antibody of 16 any one or one of its Fab or derivative, and/or according to claim 19 to the purposes of the 27 described compositions of any one in the medicine that copies for the preparation of inhibition HIV.

30. according to claim 1 to 10 and the described antibody of 16 any one or one of its Fab or derivative, and/or according to claim 19 to the 27 described compositions of any one for the preparation of the purposes in the medicine of the prevention of HIV disease or treatment.

31. method that is used for prevention or treatment HIV, wherein said method comprises by using according to claim 1 to 10 and the described antibody of 16 any one or one of its Fab or derivative to the patient that needs are arranged, and/or the step that forms according to claim 19 to the 27 described compositions of any one.

32. method according to claim 31, wherein method also comprises by use the step that anti-CCR5 compound forms to described patient.