WO2012113348A1 - 抗cd4蛋白的单克隆抗体及其活性片段及用途 - Google Patents

抗cd4蛋白的单克隆抗体及其活性片段及用途 Download PDF

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WO2012113348A1
WO2012113348A1 PCT/CN2012/071596 CN2012071596W WO2012113348A1 WO 2012113348 A1 WO2012113348 A1 WO 2012113348A1 CN 2012071596 W CN2012071596 W CN 2012071596W WO 2012113348 A1 WO2012113348 A1 WO 2012113348A1
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antibody
monoclonal antibody
cells
antigen
protein
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PCT/CN2012/071596
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English (en)
French (fr)
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顾颖
侯汪衡
方楚
高双全
曹芳
王海虹
张军
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厦门大学
厦门万泰沧海生物技术有限公司
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Publication of WO2012113348A1 publication Critical patent/WO2012113348A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen

Definitions

  • the present invention relates to a monoclonal antibody which specifically binds to a Human Cluster of Differentiation 4 (CD4) protein, and a conservative variant or active fragment thereof, a related coding sequence of a polypeptide or polypeptide analog thereof, and a production site a cell strain of the monoclonal antibody, and a method and use thereof for the prevention, immunotherapy, and diagnosis using the antibody or fragment; preventing or treating a primate, including a human, the infection source is caused by a main target cell of the CD4+ cell Diseases: These diseases include Acquired Immunodeficiency Syndrome (AIDS, AIDS), AIDS-related diseases, and human immunodeficiency virus (HIV) infection.
  • CD4 Human Cluster of Differentiation 4
  • HIV human immunodeficiency virus
  • the HIV-1 virus is an enveloped virus of about 100 nm.
  • the RNA genome and some active proteins are encapsulated in a scorpion-shaped core composed of capsid proteins.
  • the inside of the viral membrane is a capsid protein, and the envelope is located at the outermost layer of the virion. Its main body is a phospholipid bilayer, embedded with env.
  • a gene-encoded spike structure composed of gpl20 and gp41. The conformational change of the HIV envelope spike-like structure with the first receptor CD4 protein, and then with the second receptor chemokine receptors (including CCR5, CXCR4, etc.), ultimately achieve membrane fusion, The gene that releases the virus invades the cell.
  • the HIV envelope glycoprotein is expressed in the cell as a precursor of gpl60, and the mature envelope glycoprotein is cleaved into about 481 g- pl20 glycoproteins and Approximately 345 amino acids of gp41 glycoprotein ( Ratner, L. et al., Nature, 1985, 313: 277-284).
  • Gpl20 and gp41 exist as functional units of trimers on the HIV envelope, involved in viral-cell receptor binding and viral-cell fusion processes (Kwong, PD et al., Nature, 2002, 420: 678-682).
  • the HIV-1 envelope glycoprotein gpl20 interacts with the cellular receptor CD4 protein (Madondon, Fi et al, Cell, 1986, 47: 333-348) and with co-receptors (mainly CCR5 or CXCR4 proteins).
  • CD4 protein cellular receptor CD4 protein
  • co-receptors mainly CCR5 or CXCR4 proteins.
  • Infection of host cells Alkhatib, G. et al, Science, 1996, 272: 1955-1958; Deng, ⁇ et al, Nature, 1996, 381: 661-666; Dragic, ⁇ et al, Nature, 1996, 381: 667-673; Feng, Y. et al., 5W ⁇ I, 1996, 272: 872-877).
  • Binding to cellular receptors causes a conformational change in the viral envelope glycoprotein g pl20, which in turn induces viral and cell fusion of the transmembrane unit gp41 rearrangements of the envelope glycoprotein (Wyatt, R. et al., Science, 1998, 280: 1884 - 1888; Pierson, TC et al, Curr Top Microbiol Immunol, 2003, 281:1 - 27).
  • CD4 the full name of Cluster of Differentiation 4
  • auxiliary sputum cells auxiliary sputum cells, regulatory sputum cells, monocytes, macrophages, and dendritic cells.
  • One of the surface markers is an important receptor for its function, which is involved in regulating the proliferation of T immune cells, the release of lymphokines, and the interaction of immune cells, regulating the production of antibodies. It is also a receptor glycoprotein that infects CD4+ cells by many pathogens (including human immunodeficiency virus, etc.) (Isobe, et al., Proc Natl Acad Sci USA, 1986; 83(12): 4399-402; Ansari-Lari, MA Et al, Genome Res.
  • CD4 proteins are also members of the immunoglobulin superfamily.
  • the complete CD4 is a 433 amino acid membrane glycoprotein encoded by the CD4 gene with a molecular weight of approximately 55 kDa-62 kDa, including four cell membrane outer domains consisting of 1-375 amino acids.
  • domain a transmembrane region consisting of amino acids 376-395 and a cytoplasmic tail composed of amino acids 396-433 (Madondon PJ et al, Cell, 1985, 42: 93-104; Littman, DR ⁇ - , Cell, 55: 541).
  • the four extracellular domains are the first domain (domain 1 , abbreviated as D1, including amino acids 1-100), and the second domain (domain 2, referred to as D2, including amino acids 101-180).
  • the third domain domain 3, referred to as D3, including amino acids 181-290
  • D4 including amino acids 291-375
  • CD4 proteins play an important role in the replication cycle of HIV (Dalgleish, A. G. et al., Nature, 1984; 312:763).
  • CDR3-like region and the second immunoglobulin-like complementarity determining region (CDR2-like region) of the HIV-1 envelope glycoprotein gpl20 are directly involved in the interaction (Arthos, J. et al., Cell, 1989). , 57:469 - 481; Clayton, L.
  • CD4+ cells After CD4+ cells are infected with HIV, the function of the cells and the number of cells are significantly affected. The decrease in the number of T cells is due to the lytic infection of the virus, and also to the infection of CD4+ cells after HIV infection. Integration of uninfected CD4++ cells (Sodroski, J. et al., Nature, 1986, 322: 470-474). Exhaustion of CD4+ cells can cause the patient's body to be immunosuppressed, making it more susceptible to opportunistic infections and malignant tumors. This immunosuppression is widespread in patients with acquired immunodeficiency syndrome (AIDS, AIDS).
  • AIDS acquired immunodeficiency syndrome
  • AIDS is clinically characterized by a combination of HIV-associated symptoms, such as persistent systemic lymphadenopathy, fever, and weight loss.
  • AIDS is associated with central nervous system disorders due to HIV infection of CD4+ brain cells (Popovic, M. et al., Sdence, 1984, 224: 497-500).
  • the target of human immunodeficiency virus is CD4+ cells.
  • the number of CD4+ cells in patients is closely related to the development of the disease. Therefore, the detection of the number of CD4+ cells is effective in judging the effect of AIDS treatment and immunizing patients. The judgment of function plays an important role.
  • anti-CD4 antibodies have the effect of preventing HIV infection and/or treatment of HIV infection by interfering with the interaction of HIV with CD4 molecules and/or second receptors, some researchers are investigating anti-CD4 antibodies to block AIDS. The relationship and application of virus-cell interactions.
  • the anti-CD4 antibodies such as 5A8 and DB-81, have a promising role in blocking HIV-infected CD4+ receptor cells, and their epitopes are located.
  • the D2 domain of the CD4 protein (Song, R. et al., J Virol. 2010, 84: 6935-6942; Burastero, SE et al., JT Awkward si Med. 2009 Nov 28; 7:101).
  • monoclonal antibodies against other epitopes or domains of CD4 have been reported, such as monoclonal antibodies in specific fields such as monoclonal antibodies that can be used for peripheral blood CD4 antigen detection, but these antibodies are also reported in the relevant reports. Treatment deficiencies.
  • antibodies Leu3A and OKT4A that bind to the D1 domain of the CD4 molecule have a role in blocking the formation of cell syncytia caused by HIV infection (Sattentau QJ et al, Science, 1986, 234: 1120-1123; Jameson ⁇ Et al, Science. 1988, 240: 1335-1339; Peterson et al., Cell. 1988, 54: 65-72).
  • these antibodies since the recognition epitopes of these antibodies overlap with the epitope recognized by the HIV envelope glycoprotein gpl20, these antibodies cannot react with the CD4 protein molecules that bind to the envelope glycoprotein g pl20, so these antibodies are used as therapeutic drugs for HIV infection.
  • There are serious shortcomings (Bates ⁇ et al, Protein Eng.
  • the monoclonal antibody OKT4B recognizing the D2 domain of the CD4 molecule has been reported (T. Kieber-Emmons et al., Biochim Biophys Acta. 1989, 989:281-300) Significant interference with the binding of the HIV envelope glycoprotein gpl20 to the CD4 protein ( McDougal JS et al, J. Immunol., J Immunol. 1986, 137: 2937-2944;
  • Anti-CD4 antibodies of other epitopes also include MT151, VIT4, and MT321 (Sattentau QJ et al, Science. 1986, 234: 1120-1123), a table in which these antibodies recognize epitopes that bind to the HIV envelope glycoprotein g pl20. There is some overlap in the conformation (Sattentau QJ et al, J Exp Med, 1989, 170: 1319-1334; Bates PA et al, Protein Eng. 1989, 3: 13-21; Landau NR et al, Nature.
  • the antibody of the present invention recognizes an epitope on the D1 (Dl-2) domain of the CD4 protein, and when an HIV is present/CD4 protein binds to the HIV envelope glycoprotein gpl20, the epitope can be expressed with the monoclonal antibody of the present invention.
  • Better reactivity significantly blocking HIV from invading cells, preventing the virus from replicating in cells, blocking cytopathic effects caused by HIV infection, and preventing the spread of viruses between cells.
  • Such antibodies have application value in blocking new treatments for HIV infection and related diseases such as AIDS. Summary of the invention
  • the present invention provides a monoclonal antibody which specifically binds to the Human Cluster of Differentiation 4 (CD4) protein, thereby blocking the invasion of human immunodeficiency virus (HIV) into CD4+ cells.
  • CD4 Human Cluster of Differentiation 4
  • the monoclonal antibody of the present invention may bind to an epitope on the first domain (D1, amino acid 1-113) of the human surface antigen differentiation cluster 4 protein, or to the first 1-2 domain including the domain. (D1-2, amino acid 1-180) binds, and binds to other forms of truncated human surface antigen differentiation cluster 4 protein comprising the first domain, recombinantly expressed CD4, cell surface CD4 protein.
  • the antibody of the present invention does not significantly block the interaction between the human immunodeficiency virus gpl20 and the CD4 protein, and the monoclonal antibody of the present invention blocks the HIV virus-infected cells and blocks the replication of the HIV virus in the recipient cells.
  • the invention provides various homologous forms of anti-CD4 antibodies: monoclonal antibodies, fragments of immunoglobulins (eg, at least one immunologically active portion of an immunoglobulin molecule), such as Fab, Fab ⁇ F(ab' 2) A Fv fragment, a single chain antibody molecule or a multispecific antibody formed from any fragment of an immunoglobulin molecule comprising one or more CDR regions.
  • the present invention also provides a recombinant antibody, a recombinant chimeric antibody, a recombinant humanized antibody or the like formed by binding one or more CDR regions of a human immunoglobulin to one or more different human immunoglobulin framework regions. antibody. And a mimetic substance of the CDR functional region of the antibody of the invention.
  • the present invention provides hybridoma cell lines, isolated nucleic acid molecules and short peptides, and pharmaceutical compositions and medical diagnostic devices and kits comprising the monoclonal antibodies of the present invention.
  • the present invention also provides a method for detecting, diagnosing, preventing, and treating a mammal, including a human, using a monoclonal antibody of the present invention, and the infection source is a CD4+ cell as a main target cell: these diseases include acquired immunodeficiency syndrome ( AIDS, AIDS), AIDS-related diseases, human immunodeficiency virus (HIV) infections.
  • AIDS, AIDS acquired immunodeficiency syndrome
  • HIV human immunodeficiency virus
  • the invention provides a monoclonal antibody or antigen binding portion thereof, It has at least one feature selected from the group consisting of:
  • the invention provides a monoclonal antibody or antigen binding portion thereof, wherein the CDR1, CDR2 and CDR3 of the heavy chain of the antibody and the CDR1, CDR2 and CDR3 of the light chain of the antibody are respectively CTCCC deposited Hybridoma No. C201098 produced heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of antibody 15A7.
  • the invention provides a monoclonal antibody or antigen binding portion thereof, wherein the CDR1, CDR2 and CDR3 of the heavy chain of the antibody and the CDR1, CDR2 and CDR3 of the light chain of the antibody are hybridomas, respectively The heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of the produced antibody 14G7.
  • the invention provides a monoclonal antibody or antigen binding portion thereof, wherein the heavy chain variable region and the light chain variable region of the antibody are the weight of antibody 15A7 produced by the hybridoma of CCTCC Accession No. C201098, respectively. Chain variable region and light chain variable region.
  • the invention provides a monoclonal antibody or antigen binding portion thereof, wherein the heavy chain variable region and the light chain variable region of the antibody are the heavy chain variable region of antibody 14G7 produced by hybridoma, respectively Light chain variable region.
  • the invention provides a monoclonal antibody or antigen binding portion thereof that specifically binds to a human surface antigen differentiation cluster CD4 protein, wherein the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: Nucleotide shown or by SEQ ID ⁇ :1 Sequence encoding; wherein the amino acid sequence of the light chain variable region of the antibody is as set forth in SEQ ID NO: 4 or encoded by the nucleotide sequence of SEQ ID NO: 3.
  • the invention provides a monoclonal antibody or antigen binding portion thereof that specifically binds to a human surface antigen differentiation cluster CD4 protein, wherein the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: Shown or encoded by the nucleotide sequence of SEQ ID NO: 17; wherein the amino acid sequence of the light chain variable region of the antibody is as set forth in SEQ ID NO: 20 or encoded by the nucleotide sequence of SEQ ID NO: 19.
  • an antibody of the invention comprises one or more heavy chain CDRs selected from the group consisting of SEQ ID NOs: 5-7; and/or comprises one or more light chain CDRs selected from the group consisting of SEQ ID NOs: 8-10 ;
  • the heavy chain CDR1, CDR2 and CDR3 of the antibody of the invention are one or more of the nucleotide sequences of SEQ ID NOs: 5-7 or SEQ ID ⁇ : 11-13, respectively.
  • the light chain CDR1, CDR2 and CDR3 of the antibody of the invention are SEQ ID NOs: 8-10 or one of the nucleotide sequences of SEQ ID NOs: 14-16, respectively. Multiple encodings;
  • the monoclonal antibody or antigen binding portion thereof of the invention is a Fab, Fab', F(ab') 2 , Fv or single chain antibody.
  • an antibody of the invention comprises one or more heavy chain CDRs selected from the group consisting of SEQ ID NOs: 21-23; and a light chain CDR comprising one or more selected from the group consisting of SEQ ID NOs: 24-26;
  • amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of the antibodies of the invention are SEQ ID NOs: 21-23 or one or more of the nucleotide sequences of SEQ ID NOs: 27-29, respectively. Coding
  • amino acid sequences of the light chain CDR1, CDR2 and CDR3 of the antibodies of the invention are SEQ ID NOs: 24-26 or by SEQ ID, respectively. NO: one or more of the nucleotide sequences of 30-32;
  • the monoclonal antibody or antigen binding portion thereof of the invention is a Fab, Fab', F(ab') 2 , Fv or single chain antibody.
  • the monoclonal antibody or antigen binding portion binds human surface antigen protein cluster of differentiation 4 K D value of less than ⁇ ⁇ 5 [mu] according to the present invention.
  • a monoclonal antibody of the invention comprises a non-CDR region, which is from a species other than a murine.
  • the present invention provides a monoclonal antibody that specifically binds to a human surface antigen differentiation cluster 4 protein, which is a hybridoma producing antibody 15A7 of CCTCC Accession No. C201098, and the corresponding hybridoma cell line is CCTCC Deposit No. C201098 Hybridization
  • the invention provides an isolated nucleic acid molecule encoding a heavy chain variable region of a monoclonal antibody or antigen binding portion thereof of the invention.
  • the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO:2.
  • the nucleic acid molecule sequence is SEQ ID NO: 1.
  • the invention provides an isolated nucleic acid molecule encoding a light chain variable region of a monoclonal antibody or antigen binding portion thereof of the invention.
  • the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO:4.
  • the nucleic acid molecule sequence is SEQ ID NO:3.
  • the invention provides an isolated nucleic acid molecule encoding a heavy chain variable region of a monoclonal antibody or antigen binding portion thereof of the invention.
  • the heavy chain variable region of the antibody is listed as SEQ ID NO: 18.
  • the nucleic acid molecule sequence is SEQ ID NO: 17.
  • the invention provides an isolated nucleic acid molecule encoding a light chain variable region of a monoclonal antibody or antigen binding portion thereof of the invention.
  • the light chain variable region of the antibody is SEQ ID NO: 20.
  • the nucleic acid molecule sequence is SEQ ID NO: 19.
  • the invention provides an expression vector comprising a nucleic acid molecule of the invention. In another aspect, the invention provides a host cell comprising an expression vector of the invention.
  • the invention provides a method of detecting a human surface antigen differentiation cluster 4 protein in a sample, comprising the steps of:
  • the monoclonal antibody is attached to a solid support.
  • the solid support is selected from the group consisting of: microtiter plates, magnetic particles, latex particles, and nitrocellulose membranes.
  • the monoclonal antibody is attached to the solid phase in such a direction that the binding efficiency of the monoclonal antibody to the sample is increased.
  • the monoclonal antibody is attached to the solid phase by its constant region.
  • the reaction is determined by enzymatic color development.
  • the reaction is determined by fluorescence.
  • the reaction is measured by chemiluminescence.
  • the monoclonal antibody described in the above method is Fab, Fab', F(ab') 2 or Fv.
  • the sample is from a bird or a human.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a monoclonal antibody of the invention, or an antigen binding portion thereof, and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions of the invention further comprise other antiviral components.
  • the antibody in the pharmaceutical composition of the invention is Fab, Fab', F(ab') 2 Or Fvo
  • the invention provides a method of treating a condition elicited by a human immunodeficiency virus infection in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the invention.
  • the invention provides the use of a monoclonal antibody of the invention for the manufacture of a medicament for the treatment or prevention of a disease (e.g., AIDS) targeting a CD4+ cell.
  • a disease e.g., AIDS
  • the invention provides the use of a monoclonal antibody of the invention for the manufacture of a medicament for blocking HIV-infected CD4+ cells and for reinfection.
  • the invention provides the use of a monoclonal antibody of the invention for screening human surface antigen-inducing cluster 4 protein mimotope peptides.
  • the present invention provides the use of the above mimotope peptide for the preparation of a vaccine for preventing a disease (e.g., AIDS) targeting CD4+.
  • a disease e.g., AIDS
  • the present invention provides the use of the above mimotope peptide for the preparation of a kit for treating or diagnosing a disease (e.g., AIDS) targeting CD4+.
  • a disease e.g., AIDS
  • Figure 1 shows the amino acid sequence of the extracellular domain of human CD4.
  • the map shows the start and stop positions of the four domains of D1 domain, D2 domain, D3 domain and D4 domain.
  • Figure 2 is a SDS-PAGE result of recombinant human CD4 protein expression purification, and each figure is A: CD4 (dl); B: CD4 (d2); C: CD4 (d3); D: CD4 (d4); E: CD4 (dl-2); F: sCD4 (ie D1-D4 domain); each sample of each small image from left to right: 1: protein molecular weight marker; 2: expression after induction; 3: 2M urea Clear; 4: 4M urea supernatant; 5: 8M urea supernatant; 6: purified sample; 7: BL21 empty bacteria.
  • Figure 3 is a graph showing that each dilution of monoclonal antibody 15A7 blocks infection of Tzm-bl cells by different subtypes of HIV strains, including HIV NL 4- 3 (subtype B), HIV 89. 6 (B Subtype), HIV Right G114 (Subtype D), HIV M j 4 ( Type C), HIV WCML249 (D/C subtype).
  • Figure 4 is a graph showing that each dilution of monoclonal antibody 14G7 blocks infection of Tzm-bl cells by different subtypes of HIV strains, including HIV NL 4- 3 (subtype B), HIV 89. 6 (B Subtype), HIV G114 (Subtype D), HIV M j 4 (Subtype C), HIV WCML249 (D/C subtype).
  • Figure 5 is a result of immunofluorescence laser confocal microscopy of monoclonal antibody 15A7 binding to Tzm-bl cells, U87.CD4.CXCR4 cells and U87.CD4.CCR5 cells, wherein the control used is an anti-p24 monoclonal antibody.
  • H5F4
  • Figure 6 is a result of immunofluorescence laser confocal microscopy of monoclonal antibody 14G7 in combination with Tzm-bl cells, U87.CD4.CXCR4 cells and U87.CD4.CCR5 cells, wherein the control used is an anti-p24 monoclonal antibody.
  • H5F4
  • Figure 7 shows the results of flow cytometry analysis of recombinant protein CD4 (dl), CD4 (d2), CD4 (d3), CD4 (d4), CD4 (dl-2) and sCD4 blocking 15A7 antibody-cell reaction.
  • Figure 8 shows the results of flow cytometry of the recombinant proteins CD4(dl), CD4(d2), CD4(d3), CD4(d4), CD4(dl-2) and sCD4 blocking 14G7 antibody-cell reaction.
  • Figure 9 is a diagram showing the single-chain antibody expression plasmid of monoclonal antibodies 15A7 and 14G7.
  • Figure 10 is the results of SDS-PAGE purification of monoclonal antibody.
  • the samples from left to right are: 1: protein molecular weight Marker; 2: monoclonal antibody 15A7 reduction treatment sample; 3: 15A7 antibody Fab fragment reduction treatment sample; 4: 15A7 Antibody F(ab') 2 fragment reduction; 5: Monoclonal antibody 15A7 non-reducing sample; 6: 15A7 antibody Fab non-reducing sample; 7: 15A7 antibody F(ab') 2 non-reducing sample.
  • Figure 11 shows the results of the reaction of monoclonal antibody 15A7 and its F(ab') 2 Fab antibody fragment with HIV NL4 -3 virus, blocking its entry into Tzm-bl cells, and the control was a buffer.
  • Figure 12 is a flow cytometry test result of different receptors on the surface of Tzm-bl cells
  • Medium A is a monoclonal antibody 15A7 as a primary antibody, FITC-labeled rabbit anti-mouse antibody (RAM-FITC, Cat. No. F9006 Sigma) is labeled as a secondary antibody;
  • B is a FITC-labeled anti -CD4 (Cat. No. 555346, BD Biosciences) Labels the CD4 receptor protein on the surface of the cell to be tested;
  • C is a label labeled with PE-labeled anti-CXCR4 (Cat. No.
  • D panel is labeled with PE-Cy5-labeled anti-CCR5 (Cat. No. 556889, BD Biosciences) to label the CCR5 receptor protein on the surface of the cell to be tested;
  • E panel is an unlabeled cell control.
  • Figure 13 shows the results of flow cytometry of different receptors on the surface of MT4 cells, wherein the panel A is a monoclonal antibody 15A7 as a primary antibody, FITC-labeled rabbit anti-mouse antibody (RAM-FITC, Cat. No. F9006 Sigma) Mark the cells to be tested for the secondary antibody; B-map is to label the CD4 receptor protein on the surface of the cell to be tested with FITC-labeled anti-CD4 mAb (Cat. No. 555346, BD Biosciences); C-map is labeled with PE anti-CXCR4 mAb (Cat. No.
  • D is a PE-Cy5-labeled anti-CCR5 mAb (Cat. No. 556889, BD Biosciences) Marking the CCR5 receptor protein on the surface of the cell to be tested;
  • E panel is an unlabeled cell control.
  • Figure 14 shows the results of flow cytometry of different receptors on the surface of H9 cells.
  • the small panel is a monoclonal antibody 15A7 as a primary antibody, FITC-labeled rabbit anti-mouse antibody (RAM-FITC, Cat. No. F9006 Sigma) Mark the cells to be tested for the secondary antibody;
  • B-map is to label the CD4 receptor protein on the surface of the cell to be tested with FITC-labeled anti-CD4 mAb (Cat. No. 555346, BD Biosciences);
  • C-map is labeled with PE anti-CXCR4 mAb (Cat. No.
  • CXCR4 receptor protein on the surface of the cell to be tested
  • D is a PE-Cy5-labeled anti-CCR5 mAb (Cat. No. 556889, BD Biosciences)
  • the CCR5 receptor protein of the test cell is labeled
  • the E panel is an unlabeled cell control.
  • Figure 15 shows the results of flow cytometry of different receptors on the surface of U87.CD4.CXCR4 cells, wherein the panel A is a monoclonal antibody 15A7 as a primary antibody, FITC-labeled rabbit anti-mouse antibody (RAM-FITC, Cat.No) F9006 Sigma) labeled the test cells for the secondary antibody; B is the FI4-labeled anti-CD4 mAb (Cat. No. 555346, BD Biosciences) to label the CD4 receptor protein on the surface of the test cell; The CXCR4 receptor protein on the surface of the cells to be tested was labeled with PE-labeled anti-CXCR4 mAb (Cat. No.
  • the small panel is the anti-CCR5 monoclonal antibody labeled with PE-Cy5 (Cat. No. 556889, BD Biosciences) marks the CCR5 receptor protein on the surface of the cell to be tested; the E panel is an unlabeled cell control.
  • Figure 16 shows the results of flow cytometry of different receptors on the surface of U87.CD4.CCR5 cells, wherein the panel A is a monoclonal antibody 15A7 as a primary antibody and a FITC-labeled rabbit anti-mouse antibody (RAM-FITC, Cat.No). F9006 Sigma) labeled the test cells for the secondary antibody; B is the CD4 receptor protein on the surface of the test cell labeled with FITC-labeled anti-CD4 mAb (Cat. No. 555346, BD Biosciences); The CXCR4 receptor protein on the surface of the cells to be tested was labeled with PE-labeled anti-CXCR4 mAb (Cat. No.
  • the small panel is the anti-CCR5 monoclonal antibody labeled with PE-Cy5 (Cat. No. 556889, BD Biosciences) marks the CCR5 receptor protein on the surface of the cell to be tested; the E panel is an unlabeled cell control.
  • Figure 17 shows the results of flow cytometry of different receptors on the surface of human peripheral blood mononuclear cells (PBMC).
  • the panel A is a monoclonal antibody 15A7 as a primary antibody and a FITC-labeled rabbit anti-mouse antibody (RAM-FITC). , Cat.No. F9006 Sigma) labeled the test cell for the secondary antibody;
  • B is the CD4 receptor protein on the surface of the test cell labeled with FITC-labeled anti-CD4 mAb (Cat. No. 555346, BD Biosciences);
  • Panel C is a C-CR4 receptor protein labeled with PE-labeled anti-CXCR4 mAb (Cat. No.
  • D-map is an anti-CCR5 monoclonal antibody labeled with PE-Cy5 ( Cat.No ⁇ 556889, BD Biosciences )
  • the CCR5 receptor protein on the surface of the cell to be tested is labeled; the E panel is an unlabeled cell control.
  • Fig. 18 shows the case where the monoclonal antibody 15A7 detected by flow cytometry was labeled with 293FT cells expressing HIV, wherein the control cells used were blank 293FT cells, and the control antibody was anti-p24 monoclonal antibody H5F4.
  • Figure 19 shows the results of laser confocal microscopy of immunofluorescence of monoclonal antibody 15A7 and HIV-expressing 293FT cells, wherein the control cells used were blank 293FT cells and the control antibody was anti-p24 monoclonal antibody H5F4.
  • Figure 20 shows various truncated CD4 proteins including recombinant proteins CD4 (dl), CD4 (d2), CD4 (d3), CD4 (d4), CD4 (dl-2) and sCD4 blocking HIV NL4 - 3 infection of Tzm -bl cell effect.
  • Figure 21 is a result of immunoblotting of purified truncated CD4 protein and monoclonal antibody 15A7.
  • the A1 thumbnail is the truncated CD4 protein reduction SDS-PAGE map
  • the A2 thumbnail is the truncated CD4 protein reducing gel immunoblotting reaction map
  • the B1 thumbnail is the truncated CD4 protein non-reducing gel map
  • the B2 thumbnail is the truncated CD4 Protein non-reductive plaque reaction.
  • Each sample in each small image is 1: protein molecular weight marker; 2: CD4 (dl); 3: CD4 (d2); 4: CD4 (d3); 5: CD4 (d4); 6: CD4 (dl-2) 7: sCD4; 8: control protein; 9: BL21 empty bacteria.
  • Figure 22 shows the results of flow cytometry detection of TZM-bl cells labeled with 15A7, including the addition of HIV NL4 _ 3 and no HIV NL 4-3.
  • FIG 23 is a recombinant protein CD4 (dl), CD4 (dl -2) and recombinant sCD4 and blocking assay results in infection and HIV NIj4 _ 3 Tzm-bl cells 15A7.
  • Figure 24 is a reaction of CD4 (dl), CD4 (dl-d2) and recombinant sCD4 with 293FT cells transfected with pNL4-3, using monoclonal antibody 15A7 as a secondary antibody, and then labeled with FITC-labeled rabbit anti-mouse antibody.
  • the results of flow cytometry were performed, in which the A small image is the reaction result of monoclonal antibody 15A7; the B small image is the anti-CD4 monoclonal antibody (clone number Q4120, Cat. No. C1805 Sigma) as a control label for the test cell.
  • Surface CD4 The result of the reaction of body protein.
  • Figure 25 shows the results of immunoblotting of monoclonal antibody 15A7 and point mutation CD4 (dl-2) protein.
  • the A1 panel is a SDS-PAGE map of each point mutation CD4(dl-2) protein reduction; the A2 panel is a non-reducing SDS-PAGE map of each point mutation CD4(dl-2) protein; CD4 (dl-2) protein reduction-like immunoblot hybridization map; B2 panel is a non-reducing immunoblot hybridization map of each point mutation CD4 (dl-2) protein.
  • the sequence of each sample in each small image is 1: protein molecular weight marker; 2: CD4(dl-2)-N73A; 3: CD4(dl-2)-R59A; 4: CD4(dl-2)-K46A; 5: CD4(dl-2)-F43A; 6: Control protein CD4 (dl-2).
  • surface antigen differentiation cluster 4" in the present invention refers to any surface antigen differentiation cluster 4 protein encoded by the surface antigen differentiation cluster 4 gene.
  • CD4+ cells in the present invention refers to cells having a CD4 glycoprotein on the cell surface, including CD4+ T lymphocytes and CD4+ cell lines such as TZM-bl, H9 and C8166.
  • CD4 first domain (D1) refers to the truncated surface antigen differentiation cluster 4 polypeptide of positions 1 to 98.
  • CD4 second domain (D2) in the present invention - the term refers to the truncated surface antigen differentiation cluster 4 polypeptide at positions 99 to 183.
  • CD4 first domain (D3) refers to the truncated surface antigen differentiation cluster 4 polypeptide at positions 184 to 291.
  • CD4 first domain (D4) refers to the truncated surface antigen differentiation cluster 4 polypeptide at positions 292 to 365 in the present invention.
  • CD4 first/second domain (D1-2) in the present invention - the words refer to the first to the 183th A truncated surface antigen differentiation cluster 4 polypeptide.
  • recombinant soluble CD4 in the present invention refers to the truncated surface antigen differentiation cluster 4 polypeptide of positions 1 to 365.
  • anti-CD4 antibody in the present invention refers to an antibody which recognizes an epitope located on a surface antigen-differentiating cluster 4 protein molecule, that is, an antibody which binds to an epitope on the surface antigen-differentiating cluster 4 protein.
  • envelope glycoprotein in the present invention refers to an envelope glycoprotein of human immunodeficiency virus.
  • the envelope glycoprotein mediates the process of adsorption and entry of human immunodeficiency virus against host cells. Its precursor form is gpl60, which is processed to be gpl20 and gp41, and exists in the functional unit of the polymer on the human immunodeficiency virus envelope.
  • antibody refers to any immunoglobulin, including monoclonal antibodies, polyclonal antibodies, bispecific or multispecific antibodies that bind to specific antigens.
  • a complete antibody contains two heavy chains and two light chains. Each heavy chain contains a variable region and three constant regions of the first, second, third, etc.; each light chain comprises a variable region and a constant region.
  • the antibody is of the "Y" type, and the neck of the "Y” type structure contains two heavy and second constant regions of the heavy chain which are formed by disulfide bonding. Each arm of the "Y"-type structure contains a first constant region and a variable region of one of the heavy chains, and a variable region and a constant region of a light chain.
  • variable regions of the light and heavy chains determine the binding of the antigen; the variable region of each chain contains three hypervariable regions, termed the complementarity determining regions (CDRs) (the CDRs of the light chain (L) comprise LCDR1, LCDR2 The CDR color of LCDR3, heavy chain (H) contains HCDR1, HCDR2, HCDR3. It is named by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Vol. 1-3, NIH Publication 91-3242, Bethesda MD). Among them, the three CDRs are separated by a framework region (FR). The framework regions are more conserved than the CDR regions and form a shelf-like structure to support the hypervariable regions.
  • CDRs complementarity determining regions
  • the constant regions of the heavy and light chains are not associated with antigen binding, but have multiple effector functions.
  • Antibodies can be divided into several categories based on the amino acid sequence of the heavy chain constant region, mainly: IgA, IgD, IgE, IgG, and IgM, Some of these classes are further divided into subclasses such as IgG1, IgG2, IgG3, IgG4, IgAl or IgA2.
  • antibody as used in the present invention, unless specifically referred to as an intact immunoglobulin, also refers to a fragment of an immunoglobulin (eg, at least an immunologically active portion of an immunoglobulin molecule), such as Fab, Fab ⁇ F ( Ab') 2, an Fv fragment, a single chain antibody molecule or a multispecific antibody formed from any fragment of an immunoglobulin molecule containing one or more CDR regions.
  • the antibody of the present invention may also be an antibody formed by binding one or more CDR regions of a specific human immunoglobulin to one or more different human immunoglobulin framework regions.
  • An antibody-associated "Fab” fragment refers to a portion of an antibody molecule comprising a variable region and a constant region of a light chain and a variable region and a constant region of a heavy chain that are disulfide-bonded.
  • a "Fab” fragment refers to a Fab fragment that contains a portion of the hinge region.
  • F(ab') 2 refers to a dimer of Fab'.
  • Fc of an antibody refers to a portion of an antibody in which the second and third constant regions of the first heavy chain are joined to the second and third constant regions of the second heavy chain by disulfide bonds.
  • the Fc portion of an antibody has many different functions but does not participate in antigen binding.
  • the "Fv” segment of an antibody refers to the smallest fragment of an antibody that binds to the entire antigen binding site.
  • An Fv fragment comprising a variable region of a light chain binds to a variable region of a heavy chain.
  • single-chain antibody or “scFv” in the present invention refers to an engineered antibody in which a light chain variable region is directly linked to a heavy chain variable region or linked by a peptide chain (Houston 1988)
  • the "single-chain antibody Fv-Fc" or “scFv-Fc” in the present invention also includes an engineered antibody formed by the Fc segment of the scFv-linked antibody.
  • antigenic determinant refers to the portion of the amino acid or atomic group in the antigen molecule that binds to the antibody.
  • the term "monoclonal antibody” as used in the present invention refers to a fragment of an antibody or antibody from a group of highly homologous antibody molecules, that is, a group of identical except for natural mutations that may occur in only a few cases.
  • the mAb has a high specificity for a single epitope on the antigen.
  • Monoclonal antibodies differ from polyclonal antibodies, which are antibody molecules that comprise different epitopes on the recognition antigen.
  • the monoclonal antibody to which the present invention relates is not limited to this preparation method.
  • the monoclonal antibodies involved in the present invention can be obtained by the hybridoma technique first reported by Kohler et al. (KShler G. et al., Nature, 1975, 256: 495-497), and can also be obtained by recombinant DNA techniques (see, for example, U.S. Patent 4,816,567).
  • chimeric antibody as used in the present invention means that a part of an antibody light chain or/and a heavy chain is derived from a particular species or belongs to a particular antibody class or subclass having the same sequence or A homologous antibody, while another portion of the antibody light chain or/and heavy chain is an antibody that is derived from another species or belongs to another antibody class or subclass of the same or homologous sequence. In any event, such antibody fragments retain the binding activity to the antigen of interest (USP 4,816,567; Morrison et al, Pro Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • the term "humanized antibody” refers to an antibody obtained by replacing all or part of the CDR regions of a human immunoglobulin (receptor antibody) with a CDR region of a non-human antibody (donor antibody). Or an antibody fragment, wherein the donor antibody can be a mouse, rat or rabbit antibody having the desired specificity, affinity and reactivity.
  • the amino acid sequence of the framework region (FR) of the human immunoglobulin can be replaced by the amino acid sequence of the corresponding non-human antibody.
  • the amino acid residue of the humanized antibody may also be derived from neither the receptor antibody nor the CDR region or framework region sequence of the donor antibody. The purpose of these artificial modifications is to further refine or optimize antibody performance.
  • a humanized antibody refers to a variable region containing at least one, usually two, nearly complete, wherein all or nearly all of the corresponding CDR regions are derived from a non-human antibody, wherein all or almost all of the FR regions are From human antibodies.
  • An ideal humanized antibody contains at least a portion of the Fc region of an immunoglobulin, typically the Fc region of a human immunoglobulin.
  • isolated refers to that obtained by artificial means in a natural state. If a certain "separated” substance or component appears in nature, it may be that the natural environment in which it is located has changed or that it has been isolated from the natural environment, or both. For example, a certain living animal has a naturally isolated polynucleotide or polypeptide that is not isolated, and the same high-purity polynucleotide or polypeptide isolated from this natural state is called Separation.
  • the “separated” here does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of substances that do not affect the activity of the substance. It is not pure matter.
  • vector refers to a nucleic acid delivery vehicle which can insert a polynucleotide encoding a protein into it and obtain expression of the protein.
  • the vector can be expressed in a host cell by transforming, transducing or transfecting the host cell with the genetic material element it carries.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PACs); phage such as lambda phage or M13 phage And animal viruses, etc.
  • the animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, luxury viruses (such as simple oozing viruses), poxviruses, baculoviruses, papillomaviruses, papilloma polyves Virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses adeno-associated viruses
  • luxury viruses such as simple oozing viruses
  • poxviruses such as simple oozing viruses
  • baculoviruses such as simple oozing viruses
  • papillomaviruses such as SV40
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • the vector may also include components that assist it in entering the cell, such as viral particles, lipo
  • host cell refers to a cell into which a vector is introduced, including many cell types such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, such as S2 Drosophila cells or Sf. Insect cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus, such as S2 Drosophila cells or Sf. Insect cells
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • neutralizing antibody refers to an antibody or antibody fragment that purifies or significantly reduces the virulence of the target viral antigen binding.
  • sequence percent in sequence refers to the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acid or amino acid in the corresponding nucleic acid or polypeptide sequence, respectively.
  • sequence similarity percentage as used herein in connection with a nucleic acid sequence or polypeptide sequence is defined as a similar percentage of a candidate nucleic acid sequence or amino acid residue sequence to a nucleic acid sequence or amino acid sequence of interest, respectively. For a sequence, compare it to the destination sequence, Mutation gaps can be skipped if necessary to achieve maximum gene similarity percentages without regard to any conservative mutations of similar sequences.
  • a variety of alignment methods in the art can be used to determine the similarity of nucleic acid or amino acid sequences, such as useful BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) and the like. Those skilled in the art will appreciate that the appropriate measurement parameters can be set for comparison, including the comparison of full length sequences for some algorithms that use maximum comparability.
  • binding affinity refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen from which the antibody is produced.
  • the binding affinity of the antibody that binds the first antigen to the second antigen is undetectable or weak.
  • an antigen-specific antibody is associated with an affinity (KD) ⁇ 10 5 M (eg, 10.6 M, 10 7 M, 10 8 M, 10 9 M, 10 -1 ° M, etc.)
  • KD refers to the ratio of the dissociation rate to the binding rate (k Qff / k Qn ), which can be determined by methods familiar to those skilled in the art.
  • the monoclonal antibody of the present invention is capable of specifically binding to a surface antigen differentiation cluster 4 receptor.
  • One aspect of the present invention relates to a monoclonal antibody capable of specifically binding to a surface antigen differentiation cluster 4 receptor and a corresponding antigen-binding fragment thereof.
  • the monoclonal antibody of the present invention is secreted by mouse hybridoma cell line 15A7.
  • the names of these monoclonal antibodies are named after their corresponding hybridoma cell lines. Namely, these monoclonal antibodies 15A7 were produced by hybridoma cell line 15A7 and designated as 15A7.
  • the monoclonal antibody 15A7 specifically binds to the surface antigen differentiation cluster 4 protein.
  • Mouse hybridoma cell line 15A7 has been deposited with the China Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) under the accession number CCTCC - C201098 (hybridoma).
  • the monoclonal antibody of the present invention further includes a monoclonal antibody capable of blocking the binding of the monoclonal antibody 15A7 to the surface antigen differentiation cluster 4 protein.
  • the epitope of the surface antigen-inducing cluster 4 protein of these monoclonal antibodies can be combined with a single
  • the epitopes recognized by anti-15A7 are the same.
  • Epitopes recognized by these monoclonal antibodies can also be combined with monoclonal antibodies
  • the epitopes recognized by 15A7 overlap in space.
  • Such a monoclonal antibody can reduce the binding of mAb 15A7 to a surface antigen differentiation cluster 4 receptor protein by at least 30%, or at least 40%, or preferably at least 50%, or more preferably at least 60%, or more preferably at least 70%. Or more preferably at least 80%, or more preferably at least 90%, or more preferably 95%, or most preferably 99%.
  • the monoclonal antibody of the present invention does not significantly inhibit the binding of the HIV envelope glycoprotein g pl20 to the human surface antigen differentiation cluster 4 protein.
  • These antibodies affect the HIV envelope glycoprotein g pl20 and the human surface antigen differentiation cluster 4 protein (or recombinant human surface antigen differentiation cluster 4 protein sCD4 and truncated surface antigen differentiation cluster 4 protein) by up to 30%, or more preferably 20%, Or most preferably 0%.
  • Binding of the monoclonal antibody of the present invention to human surface antigen differentiation cluster 4 protein is not affected in the presence of HIV envelope glycoprotein g pl20
  • the binding force of such a monoclonal antibody to the complex of the HIV envelope glycoprotein gpl20 and the surface antigen differentiation cluster 4 receptor protein is 30%, or preferably 20%, or more preferably 5%.
  • a known method such as the method described in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) can be used to determine that an unknown monoclonal antibody reduces the binding of a surface antigen to the surface antigen 4 of a known monoclonal antibody. ability.
  • the antigen is pre-coated on a microplate, and then serially incubated in the pre-coated microplates, and the amount of the antibody to be tested at different dilutions is determined to be bound to the plate.
  • the antibody to be tested competes for the ability of known antibodies to bind antigen, and the ability of antibodies to bind antigen is known to be weaker.
  • the antigen is pre-coated on a 96-well microtiter plate and the ability of the unlabeled mAb to block the labeled mAb is determined by radiolabeling or enzymatic labeling.
  • Hybridomas as reported by Kohler et al., Nature 256: 495 (1975) can be used.
  • the preparation method is to prepare a monoclonal antibody.
  • the immunogen (addition of an adjuvant if necessary) is first immunized with a mouse or other suitable host animal.
  • the method of injection of the immunogen or adjuvant is usually subcutaneous injection or intraperitoneal injection.
  • Pre-coupling of the immunogen to certain known proteins, such as serum albumin or soybean trypsin inhibitors may help to enhance the immunogenicity of the antigen in the host.
  • the adjuvant may be a Freund's adjuvant or an MPL-TDM or the like.
  • lymphocytes secreting antibodies that specifically bind to the immunogen are produced in the body.
  • lymphocytes can also be obtained by in vitro immunization.
  • the lymphocytes of interest are collected from myeloma cells and fused with a suitable fusing agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996).
  • the hybridoma cells prepared above may be inoculated into a suitable culture medium, and the culture solution preferably contains one or more substances capable of inhibiting the growth of unfused, maternal myeloma cells.
  • the culture solution preferably contains one or more substances capable of inhibiting the growth of unfused, maternal myeloma cells.
  • HGPRT or HPRT hypoxanthine guanine phosphotransferase
  • addition of hypoxanthine, ⁇ , and thymine (HAT medium) to the culture medium will inhibit HGPRT.
  • HGPRT hypoxanthine guanine phosphotransferase
  • HAT medium hypoxanthine, ⁇ , and thymine
  • Preferred myeloma cells should have a high fusion rate, stable antibody secretion capacity, and sensitivity to HAT culture.
  • murine myeloma is preferred for myeloma cells, such as MOP-21 and MC-11 mouse tumor-derived strains (THE Salk Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2/0 or X63-Ag8. -653 cell line (American Type Culture Collection, Rockville, Md. USA).
  • human myeloma and human murine heterologous myeloma cell lines to prepare human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51 -63, Marcel Dekker, Inc., New York, 1987).
  • Hybridoma cell growth medium is used to detect the production of monoclonal antibodies against specific antigens Health.
  • Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells are immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and flow cytometry (FACS).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • FACS flow cytometry
  • Scatchard analysis described by Munson et al., Anal. Biochem. 107: 220 (1980) can be used to determine the affinity of a monoclonal antibody.
  • the target cell forest can be subjected to standard limiting dilution as described by Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996. The method is subcloned.
  • a suitable culture solution may be DMEM or RPMI-1640 or the like.
  • hybridoma cells can also be grown in animals in the form of ascites.
  • the monoclonal antibodies secreted by the subcloned cells can be cultured from the cell culture medium, Separated from ascites or serum.
  • the monoclonal antibodies of the invention can also be obtained by genetic engineering recombination techniques.
  • a DNA molecule encoding a monoclonal antibody heavy chain and a light chain gene can be isolated from a hybridoma cell by PCR amplification using a nucleic acid primer that specifically binds to the monoclonal antibody heavy chain and light chain genes.
  • the resulting DNA molecule is inserted into an expression vector and then transfected into a host cell, such as an E. coli cell, a simian COS, a CHO cell, or other myeloma cell that does not produce an immunoglobulin.
  • the transfected host cells are cultured under specific conditions and express the antibody of interest.
  • the antibody of the present invention has high specificity and high affinity for human surface antigen differentiation cluster 4 protein binding. These antibodies are cross-reactive with other primate surface antigen differentiation cluster 4 receptor proteins.
  • the monoclonal antibody of the present invention may be an antibody having a conventional "Y" type structure comprising two heavy chains and two light chains.
  • the antibody may also be a Fab fragment, Fab', F(ab') 2 , Fv, or other on a conventional "Y"-type antibody that retains affinity for a surface antigen-inducing cluster 4 receptor protein.
  • Antibody fragments of the invention can be obtained by hydrolyzing intact antibody molecules (see
  • Fab' fragments can be obtained directly from E. coli cells or chemically coupled to form F(ab') 2 fragments (Carter et al, Bio/Technology, 10: 163-167 (1992)).
  • the F(ab') 2 fragment can be obtained by leucine zipper GCN4 linkage.
  • Fv, Fab, Fab' or F(ab') 2 fragments can also be isolated directly from recombinant host cell culture media. Other techniques for making antibody fragments are well known to those of ordinary skill in the art.
  • the present invention relates to a nucleic acid molecule encoding an antibody or antibody fragment that specifically binds to a surface antigen-differentiating cluster 4 protein (cell surface-containing, recombinantly expressed and recombinantly expressed truncated human surface antigen differentiation cluster 4 protein).
  • the nucleic acid molecule encoding the antibody can be isolated from hybridoma cells.
  • One of ordinary skill in the art is well aware of the nucleic acid sequences that can be determined using conventional techniques.
  • the antibody nucleic acid molecules to which the present invention relates can also be obtained by conventional genetic engineering recombinant techniques or chemical synthesis methods.
  • the sequence of the antibody nucleic acid molecule of the invention comprises a heavy chain variable region of an anti-human surface antigen differentiation cluster 4 protein antibody or a partial nucleic acid sequence of an antibody molecule.
  • the sequence of the antibody nucleic acid molecule of the present invention also includes a light chain variable region of an anti-human surface antigen differentiation cluster 4 protein antibody or a partial nucleic acid sequence of an antibody molecule.
  • the sequences of the antibody nucleic acid molecules of the invention further comprise CDR sequences of the heavy or light chain variable regions.
  • the complementary determinant region is the site that binds to the epitope, and the CDR sequences in this study were determined by IMGT/V-QUEST (http://imgt.cmes.fr/textes/vquest/). But different partitions The CDR sequences obtained by the method are slightly different.
  • nucleic acid molecules encoding monoclonal antibody 15A7 heavy and light chain variable region sequences The nucleic acid molecule of the monoclonal antibody 15A7 heavy chain variable region sequence corresponds to SEQ ID NO: 1. Nucleic acid molecules of the monoclonal antibody 15A7 light chain variable region sequence correspond to SEQ ID NO: 3 combat The present invention also relates to nucleic acid molecule variants or analogs comprising the monoclonal antibody 15A7 heavy and light chain variable region sequences.
  • the invention also relates to variants of various isolated nucleic acid molecules.
  • the sequence of the nucleic acid variant has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85% identity with the nucleic acid sequence SEQ ID NO: l. SEQ ID NO: 3. More preferably it is at least 90%, most preferably at least 95%.
  • the present invention also provides a nucleic acid molecule capable of specifically binding to a coding sequence of an antibody fragment of a surface antigen differentiation cluster 4 protein.
  • the invention further relates to an isolated nucleic acid molecule encoding an antibody heavy chain variable region of amino acid sequence SEQ ID NO: 2.
  • the invention further relates to a nucleic acid molecule encoding an antibody light chain variable region of amino acid sequence SEQ ID NO: 4.
  • the present invention relates to recombinant expression vectors containing the nucleic acid molecules, and to host cells transformed with these molecules. Moreover, the present invention also relates to a method of culturing and isolating the antibody of the invention under specific conditions using a host cell comprising the nucleic acid molecule.
  • the monoclonal antibody 15A7 heavy and light chain variable region amino acid sequences can be deduced from the corresponding nucleic acid sequences.
  • the monoclonal antibody 15A7 heavy chain variable region amino acid sequence is SEQ ID NO: 2.
  • the monoclonal antibody 15A7 light chain variable region amino acid sequence is SEQ ID NO: 4.
  • the invention provides that the amino acid sequence of the heavy chain variable region has a sequence similarity to SEQ ID NO: 2 of at least 70%, preferably at least 75%, preferably at least 80%, preferably 85%, more preferably It is at least 90%, most preferably at least 95% of the antibody.
  • the invention provides an amino acid sequence of a light chain variable region and SEQ ID
  • the sequence similarity of NO:4 is at least 70%, preferably at least 75%, preferably at least 80%, preferably 85%, still more preferably at least 90%, and most preferably at least 95%.
  • the amino acid sequences of the CDRs of the heavy and light chain variable regions of mAb 15A7 are: CDR1, CDR2 and CDR3 of the heavy chain 15A7 heavy chain are respectively SEQ ID NOs: 5-7o CDR1 of the 15A7 light chain of mAb
  • the acid sequences of CDR2 and CDR3 are SEQ ID NOs: 8-10o, respectively.
  • the invention provides an anti-CD4 mAb heavy chain or fragment comprising one or more CDRs selected from the group consisting of SEQ ID NOs: 5-7.
  • the CDRs of the heavy chain or fragment of the anti-CD4 mAb contain
  • the column may have a mutation or addition or deletion of one or more amino acids at SEQ ID NOs: 5-7.
  • the amino acid that is mutated or added or deleted does not exceed 3 amino acids. More preferably, the amino acid that is mutated or added or deleted does not exceed 2 amino acids. Most preferably, the amino acid that is mutated or added or deleted does not exceed one amino acid.
  • the invention provides an anti-CD4 mAb light chain or fragment comprising one or more CDRs selected from the group consisting of SEQ ID NOs: 8-10.
  • the CDRs of the light chain or fragment of the anti-human CD4 mAb contain amino acid sequences which may exhibit mutations, additions or deletions of one or more amino acids at SEQ ID NOs: 8-10.
  • the amino acid that is mutated, added or deleted does not exceed 3 amino acids. More preferably, the amino acid that is mutated, added or deleted does not exceed 2 amino acids. Most preferably, the amino acid that is mutated, added or deleted does not exceed one amino acid.
  • Variants after mutation, addition or deletion of the amino acid of the variable region of the above antibody or CDR still retain the ability to specifically bind to CD4.
  • the invention also encompasses variants of such antigen-binding fragments.
  • the monoclonal antibody variants of the invention can be obtained by conventional genetic engineering methods. Those skilled in the art are fully aware of methods for engineering DNA molecules using nucleic acid mutations. In addition, nucleic acid molecules encoding heavy and light chain variants can also be obtained by chemical synthesis. Chimeric, humanized, and fusion proteins
  • the invention also provides a chimeric antibody consisting of a heavy or/and a light chain integral or partial variable region of a murine monoclonal antibody 15A7 or variant thereof, which binds to a constant region of a human monoclonal antibody .
  • the invention also encompasses humanized antibodies which are constructed by grafting one or more CDRs of murine monoclonal antibody 15A7 or a variant thereof onto a framework of a human antibody.
  • the invention also provides a fusion protein comprising a certain molecule, in whole or in part, comprising a monoclonal antibody of the invention.
  • Chimeric antibodies, humanized antibodies and fusion proteins can all be obtained using conventional genetic engineering techniques.
  • DNA encoding a monoclonal antibody can be engineered by mutation to replace the sequence of the constant region of the heavy and light chains of the human antibody with a homologous mouse sequence (Morrison, et al., Proc. Nat. Acad). Sci. 81: 6851 (1984)), or a chimeric or humanized antibody or fusion protein obtained by covalently coupling all or part of an immunoglobulin coding sequence to a non-immunoglobulin coding sequence.
  • the invention provides an anti-CD4 antibody capable of neutralizing a human immunodeficiency virus.
  • the neutralizing antibody is capable of neutralizing at least 60%, or at least 70%, or preferably at least 75%, or preferably at least 80%, or preferably at least 85% of the activity of the human immunodeficiency virus-infected CD4+ cells. Or preferably at least 90%, more preferably at least 95%, and most preferably at least 99.99%.
  • the IC50 neutralizing active concentration is preferably between 0.00 Ol - 10 g / mL, or preferably 5 ( ⁇ g / mL.
  • the invention also provides a method for detecting a specimen by using the monoclonal antibody of the invention Method of CD4 antigen or mimetic and/or antibody thereof.
  • the invention provides a method of detecting CD4 and CD4 related treatment comprising the steps of: (i) binding a monoclonal antibody or fragment thereof of the invention to an antigen in the above sample to form an antibody-antigen or antibody fragment- An antigen complex; (ii) detecting the sample complex to determine if there is a CD4 antigen or mimetic thereof in the sample.
  • the detection method can use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescence immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competition method, and the like.
  • ELISA enzyme-linked immunosorbent assay
  • the above detection method can be used to detect a target antigen or antibody by means of a competition method or a sandwich method.
  • the competition method compares the antigen in the sample with a known amount of labeled antigen to compete for the quantitative relationship of the monoclonal antibodies described in the present invention.
  • An immunological test based on the competition method is carried out by adding a sample containing an unknown amount of the target antigen to a conventionally known physical or chemical method for coating the monoclonal antibody of the present invention onto a solid support.
  • a predetermined amount of the labeled target antigen is added to carry out the reaction. After incubation, the solid support is rinsed and the activity of the label bound to the support is detected.
  • the target antigen in the sample is sandwiched between the coated mAb and the labeled mAb, and then a substrate such as an enzyme substrate is added, and the presence of the antigen is detected by the change in the color of the substrate.
  • Immunological detection based on the sandwich method is carried out, for example, by first adding a sample containing an unknown amount of the target antigen to a solid phase support which has been previously coated with the monoclonal antibody described in the present invention by physical or chemical means. Then, the labeled monoclonal antibody of the present invention is added to carry out the reaction. After incubation, the support is rinsed and the activity of the label bound to the support is detected.
  • the label may be a radioactive isotope such as 125 iodine, an enzyme, a substrate for the enzyme, a luminescent substance such as isoluminol and acridinium ester, a fluorescent substance such as fluorescein and rhodamine, biotin and a colored substance such as latex particles and colloidal gold.
  • the enzyme for labeling may be a peroxidase (such as horseradish peroxidase HRP), alkaline phosphatase, P-galactosidase, and glucose oxidase.
  • Suitable substrate for these reactions There are 2,2'-azino-bis(3-ethylbenzothiapyrrolidin-6sulfonic acid), luminol-hydrogen peroxide, o-phenylenediamine-hydrogen peroxide (for peroxidase) , p-nitrophenyl phosphate, 4-methylammonium ketone, 3-(2'-helix adamantane)-4-methoxy-4-(3"-phosphoryl)phenyl-1,2- Diethoxyalkane (for alkaline phosphatase), p-nitrophenyl-P-D-galactose and methylumbelliferyl- ⁇ -D-galactose (for beta-galactosidase).
  • markers include Quantum dot labeling, chromophore labeling, enzyme labeling, affinity ligand labeling, electromagnetic spin labeling, heavy atom labeling, probes labeled with nanoparticle light scattering labels or other nanoparticles, fluorescein isothiocyanate (FITC) ), TRITC, rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red, Phar-Red, isophycoerythrin (APC), epitope markers FLAG or HA epitope, and enzyme labeling such as alkaline phosphatase, horseradish peroxidase, I 2 -galactosidase, alkaline phosphatase, P-galactosidase or acetylcholinesterase and hapten coupling Object like ocean Yellow digitoxin or dinitrophenol, or a combination being able to form a complex pair, such as streptavidin
  • the label comprises a semiconductor nanocrystallite such as a quantum dot (ie, Qdots), see USP 6,207,392uß Qdots available from Quantum
  • Semiconductor nanocrystallites for use in the present invention include Group
  • Nanocrystallites of II-V semiconductors such as MgS, MgSe, MgTe, CaS, CaSe, CaTe,
  • Nanocrystallites of CdSe, CdTe, HgS, HgSe, HgTe, and mixtures thereof and Group III-V semiconductors such as GaAs, InGaAs, InP, InAs, and mixtures thereof.
  • Group III-V semiconductors such as GaAs, InGaAs, InP, InAs, and mixtures thereof.
  • IV semiconductors such as germanium or silicon, or the use of organic semiconductors, may be convenient and feasible under certain conditions.
  • the semiconductor nanocrystallites may also include alloys containing two or more semiconductors selected from the group consisting of Group III-V compounds, Group II-VI compounds, Group IV elements, and combinations thereof.
  • the fluorescent energy acceptor is attached to the detection probe as a label.
  • the fluorescent energy acceptor can be formed by reacting a compound with singlet oxygen to form a fluorescent compound, or by reacting a compound with an auxiliary compound and converting it to a fluorescent compound. Such compounds may be included in the buffer in the device of the invention.
  • the fluorescent energy acceptor can be part of a compound comprising a chemiluminescent agent or group, for example, the fluorescent energy acceptor can comprise a metal complex such as a rare earth metal such as ruthenium, osmium, iridium.
  • lanthanide markers such as cerium (III) provide effective long-term signal emission while being less susceptible to photobleaching, thus allowing processing/reaction samples to be included
  • the test set can be placed for a longer period of time if needed.
  • Long-lived fluorescent ruthenium nanoparticles have been used as markers in various heterologous and homologous immunoassays, see, for example, Huhtinen et al. Clin. Chem. 2004 Oct; 50(10): 1935- 6. However, when these internally labeled nanoparticles are used together with time-resolved fluorescence detection, the assay performance can be improved.
  • the dynamic range measured at low concentrations can be extended; moreover, by using highly specific active nanoparticles coated with detection antibodies
  • the kinetic properties of the assay can also be improved by using particulate markers instead of conventionally labeled detection antibodies.
  • ruthenium (III) nanoparticles are highly effective donors for fluorescence resonance energy transfer, allowing simple, rapid, and efficient screening.
  • the label such as the fluorescent label disclosed herein, comprises a nanoparticle label coupled to a biomolecule. In other words, nanoparticles can be used as detection or capture probes.
  • ruthenium (III)-labeled nanoparticles attached to a monoclonal antibody or streptavidin (SA) can be used to detect a specific analyte in a sample, such as a nanoparticle-based immunoassay.
  • Nanoparticles can serve as substrates for attachment of specific binding agents to analytes and assays (such as labels) or capture components. Examples of the labeling can be found in USP 4,695, 554; 4, 863, 875; 4, 373, 932; and 4, 366, 241, US Patent No. 4, 313, 734, and 4, 373, 932, which are incorporated herein by reference to U.S. Pat.
  • Organic polymer latex particles for use as labels are disclosed.
  • the method of binding a label to an antigen or antibody can be carried out by the maleimide method (J. Biochem. (1976), 79, 233), biotin activation method (J. Am. Chem. Soc. 1978), 100, 3585), hydrophobic bonding, ester activation or isocyanate (Igaku Shoin “Enzyme immune assay techniques", 1987).
  • the above markers are radioisotopes, use a good radiation-proof work surface or liquid protection equipment. If the above marker is an enzyme, a substrate is added, and the activity of the enzyme is measured by a colorimetric method or a fluorometer. If the above label is a fluorescent substance, a luminescent substance or a colored substance, the method of measurement can be carried out correspondingly using methods well known in the art.
  • the antibodies, active fragments thereof and homologs thereof of the present invention provide a method for preventing and/or treating CD4+ cell-associated viral infections and related diseases, particularly human immunodeficiency virus (HIV) infection and related diseases thereof.
  • HIV human immunodeficiency virus
  • Including AIDS, including applying to patients A certain amount of a pharmaceutically active pharmaceutical ingredient comprising the monoclonal antibody of the present invention is used.
  • the present invention also provides a pharmaceutical composition comprising the monoclonal antibody of the present invention or a salt drug obtained thereon.
  • the pharmaceutical composition of the present invention may further comprise other agents for preventing and/or treating HIV infection, which may be administered simultaneously, separately or continuously with these antiviral agents.
  • the antibody of the present invention and active fragments thereof and homologs thereof can be used together with an anti-reverse transcription agent capable of inhibiting reverse transcriptase, such as AZT, or with an agent capable of inhibiting HIV protease;
  • the pharmaceutical composition of the invention further comprises, but is not limited to, an antiviral agent such as an interferon, an immunosuppressive agent such as cyclosporin.
  • one or more antibody homologs can also be used with one or more of the foregoing therapeutic agents.
  • An advantage of such combination therapy is that lower doses of the therapeutic agent can be used, thus avoiding toxicity or side effects that may be present when using a single agent.
  • compositions of the present invention comprise one or more immunotherapeutic effective amounts of antibody homologs, or derivatives thereof, in accordance with the present invention, and are preferably pharmaceutically acceptable carriers.
  • Immunotherapy effective amount means an amount sufficient to prevent an immune response caused by other diseases caused by HIV infection or AIDS, or by an infectious agent of a cell which originally expresses CD4.
  • “Pharmaceutically acceptable carrier” refers to a carrier that does not elicit an allergic or other uncomfortable effect on the patient to whom it is administered.
  • Suitable pharmaceutically acceptable carriers include, for example, one or more of water, physiological saline, phosphate buffer, levulose, glycerol, ethanol, and the like, as well as combinations of the foregoing.
  • the pharmaceutically acceptable carrier may further comprise minor auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf life or utility of the antibody or its active fragments or homologs thereof.
  • the pharmaceutical composition of the present invention can be administered by conventional methods including intravenous drip, intramuscular injection, vaginal, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic sulcus. Internal, groin, intravesical, local (eg, Powder, ointment or drops), or nasal route, but not limited to this. Preferred are injection and infusion forms.
  • composition of the invention can take many different forms. These include, for example, solid, semi-solid, and liquid dosage forms such as tablets, pills, powders, solutions, dispersion suspensions, liposomes, suppositories, solutions for injection and infusion.
  • solid dosage forms such as tablets, pills, powders, solutions, dispersion suspensions, liposomes, suppositories, solutions for injection and infusion.
  • the preferred form depends on the mode of administration and its prophylactic or therapeutic application.
  • Preferred compositions are in the form of solutions for injection and infusion.
  • compositions suitable for parenteral injection may contain sterile water or non-aqueous solutions, aerosols, suspensions or emulsions which meet the requirements for pharmaceutical preparation, and can be resuspended as an injectable solution or aerosol aseptic powder when ready for use. .
  • Suitable aqueous and non-aqueous vehicles, tools and various diluents such as water, ethanol, polyols (such as propylene glycol, polyethylene glycol, glycerol and the like), suitable mixtures, vegetable oils (such as Olive oil), and organic fats that can be used for injection, such as ethane oleic acid, such as the use of lecithin capsids to maintain proper fluidity of the drug, such as the use of aerosols, surfactants to maintain a suitable particle size.
  • the pharmaceutical composition of the present invention may further contain some protective, moisturizing, emulsifying and aerosolizing adjuvants, and may also contain instant ingredients for preventing microbial contamination, such as various antibacterial agents, antifungal agents, such as parabens, Chlorobutanol, benzophenone, sorbic acid and the like. Agents that maintain osmotic pressure, such as sugar, NaCl, and the like, may also be included. Prolonged adsorption reagents can be used to prolong the adsorption time of the injectable drug components, such as monostearate and gel.
  • Oral solid phase dosage forms include capsules, tablets, powders, granules and the like.
  • the active ingredients in these solid phase dosage forms are admixed with at least one conventional inert pharmaceutical excipient (or carrier) such as sodium citrate, phosphoric acid, or (a) fillers or additives such as starch, lactose, sucrose, mannose and silicic acid.
  • binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;
  • humectants such as glycerol;
  • fragmentation agents such as agar, carbonic acid Calcium, potato flour or tapioca flour;
  • retarder such as paraffin;
  • promote absorption e ) a humectant such as cetyl alcohol and glyceryl monostearate;
  • an adsorbent such as kaolin and bentonite;
  • a lubricant such as talc, stearin Acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or a mixture thereof.
  • buffers may also be included.
  • the solid phase dosage form can be formed into a modified release or pulse release dosage form by adding some excipients which can change the release rate of the drug to the various direct release excipients mentioned above, and can be included in the dosage form.
  • Rate dry release modifiers include carboxypropyl methylcellulose, methylcellulose, sodium carboxymethylcellulose, cellulose ethane, cellulose acetate, polyethylene oxide, xanthan gum, amino acrylate copolymer , hydrogenated seasoning oil, bran wax, paraffin, cellulose acetate phthalate, carboxypropyl methylcellulose phthalate, methacrylic acid copolymer or a mixture of the above.
  • Modified release and pulse release dosage forms may contain one or a group of excipients with improved dry release rates.
  • the pharmaceutical composition of the present invention may also consist of a rapid atomizing agent or an anti-solvent (FDDFs) comprising the following components: aspartame, potassium sulfamate, citric acid, croscarmellose sodium, crospovidone, ascorbic acid, B Acryl propylate, ethane-based cellulose, gelatin, hydroxypropylmethylcellulose, magnesium stearate, mannitol, methylmethacrylate, flavored peppermint, polyethylene glycol, gas Silica gel, silica, sodium starch glycolate, sodium stearate stearate, sorbitol, xylitol.
  • FDDFs anti-solvent
  • atomization and dissolution depends on the solubility of the drug used. For example, if the drug is insoluble, it can be made into a rapid aerosolized form. If the drug is soluble, it can be made. Fast solvent type.
  • Similar forms of solid phase ingredients are also made into a soft gelatin or hard gelatin filling form using, for example, lactose or milk sugar or other high molecular weight polyethylene glycols and similar excipients.
  • Solid dosage forms such as tablets, dragees, capsules and granules and the like can be made by means of an outer casing such as a casing or other known to those skilled in the art. It may also contain an opacifying agent, or it may contain similar ingredients which can slow, delay, and control the release of the active drug. It is also possible to use a component such as a polymer and a paraffin wax for embedding. If appropriate, the active ingredient may also be formulated in the form of a microterpene using one or more of the excipients described above.
  • Liquid dosage forms for oral administration including emulsions, solutions, suspensions, syrups, and medicinal agents that meet pharmaceutical requirements.
  • the liquid dosage form may contain inert solutions such as water or other solvents, soluble agents and emulsifiers such as ethane alcohol, isopropyl alcohol, ethane carbonate, phenyl benzoic acid, which are commonly used in the art. Salt, propylene glycol, 1,3-butene glycol, oil, in particular, cottonseed oil, groundnut oil, corn oil, olive oil, seasoning oil and sesame oil, glycerin, hydroquinone alcohol, polyethylene Alcohol and fatty acid sorbitol esters, and mixtures of the above or similar materials.
  • the pharmaceutical ingredient may also include adjuvants such as humectants, emulsifiers, suspending agents, saccharifying agents, flavoring agents, and flavoring agents.
  • the pharmaceutical ingredient may further comprise ethoxylated homopolyethanol, polyoxyethylene alkyl sorbitol and sorbitan lipid, microcrystalline cellulose, meta-alumina, bentonite, agar polymer and tragacanth, or these substances a suspending agent such as a mixture.
  • the pharmaceutical composition of the present invention can also be made into a mixture suitable for veterinary treatment, or a veterinary salt, or a veterinary solvent or a preliminary medicine, and is made to suit the requirements of ordinary veterinarians and veterinarians.
  • a suitable dosage form for the administration of a particular animal and the route of administration of the drug can also be made into a mixture suitable for veterinary treatment, or a veterinary salt, or a veterinary solvent or a preliminary medicine, and is made to suit the requirements of ordinary veterinarians and veterinarians.
  • a suitable dosage form for the administration of a particular animal and the route of administration of the drug can also be made into a mixture suitable for veterinary treatment, or a veterinary salt, or a veterinary solvent or a preliminary medicine, and is made to suit the requirements of ordinary veterinarians and veterinarians.
  • Preferred pharmaceutical compositions of the invention are similar to those of other antibodies for passive immunization of humans, such as tumor therapeutic antibodies.
  • a preferred mode of administration is parenteral administration.
  • the one or more monoclonal antibodies of the invention may be combined with other antiviral agents for the prevention and or treatment of CD4+ cell related diseases including, but not limited to, human immunodeficiency infections and related diseases.
  • the monoclonal antibody can be administered simultaneously, separately or continuously with these antiviral agents.
  • Other antiviral agents include ribavirin, adamantane, urea, IL-2, IL-12 and pentacarboxylate Chain cyanoacid, but not limited to these.
  • an antibody homolog or active fragment thereof of the invention will depend on the administration procedure, the unit dose of the antibody homolog administered, and whether the antibody homologue is used with other agents. , the patient's immunity and health, and the antiviral activity of the particular antibody homolog administered.
  • mice 6-week-old female BALB/c mice were provided at the Experimental Animal Center of the College of Life Sciences, Xiamen University.
  • mice immunization The recombinant sCD4 antigen solution dissolved in PBS was emulsified in equal volume with Freund's complete adjuvant (CFA), and injected into the muscles of the extremities, each injection of the purified recombinant sCD4 antigen prepared in Example 2 g (total volume 50 ⁇ ). On the 15th and 29th day after the first immunization, booster immunization was performed with the same dose of recombinant antigen solution plus Freund's incomplete adjuvant (IFA). 72 hours before the fusion, the tail vein was boosted with 5 g of antigen without adjuvant for 1 boost.
  • CFA Freund's complete adjuvant
  • the spleen cells of the mouse with the highest serum neutralization titer were fused with mouse myeloma cells, and the spleen was first ground to obtain a spleen cell suspension, and the cells were counted. Press 1/6 on splenocytes The number of cultured SP2/0 mouse myeloma cells was mixed, centrifuged, and subjected to 50% polyethylene glycol (PEG2000) for 1 minute to fuse the spleen cells with mouse myeloma cells SP2/0.
  • PEG2000 polyethylene glycol
  • HT medium containing 20% FBS 1.361 mg of hypoxanthn (H), 0.388 mg of thymidine (T), plus RPMI 1640 (GIBCO) medium to 100 mLo 45 ⁇ 50 conditions. After dissolution, filter and sterilize. Semi-retained liquid change.
  • ELISA plates were coated with purified sCD4 prepared in Example 2. Monoclonal antibody-added cells were incubated in each well for 30 minutes; washed on PBST for 5 times on a TECAN automatic plate washer, at intervals of 20 seconds, decanted, and added to the appropriate dilution of HRP- Goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3 antibody (Serotec) enzyme-labeled secondary antibody, incubated at 37"C for 30 minutes; washed 5 times with PBST on TEC AN automatic plate washer, at intervals of 20 Seconds, deducted, add 1 drop of each of the coloring solutions A (H 2 0 2 ) and B ( TMB ), develop color at 37* € for 10 minutes, add 1 drop of stop solution (2M H 2 S0 4 ); The OD 45 () nm (reference wavelength is 620 nm), the threshold is 2 times the negative mean, the
  • Hybridoma culture The stable hybridoma monoclonal antibody cell line was first expanded and cultured in a carbon dioxide incubator, cultured in 96 wells, transferred to 24 wells, and transferred to a 50 mL cell bottle for expansion culture. 10 weeks old healthy BALB/c mice were intraperitoneally injected with 0.5 mL/liquid paraffin. After 1-2 weeks, each mouse was intraperitoneally injected with lxlO 6 hybridoma cells, and ascites was collected 7-10 days later. Centrifuge at 3000 rpm for 15 minutes, aspirate the middle clarified portion of the liquid, filter the 0.45 ⁇ m microporous membrane, and store it after -20.
  • the precipitate was dissolved in an appropriate amount of PBS, placed in a dialysis bag, and desalted for about 12 hours in a 120-100 ⁇ 20 mM Tris-HCl buffer containing 20 mM NaCl, H 7.8, and stirred for 3 hours. Above dialysate. After taking it out, pack -20 and save.
  • CD4(dl)F 5'- CGG CAT ATG AAG AAA GTG GTG CTG GGC -3'
  • CD4(d4)R 5'-
  • GCGAATTCTTACCATGTGGGCAGAACCTT -3' is a reverse primer, and the PCR reaction was carried out in a PCR thermocycler (Biometra T3) under the following conditions: 94 * € 10 minutes; followed by 94 30 seconds, 56 30 seconds, C 25 cycles of 1 minute, and finally 72 extensions of 10 minutes. A specific lllkb size was obtained for the preparation of each truncated recombinant human CD4 polypeptide.
  • the PCR product obtained above was ligated with a commercially available pMD18-T vector (manufactured by TaKaRa Co., Ltd.), and identified by Nde I/EcoR I digestion to obtain a positive clone into which the sCD4 gene was inserted.
  • the forward primer in Table 1 (the 5' end introduced restriction endonuclease Ndel) and the reverse Primer (the 5' end introduces a restriction endonuclease EcoRI site).
  • the PCR reaction was carried out on a PCR thermocycler (Biometra T3) under the following conditions: 94 * € 10 minutes; followed by 94 30 seconds, 56 30 seconds, C 45 cycles of 25 cycles, and finally, 1/C extension for 10 minutes .
  • the forward primer used for CD4(dl) is CD4(dl)F
  • the reverse primer is CD4(dl)R
  • the forward primer used for CD4(d2) is CD4(d2)F
  • the reverse primer is CD4 ( D2)R
  • CD4(d3) The forward primer used is CD4(d3)F
  • the reverse primer is CD4(d3)R
  • the forward primer used for CD4(d4) is CD4(d4)F
  • the reverse primer is CD4(d4)R
  • the forward primer used for CD4(dl-2) is CD4(dl)F
  • the reverse primer CD4(d2)R gives a specific DNA fragment for encoding the truncated human CD4 of the present invention.
  • the polynucleotide sequence has an amino acid sequence as shown in FIG.
  • the expression vector pTO-T7 was constructed according to the literature (Luo et al., Journal of Bioengineering, 2000, 16: 53-57).
  • the specific step of constructing the vector for expressing the polypeptide of the present invention comprises: linking the PCR product obtained as described above to a commercially available pMD 18-T vector (manufactured by TaKaRa Co., Ltd.), and identifying by Nde I/EcoR I digestion, A positive subcloning of the CD4 gene was inserted into each segment; Nde I/EcoR I was digested to obtain the CD4 gene fragment of each segment, and then ligated with the NTO I/EcoR I digested pTO-T7 expression vector, Nde I/EcoR I enzyme The recombinant expression clones pTO-T7-CD4(dl), pTO-T7-CD4(d2), pTO-T7-CD4(d3), pTO-T7-CD4(d
  • ⁇ recombinant expression vector plasmid (0.15 mg/mL) was transformed into 40 ⁇ calcium chloride prepared competent Escherichia coli BL21 (purchased from New England Biolabs), coated Solid LB medium resistant to kanamycin (final concentration 25 mg/mL, the same below), 37* € static culture for 10 ⁇ 12 hours, until the single colony is clearly identifiable.
  • Each 1 L of the triangular culture flask was filled with 500 mL of fresh LB liquid medium, and each was transferred to a storage solution of 200 ⁇ , and shaken for 37 hours at 37* €, to a cell concentration of OD 600 «1.0. Then, 500 ⁇ 0.8 ⁇ IPTG was added to each flask to a final concentration of 0.8 mM, and culture was induced by shaking for 4 hours, and the cells were collected by centrifugation.
  • the cells were disrupted by a sonicator (Uilbra-Cell VCX500 type from SONICS & MATERIALS), and 20 mL of the cell suspension obtained in 500 mL of the culture solution was used as an example.
  • the crushing conditions were as follows: 55% power was set, water bath, and the pulse breaking time was 2 seconds per interval.
  • Samples were numbered CD4(dl) 2M, CD4(d2) 2M, CD4(d3) 2M, CD4(d4) 2M, CD4(dl-2) 2M and sCD4 2M; equal volume of 4M urea/buffer for precipitation I resuspend; 37* € 200rpm for 1 hour, 4* € for overnight (10 hours); 4"C 12,000rpm Centrifuge for 10 minutes, leave the supernatant, sample number is CD4 (dl) 4M, CD4 (d2) 4M, CD4 (d3) 4M, CD4 (d4) 4M, CD4 (dl-2) 4M and sCD4 4M; equal volume for precipitation 8 M urea/buffer I was resuspended; shaken at 37 200 rpm for 1 hour, placed overnight (10 hours); centrifuged at 12,000 rpm for 10 minutes at 4 ° C, leaving the supernatant clear.
  • Sample numbers were CD4 (dl) 8M, CD4 (d2) 8M, CD4 (d3) 8M, CD4 (d4) 8M, CD4 (dl-2) 8M and sCD4 8M; Take 2M/4M/8M supernatants of CD4(dl), CD4(d2), CD4(d3), CD4(d4), CD4(dl-2) and sCD4, respectively, and add the condensate loading buffer ( 50 mM Tris Cl (pH 6.8), 100 mM dithreitol (DTT), 2% SDS, 0.1% bromophenol blue, 10% glycerol); boiling water bath for 10 minutes; centrifugation at 12,000 rpm for 10 minutes; 15% acrylamide SDS-PAGE analysis was performed to determine the specific gravity of the target protein in 2 ⁇ /4 ⁇ /8 ⁇ urea/buffer I.
  • condensate loading buffer 50 mM Tris Cl (pH 6.8), 100 mM dithreitol (DTT), 2%
  • the urea content of the sample was 4xl (T 6 M; the dialyzed sample was centrifuged at 4*C 12,000 rpm for 10 minutes; the supernatant was filtered through a 0.22 ⁇ microporous membrane for further purification.
  • CD4 (dl), CD4 (d2), CD4 (d3), CD4 (d4), CD4 (dl-2) and sCD4 by HPLC gel filtration chromatography.
  • the renatured samples were further purified using the following HPLC system and method.
  • Figure 2 shows the SDS-PAGE after induction of E. coli cells transformed with each expression plasmid
  • a small image is CD4 (dl)
  • B small image is CD4 (d2)
  • C small image is CD4 (d3)
  • D small The picture shows CD4(d4)
  • E is small picture CD4(dl-2)
  • F picture is sCD4) shows CD4(dl), CD4(d2), CD4(d3), CD4(d4) and CD4(dl-2 sCD4 has an expression band at the corresponding position, and the expression amount is between 10% and 40%
  • the second track of each AF map mainly in the form of inclusion bodies. After washing with 2M, 4M and 8M urea, 2M urea supernatant can be seen (Fig.
  • HIVMJ4 0.071 0.235
  • Incubation of the secondary antibody Carefully remove the coverslip, flip the coverslip so that the cells are facing up, place them in a 24-well plate, and add 1 mL of PBS for 5 minutes at room temperature. The PBS was then aspirated and washed twice with PBS. Take a new sealing film over the 24-well plate cover and add 25 ⁇ of secondary antibody to the position of the well on the 24-well plate.
  • the secondary antibody is anti-Mouse IgG (whole molecule)-FITC (Cat. No. F9006, Sigma) diluted 1:200 in PBS. Place the coverslip in the droplet of the secondary antibody in the same way as the closure, ensuring that the cells are facing down. Place a 24-well plate with a coverslip on a wet box (saturated humidity) and incubate at room temperature for 30 minutes in the dark.
  • DAPI staining Carefully remove the coverslip, flip the coverslip so that the cells are facing up, place them in a 24-well plate, and add 1 mL of PBS for 5 minutes at room temperature. The PBS was then aspirated and washed twice with PBS. Take a new sealing film and cover it on the 24-well plate cover. According to the position of the hole on the 24-well plate, add 25 ⁇ DAPI diluted with PBS (diluted 1:2000), and cover the glass according to the same operation. Place the piece in the DAPI droplets, making sure the cells are facing down. Place the 24-well plate cover with the cover slip in the wet box (maintaining saturated humidity) Incubate for 5 minutes at room temperature in the dark.
  • the monoclonal antibodies 15A7, 14G7 were able to bind to the cell membrane of the three cells. This indicates that the monoclonal antibody can bind to the CD4 protein expressed on the surface of the three cells.
  • Example 5 Each truncated CD4 polypeptide block/inhibit monoclonal antibody Flow cytometry detection of 15A7, 14G7-labeled CD4+ cells
  • Tzm-bl was plated in 96-well cell culture plates at 1.5 ⁇ 10 4 cells/well, and incubated in a 5% carbon dioxide incubator for 12 hours before use in experiments.
  • the primary purified CD4(dl), CD4(d2), CD4(dl-2), CD4(d3), CD4(d4), sCD4 protein were diluted with DMEM medium of 10% FBS to a final concentration of l ( ⁇ g/ mL, then add the diluted monoclonal antibodies 15A7, 14G7, respectively, so that the final concentration of the antibody reaches the labeled concentration in the abscissa axis of Figure 7 and Figure 8, and then incubate for 1 hour at 37.
  • One tube of the added reverse transcription primer is MVJkR (5'-CCg TTT (T/g)AT (T/C)TC CAg CTT ggT (g/C)CC-3'), which is used to amplify light chain variable
  • the region gene, the reverse transcription primer added to the other tube is MVDJhR (5,-C ggT gAC Cg(T/A) ggT (C/g/T)CC TTg (g/A)CC CCA-3'),
  • the heavy chain variable region gene was amplified.
  • Each tube was further added with luL dNTP (10 mM, TAKARA), placed in a water bath at 72 ° C for 10 minutes, immediately placed in an ice bath for 5 minutes, and added to ⁇ 5x reverse transcription buffer.
  • luL dNTP 10 mM, TAKARA
  • luL dNTP 10 mM, TAKARA
  • luL RNasin 40unit/ L, Promega
  • the antibody variable region was isolated by polymerase chain reaction (PCR) using primer sets synthesized according to Novagen's Ig-Prime kits and additionally designed and synthesized.
  • MVJkR is a downstream primer for light chain variable region gene amplification
  • MVDJhR is a downstream primer for heavy chain variable region gene amplification.
  • the template is the two cDNAs synthesized above.
  • the PCR conditions were: 94 ° C for 5 minutes, 94 ° C for 40 seconds, 53 ° C for 1 minute, 72 ° C for 50 seconds, 35 cycles, and 72 ° C for 15 minutes.
  • the fragment of interest was recovered and cloned into the pMD18-T vector (TaKaRa), and sequenced, and the sequence of the antibody variable region was determined by alignment of the Blast analysis, and the corresponding amino acid sequence was deduced.
  • the antibody variable region genes were cloned from the monoclonal antibody 15A7, 14G7 hybridoma cell lines by the above methods, and the corresponding amino groups were deduced.
  • Table 3 shows the upstream primer sequence used, monoclonal antibody 15A7 heavy chain variable region nucleotide SEQ ID ⁇ : 1; heavy chain amino acid sequence number SEQ ID NO: 2; light chain variable region nucleotide SEQ ID NO: 3; Light chain amino acid sequence number SEQ ID NO: 4.
  • CDR complementary determinant region
  • variable region heavy chain gene sequence was input into the VBASE2 database (http://www.vbase2.org/) for alignment analysis.
  • the results showed that the cloned V fragments were all Igh-VJ558 VH1 family, and the Vh gene size was 351 bp, encoding a polypeptide consisting of 117 amino acid residues, which had structural features corresponding to the KABAT structure.
  • the heavy chain CDR regions are shown in Table 4.
  • variable region light chain gene sequences were imported into the VBASE2 database for alignment analysis.
  • the results showed that the cloned V fragments were all IGKV21, the Vk gene was 15A7 Vk: 339 bp, encoding a polypeptide consisting of 113 amino acid residues, 14G7 Vk: 324 bp, encoding a polypeptide consisting of 108 amino acid residues. It has structural features that correspond exactly to the KABAT structure.
  • the light chain CDR regions are shown in Table 4.
  • the heavy and light chain variable regions of the antibody gene are joined by a (GGGGS) 3 short peptide into a single-chain antibody DNA fragment.
  • 15A7 VHF was used as the forward primer
  • 15A7 VHR was used as the reverse primer to amplify the 15A7 heavy chain variable region fragment
  • 15A7 VKF was used as the forward primer
  • 15A7 VKR amplifies the 15A7 light chain variable region fragment for the reverse primer.
  • the primer sequences are shown in Table 5. Two antibody gene fragments were recovered separately. The heavy chain variable region and the light chain variable region fragment were used as primers and templates to overlap and extend in a new PCR system to obtain a small amount of intact single-chain antibody fragments.
  • 15A7 VHF as a forward primer
  • 15A7 VKR as a reverse primer
  • the single-chain antibody fragment was recovered, and the single-chain antibody fragment was digested with Nde l/Xho l and cloned into the same digested pET-30a-c(+) prokaryotic expression vector, and the vector is shown in Fig. 9.
  • ER2566 E. coli as an expression strain, it was expressed by a conventional method, and the resulting protein was present in the form of insoluble inclusion bodies.
  • the purified inclusion bodies were washed by a conventional method, and as a result, the single-chain antibody was mainly dissolved in 8 M urea.
  • the single-chain antibody protein dissolved in 8 M urea was gradually dialyzed against lxPBS, and the precipitate was removed by centrifugation at 12,000 rpm for 10 minutes, and the finally obtained preliminary purified single-chain antibody solution was subjected to activity measurement.
  • 14G7VKR S'-AAGCTTCTCGAGTTACCGTTTCATTTCCAGCTT -3'
  • the activity of the initially purified 15A7 single-chain antibody and 14G7 single-chain antibody was detected by competitive ELISA.
  • CD4 antigen was coated in the wells of polystyrene strips, and blocked with lxED (2% gelatin, 0.5% casein and 2% sucrose in lxPBS solution), and the above single-chain antibody solution was added to the wells.
  • Positive control In the wells, ⁇ monoclonal 15 ⁇ 7, monoclonal antibody 14G7 were added, and negative control wells were added with ⁇ unrelated monoclonal antibody 1F2 dilution or 10 ( ⁇ L PBS, three-well repeat.
  • CD4(dl-2) 1.159 1.080 1.209 1.095 sCD4 0.395 0.338 0.236 0.237
  • CD4(dl-2) 1.052 0.989 1.119 1.120 sCD4 0.412 0.323 0.225 0.253
  • CD4(d2) 0.076 0.058 0.049 0.058
  • the reactivity of the initially purified 15A7 single-chain antibody, 14G7 single-chain antibody and CD4 protein was detected by ELISA.
  • the 15A7 single-chain antibody, 14G7 single-chain antibody was labeled with biotin biotin and diluted with HCV-ED.
  • CD4 antigen CD4 (dl), CD4 (d2), CD4 (dl-2), CD4 (d3), CD4 (d4) and sCD4 were coated in the wells of polystyrene strips and blocked with lxED in the wells to be tested.
  • CD4(d4) 0.227 0.197 0.045 sCD4 0.438 0.428 0.091
  • CD4(d4) 0.212 0.232 0.051 sCD4 0.525 0.521 0.089
  • the cleaved monoclonal antibody was dialyzed against 20 mM ⁇ ( ⁇ 8 ⁇ 0) buffer overnight and then purified by DEAE-HPLC.
  • the column was equilibrated with 20 mM TB (pH 8.0), eluted linearly with 20 mM TB (pH 8.0) containing 0.05 M NaCl for 15 minutes, and washed with 20 mM TB (pH 8.0) containing 1 M NaCl and a small amount of Fc. Cut the total resistance.
  • the loading amount was 10-100 mg of antibody, the flow rate was lmL/min, and the detection wavelength was 280 nm.
  • the purity of the collected FabSDS-PAGE was measured and concentrated in a 10 kD concentrating tube. After dialysis against 20 mM PBS (pH 7.4), the sterilized (0 ⁇ 22 ⁇ microporous membrane) was dispensed, and the Fab pure product of the monoclonal antibody 15A7 was obtained.
  • the column was equilibrated with 20 mM TB (pH 8.0) and linearly eluted with 20 mM TB (pH 8.0) containing 1 M NaCl for 30 minutes to wash down F(ab') 2 and a small amount of uncut total antibody, respectively.
  • the loading amount was 10-100 mg of antibody, the flow rate was 1 mL/min, and the detection wavelength was 280 nm.
  • the collected F(ab') 2 was purified by SDS-PAGE and concentrated in a 10 kD concentrating tube. After dialysis against 20 mM PBS (pH 7.4), the sterilized (0 ⁇ 22 ⁇ microporous membrane) was dispensed. The F(ab') 2 pure product of the antibody 15A7 was cloned.
  • the SDS-PAGE results of the purified monoclonal antibody 15A7, monoclonal antibody 15A7 Fab fragment and monoclonal antibody F(ab') 2 fragment are shown in Figure 10: wherein the first lane is the protein molecular weight marker; the second lane is the reduction Monoclonal antibody 15A7 treated with ⁇ -mercaptoethanol in a boiling water bath for 10 minutes; Lane 3 is a monoclonal antibody 15A7 Fab fragment treated with a reducing agent ⁇ -mercaptoethanol in a boiling water bath for 10 minutes; the fourth channel is a reducing agent ⁇ Monoclonal antibody 15A7 F(ab') 2 fragment treated with mercaptoethanol in a boiling water bath for 10 minutes; lane 5 is monoclonal antibody 15A7; lane 6 is 15A7 Fab fragment; lane 7 is monoclonal antibody 15A7 F (ab ') 2 fragments.
  • Example 9 Measurement of neutralizing active IC50 of antibody 15A7 and its active fragment
  • the subtype B virus strain HIV NL4 -3 was selected, and the antibody ICA was neutralized by a cell neutralization assay to detect antibody 15A7 and its active fragment.
  • TZM-bl cells were seeded in 96-well cell culture plates at a concentration of 1.5 ⁇ 10 4 cells/well, and were used for detection 12 hours later. Dilute antibody 15A7 and its Fab, F(ab') 2 fragments 2 times at a dilution concentration of ⁇ , dilute 20 gradients, add ⁇ antibody dilution and 50 ⁇ HIV virus suspension to each well of 96-well U-bottom plate (diluted) Incubate to 100 TCID50), 37" € for 1 hour.
  • Xm is the maximum dose of l g ; I is l g (maximum dose / adjacent dose); P is the sum of positive reaction rates; Pm is the maximum positive reaction rate; Pn is the minimum positive reaction rate.
  • HIVMJ4 0.071 NT NT
  • Example 10 Flow cytometry of monoclonal antibody 15A7-labeled CD4+ cells
  • the CD4-expressing cell line used in this experiment Tzm-bl (Cat. No. 8129 National Institutes of Health AIDS Research and Reference Reagent Program; U87.CD4.CXCR4 (Cat. No. 4036 National Institutes of Health AIDS Research and Reference Reagent Program); U87.CD4.CCR5 (Cat. No. 4035 National Institutes of Health AIDS Research and Reference Reagent Program); MT4 (Cat.No. 120 National Institutes of Health AIDS Research and Reference Reagent Program); H9 (Cat. No. 87 National Institutes of Health AIDS Research and Reference Reagent Program).
  • the above 5 forest cells can stably express CD4, wherein Tzm-bl U87.CD4.CXCR4, U87.CD4.CCR5 3 strains are adherent cells, and MT4 and H9 are suspension cells.
  • PBMC peripheral blood mononuclear cells
  • the stratification is formed in the centrifuge tube, the uppermost is plasma, and the PBMC is separated between the plasma layer and the lymphocyte separation solution, and the uppermost layer of plasma is aspirated, and the PBMC at the interface of the plasma layer and the lymphocyte separation solution is collected to a 50 mL centrifuge tube.
  • Cell viability (should be >95%) was measured by 1% trypan blue staining and cells were counted and cultured in AIM-V Medium (Cat. No. 12055 Invitrogen) medium.
  • Antibodies used in the experiments FITC-labeled anti-CD4 (Cat. No. 555346, BD Biosciences), PE-labeled CXCR4 (Cat. No. 555974, BD Biosciences), PE-Cy5-labeled CCR5 (Cat. No. 556889, BD Biosciences) All of the above antibodies were diluted with PBS 1:200.
  • Monoclonal antibody 15A7 (diluted 1:1000) was used as a primary antibody, and Anti-Mouse IgG (whole molecule)-FITC (Cat. No. F9006 Sigma) was diluted with PBS 1:200 as a secondary antibody.
  • Tzm-bl U87.CD4.CXCR4, U87.CD4.CCR5 and other 3 adherent cells were prepared by single-cell suspension with 0.25% trypsin.
  • MT4, H9 and PBMC were suspended cells.
  • the tube is blown into a single cell suspension, cell concentration All were adjusted to lxlo mL, and 4 mL of cell suspension was dispensed into 8 tubes, and randomly divided into four groups of B, C, and D.
  • Two tubes in each group one tube for antibody labeling and one tube for negative control.
  • Each tube of cells was mixed with 1.5 mL of PBS, inverted, centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was collected.
  • Labeled antibody Resuspend the cells with a diluted antibody solution at 200 ⁇ and mix. ⁇ , ⁇ , and C groups were added to FITC-anti-CD4, PE-anti-CXCR4, and PE-Cy5-anti-CCR5 antibodies, and incubated at room temperature for 30 minutes in the dark. The mixture was mixed by adding 2 mL of PBS, centrifuged at 500 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was collected and resuspended in 500 ⁇ of PBS. Flow cytometry was used to detect antibody labeling.
  • the group D primary antibody was resuspended in a monoclonal antibody 15A7 solution, incubated at room temperature for 1 hour, mixed with 2 mL of PBS, centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the above operation was repeated once to collect the cells. Resuspend the cells in 200 L secondary anti-Mouse IgG (whole molecule)-FITC solution and mix for 30 minutes at room temperature in the dark. After adding 2 mL of PBS, centrifuged at 500 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was collected and resuspended in 500 ⁇ PBS.
  • FIG. 12 shows the results of flow cytometry detection after Tzm-bl cell labeling;
  • Figure 13 shows the results of flow cytometry detection after MT4 cell labeling;
  • Figure 14 shows the H9 cell labeling
  • the flow cytometry detection results are shown by flow cytometry;
  • Figure 15 is the flow cytometry detection result after U87.CD4.CXCR4 cell labeling;
  • Figure 16 is the flow cytometry detection result after U87.CD4.CCR5 cell labeling;
  • 17 is the result of flow cytometry detection after PBMC cell labeling.
  • the positive rate results after labeling of each cell were counted in Table 11.
  • the flow test results set the negative control value to 1%, and the binding of the 15A7 antibody to the surface receptors of each cell line was significantly correlated with the anti-CD4 antibody. Table 11. Flow detection results of surface receptors on each cell line by monoclonal antibody 15A7
  • Example 11 Monoclonal antibody Flow cytometry detection of binding of 15A7 to HIV-expressing cells
  • 293-FT cells were seeded in 24-well cell culture plates at a concentration of 5 ⁇ 10 4 cells/well, incubated in a 5% carbon dioxide incubator at 37* € for 12 hours, using 0.6 ⁇ ⁇ pNL4-3 plasmid (HIV NIj4 _ 3 infectious clone)
  • the whole gene plasmid was transfected into cells, and the transfection reagent was Sofast (Xiamen Sun Horse Bioengineering Co., Ltd.).
  • Sofast Xiamen Sun Horse Bioengineering Co., Ltd.
  • the medium in the 24-well plate was aspirated, and a single cell suspension was prepared by digesting with 0.25% trypsin, and the cells were transferred to a 4 mL centrifuge tube, one tube per well. Each tube cell suspension was mixed with 1.5 mL of PBS, centrifuged at 500 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was collected.
  • 293FT cells were digested with 0.25% trypsin and prepared as a single cell suspension.
  • the cells were inoculated into a 24-well plate pre-plated with sterile coverslips at a concentration of 5 ⁇ 10 4 cells/well, and then supplemented with 400 ⁇ DMEM complete medium per well (addition of 10% FBS, 2 mM L-glutamine, O. lmM non-essential J ⁇ acid and 1% double antibody) 5% carbon dioxide incubator for 12 hours.
  • the cells were transfected with 0.6 g pNL4-3 plasmid (HIV NIj4 _ 3 infectious clone whole gene plasmid), and the transfection reagent was Sofast (Xiamen Sun Horse Bioengineering Co., Ltd.). For the transfection method, see the reagent guide. Untransfected cells were used as controls. Cell fixation was performed 48 hours after transfection.
  • the PBS was aspirated, and 500 ⁇ M of 0.3% Triton-X100 (100 ⁇ L of Triton-X100) in 100 mL per well was added to each well for 5 minutes at room temperature, and 0.3% Triton-X100 was aspirated. 1 mL of PBS was added to each well, allowed to stand at room temperature for 5 minutes, then aspirated, and washed twice with PBS. Closed: Place a parafilm on the 24-well plate cover. Add 25 ⁇ l of goat serum to each well of the 24-well plate. Carefully remove the coverslip from the 24-well plate with the eyelids and remove the cover with absorbent paper. Remove excess liquid from the slide and place the coverslip in the droplets of the goat's serum to ensure that the cells are facing down. Place the 24-well plate cover with the coverslip in a wet box (keep saturated humidity) and incubate for 1 hour at room temperature.
  • Incubation of the secondary antibody Carefully remove the coverslip, flip the coverslip so that the cells are facing up, place them in a 24-well plate, and add 1 mL of PBS for 5 minutes at room temperature. The PBS was then aspirated and washed twice with PBS. Take a new sealing film over the 24-well plate cover and add 25 ⁇ of secondary antibody to the position of the well on the 24-well plate.
  • the secondary antibody is anti-Mouse IgG (whole molecule)-FITC (Cat. No. F9006, Sigma) diluted 1:200 in PBS. Place the coverslip in the droplet of the secondary antibody in the same way as the closure, ensuring that the cells are facing down. Place a 24-well plate with a coverslip on a wet box (saturated humidity) and incubate at room temperature for 30 minutes in the dark.
  • DAPI staining Carefully remove the coverslip, flip the coverslip so that the cells are facing up, place them in a 24-well plate, and add 1 mL of PBS for 5 minutes at room temperature. The PBS was then aspirated and washed twice with PBS. Take a new sealing film and cover it on the 24-well plate cover. According to the position of the hole on the 24-well plate, add 25 ⁇ DAPI diluted with PBS (diluted 1:2000), and cover the glass according to the same operation. Place the piece in the DAPI droplets, making sure the cells are facing down. Place the 24-well plate cover with the cover slip in the wet box (maintaining saturated humidity) Incubate for 5 minutes at room temperature in the dark.
  • the primary purified CD4(dl), CD4(dl-2), CD4(d2), CD4(d3), CD4(d4) were diluted by gradient and mixed with 100 TCID50 of HIV NL4 _ 3 after 37* € After incubating for 1 hour, the above mixture was added to pre-plated Tzm-bl cells, incubated in a 5% carbon dioxide incubator 37 for 48 hours, developed after color development, and counted with Elispot, and the neutralization efficiency was calculated. As a result, as shown in Fig.
  • CD4 (dl) has an IC50 of 0.143 g/mL
  • CD4 (dl- 2) The IC50 is 0.25 ( ⁇ g/mL
  • the IC50 of sCD4 is 1.815 g/mL.
  • TZM-bl cells were seeded in 24-well cell culture plates at a concentration of 1 x 10 cells/well, and used for experiments 12 hours later.
  • Virus was added to the 24-well plate (diluted to 100 TCID50) and the control was replaced with DMEM medium. After incubation for 2 hours, the supernatant containing the virus was discarded and washed 3 times with PBS. A dilution of monoclonal antibody 15A7 was added and incubated for 1 hour at 37*.
  • TZM-bl cells were seeded in 96-well cell culture plates at a concentration of 1.5 ⁇ 10 4 /well, and incubated in a 5% carbon dioxide incubator for 12 hours for detection.
  • the medium in the 96-well plate was aspirated, and added to ⁇ phenol red-free DMEM medium, and incubated in a 5% carbon dioxide incubator at 37* € for 2 hours, according to the Cell Counting Kit-8 kit (Biyuntian Biotechnology Research Institute) instructions.
  • the wells were incubated with ⁇ CCK-8, 5% carbon dioxide incubator, and after 20, 40, and 60 minutes of incubation, the plates were read with a microplate reader and the absorbance was measured.
  • HIV subtype strain HIV virus NL4 _ 3 was selected for cell infection and Tzm-bl cells were infected by incubation with monoclonal antibody 15A7 and truncated CD4 polypeptide. happening. Tzm-bl cells were plated at 1.5 x 10 4 /well in 96-well plates and used for neutralization experiments 12 hours later. The monoclonal antibody 15A7 was diluted according to a dilution gradient of 100 g/mL, and 20 gradients were diluted. The corresponding CD4 (dl), CD4 (dl-2), sCD4 polypeptide were added to the diluted medium, and the control was PBS.
  • the experiment used sCD4 polypeptide at a concentration of 25 pM, and the volume of the dilution was ⁇ well.
  • PBS was used instead of 15A7 antibody to dilute the antibody, and the corresponding CD4(dl), CD4(dl-2) sCD4 polypeptide was also added to the diluted medium to verify that the sCD4 polypeptide itself blocked HIV infection of Tzm-bl.
  • the amount of HIV used was 100 TCID50/well and the volume was 50 ⁇ well, mixed with the above dilution at 37.
  • the cells were incubated for 1 hour in a C incubator, and the mixture was added to pre-plated Tzm-bl cells, and then incubated in a 37 5% carbon dioxide incubator for 48 hours, then fixedly permeabilized and developed. Count and calculate the neutralization efficiency with Elispot.
  • the experimental results are shown in Figure 23: From the experimental results, it can be seen that CD4(dl), CD4(dl-2), sCD4 polypeptide have a significant effect on the blocking of HIV-infected TZM-bl by monoclonal antibody 15A7 ( ⁇ 0 ⁇ 01) ).
  • 293-FT cells were seeded in 24-well cell culture plates at a concentration of 5 ⁇ 10 4 cells/well, incubated in a 5% carbon dioxide incubator at 37* € for 12 hours, using 0.6 ⁇ ⁇ pNL4-3 plasmid (HIV NIj4 _ 3 infectious clone)
  • the whole gene plasmid was transfected into cells, and the transfection reagent was Sofast (Xiamen Sun Horse Bioengineering Co., Ltd.).
  • Sofast Xiamen Sun Horse Bioengineering Co., Ltd.
  • the medium in the 24-well plate was aspirated, and a single cell suspension was prepared by digesting with 0.25% trypsin, and the cells were transferred to a 4 mL centrifuge tube, one tube per well. Each tube cell suspension was mixed with 1.5 mL of PBS, centrifuged at 500 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was collected. The cells were resuspended in 200 ⁇ M PBS, and CD4 (dl), CD4 (dl-2), and sCD4 proteins were added at a final concentration of 0.5 ⁇ M; the control group was replaced with PBS for protein solution and incubated for 1 hour at room temperature.
  • FIG. 24 A panel is the result of 293FT cells and 293FT-HIV+ cells incubated with CD4 (dl), CD4 (dl-2), and sCD4 proteins by monoclonal antibody antibody 15A7 as the first antibody;
  • the figure is the result of anti-Human CD4 (Clone Q4120) as 293FT cells and 293FT-HIV+ cells incubated with CD4 (dl), CD4 (dl-2), and sCD4 proteins, respectively.
  • the positive rate results are summarized in Table 15.
  • p24 expression is expressed in 293FT-HIV+ cells, and the positive rate is 7.2%, indicating that pNL4-3 plasmid is transfected into cells and expressed in cells, but since p24 protein is mainly present in cells, there is no In the case of transfusion, the positive rate of detection was low; the positive rate of the labeled antibody anti-Human CD4 (Clone Q4120) labeled cells showed that CD4 (dl), CD4 (dl-2) and sCD4 were bound to 293FT-HIV+ cells.
  • the CD4 (dl-2) and sCD4 proteins purified in Example 2 were labeled with biotin, and the specific operation was as follows: The above protein was diluted with PBS to a final concentration of 10 ⁇ M in 10 mL, and biotin (4 mM) was added to EZ-Link. Sulfo-NHS-LC-Biotin, Cat.No. 21335, Thermo Scientific), mix and incubate for 2 hours at room temperature, then load into a dialysis bag, place in 1L PBS, dialysis under stirring for 4", and replace dialysis for 3 hours. The liquid was repeated 4 times.
  • CD4(dl-2) 1.18X10 -9 4.22x10 s S.OOxlO- 4 sCD4 1.20X10 -9 3.37x10 s 4.04xl0 4
  • Example 20 Expression of point-mutated CD4(dl-2) polypeptide
  • the recombinant CD4 gene polypeptide recombinant expression clone pTO-T7-CD4(dl-2) was subjected to site-directed mutagenesis, and the target site amino acid was mutated to alanine.
  • the primer sequence is shown in Table 17.
  • the PCR reaction was carried out using a PCR thermocycler (Biometra T3) under the following conditions: 94 X: lOmin; followed by 94 X: 2 min, 56 * € 50 s, 72 X: 7 cycles of 17 cycles, and finally 72* € extension for 10 min.
  • the endonuclease Dpn I was added to the template solution at 37* € for more than 3 hours, and the ER2566 E.
  • coli was used as an expression strain, which was expressed by a conventional method, and the expressed protein was in the form of insoluble inclusion bodies.
  • the inclusion bodies were purified by a conventional method, and the target protein was mainly dissolved in 8 M urea.
  • the protein dissolved in 8 M urea was gradually dialyzed into 1 X PBS to complete renaturation, and the precipitate was removed by centrifugation at 12,000 rpm for 10 minutes to obtain a preliminary purified site-directed mutant protein CD4(dl-2)-F43A, CD4(dl-2). -K46A, CD4(dl-2)-R59A, CD4(dl-2)-N73A.
  • Example 21 Indirect ELISA for detection of reactivity of monoclonal antibody 15A7 with point mutation CD4 (dl-2) polypeptide
  • CD4(dl-2)-F43A, CD4(dl-2)-K46A, CD4(dl-2)-R59A, CD4(dl-2)-N73A and CD4(dl-d2) proteins were used as 1 x PBS
  • the coating buffer was diluted to a final concentration of 0.5 g/mL, and added to a 96-well polyethylene microtiter plate for 2 hours according to the ⁇ well, and then placed at 4 overnight.
  • CD4(dl-2)-F43A 0.962 0.514 0.295 0.191 0.111 0.064 0.043 0.03 0.029 0.016 0.041
  • CD4(dl-2)-K46A 0.265 0.128 0.157 0.165 0.100 0.087 0.089 0.056 0.053 0.160 0.095 0
  • CD4(dl-2)-R59A 0.696 0.581 0.348 0.198 0.125 0.067 0.050 0.044 0.027 0.022 0.027 0
  • CD4(dl-2)-N73A 1.658 0.539 0.392 0.24 0.121 0.087 0.139 0.094 0.073 0.082 0.052 0
  • Example 22 Western blot hybridization assay for the reactivity of monoclonal antibody 15A7 with point mutation CD4 (dl-2) polypeptide
  • CD4(dl-2)-F43A, CD4(dl-2)-K46A, CD4(dl-2)-R59A, CD4(dl-2)-N73A and CD4(dl-d2) proteins Non-reducing samples (excluding DTT) were separated by 13.5% SDS-PAGE electrophoresis, electrotransferred onto nitrocellulose membrane, and 5% incubated with defatted milk at room temperature for 1.5 hours; the membrane after blocking was added with primary antibody 15A7 ( 1: 200 diluted monoclonal antibody in skim milk), 4 reaction overnight; TNT ( 8.765g NaCl, 1.21g Tris Base and 0.5ml Tween-20, add deionized water to 1L, adjust the pH to 8.0) three times, each 5 minute intervals; AP-labeled goat anti-mouse (GAM H+L- AP) diluted 1:5000 in 5% skim milk as enzyme-labeled secondary antibody (Protos), reacted at room temperature for
  • CD4(dl-2)-F43A 0.962 0.514 0.295 0.191 0.111 0.064 0.043 0.03 0.029 0.016 0.041
  • CD4(dl-2)-K46A 0.265 0.128 0.157 0.165 0.100 0.087 0.089 0.056 0.053 0.160 0.095
  • CD4(dl-2)-R59A 0.696 0.581 0.348 0.198 0.125 0.067 0.050 0.044 0.027 0.022 0.027
  • CD4(dl-2)-N73A 1.658 0.539 0.392 0.24 0.121 0.087 0.139 0.094 0.073 0.082 0.052
  • CD4(dl-2) 1.885 1.382 1.246 0.986 0.776 0.624 0.511 0.262 0.25 0.266 0.165

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Abstract

本发明提供了抗CD4蛋白的单克隆抗体及其活性片段及用途，本发明的抗体可阻断人免疫缺陷病毒通过与人CD4蛋白结合侵入细胞。本发明的抗体可用于探测、诊断、预防和治疗以CD4细胞为目标细胞的疾病，特别是人免疫缺陷病毒（HIV）病毒引起的艾滋病。本发明还提供了相关的杂交瘤细胞株,分离的核酸分子和短肽，以及包含该单克隆抗体的药物组合物和试剂盒。

Description

抗 CD4蛋白的单克隆抗体及其活性片段及用途 发明领域
本发明涉及可特异性结合人表面抗原分化簇 4 ( Cluster of Differentiation 4, CD4 )蛋白的单克隆抗体， 及其保守性变体或活 性片段， 其多肽或多肽类似物的相关编码序列， 产生所述单克隆抗 体的细胞株， 及应用该抗体或片段用于预防、 免疫治疗和诊断的方 法和用途； 预防或治疗灵长类， 包括人类在内的， 感染源以 CD4+ 细胞的主要目标细胞引起的疾病： 这些疾病包括获得性免疫缺陷综 合症 （艾滋病， AIDS ) , 艾滋病相关的疾病， 人类免疫缺陷病毒 ( human immunodeficiency virus, HIV )感染。 背景技术
上世纪发现 HIV以来，许多研究者致力于 HIV的病毒分子生物 学、 免疫、 疫苗等方面的研究， 但对于 HIV疫苗及其免疫疗法的发 展仍存在许多的困难， 包括 HIV-1的变异性， 病毒的多种传播途径 / 方式， 以及有关预防 HIV感染所必需的免疫反应仍有分歧等。
HIV-1病毒是约 100纳米的包膜病毒。其 RNA基因组及一些活 性蛋白包裹在由衣壳蛋白组成的雉形核心中， 病毒膜的内部为衣壳 蛋白， 包膜位于病毒颗粒的最外层， 其主体为磷脂双分子层， 嵌有 由 env基因编码的 gpl20和 gp41组成的刺突状（spike )结构。 HIV 包膜刺突状结构与细胞第一受体 CD4蛋白接触后发生构象变化， 然 后与第二受体趋化因子受体蛋白（ chemokine receptors,包括 CCR5、 CXCR4等）作用， 最终实现膜融合， 释放病毒的基因侵入细胞。
HIV病毒包膜糖蛋白是以 gpl60的前体形式在细胞中表达出来 的， 成熟的包膜糖蛋白被剪切为约 481个^ ^酸的 gpl20糖蛋白和 约 345 个氨基酸的 gp41 糖蛋白 （ Ratner, L.等人， Nature, 1985,313:277-284 ) 。 gpl20和 gp41在 HIV包膜上以三聚体的功能 单位形式存在， 参与病毒与细胞受体的结合及病毒与细胞融合的感 染过程（ Kwong, P.D.等人， Nature, 2002,420:678-682; Diskin, R.等人， Nat Struct Mol Biol, 2010,17:608-613; Buzon, V·等人， PLoS Pathog, 2010, 6(5):el000880. ) 。
HIV-1包膜糖蛋白 gpl20通过与细胞受体 CD4蛋白（ Maddon, F.i.等人， Cell, 1986,47:333-348 ) 以及与共受体（co-receptor, 主要 为 CCR5 或 CXCR4蛋白）的相互作用感染宿主细胞 ( Alkhatib, G. 等人， Science , 1996,272:1955-1958; Deng, Η·等人， Nature , 1996,381:661-666; Dragic, Τ·等人， Nature, 1996,381: 667-673; Feng, Y.等人， 5W^I ， 1996,272:872 - 877 ) 。 与细胞受体的结合引起病毒 包膜糖蛋白 gpl20 构象改变， 进而诱导与包膜糖蛋白的跨膜单位 gp41结构重排 ( rearrangements )使病毒与细胞融合 ( Wyatt, R.等 人， Science, 1998,280:1884 - 1888; Pierson, T.C.等人， Curr Top Microbiol Immunol, 2003,281:1 - 27 ) 。
CD4全称表面抗原分化簇 4 ( Cluster of Differentiation 4 ) , 是 表达在包括辅助 Τ细胞、 调节 Τ细胞、 单核细胞、 巨噬细胞和树突 细胞等细胞表面的糖蛋白， 是辅助 Τ 细胞的表面标记 （surface markers )之一， 是其行使其功能的重要受体， 参与调节 T免疫细胞 的增殖、 淋巴因子的释放以及参与免疫细胞的相互作用， 调节抗体 的产生。 同时是许多病原体（包括人免疫缺陷病毒等）感染 CD4+ 细胞的受体糖蛋白 （ Isobe, Μ·等人， Proc Natl Acad Sci USA, 1986;83(12):4399-402； Ansari-Lari, M.A.等人， Genome Res. 1996;6(4):314-26 ； Bofill, M. 等 人 ， Clin Exp Immunol. 1992;88(2):243-52 ) 。 与很多其他细胞表面受体 /标记分子相似， CD4蛋白也属于免疫 球蛋白超家族 ( Immunoglobulin superfamily )的成员。 完整的 CD4 是由 CD4 基因编码的 433 个氨基酸膜糖蛋白， 其分子量约为 55kDa-62kDa,包括由 1-375位氨基酸组成的 4个细胞膜外部结构域
( domain ) , 376-395位氨基酸构成的跨膜区和 396-433位氨基酸构 成的细胞质内尾部（ cytoplasmic tail )( Maddon P. J.等人， Cell, 1985, 42: 93-104; Littman, D.R.^- , Cell, 55: 541 )。 其中细胞外的 4个 结构域分别为第一结构域（domain 1 , 简称 D1 , 包括第 1-100位氨 基酸）， 第二结构域（domain 2, 简称 D2, 包括第 101-180位氨基 酸）， 第三结构域（domain 3， 简称 D3, 包括第 181-290位氨基酸） 和第四结构域（domain 4, 简称 D4, 包括第 291-375位氨基酸）
( Maddon, P.J.等人， Cell, 1985;42:93-104; Maddon, P.J.等人， Cell, 1986;47:333-348; Maddon, P.J.等人， Cell, 1988;54:865-874; Wang, 等人， Nature, 1990, 348: 411-418 ) ； 其中 Dl和 D3在结构上 类似于免疫球蛋白可变区结构域 ( immunoglobulin variable domain, IgV domain ) , D2和 D4在结构上类似于免疫球蛋白恒定区结构域
( immunoglobulin constant domains » IgC domain ) 。
作为 HIV感染细胞的受体蛋白， CD4蛋白在艾滋病毒的复制周 期中发挥重要的作用 （Dalgleish, A. G.等人， Nature, 1984;312:763
- 767; Klatzmann, D.等人, Science, 1984; 225:59 - 63； Klatzmann, Ό.等人, Nature, 1984, 312: 767 - 768 ) 0 CD4的第一个结构域（ Dl ) 中的 CDR3 样区 （CDR3-like region )和 HIV-1包膜糖蛋白 gpl20 的第二免疫球蛋白样互补决定区（ CDR2-like region )直接参与了相 互作用（ Arthos, J.等人， Cell, 1989,57:469 - 481； Clayton, L. Κ·等人， Nature, 1989， 339:548 - 551； Mizukami, Τ·等人， Proc Natl Acad Sci U S A. 1988,85:9273-9277； Batinic, D.等人， J Biol Chem. 1992， 267:6664-6671； Camerini, D.等人， Cell. 1990,60:747-754； Kalyanaraman, V. S.等人， J Immunol. 1990,145:4072-4078; Lifson, J. D.等人， Science. 1988,241:712-716； Truneh, Α·等人， J Biol Chem. 1991,266:5942-5948; Diskin, R.等人， Nat Struct Mol Biol. 2010,17:608-613 ) , 两个蛋白分子的相互作用引起 CD4-gpl20复合 物发生构象变化， 使 gpl20蛋白可以进一步与细胞第二受体， 即趋 化因子受体 5 ( chemokine receptor-5, CCR5 )或 CX趋化因子受体 4 ( CX chemokine receptor-4, CXCR4 )结合。 与细胞第二受体的结 合引发糖蛋白三聚体构象改变， 使 HIV表面的 gp41蛋白插入细胞 膜， 最终导致病毒包膜和细胞膜的融合（ Kuritzkes, D.R., Curr Ορίη HIV AIDS. 2009,4:82-87； Briz, V·等人， J Antimicrob Chemother. 2006,57:619-627； Castagna, Α·等人， Drugs. 2005,65:879-904 ) 。
CD4+细胞被 HIV感染后，细胞功能和细胞的数量都受到明显的 影响， 其中 T细胞数量的减少一方面是由于病毒的裂解型感染， 同 时也与 HIV 感染后的 CD4+细胞发生与其他感染的或未感染的 CD4++细胞的融合现象有关（ Sodroski, J.等人， Nature, 1986, 322: 470-474 ) 。 CD4+细胞耗尽， 会导致患者机体表现为免疫抑制， 从而 越来越容易受到机会性感染和发生恶性肿瘤。 这种免疫抑制现象在 后天免疫缺损综合症（艾滋病， AIDS ) 患者中广泛存在。 通常艾滋 病临床表现为与艾滋病毒相关的复合症状特点， 如持续性全身淋巴 结肿大， 发烧和体重下降等的综合征。 在某些情况下， 由于艾滋病 毒感染 CD4+脑细胞使艾滋病伴随有中枢神经系统紊乱（ Popovic, M. 等人， Sdence, 1984, 224: 497-500 ) 。 人类免疫缺陷病毒的攻击对 象是 CD4+细胞， 在病毒引起艾滋病的过程中， 患者体内 CD4+细胞 的数量与病情的发展有着密切的关系， 所以 CD4+细胞数量的检测对 艾滋病治疗效果的判断和对患者免疫功能的判断有重要作用。 由于抗 CD4抗体具有通过干扰 HIV与 CD4分子和 /或第二受体 相互作用进而影响 HIV侵入受体细胞， 发挥预防和 /或治疗 HIV感 染的作用， 一些研究者正在研究抗 CD4抗体阻断艾滋病毒与细胞相 互作用的关系和应用。 目前^ I道的在阻断艾滋病毒感染 CD4+受体细 胞中作用明显， 同时具有应用于 HIV感染引发的艾滋病方面具有良 好前景的抗 CD4抗体如 5A8和 DB-81 , 其识别的表位均位于 CD4 蛋白的 D2 结构域 （Song, R.等人， J Virol. 2010， 84:6935-6942; Burastero, S.E.等人， J T醒 si Med. 2009 Nov 28;7:101 ) 。
已经报道了关于抗 CD4 其他表位或结构域的单克隆抗体的研 究， 如可用于外周血 CD4抗原检测的单克隆抗体等特定领域的单克 隆抗体， 但相关报告中也报道了这些抗体在艾滋病治疗上的不足之 处。
例如， 与 CD4分子 D1结构域结合的抗体 Leu3A和 OKT4A具 有阻断感染艾滋病毒引起的细胞合胞体形成现象的作用 （ Sattentau Q.J.等人， Science, 1986,234:1120-1123; Jameson Β·Α·等人， Science. 1988,240:1335-1339; Peterson Α·等人， Cell. 1988,54:65-72 )。 但是由 于这些抗体的识别表位与 HIV包膜糖蛋白 gpl20识别的表位重叠， 这些抗体无法与结合了包膜糖蛋白 gpl20的 CD4蛋白分子反应， 因 此这些抗体作为艾滋病毒感染的治疗药物使用方面存在严重缺陷 ( Bates Ρ·Α·等人， Protein Eng. 1989,3:13-21; McDougal J.S.等人， J Immunol. 1986,137:2937-2944; Landau N.R. 等 人 ， Nature. 1988,334:159-162; Dalgleish A.G.等人， Lancet. 1987,2:1047-1050 ) , 同时在机体内也会由于机体的免疫抑制而存在应用缺陷 （ Samuele Ε·Β·等人， J Transl Med. 2009,7:101 ) 。
据报道， 识别 CD4分子 D2结构域的单克隆抗体 OKT4B ( T. Kieber-Emmons Τ·等人， Biochim Biophys Acta. 1989， 989:281-300 ) 对于 HIV包膜糖蛋白 gpl20与 CD4蛋白的结合具有明显的干扰作用 ( McDougal J.S.等人， J. Immunol., J Immunol. 1986， 137:2937-2944;
Lundin Κ·等人， J Immunol Methods. 1987,97:93-100; Lamarre D.等 人， EMBO J.， 1989,8:3271-3277 ) ,但是其阻止 HIV感染的细胞形成 合胞体现象的作用明显比 OKT4A 弱， 在治疗中的弱点更加突出 ( Sattentau Q.J.等人， Science. 1986,234:1120-1123; Moore J.P.等人，
Science. 1990,250:1139-42 ) 。
其他表位的抗 CD4抗体还包括 MT151, VIT4, 和 MT321等 ( Sattentau Q.J.等人， Science. 1986， 234:1120-1123 ) , 这些抗体识 别的表位与 HIV包膜糖蛋白 gpl20结合的表位在构象上有一定的重 叠（ Sattentau Q.J.等人， J Exp Med, 1989， 170:1319-1334; Bates P.A. 等人， Protein Eng. 1989,3:13-21; Landau N.R.等人， Nature.
1988,334:159-162; Merkenschlager M. 等 人 ， J Immunol.
1990,145:2839-2845; Benkirane M. 等人， J Virol. 1995,69:
6898-6903 ) 。
本发明的抗体识别 CD4蛋白 Dl ( Dl-2 )结构域上的表位， 当 有 HIV存在 /CD4蛋白与 HIV包膜糖蛋白 gpl20结合后， 该表位可 与本发明之单克隆抗体表现出更好的反应活性， 显著阻断 HIV侵入 细胞， 使病毒不能在细胞中复制， 阻断由于 HIV感染引起的细胞病 变， 从而阻止病毒在细胞间的传播。 这样的抗体在阻断 HIV感染及 艾滋病等相关疾病治疗新方式方面具有应用价值。 发明内容
本发明提供了可特异性结合人表面抗原分化簇 4 ( Cluster of Differentiation 4, CD4 )蛋白的单克隆抗体， 即可阻断人类免疫缺 陷病毒 ( human immunodeficiency virus » HIV )侵入 CD4+细胞， 抑制 HIV感染、 阻断 HIV在 CD4+间传播， 治疗艾滋病活性的单克 隆抗体。
本发明的单克隆抗体可与人表面抗原分化簇 4蛋白的第一结构 域（D1 , 第 1-113位氨基酸）上的表位结合， 也可与包含此结构域 的第 1-2结构域（D1-2, 第 1-180位氨基酸）结合， 以及与包含第一 结构域的其他形式的截短的人表面抗原分化簇 4蛋白、 重组表达的 CD4、 细胞表面的 CD4蛋白结合。 同时本发明之抗体对人类免疫缺 陷病毒 gpl20与 CD4蛋白的相互作用无明显阻断作用， 本发明之单 克隆抗体可阻断 HIV病毒感染细胞，阻断 HIV病毒在受体细胞中的 复制。
另外， 本发明提供了抗 CD4抗体的多种同源形式： 单克隆抗体、 免疫球蛋白的片段（如至少是免疫球蛋白分子的一个免疫活性区 段） ， 如 Fab、 Fab\ F(ab')2、 Fv片段、 单链抗体分子或由含有一 个或多个 CDR 区的免疫球蛋白分子的任意片段形成的多特异性抗 体。 另外， 本发明还提供了重组抗体、 重组嵌合抗体、 重组人源化 抗体等由人免疫球蛋白中的一个或多个 CDR 区结合一个或多个不 同的人免疫球蛋白的构架区形成的抗体。以及本发明抗体的 CDR功 能区域的模拟物质。
本发明提供了杂交瘤细胞株，分离的核酸分子和短肽， 以及包含 本发明的单克隆抗体的药物组合物和医药诊断设备和试剂盒。 本发 明也提供了利用本发明的单克隆抗体探测、 诊断、 预防和治疗哺乳 动物，包括人类在内的，感染源以 CD4+细胞为主要目标细胞的疾病： 这些疾病包括获得性免疫缺陷综合症（艾滋病， AIDS ) ， 艾滋病相 关的疾病,人类免疫缺陷病"^ human immunodeficiency virus, HIV ) 感染的疾病。
在一个方面， 本发明提供了一种单克隆抗体或其抗原结合部分， 其具有至少一项选自以下的特征：
(i) 特异性结合人表面抗原分化簇 4蛋白；
(ii) 与含有人表面抗原分化簇 4蛋白 D1结构域的截短形式的人 表面抗原分化簇 4多肽特异性反应；
(iii)对人免疫缺陷病毒包膜糖蛋白 gpl20与 面抗原分化簇 4 蛋白的反应无明显影响； 和
(iv) 阻断人免疫缺陷病毒感染 CD4+细胞及再感染， 和 /或阻断由 此引起的疾病， 例如艾滋病。
在一个实施方式中， 本发明提供了单克隆抗体或其抗原结合部 分， 其中所述抗体的重链的 CDR1、 CDR2和 CDR3和其中所述抗 体的轻链的 CDR1、CDR2和 CDR3分别是 CCTCC保藏号 C201098 的杂交瘤产生的抗体 15A7的重链 CDR1、 CDR2和 CDR3和轻链 CDR1、 CDR2和 CDR3。
在一个实施方式中， 本发明提供了单克隆抗体或其抗原结合部 分， 其中所述抗体的重链的 CDR1、 CDR2和 CDR3和其中所述抗 体的轻链的 CDR1、 CDR2和 CDR3分别是杂交瘤产生的抗体 14G7 的重链 CDR1、 CDR2和 CDR3和轻链 CDR1、 CDR2和 CDR3。
在一个实施方式中， 本发明提供了单克隆抗体或其抗原结合部 分， 其中所述抗体的重链可变区和轻链可变区分别是 CCTCC保藏 号 C201098的杂交瘤产生的抗体 15A7的重链可变区和轻链可变区。
在一个实施方式中， 本发明提供了单克隆抗体或其抗原结合部 分， 其中所述抗体的重链可变区和轻链可变区分别是杂交瘤产生的 抗体 14G7的重链可变区和轻链可变区。
在一个实施方式中，本发明提供了特异性结合人表面抗原分化簇 CD4蛋白的单克隆抗体或其抗原结合部分， 其中所述抗体的重链可 变区的氨基酸序列如 SEQ ID NO:2所示或由 SEQ ID ΝΟ:1的核苷酸 序列编码； 其中所述抗体的轻链可变区的氨基酸序列如 SEQ ID NO:4所示或由 SEQ ID NO:3的核苷酸序列编码。
在一个实施方式中，本发明提供了特异性结合人表面抗原分化簇 CD4蛋白的单克隆抗体或其抗原结合部分， 其中所述抗体的重链可 变区的氨基酸序列如 SEQ ID NO:18所示或由 SEQ ID NO:17的核苷 酸序列编码； 其中所述抗体的轻链可变区的氨基酸序列如 SEQ ID NO:20所示或由 SEQ ID NO:19的核苷酸序列编码。
在一个实施方式中， 本发明的抗体包含一个或多个选自 SEQ ID NOs: 5-7的重链 CDR;和 /或包含一个或多个选自 SEQ ID NOs: 8-10 的轻链 CDR;
在一个实施方式中， 本发明所述抗体的重链 CDR1、 CDR2 和 CDR3的^^ 列分别为 SEQ ID NO:5-7或由 SEQ ID ΝΟ:11-13 的核苷酸序列中一个或多个编码；
在一个实施方式中， 本发明所述抗体的轻链 CDR1、 CDR2 和 CDR3的 ^^^列分别为 SEQ ID NO:8-10或由 SEQ ID NO:14-16 的核苷酸序列中一个或多个编码；
在一个实施方式中，本发明所述的单克隆抗体或其抗原结合部分 为 Fab、 Fab' 、 F(ab')2 、 Fv或单链抗体。
在一个实施方式中， 本发明的抗体包含一个或多个选自 SEQ ID NOs: 21-23的重链 CDR; 以及包含一个或多个选自 SEQ ID NOs: 24-26的轻链 CDR;
在一个实施方式中， 本发明所述抗体的重链 CDR1、 CDR2 和 CDR3 的氨基酸序列分别为 SEQ ID NO:21-23 或由 SEQ ID NO:27-29的核苷酸序列中一个或多个编码；
在一个实施方式中， 本发明所述抗体的轻链 CDR1、 CDR2 和 CDR3 的氨基酸序列分别为 SEQ ID NO:24-26 或由 SEQ ID NO:30-32的核苷酸序列中一个或多个编码；
在一个实施方式中，本发明所述的单克隆抗体或其抗原结合部分 为 Fab、 Fab' 、 F(ab')2 、 Fv或单链抗体。
在一个实施方式中，本发明所述的单克隆抗体或其抗原结合部分 结合人表面抗原分化簇 4蛋白的 KD值小于 ΙχΙΟ·5 Μ。
在一个实施方式中， 本发明所述的单克隆抗体包括非 CDR区， 该非 CDR区是来自不是鼠类的物种。
在另一个方面，本发明提供了特异性结合人表面抗原分化簇 4蛋 白的单克隆抗体， 其是 CCTCC保藏号 C201098的杂交瘤产生的抗 体 15A7,其相应的杂交瘤细胞株是 CCTCC保藏号 C201098的杂交 在另一个方面， 本发明提供了分离的核酸分子， 其编码本发明所 述的单克隆抗体或其抗原结合部分的重链可变区。 在一个实施方式 中， 所述抗体的重链可变区的氨基酸序列为 SEQ ID NO:2。 在另一 个实施方式中， 所述的核酸分子序列为 SEQ ID NO:l。
在另一个方面， 本发明提供了分离的核酸分子， 其编码本发明所 述的单克隆抗体或其抗原结合部分的轻链可变区。 在一个实施方式 中， 所述抗体的轻链可变区的氨基酸序列为 SEQ ID NO:4。 在另一 个实施方式中， 所述的核酸分子序列为 SEQ ID NO:3。
在另一个方面， 本发明提供了分离的核酸分子， 其编码本发明所 述的单克隆抗体或其抗原结合部分的重链可变区。 在一个实施方式 中， 所述抗体的重链可变区的^ 列为 SEQ ID NO:18。在另一 个实施方式中， 所述的核酸分子序列为 SEQ ID NO:17。
在另一个方面， 本发明提供了分离的核酸分子， 其编码本发明所 述的单克隆抗体或其抗原结合部分的轻链可变区。 在一个实施方式 中， 所述抗体的轻链可变区的^ 列为 SEQ ID NO:20。在另一 个实施方式中， 所述的核酸分子序列为 SEQ ID NO:19。
在另一个方面， 本发明提供了包含本发明的核酸分子的表达载 体。 在另一个方面， 本发明提供了包含本发明的表达载体的宿主细 胞。
在另一个方面，本发明提供了检测样品中人表面抗原分化簇 4蛋 白的方法， 其包括如下步骤：
a )将所述的样品与本发明的单克隆抗体或其抗原结合部分接 触；
b )检测所述的单克隆抗体与所述样品中的蛋白的反应；
c )检测所述的单克隆抗体与所述样品中的蛋白及人免疫缺陷病 毒包膜蛋白的反应。
在一个实施方式中， 所述的单克隆抗体附着在固相载体上。在另 一个实施方式中， 所述的固相载体选自： 微滴定板、 磁颗粒、 胶乳 颗粒和硝化纤维素膜。 在优选的实施方式中， 所述的单克隆抗体是 按如此方向附着在所述固相上， 该方向能够增加所述单克隆抗体与 所述样品的结合效率。 在另一个实施方式中， 所述的单克隆抗体是 通过其恒定区域而附着在所述固相上的。 在一个具体实施方式中， 所述的反应是通过酶显色进行测定的。在另一个具体的实施方式中， 所述的反应是通过荧光进行测定的。 在又一个具体的实施方式中， 所述的反应是通过化学发光进行进行测定的。 在一个优选的实施方 式中， 上述方法中所述的单克隆抗体为 Fab、 Fab' 、 F(ab')2 或 Fv。 在另一个优选的实施方式中， 所述的样品来自于鸟类或人类。
在另一个方面， 本发明提供了药物组合物， 其包含本发明的单克 隆抗体或其抗原结合部分， 以及药学上可接受的载体。 在一个实施 方式中， 本发明的药物组合物进一步包括其它抗病毒成分。 在另一 个实施方式中，本发明的药物组合物中的抗体为 Fab、 Fab' 、 F(ab')2 或 Fvo
在另一个方面，本发明提供了治疗受试者中由人免疫缺陷病毒感 染引发的疾病的方法， 其包括向所述的受试者施用治疗上有效量的 本发明的药物组合物。
在另一个方面，本发明提供了本发明的单克隆抗体用于制备治疗 或预防以 CD4+细胞为目标细胞的疾病 （如艾滋病） 的药物的用途。
在另一个方面，本发明提供了本发明的单克隆抗体用于制备阻断 HIV感染 CD4+细胞及再感染的药物的用途。
在另一个方面，本发明提供了本发明的单克隆抗体用于人表面抗 原分化簇 4蛋白模拟表位肽筛选的用途。
在另一个方面， 本发明提供了上述模拟表位肽用于制备预防以 CD4+为目标细胞的疾病 （如艾滋病） 的疫苗的用途。
在另一个方面，本发明提供了上述模拟表位肽用于制备治疗或诊 断以 CD4+为目标细胞的疾病 （如艾滋病）试剂盒的用途。 附图说明
图 1是人 CD4胞外区氨基^ ψ列， 图中标注 D1结构域， D2结 构域， D3结构域， D4结构域四个结构域的起止位置。
图 2是重组人 CD4蛋白表达纯化的 SDS-PAGE结果,各图依次 为 A: CD4(dl); B: CD4(d2); C: CD4(d3); D: CD4(d4); E: CD4(dl-2); F: sCD4 (即 D1-D4结构域）； 各小图各道样品从左至右依次为： 1: 蛋白分子量 marker; 2: 诱导后表达菌体； 3: 2M尿素上清； 4: 4M 尿素上清； 5: 8M尿素上清； 6: 纯化后样品； 7: BL21空载菌体。
图 3是各稀释度的单克隆抗体 15A7阻断不同亚型 HIV实验株 病毒感染 Tzm-bl细胞的曲线图，实验株病毒包括 HIVNL4-3( B亚型）， HIV89.6( B亚型）， HIV權 G114( D亚型）， HIVMj4( C亚型）， HIVWCML249 ( D/C亚型） 。
图 4是各稀释度的单克隆抗体 14G7阻断不同亚型 HIV实验株 病毒感染 Tzm-bl细胞的曲线图，实验株病毒包括 HIVNL4-3( B亚型）， HIV89.6( B亚型）， HIV G114( D亚型）， HIVMj4( C亚型）， HIVWCML249 ( D/C亚型） 。
图 5是单克隆抗体 15A7与 Tzm-bl细胞， U87.CD4.CXCR4细 胞和 U87.CD4.CCR5细胞结合的免疫荧光的激光共聚焦显微镜检测 结果， 其中使用的对照为 anti-p24的单克隆抗体 H5F4
图 6是单克隆抗体 14G7与 Tzm-bl细胞， U87.CD4.CXCR4细 胞和 U87.CD4.CCR5细胞结合的免疫荧光的激光共聚焦显微镜检测 结果， 其中使用的对照为 anti-p24的单克隆抗体 H5F4
图 7 是重组蛋白 CD4(dl)、 CD4(d2)、 CD4(d3)、 CD4(d4)、 CD4(dl-2)和 sCD4阻断 15A7抗体与细胞反应的流式细胞仪检测结 果。
图 8 是重组蛋白 CD4(dl)、 CD4(d2)、 CD4(d3)、 CD4(d4)、 CD4(dl-2)和 sCD4阻断 14G7抗体与细胞反应的流式细胞仪检测结 果。
图 9是单克隆抗体 15A7和 14G7的单链抗体表达质粒图。
图 10是单抗纯化 SDS-PAGE结果， 各道样品从左至右依次为： 1:蛋白分子量 Marker; 2:单克隆抗体 15A7还原处理样品； 3: 15A7 抗体 Fab片段还原处理样品； 4: 15A7抗体 F(ab')2片段还原； 5: 单克隆抗体 15A7非还原处理样品； 6: 15A7抗体 Fab非还原处理样 品； 7: 15A7抗体 F(ab')2非还原处理样品。
图 11是单克隆抗体 15A7及其 F(ab')2 Fab抗体片段和 HIVNL4-3 病毒反应， 阻断其进入 Tzm-bl细胞的结果， 对照为緩冲液。
图 12是 Tzm-bl细胞表面不同受体的流式细胞仪检测结果， 其 中 A小图是用单克隆抗体 15A7为一抗， FITC标记的兔抗鼠抗体 ( RAM-FITC, Cat.No. F9006 Sigma )为二抗标记待测细胞； B小 图是用 FITC标记的 anti-CD4 ( Cat.No. 555346, BD Biosciences ) 标记待测细胞表面的 CD4 受体蛋白； C 小图是用 PE 标记的 anti-CXCR4 ( Cat.No. 555974, BD Biosciences )标记待测细胞表面 的 CXCR4受体蛋白； D小图是用 PE-Cy5标记的 anti-CCR5( Cat.No. 556889, BD Biosciences )标记待测细胞表面的 CCR5受体蛋白； E 小图为无标记的细胞对照。
图 13是 MT4细胞表面不同受体的流式细胞仪检测结果， 其中 A 小图是用单克隆抗体 15A7 为一抗， FITC 标记的兔抗鼠抗体 ( RAM-FITC, Cat.No. F9006 Sigma )为二抗标记待测细胞； B小 图是用 FITC标记的 anti-CD4单抗（ Cat.No. 555346, BD Biosciences ) 标记待测细胞表面的 CD4 受体蛋白； C 小图是用 PE 标记的 anti-CXCR4单抗（ Cat.No· 555974, BD Biosciences )标记待测细胞 表面的 CXCR4受体蛋白； D小图是用 PE-Cy5标记的 anti-CCR5 单抗（ Cat.No· 556889, BD Biosciences )标记待测细胞表面的 CCR5 受体蛋白； E小图为无标记的细胞对照。
图 14是 H9细胞表面不同受体的流式细胞仪检测结果， 其中 A 小图是用单克隆抗体 15A7 为一抗， FITC 标记的兔抗鼠抗体 ( RAM-FITC, Cat.No. F9006 Sigma )为二抗标记待测细胞； B小 图是用 FITC标记的 anti-CD4单抗（ Cat.No. 555346, BD Biosciences ) 标记待测细胞表面的 CD4 受体蛋白； C 小图是用 PE 标记的 anti-CXCR4单抗（ Cat.No· 555974, BD Biosciences )标记待测细胞 表面的 CXCR4受体蛋白； D小图是用 PE-Cy5标记的 anti-CCR5 单抗（Cat.No· 556889, BD Biosciences )标记待测细胞的 CCR5受 体蛋白； E小图为无标记的细胞对照。 图 15是 U87.CD4.CXCR4细胞表面不同受体的流式细胞仪检测 结果，其中 A小图是用单克隆抗体 15A7为一抗， FITC 标记的兔抗 鼠抗体 ( RAM-FITC, Cat.No. F9006 Sigma )为二抗标记待测细胞； B 小图是用 FITC 标记的 anti-CD4 单抗 （Cat.No. 555346 , BD Biosciences )标记待测细胞表面的 CD4受体蛋白； C小图是用 PE 标记的 anti-CXCR4单抗（ Cat.No· 555974, BD Biosciences )标记待 测细胞表面的 CXCR4 受体蛋白； D 小图是用 PE-Cy5 标记的 anti-CCR5单抗（Cat.No· 556889， BD Biosciences )标记待测细胞 表面的 CCR5受体蛋白； E小图是无标记的细胞对照。
图 16是 U87.CD4.CCR5细胞表面不同受体的流式细胞仪检测结 果，其中 A小图是用单克隆抗体 15A7为一抗， FITC 标记的兔抗鼠 抗体（RAM-FITC, Cat.No. F9006 Sigma )为二抗标记待测细胞； B 小图是用 FITC 标记的 anti-CD4 单抗 （ Cat.No. 555346 , BD Biosciences )标记待测细胞表面的 CD4受体蛋白； C小图是用 PE 标记的 anti-CXCR4单抗（ Cat.No· 555974, BD Biosciences )标记待 测细胞表面的 CXCR4 受体蛋白； D 小图是用 PE-Cy5 标记的 anti-CCR5单抗（Cat.No· 556889， BD Biosciences )标记待测细胞 表面的 CCR5受体蛋白； E小图是无标记的细胞对照。
图 17是人外周血单个核细胞（PBMC )细胞表面不同受体的流 式细胞仪检测结果，其中 A小图是用单克隆抗体 15A7为一抗， FITC 标记的兔抗鼠抗体（RAM-FITC, Cat.No. F9006 Sigma )为二抗标 记待测细胞； B 小图是用 FITC 标记的 anti-CD4 单抗（Cat.No. 555346, BD Biosciences )标记待测细胞表面的 CD4 受体蛋白； C 小图是用 PE 标记的 anti-CXCR4 单抗 （ Cat.No. 555974 , BD Biosciences )标记待测细胞表面的 CXCR4 受体蛋白； D 小图是用 PE-Cy5标记的 anti-CCR5单抗（ Cat.No· 556889, BD Biosciences ) 标记待测细胞表面的 CCR5受体蛋白； E小图是无标记的细胞对照。 图 18是流式细胞仪检测的单克隆抗体 15A7标记表达了 HIV的 293FT细胞的情况， 其中使用的对照细胞为空白的 293FT细胞， 对 照抗体为 anti-p24的单克隆抗体 H5F4。
图 19是单克隆抗体 15A7与表达了 HIV的 293FT细胞的免疫荧 光的激光共聚焦显微镜检测结果， 其中使用的对照细胞为空白的 293FT细胞， 对照抗体为 anti-p24的单克隆抗体 H5F4。
图 20 是各种截短的 CD4 蛋白， 包括重组蛋白 CD4(dl)、 CD4(d2)、 CD4(d3)、 CD4(d4)、 CD4(dl-2)和 sCD4阻断 HIVNL4-3感 染 Tzm-bl细胞的效果。
图 21是纯化的截短 CD4蛋白与单克隆抗体 15A7的免疫印迹反 应结果。 其中 A1小图是截短 CD4蛋白还原 SDS-PAGE图， A2小 图是截短 CD4蛋白还原胶免疫印迹反应图； B1小图是截短 CD4蛋 白非还原胶图， B2小图是截短 CD4蛋白非还原 疫印迹反应。各 小图各道样品依次为 1:蛋白分子量 marker; 2:CD4(dl); 3:CD4(d2); 4:CD4(d3); 5:CD4(d4); 6:CD4(dl-2); 7:sCD4; 8:对照蛋白； 9:BL21 空载菌体。
图 22是用 15A7标记 TZM-bl细胞的流式细胞仪检测结果， 其 中包括加入 HIVNL4_3和不加 HIV NL4-3两种情况。
图 23是重组蛋白 CD4(dl)、 CD4(dl-2)和重组 sCD4阻断 15A7 中和 HIVNIj4_3感染 Tzm-bl细胞的中和实验检测结果。
图 24是用 CD4(dl)、CD4(dl-d2)和重组 sCD4与转染了 pNL4-3 的 293FT细胞反应，用单克隆抗体 15A7作为二抗，再用 FITC标记 的兔抗鼠抗体标记后进行流式细胞仪检测的结果， 其中 A小图为单 克隆抗体 15A7 的反应结果； B 小图为用 anti-CD4单抗（克隆号 Q4120, Cat.No. C1805 Sigma )作为对照标记待测细胞表面 CD4受 体蛋白的反应结果。
图 25是单克隆抗体 15A7与点突变 CD4(dl-2)蛋白的免疫印迹 反应结果。其中 A1小图是各点突变 CD4(dl-2)蛋白还原 SDS-PAGE 图； A2小图是各点突变 CD4(dl-2)蛋白非还原样 SDS-PAGE图； B1 小图是各点突变 CD4(dl-2)蛋白还原样免疫印迹反应杂交图； B2小 图是各点突变 CD4(dl-2)蛋白非还原样免疫印迹反应杂交图。各小图 各道样品顺序依次为 1: 蛋白分子量 marker; 2: CD4(dl-2)-N73A; 3: CD4(dl-2)-R59A; 4: CD4(dl-2)-K46A; 5: CD4(dl-2)-F43A; 6: 对照蛋白 CD4(dl-2)。 具体实施方式
本发明申请中涉及的有关术语的定义如下：
本发明中的 "表面抗原分化簇 4"一词指任何由表面抗原分化簇 4基因编码的表面抗原分化簇 4蛋白。
本发明中的 "CD4+细胞"一词指细胞表面具有 CD4糖蛋白的细 胞， 其中包括 CD4+ T淋巴细胞和 CD4+细胞系， 如 TZM-bl、 H9和 C8166等。
本发明中的 "CD4第一结构域（D1 ) " —词指第 1至第 98位 的截短的表面抗原分化簇 4多肽。
本发明中的 "CD4第二结构域（D2 ) " —词指第 99至第 183 位的截短的表面抗原分化簇 4多肽。
本发明中的 "CD4第一结构域（D3 ) " —词指第 184至第 291 位的截短的表面抗原分化簇 4多肽。
本发明中的 "CD4第一结构域（D4 ) " —词指第 292至第 365 位的截短的表面抗原分化簇 4多肽。
本发明中的 "CD4第一 /二结构域（D1-2 ) "—词指第 1至第 183 位的截短的表面抗原分化簇 4多肽。
本发明中的 "重组可溶性 CD4 ( sCD4, rsCD4, D1-4 ) " 一词 指第 1至第 365位的截短的表面抗原分化簇 4多肽。
本发明中的 "抗 CD4抗体" 一词是指识别位于表面抗原分化簇 4蛋白分子上的表位的抗体，即与表面抗原分化簇 4蛋白上的表位结 合的抗体。
本发明中的 "包膜糖蛋白" 一词指人类免疫缺陷病毒的包膜糖 蛋白。 包膜糖蛋白介导人类免疫缺陷病毒针对宿主细胞的吸附和进 入过程。 其前体形式为 gpl60, 经过加工后为 gpl20和 gp41 , 在人 类免疫缺陷病毒包膜上以聚合体的功能单位形式存在。
本发明中的 "抗体" 一词指任意一种免疫球蛋白， 包括能结合 特异性抗原的单抗、 多抗、 双特异性或多特异性抗体。 一个完整的 抗体包含两条重链和两条轻链。 每条重链含有一个可变区和第一、 第二、 第三等三个恒定区； 每条轻链包含一个可变区和一个恒定区。 抗体呈 "Y" 型， "Y" 型结构的颈部含有两条重链的第二和第三恒 定区， 其通过二硫键结合形成。 "Y" 型结构的每条臂含有其中一 条重链的第一恒定区和可变区， 和一条轻链的可变区和恒定区。 轻 链和重链的可变区决定抗原的结合； 每条链的可变区均含有三个高 变区， 称为互补决定区 （CDR ) (轻链 (L)的 CDR 包含 LCDR1、 LCDR2、 LCDR3,重链 (H)的 CDR色含 HCDR1、 HCDR2、 HCDR3. 其由 Kabat等人命名, 见 Sequences of Proteins of Immunological Interest, Fifth Edition (1991)， 第 1-3卷， NIH Publication 91-3242, Bethesda MD )。 其中， 三个 CDR由构架区（FR )间隔开。 构架区 比 CDR区更保守并形成一个架子状结构支撑超变区。重链和轻链的 恒定区与抗原结合无关， 但具有多种效应功能。 抗体依据重链恒定 区的氨基酸序列可以分成几类，主要是： IgA、 IgD、 IgE、 IgG和 IgM, 其中有些类还进一步分成亚类， 如 IgGl、 IgG2、 IgG3、 IgG4、 IgAl 或 IgA2等。
本发明中的 "抗体" 一词， 除特指完整的免疫球蛋白外， 也指 免疫球蛋白的片段（如至少是免疫球蛋白分子的一个免疫活性区 段） ， 如 Fab、 Fab\ F(ab')2、 Fv片段、 单链抗体分子或由含有一 个或多个 CDR 区的免疫球蛋白分子的任意片段形成的多特异性抗 体。 另外， 本发明涉及的抗体也可以是由一个特定的人免疫球蛋白 中的一个或多个 CDR 区结合一个或多个不同的人免疫球蛋白的构 架区形成的抗体。 在片段蛋白分子中仅包含重链 CDR 中的 1 个 CDR, 2个 CDR, 3个 CDR可实现相同表位的识别作用。 在片段蛋 白分子中仅包含轻链 CDR中的 1个 CDR, 2个 CDR至 3个 CDR 区段同样可实现实现相同表位的识别作用。只保留鼠单抗的 1个或 2 个 CDR区， 其余都是全新的氨基酸序列， 获得的新抗体仍具有亲本 鼠单抗的活性。 1998 年， Rader 等将鼠单抗 LM609 可变区上的 HCDR3 和 LCDR3 移植到人源抗体序列中， 成功获得仅保留鼠 HCDR3和 LCDR3的人源化抗体（ Rader C， Cheresh DA, Barbas CF 3rd.A phage display approach for rapid antibody humanization: designed combinatorial V gene libraries.Proc Natl Acad Sci U S A. 1998, 95(15):8910-5 ) 。 2000年, Klimka等获得只保留鼠 HCDR3 的抗 CD-30的人源化抗体（ Klimka A, Matthey B， Roovers RC， et al, Human anti-CD30 recombinant antibodies by guided phage antibody selection using cell panning. Br J Cancer. 2000， 83(2):252-60 ) 。
抗体相关的 "Fab"片段是指含有一条轻链的可变区和恒定区和 一条重链的可变区和恒定区经二硫键结合起来的抗体分子的一部 分。 "Fab'" 片段是指包含了部分铰链区的 Fab片段。
F(ab')2指的是 Fab'的二聚体。
抗体的 "Fc" 指的是第一重链的第二、 第三恒定区与第二重链 的第二、 第三恒定区经二硫键结合成的抗体的一部分。 抗体的 Fc段 有多种不同的功能， 但不参与抗原的结合。
抗体的 "Fv" 段指的是能结合完整的抗原结合位点的抗体的最 小片段。一个 Fv片段包括一条轻链的可变区结合到一条重链的可变 区。
本发明中的 "单链抗体" 或 "scFv" 指的是由轻链可变区与重 链可变区直接相连或通过一个肽链连接而成的工程抗体（ Houston 1988 )
本发明中的 "单链抗体 Fv-Fc" 或 "scFv-Fc" 也包括 scFv连接 抗体的 Fc段形成的工程抗体。
本发明中的 "抗原决定簇" （或称表位）指的是抗原分子中与 抗体结合的那部分氨基酸或原子基团。
本发明中的 "单抗" 一词指的是来自一群高度同源的抗体分子 中的一个抗体或抗体的一个片段， 也即除仅在少数情况下可能出现 的自然突变外， 一群完全相同的抗体分子。 单抗对抗原上的单一表 位具有高特异性。 单抗与多抗不同， 多抗是包含了识别抗原上的不 同表位的抗体分子。 虽然传统的单抗是由杂交瘤细胞分泌的， 但本 发明涉及的单抗并不仅限于此制备方法。 本发明涉及的单抗可采用 Kohler 等首次报道的杂交瘤技术获得 （ KShler G.等人， Nature, 1975,256:495-497 ) , 也可采用重组 DNA技术获得（如参见 U.S.P 4,816,567 ) 。
本发明中的 "嵌合抗体" 一词指的是抗体轻链或 /和重链的一部 分是源自某一特定物种或属于某一特定抗体类或亚类的序列相同或 同源的抗体， 而抗体轻链或 /和重链的另一部分是源自另一物种或属 于另一抗体类或亚类的序列相同或同源的抗体。 不管怎样， 这种抗 体片段仍保留了对目标抗原的结合活性（ U.S.P 4,816,567; Morrison 等人， Pro Natl. Acad. Sci. USA, 81:6851 6855 (1984) ) 。
本发明中， "人源化抗体" 一词指的是人源免疫球蛋白 （受体 抗体） 的全部或部分 CDR区被一非人源抗体（供体抗体） 的 CDR 区替换后得到的抗体或抗体片段， 其中的供体抗体可以是有预期特 异性、 亲和性和反应性的小鼠、 大鼠或兔抗体。 另外， 人源免疫球 蛋白的构架区 （FR ) 的氨基酸序列也可被相应的非人源抗体的氨基 酸序列所替换。 而且， 人源化抗体的氨基酸残基也可以既非来源于 受体抗体， 也非来源于供体抗体的 CDR区或构架区序列。 这些人工 修饰的目的是进一步完善或优化抗体性能。 总之， 人源化抗体是指 含有至少一个、 通常是两个几乎完整的可变区， 其中对应的所有或 几乎所有的 CDR 区是来自非人源抗体， 其中的全部或几乎全部的 FR 区是来自人源抗体。 理想的人源化抗体至少含有免疫球蛋白的 Fc区的一部分，通常是人源免疫球蛋白的 Fc区。更多详细内容请参 阅： Jones Ρ·Τ·等人， Nature, 1986,321:522-525； Reichmann L.等人， Nature. 1988,332:323-327; Presta, Curr Opin Struct Biol, 1992,2:593-596 and Clark M. Immunol Today 2000,21: 397-402.
本发明涉及的 "被分离" 一词， 指的是天然状态下经人工手段 获得的。 如果自然界中出现某一种 "被分离" 的物质或成分， 那么 可能是其所处的天然环境发生了改变或从天然环境下分离出该物 质， 或二者情况均有发生。 比如， 某一活体动物体内天然存在某种 未被分离的多聚核苷酸或多肽， 而从这种天然状态下分离出来的高 纯度的相同的多聚核苷酸或多肽即称之为被分离。 这里的 "被分离" 不排除混有人工或合成的物质， 也不排除存在不影响物质活性的其 它不纯物质。
本发明中 "载体" 一词指的是， 可将编码某蛋白的多聚核苷酸 插入其中并使蛋白获得表达的一种核酸运载工具。 载体可通过转化、 转导或转染宿主细胞， 使其携带的遗传物质元件在宿主细胞内得以 表达。 举例来说， 载体包括： 质粒； 噬菌粒； 柯斯质粒； 人工染色 体如酵母人工染色体（ YAC )、 细菌人工染色体 ( BAC )或 P1来源 的人工染色体 ( PAC )； 噬菌体如 λ噬菌体或 M13噬菌体及动物病 毒等。 用作载体的动物病毒种类有逆转录病毒（包括慢病毒） 、 腺 病毒、 腺相关病毒、 奢病毒（如单纯疾渗病毒） 、 痘病毒、 杆状 病毒、 乳头瘤病毒、 乳头多瘤空泡病毒（如 SV40 )。 一种载体可能 含有多种控制表达的元件， 包括启动子序列、 转录起始序列、 增强 子序列、 选择元件及报告基因。 另外， 载体还可含有复制起始位点。 载体还有可能包括协助其进入细胞的成分， 如病毒颗粒、 脂质体或 蛋白外壳， 但不仅仅只有这些物质。
本发明中 "宿主细胞" 一词指的是导入载体的细胞， 包括如下 许多细胞类型， 如大肠杆菌或枯草菌等原核细胞， 如酵母细胞或曲 霉菌等真菌细胞， 如 S2果蝇细胞或 Sf 等昆虫细胞， 或者如纤维原 细胞， CHO细胞， COS细胞， NSO细胞， HeLa细胞， BHK细胞， HEK 293细胞或人细胞的动物细胞。
"中和抗体" 一词指的是能清除或显著降低目标病毒抗原结合 毒力的抗体或抗体片段。
"序列相同百分比" 一词指的是候选序列中的核酸或氨基酸分 别与对应的核酸或多肽序列中核酸或氨基酸相同性的百分比。 这里 涉及到的与核酸序列或者多肽序列有关的术语 "序列相似百分比" 定义为候选核酸序列或氨基酸残基序列分别与目的核酸序列或氨基 酸序列的相似百分比。 对于某一个序列， 将它和目的序列进行比对， 必要时可跳过突变缺口， 以达到最大的基因相似百分比， 而不去考 虑相似序列的任意保守性突变。 本领域的多种比对方法可用于确定 核酸或氨基酸序列的相似性， 如可用的计算机软件包括 BLAST, BLAST-2, ALIGN, ALIGN-2 或 Megalign ( DNASTAR )等。 熟悉 本领域技术的工作人员懂得为比对设定合适的测量参数， 包括为使 用最大可比性的一些运算法则达到全长序列的比较。
"特异性结合" 一词指的是， 两分子间的非随机结合反应， 如 抗体和产生该抗体的抗原间的反应。 此处， 结合第一种抗原的抗体 对第二种抗原的结合亲和力是检测不到的或很弱。 在某些实施方式 中， 某抗原特异性抗体是指以亲和力 ( KD ) < 10 5 M (如 10·6 M、 10 7 M, 10 8 M, 10 9 M, 10-1° M等）结合该抗原， 其中 KD指解离 率与结合率的比值（kQff/kQn ) , 其可以采用本领域技术人员熟悉的方 法进行测定。
抗体
本发明所述单抗能够特异性结合表面抗原分化簇 4受体。 本发 明的一个方面涉及能特异性结合表面抗原分化簇 4受体的单抗及其 相应的抗原结合片段。
在一个实施方式中， 本发明所述单抗是由小鼠杂交瘤细胞株 15A7所分泌的。这些单抗的名称以其相应的杂交瘤细胞株进行命名。 也就是，这些单抗 15A7由杂交瘤细胞株 15A7产生，并命名为 15A7。 单抗 15A7能特异性结合表面抗原分化簇 4蛋白。小鼠杂交瘤细胞株 15A7 已在中国典型培养物保藏中心（CCTCC, Wuhan University, Wuhan, China )进行保藏， 保藏号是 CCTCC - C201098 (杂交瘤细
15A7, 保藏时间 2010年 10月 22日） 。
本发明的单抗还包括能阻断单抗 15A7结合表面抗原分化簇 4蛋 白的单抗。 这些单抗结合的表面抗原分化簇 4蛋白的表位可以与单 抗 15A7 所识别的表位相同。 这些单抗所识别的表位也可以和单抗
15A7所识别的表位在空间上有重叠。这样的单抗可以降低单抗 15A7 与表面抗原分化簇 4受体蛋白的结合力至少 30 % , 或至少 40 % , 或 优选至少 50 % , 或更优选至少 60 % , 或更优选至少 70 % , 或更优选 至少 80 % , 或更优选至少 90 % , 或更优选 95 % , 或最优选 99 %。
本发明的单抗不能显著抑制 HIV包膜糖蛋白 gpl20与人表面抗 原分化簇 4蛋白结合。 这些抗体影响 HIV包膜糖蛋白 gpl20与人表 面抗原分化簇 4蛋白（或重组人表面抗原分化簇 4蛋白 sCD4及截短 的 面抗原分化簇 4蛋白）至多 30%, 或更优选 20%, 或最优选 0%。
本发明的单抗与人表面抗原分化簇 4蛋白结合（含细胞表面、 重组表达和截短的人表面抗原分化簇 4蛋白）的结合在 HIV包膜糖 蛋白 gpl20存在的情况下不受影响， 这样的单抗在 HIV包膜糖蛋白 gpl20与表面抗原分化簇 4受体蛋白的复合物的结合力影响为 30 % , 或优选为 20 % , 或更优选为 5 %。
可以采用常规方法如 Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988)中描 述的方法， 测定某一未知单抗降低某一已知单抗结合表面抗原分化 簇 4蛋白的能力。 例如， 先把抗原预包被在微孔板上， 然后把系列 上述预包被后的微孔板中孵育， 洗涤后测定不同稀释度的待测抗体 已知抗体结合到板上的数量。 待测抗体竟争已知抗体结合抗原的能 力越强， 已知抗体结合抗原的能力就越弱。 通常， 抗原是预包被在 96孔微孔板上， 并利用放射标记法或酶标记法测定未标记单抗阻断 已标记单抗的能力。
可以采用 Kohler 等在 Nature 256: 495 (1975)中报道的杂交瘤 制备方法来制备单抗。 首先将免疫原 （必要时候添加佐剂）免疫注 射小鼠或其它合适的宿主动物。 免疫原或佐剂的注射方式通常为皮 下多点注射或腹腔注射。 免疫原预先偶联到某些已知蛋白， 如血清 白蛋白或大豆胰酶抑制剂上， 可能会有助于增强抗原在宿主内的免 疫原性。 佐剂可以利用福氏佐剂或 MPL-TDM等。 动物受免疫后， 体内会有分泌特异性结合免疫原的抗体的淋巴细胞产生。 另外， 淋 巴细胞也可以利用体外免疫获得。 收集目的淋巴细胞与骨髓瘤细胞 并用合适的融合剂， 如 PEG, 进行融合以获得杂交瘤细胞（Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996 ) 。
上述制备的杂交瘤细胞可以接种到合适的培养液中生长， 培养 液中最好含有一种或多种能够抑制未融合的、 母体骨髓瘤细胞生长 的物质。 例如， 对缺乏次黄嘌呤鸟嘌呤磷酸转移酶（HGPRT 或 HPRT )的母体骨髓瘤细胞， 在培养液中添加次黄嘌呤、 ^^喋呤和 胸腺嘧啶（ HAT培养基 )等物质将可以抑制 HGPRT-缺陷细胞的生 长。
优选的骨髓瘤细胞应该具有融合率高， 抗体分泌能力稳定， 对 HAT 培养液敏感等能力。 其中， 骨髓瘤细胞首选鼠源骨髓瘤， 如 MOP-21 和 MC-11 小鼠肿瘤衍生株 ( THE Salk Institute Cell Distribution Center, San Diego, Calif. USA ) , 和 SP-2/0 或 X63-Ag8-653 细胞株 ( American Type Culture Collection, Rockville, Md. USA ) 。 另外也有研究报道利用人骨髓瘤和人鼠异源骨髓瘤细 胞株制备人单抗 ( Kozbor, J. Immunol., 133: 3001 (1984); Brodeur 等 人 ， Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987 ) 。
杂交瘤细胞生长的培养液用于检测针对特异抗原的单抗的产 生。 测定杂交瘤细胞产生的单抗的结合特异性的方法有免疫沉淀或 体外结合试验,如放射免疫试^ ( RIA )、酶联免疫吸附试^ ( ELISA ), 流式细胞术 ( FACS )。例如， Munson 等在 Anal. Biochem. 107: 220 (1980)描述的 Scatchard分析法可用来测定单抗的亲和力。
当杂交瘤产生的抗体的特异性、 亲和力和反应性确定之后， 目 的细胞林可以通过 ( Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996 )所描述的标准的有限稀 释法进行亚克隆化。合适的培养液可以是 DMEM或 RPMI-1640等。 另外， 杂交瘤细胞还可以腹水瘤的形式在动物体内生长。
利用传统的免疫球蛋白纯化方法， 如蛋白 A琼脂糖凝胶、 羟基 磷灰石层析、 凝胶电泳、 透析或亲和层析等， 可以将亚克隆细胞分 泌的单抗从细胞培养液、 腹水或血清中分离出来。
本发明的单抗还可以通过基因工程重组技术获得。 利用特异性 结合单抗重链和轻链基因的核酸引物进行 PCR扩增， 可以从杂交瘤 细胞中分离得到编码单抗重链和轻链基因的 DNA分子。 所得 DNA 分子插入表达载体内， 然后转染宿主细胞， 如 E.coli 细胞、 猿猴 COS 、 CHO 细胞、 或其它不产生免疫球蛋白的骨髓瘤细胞。 转染 后的宿主细胞在特定条件下培养并表达目标抗体。
本发明的抗体对人表面抗原分化簇 4蛋白结合具有高特异性和 高亲和力。 这些抗体对其它灵长类表面抗原分化簇 4受体蛋白有交 叉反应性。
本发明的单抗可以是包含两条重链和两条轻链的传统的 "Y"型 结构状的抗体。 另外， 所述抗体也可以是保持了对表面抗原分化簇 4 受体蛋白亲和力的传统的" Y"型结构状的抗体上的 Fab片段、 Fab' 、 F(ab')2 、 Fv、 或其它类型的部分片段， 其结合血凝素蛋白的亲和力 可以高于或低于传统的 "Y" 型结构状的抗体。 本发明的抗体片段可以利用水解完整的抗体分子获得 （参见
Morimoto等人， J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan等人， Science 229:81 (1985) )。 另外， 这些抗体片段也 可以直接由重组宿主细胞产生 （reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little 等人， Immunol. Today, 21: 364-370 (2000) ) 。 比如， Fab'片段可以直接从 E. coli细胞中获得或 化学偶联形成 F(ab')2片段（ Carter等人， Bio/Technology， 10:163-167 (1992) ) 。 再如， F(ab')2 片段可以用亮氨酸拉链 GCN4 连接获得。 另外， Fv、 Fab, Fab'或 F(ab')2 片段也可以直接从重组宿主细胞培 养液中分离得到。 本领域的普通技术人员完全知晓制备抗体片段的 其它技术。
抗体核 ^^列
本发明涉及特异性结合 面抗原分化簇 4蛋白 （含细胞表面 的、 重组表达的和重组表达的截短的人表面抗原分化簇 4蛋白） 的 抗体或抗体片段的编码核酸分子。 编码抗体的核酸分子可以从杂交 瘤细胞中分离得到。 本领域的普通技术人员完全知晓利用常规技术 可以测定这些分子的核酸序列。 本发明涉及的抗体核酸分子也可以 利用传统的基因工程重组技术或化学合成方法获得。 一方面， 本发 明涉及的抗体核酸分子的序列包含了抗人表面抗原分化簇 4蛋白抗 体的重链可变区或抗体分子的部分核酸序列。 另一方面， 本发明涉 及的抗体核酸分子的序列也包括抗人表面抗原分化簇 4蛋白抗体的 轻链可变区或抗体分子的部分核酸序列。 另一方面， 本发明涉及的 抗体核酸分子的序列还包括重链或轻链可变区的 CDR序列。互补决 定区 ( complementary determinant region, CDR )是与抗原表位结 合的部位， 本研究中的 CDR 序列通过 IMGT/V-QUEST ( http://imgt.cmes. fr/textes/vquest/ )进行确定。 但是不同的划分方 法得到的 CDR序列稍有不同。
本发明的一方面涉及编码单抗 15A7 重链和轻链可变区序列的 核酸分子。 单抗 15A7 重链可变区序列的核酸分子对应于 SEQ ID NO: 1。 单抗 15A7轻链可变区序列的核酸分子对应于 SEQ ID NO: 3„ 本发明还涉及包含了单抗 15A7重链和轻链可变区序列的核酸分 子变体或类似体。
另一方面， 本发明还涉及各种分离的核酸分子的变体。 具体地， 核酸变体的序列与核酸序列 SEQ ID NO: l . SEQ ID NO: 3的同一性 至少达 70% 、 优选至少达 75 %、 更优选至少达 80 %、 更优选至少 达 85 %、 更优选至少达 90 %、 最优选至少达 95 %。
本发明还提供能特异性结合 面抗原分化簇 4蛋白的抗体片 段的编码序列的核酸分子。
本发明更进一步涉及编码氨基酸序列为 SEQ ID NO: 2的抗体 重链可变区的分离的核酸分子。 本发明还涉及编码氨基酸序列为 SEQ ID NO: 4的抗体轻链可变区的核酸分子。
本发明涉及含有所述核酸分子的重组表达载体， 也涉及转化了 这些分子的宿主细胞。 而且， 本发明还涉及利用包含所述核酸分子 的宿主细胞在特定条件下培养并分离得到发明所述抗体的方法。
抗体多肽序列
单抗 15A7 重链和轻链可变区氨基酸序列可以从对应的核酸序 列中推导得到。单抗 15A7重链可变区氨基酸序列是 SEQ ID NO: 2。 单抗 15A7轻链可变区氨基酸序列是 SEQ ID NO: 4。
另一方面， 本发明提供了重链可变区的氨基酸序列与 SEQ ID NO:2的序列相似性至少达 70 % ,优选是至少 75 % ,优选是至少 80 % , 优选是 85 %， 再优选是至少 90 %， 最优选是至少 95 %的抗体。
另一方面， 本发明提供了轻链可变区的氨基酸序列与 SEQ ID NO:4的序列相似性至少达 70 % ,优选是至少 75 % ,优选是至少 80 % , 优选是 85 %， 再优选是至少 90 %， 最优选是至少 95 %的抗体。
单抗 15A7的重链和轻链可变区的 CDR的氨基酸序列分别是： 单抗 15A7重链的 CDR1、 CDR2 和 CDR3的氨基酸序列分别 为 SEQ ID NOs:5-7o 单抗 15A7轻链的 CDR1、 CDR2和 CDR3的 ^^酸序列分别为 SEQ ID NOs:8-10o
另一方面， 本发明提供了抗 CD4单抗重链或片段， 其含有选自 SEQ ID NOs: 5-7的一个或多个 CDR。
另一方面， 抗 CD4单抗的重链或片段的 CDR含有的
列可以在 SEQ ID NOs: 5-7上出现一个或多个氨基酸的突变或增添 或缺失。 优选的是， 突变或添加或缺失的氨基酸不超过 3个氨基酸。 更优选的是， 突变或添加或缺失的氨基酸不超过 2个氨基酸。 最优 选的是， 突变或添加或缺失的氨基酸不超过 1个氨基酸。
另一方面，本发明提供了抗 CD4单抗轻链或片段含有选自 SEQ ID NOs: 8-10的一个或多个 CDR。
另一方面， 抗人 CD4单抗的轻链或片段的 CDR含有的氨基酸 序列可以在 SEQ ID NOs: 8-10上出现一个或多个氨基酸的突变、 增 添或缺失。 优选的是， 突变、 添加或缺失的氨基酸不超过 3个氨基 酸。 更优选的是， 突变、 添加或缺失的氨基酸不超过 2个氨基酸。 最优选的是， 突变、 添加或缺失的氨基酸不超过 1个氨基酸。
上述抗体或 CDR的可变区的氨基酸发生突变、添加或缺失之后 的变体仍然保留特异性结合 CD4的能力。 本发明也包含这样的抗原 结合片段的变体。
本发明的单抗变体可以通过传统的基因工程方法获得。 本领域 的技术人员完全知晓利用核酸突变改造 DNA分子的方法。 另夕卜， 编 码重链和轻链变体的核酸分子也可以通过化学合成获得。 嵌合抗体、 人源化抗体和融合蛋白
另一方面， 本发明也提供了嵌合抗体， 它是由鼠源单抗 15A7或 其变体的重链和 /或轻链完整的或部分的可变区结合人源单抗恒定区 组成的。 而且， 本发明也包括人源化抗体， 它们是由鼠源单抗 15A7 或其变体的一个或多个 CDR嫁接到人源抗体的框架上组成的。
另一方面， 本发明还提供了偶联了某种分子的完全或部分的含 有本发明所述单抗的融合蛋白。
嵌合抗体、 人源化抗体和融合蛋白都可以利用传统的基因工程 技术获得。 如， 编码单抗的 DNA可以通过突变的方法把人源抗体的 重链和轻链的恒定区的序列替换成同源性的鼠源序列而改造成 ( Morrison,等人， Proc. Nat. Acad. Sci. 81 : 6851 (1984) ) , 或通过把 整个或部分免疫球蛋白编码序列与非免疫球蛋白编码序列进行共价 偶联而获得嵌合或人源化抗体或融合蛋白。
中和抗体
另一方面， 本发明提供了能够中和人免疫缺陷病毒的抗 CD4抗 体。 在一个实施方式中， 这种中和抗体能够中和人免疫缺陷病毒感 染 CD4+细胞的活性的至少 60%, 或至少 70%, 或优选至少 75%， 或优选至少 80%, 或优选至少 85%, 或优选至少 90%， 更优选至少 95%, 最优选至少 99.99%。 在体外中和 HIV病毒实验中， IC50中 和活性浓度优选在 O.OOOl-lO g/mL之间， 或优选为 5(^g/mL。
本领域普通技术人员完全知晓利用传统的技术方法可以测定抗 体中和人免疫缺陷病毒的感染细胞活性。 如本发明的实施例中所描 述的中和试验的方法即可用于测定本发明中的某一个特定 CD4单抗 的中和活性。
检测方法
本发明还提供了一种利用本发明所述单克隆抗体检测标本中的 CD4抗原或其模拟物和 /或抗体的方法。
一方面， 本发明提供了 CD4和 CD4相关治疗的检测方法， 包 括以下步骤： （i)将本发明的单克隆抗体或其片段与上述样品中的抗 原结合而行成抗体 -抗原或抗体片段 -抗原复合物； （ii)检测该样品复 合物以确定样品中是否有 CD4抗原或其模拟物。
检测方法可以使用酶联免疫吸附 （ELISA ) 、 酶免疫检测、 化 学发光免疫检测、 放射免疫检测、 荧光免疫检测、 免疫色谱法、 竟 争法及类似检测方法。 利用竟争法或夹心法方式， 上述检测方法可 以用于检测目标抗原或抗体。
竟争法比较样品中抗原和一种已知量的标记抗原竟争结合本发 明所述单克隆抗体的数量关系。 开展基于竟争法的免疫学检测是将 含有未知数量的目标抗原的样品加入到事先用已知的物理或化学方 法把本发明所述单抗包被到固相支持物上而进行的。 同时加入预先 定量的标记后的目标抗原进行反应。 孵育后， 冲洗固相支持物， 检 测结合到该支持物上的标记物的活性。
在夹心法中， 样品中的目标抗原被夹在包被单抗和标记单抗之 间， 然后再加入标记物比如酶的底物， 通过底物颜色的变化检测并 判定抗原的存在。 开展基于夹心法的免疫学检测， 例如， 先把含有 一种未知数量目标抗原的样品加入到用物理或化学方法预先包被了 本发明中所述单克隆抗体的固相支持物上进行反应。 然后， 加入本 发明所述的标记单抗进行反应。 孵育后， 冲洗该支持物， 再对结合 到该支持物上的标记物的活性进行检测。 标记物可以是放射性同位 素如 125碘、 酶、 酶的底物、 发光物质如异鲁米诺和吖啶酯、 荧光 物质如荧光素和罗丹明、生物素和有色物质如乳胶颗粒和胶体金等。 标记用的酶可以是过氧化物酶（如辣根过氧化物酶 HRP ) 、 碱性磷 酸酶、 P半乳糖苷酶和葡萄糖氧化酶。 对于这些反应中合适的底物 有 2，2'-连氮基-双（ 3-乙基苯并噻吡咯啉 -6磺酸）、鲁米诺 -过氧化氢、 邻苯二胺-过氧化氢（针对过氧化物酶） 、 对硝基苯磷酸盐、 4-甲基 磷酸伞型酮、 3-(2'-螺旋金刚烷) -4-甲氧基 -4-(3"-磷酰基)苯基 -1，2-二 乙氧基烷（针对碱性磷酸酶）、对硝基苯 - P -D-半乳糖和甲基伞形酮 - β -D-半乳糖（针对 β半乳糖苷酶） 。 其它的标记包括量子点标记、 生色团标记、 酶标记、 亲和配体标记、 电磁自旋标记、 重原子标记、 标记有纳米微粒光散射标记或其它纳米微粒的探针、 异硫氰酸荧光 素（FITC )、 TRITC、 罗丹明、 四甲基罗丹明、 R-藻红蛋白、 Cy-3、 Cy-5、 Cy-7、 得克萨斯红、 Phar-Red、 异藻红蛋白 （APC ) 、 表位 标记如 FLAG或 HA表位、 以及酶标记如碱性磷酸酶、 辣根过氧 化物酶、 I2 -半乳糖苷酶、 碱性磷酸酶、 P -半乳糖苷酶或乙酰胆碱酯 酶和半抗原偶联物如洋地黄毒苷或二硝基苯酚、 或能够形成配合物 的结合配对如链霉抗生物素蛋白 /生物素、抗生物素蛋白 /生物素或抗 原 /抗体配合物如包括兔 IgG 和抗-兔 IgG;荧光基团如伞形三糖
( umbelliferone ) 、 荧光素、 异硫氰酸荧光素、 罗丹明、 四甲基罗 丹明、 伊红、 绿荧光蛋白、 藻红、 香豆素、 甲基香豆素、 芘、 孔雀 绿、 二苯乙烯、 荧光黄、 Cascade 蓝、 二氯三嗪基荧光素、 丹磺酰 氯、 藻红蛋白、 荧光镧系络合物如包括铕和铽、 Cy3、 Cy5、 分子信 标（ molecular beacons )和其荧光衍生物 、 发光材料如鲁米 i若； 光 散射或细胞质基因组共振材料如金或银颗粒或量子斑（ quantum dot )： 或放射性材料如 "C、 123I、 124I、 131I、 TC99m、 35S或 3H; 或 球珠 ( spherical shell ) , 以及标记有本领域已知的任何其它信号产 生标记物的探针。 例如， 可检测的分子包括但不限于荧光基团以及 前面所述其它已知的， 如在 Joseph R. Lakowicz ( Editor )所编的 Principles of Fluorescence Spectroscopy, Plenum Pub Corp, 第二版
( July 1999 )和 Richard P. Hoagland的第六版的 Molecular Probes Handbook所描述的。 在某些实施方式中， 标记物包括半导体纳米 微晶如量子斑（即 Qdots ),参见 U.S.P 6,207,392„ Qdots可从 Quantum
Dot Corporation 购得。 用于本发明的半导体纳米微晶包括 Group
II-V半导体的纳米微晶如 MgS、 MgSe、 MgTe、 CaS、 CaSe、 CaTe、
SrS、 SrSe、 SrTe、 BaS、 BaSe、 BaTe、 ZnS、 ZnSe、 ZnTe、 CdS、
CdSe、 CdTe、 HgS、 HgSe、 HgTe和其混合物以及 Group III-V半 导体的纳米微晶如 GaAs、 InGaAs, InP、 InAs和其混合物。 Group
IV半导体如锗或硅的使用， 或有机半导体的使用， 在某些条件下可 能是方便可行的。 半导体纳米微晶也可以包括合金， 其含有两种或 多种选自于 Group III-V化合物、 Group II-VI化合物、 Group IV元 素和其组合物的半导体。
在某些实施方式中， 荧光能量受体连接到检测探针作为标记物。 在一实施方式中， 荧光能量受体可以通过化合物与单线态氧反应形 成荧光化合物而形成， 或通过化合物与一辅助化合物反应并将其转 化为荧光化合物而形成。 这类化合物可以包括于本发明装置中的緩 冲液中。 在其它的实施方式中， 荧光能量受体可以是包括化学发光 剂或基团的化合物的一部分， 例如， 荧光能量受体可以包括稀土金 属如铕、 钐、 碲等金属络合物。 这些材料因其具有尖锐的发光傳带 而特别具有吸引力； 而且，镧系标记物如铕 (III)可以提供有效的长时 间的信号发射， 同时不易光漂白， 因而可以使得含有处理 /反应样品 的测试装置在需要的情况下可以放置较长一段时间。 在各种异源和 同源免疫测定法中，已经使用长寿命的荧光铕 (ΠΙ) 物纳米微粒作 为标记物， 例如可以参见 Huhtinen 等人 Clin. Chem. 2004 Oct; 50(10): 1935-6。 但这些内部标记的纳米微粒与时间分辨荧光检测一 起使用时， 测定性能可以改善。 在异源测定中， 低浓度下测定的动 态范围可以扩展； 而且， 通过使用检测抗体涂敷的高特异活性纳米 微粒标记物， 而不是使用常规标记的检测抗体， 测定的动力学特性 也可以改善。 在同源测定中， 对于荧光共振能量转移来说， 铕 (III) 纳米微粒是很有效的供体， 从而可以进行简单、 快速和高效的筛选。 在一实施方式中， 标记物如此处披露的荧光标记物， 包括与生物分 子偶联的纳米微粒标记物。 换句话说， 纳米微粒可以用作检测或捕 捉探针。 例如， 本发明中， 可以利用连接到单抗或链霉抗生物素蛋 白（SA)的铕 (III)-标记的纳米微粒来检测样品中特定的分析物， 如纳 米微粒基的免疫测定。 纳米微粒可以作为附着特定结合剂的底物， 这些特定结合剂是针对分析物和检测 (如标记物)或捕捉成分的。有关 标记物的实例可以参见 U.S.P 4,695,554; 4,863,875; 4,373,932;和 4,366,241„ U.S.P 4,313,734和 4,373,932中则披露了胶体金属和染色 颗粒。而 U.S.P 4,954,452中则披露了如何制备和使用非金属的胶体； U.S.P 4,252,459中披露了用作标记物的有机聚合物乳胶颗粒。
把标记物结合到抗原或抗体上的方法， 可以通过顺丁烯二酰亚 胺法（ J. Biochem. (1976), 79, 233 )、生物素活化法（ J. Am. Chem. Soc. (1978)， 100， 3585 )、疏水结合法、酯活化法或异氰酸酯法（ Igaku Shoin "Enzyme immune assay techniques", 1987 ) 。
如果上述标记物为放射性同位素， 则需使用好的防辐射的工作 台面或液体防护设备。 如果上述标记物为酶， 需加入底物， 酶的活 性通过比色法或荧光计测定。 如果上述标记物为荧光物质、 发光物 质或有色物质 , 测定方法可以相应的使用本领域熟知的方法进行测 定。
治疗方法和药物组合物
本发明的抗体及其活性片段及其同源物提供了一种预防和 /或治 疗 CD4+细胞相关的病毒感染及相关疾病患者的方法， 尤其是人免疫 缺陷病毒（HIV )感染及其相关的疾病包括艾滋病， 包括对患者施 用一定量的包含本发明所述单抗的有药物活性的药物成分。 本发明 还提供了一种含有本发明所述的单抗的药物成分或在此基础上得到 的盐类药物。
本发明的药物组合物可进一步包含其它用于预防和 /或治疗 HIV 感染的药剂， 可以和这些抗病毒试剂同时、 分开或连续给药。 例如， 本发明的抗体及其活性片段及其同源物可以和能抑制反转录酶的抗 反转录药剂， 如 AZT—同使用， 或与能抑制 HIV蛋白酶的药剂一 同使用； 此外， 本发明的药物组合物进一步包含抗病毒药剂例如干 扰素， 免疫抑制剂如环胞菌素， 但不仅限于这些。
此外， 一种或多种抗体同源物也可与一种或多种前述治疗剂一 同使用。 此种组合疗法的优点是可使用较低剂量的治疗药剂， 因此 可避免当使用单一药剂时可能会有的毒性或副作用。
本发明的药物组合物包含依据本发明的一种或多种免疫治疗有 效量的抗体同源物， 或其衍生物， 且优选的为药学可接受的载体。
"免疫治疗有效量" 指足够能防止因 HIV感染或艾滋病， 或由原靶 为表达 CD4的细胞的感染因子所引起的其他疾病所造成的免疫应答 作用的量。 "药学可接受的载体" 指不会在所施用的病人身上引发 过敏反应或其他不适影响的载体。
合适的药学可接受的载体包括， 例如， 一种或多种水、 生理盐 水、 磷酸緩冲液、 左旋糖、 甘油、 乙醇和其他类似物， 以及上述物 质的组合。 药学可接受的载体可进一步包括能提高抗体或其活性片 段或其同源物的保存期限或效用的微量辅助物质， 例如湿润剂或乳 化剂、 防腐剂或緩冲液。
本发明所述药物成分的施用方式可以是传统的施用途径， 包括 静脉滴注、 肌肉注射、 阴道、 口服、 口腔、 舌下、 眼球、 局部、 肠 胃外、 直肠、 叶鞘内、 内胞浆网槽内、 腹股沟、 膀胱内、 局部（如， 粉剂、 药膏或滴剂） ， 或鼻腔途径， 但不仅局限于此。 优选的是注 射及输液形式。
本发明的组成可以有许多不同形式。 这些包括， 例如， 固体、 半固体和液体的剂型， 例如片剂、 丸剂、 粉末、 溶液、 分散液货悬 浮液、 脂质体、 栓剂、 注射用及输液用溶液。 优选的形式视施用方 式及其预防或治疗应用而定。 优选的组合物是注射用及输液用的溶 液的形式。
适合肠胃外途径注射的药物成分可能含有符合药物制备要求的 无菌水或非水溶液、 气雾剂、 悬浮液或乳剂， 可在临用时重悬成可 注射的溶液或气雾剂的无菌粉剂。 如适合的水性和非水性载体， 工 具和各种稀释液如水、 乙醇、 多羟基化合物（如丙烯乙二醇、 聚乙 烯二醇、 丙三醇及其类似物） ， 合适的混合物， 菜油 （如橄榄油） ， 和可用于注射的有机脂， 如乙烷油酸， 如使用卵磷脂衣壳维持药物 的合适流动性， 如使用气雾剂、 表面活性剂以维持合适的颗粒尺寸。
本发明所述的药物组合物还可含有一些起保护性、 保湿、 乳化 和气雾化的佐剂， 也可以含有预防微生物污染的速溶成分， 如各种 抗细菌试剂、 抗真菌试剂， 如 parabens, chlorobutanol, 苯臉， 山梨 酸及类似物。 也可以包括维持渗透压的试剂， 如糖、 NaCl及其类似 物。 可使用延长吸附的试剂来延长注射用药物成分吸附时间， 如单 硬脂酸盐和凝胶等。
口服固相剂型包括胶嚢、 片剂、 粉剂、 颗粒剂等。 这些固相剂 型中的活性成分至少混有一种传统的惰性药物赋形剂 （或载体）如 柠檬酸钠、 磷酸 ， 或 (a)填充剂或添加剂如淀粉、 乳糖、 蔗糖、 甘 露糖和硅酸； （b)粘合剂， 如羧甲基纤维素、 藻酸盐、 明胶、 聚乙烯 吡咯烷酮、 蔗糖和阿拉伯树胶； （c)湿润剂， 如甘油； （d)碎裂剂， 如 琼脂， 碳酸钙， 马铃薯粉或木薯粉； （e)緩凝剂， 如石蜡； （f)促吸收 剂， 如四氨基混合物； （g)保湿剂， 如十六烷基醇和单硬脂酸甘油酯； (h)吸附剂， 如高岭土和斑脱土； （i)润滑剂， 如滑石， 硬脂酸 ， 硬 脂酸镁， 固体聚乙二醇， 硫酸十二烷醇钠， 或其上述物质的混合物。 在片剂和胶嚢剂型中， 可能还含有緩冲剂。
固相剂型可以通过做成改良释放或脉冲释放剂型， 是在上述提 到的各种直接释放赋形剂中添加一些能改变药物释放速率的赋形剂 而形成， 可以包含在剂型中也可以做成外衣的形式。 速率幹放改造 剂包括羧丙基甲基纤维素， 甲基纤维素， 碳甲基纤维素钠， 纤维素 乙烷， 醋酸纤维素， 聚乙烯氧化物， 黄原胶糖， 异丙烯酸氨共聚物， 氢化调味油， 巴西椋榈蜡， 石蜡， 邻苯二酸醋酸纤维素， 邻苯二甲 酸羧丙基甲基纤维素， 甲基丙烯酸共聚物或上述物质的混合物。 改 良释放和脉冲释放剂型可能含有一种或一组具有改良幹放速率的赋 形剂。
本发明所述药物成分还可由快速的雾化剂或消溶剂 （ FDDFs ) 组成， 包含如下成分： 天冬氨酰苯丙氨酸甲酯， 磺胺钾， 柠檬酸， croscarmellose sodium, crospovidone, 抗坏血酸, 乙燒基丙蟑酸盐, 乙烷基纤维素， 明胶， 氢氧基丙基甲基纤维素，硬脂酸镁， 甘露醇， 甲基乙丁烯酸盐， 调味薄荷， 聚乙二醇， 气化硅胶， 二氧化硅， 乙 醇酸淀粉钠， 硬脂酸延胡索酸钠， 山梨醇， 木糖醇。 这里用于描述 FDDFs的 "雾化和消溶" 一词， 依赖于所用药物的溶解性， 如药物 是不可溶的， 可制成快速的雾化剂型， 如药物是可溶的， 则可制成 快速的溶剂型。
类似形式的固相成分也使用诸如乳糖或牛奶糖或其它高分子量 的聚乙二醇及类似的赋形剂制成软明胶或硬明胶的填充剂型。
诸如片剂、 糖衣剂、 胶嚢剂和颗粒剂等之类的固体剂型可以通 过诸如肠衣或其它本领域普通人员均知晓的外包衣壳的方式制成。 也可以是含有乳浊剂、 也可以是含有能起緩慢、 延迟、 控制活性药 物释放的类似的成分。 也可以使用多聚物和石蜡等成分进行包埋。 如果合适， 也可用上述一种或多种赋形剂把活性成分制成微嚢的形 式的剂型。
用于口服的液体剂型， 包括符合药物要求的乳状剂、 溶液、 悬 浮液、 糖浆和西也剂等。 除了活性成分， 液体剂型也可含有本领域 常用的一些惰性溶液， 如水或其它溶剂， 可溶性试剂和乳化剂， 如 乙烷基醇， 异丙基醇， 乙烷基碳酸盐， 苯基安息香酸盐， 丙稀乙二 醇， 1,3-丁烯乙二醇， 油， 特别是， 棉籽油， 落花生油， 玉米油， 橄 榄油， 调味油和芝麻油， 甘油， 氢糠基醇， 聚乙二醇和脂肪酸山梨 醇酯， 以及上述物质的混合物或类似的物质。
除了这些惰性稀释液， 药物成分也可包括保湿剂、 乳化剂、 悬 浮剂、 糖化剂、 调味剂和香味剂等佐剂。 另外， 药物成分还可包括 乙氧基化均聚乙醇， 聚氧乙烯烷基山梨醇和山梨聚糖脂， 微晶纤维 素， 间氢氧化铝， 膨润土， 琼脂聚合物和黄芪胶， 或这些物质的混 合物之类的悬浮剂。
本发明所述药物成分也可制成适合兽用治疗的混合物， 或符合 兽用的盐类， 或符合兽用的溶剂或初成药， 并根据普通兽医和兽医 从业者的要求制成最适合某种特定动物的给药剂量和途径药物的合 适剂型。
本发明优选的药物组合物与那些用于人被动免疫的其他抗体的 组合物类似， 例如肿瘤治疗抗体。 优选的施用方式是非肠道施用。
本发明所述一个或多个单抗可以结合其它抗病毒试剂用于预防 和或治疗 CD4+细胞相关的疾病， 包括但不限于人类免疫缺陷感染及 其相关疾病。 单抗可以和这些抗病毒试剂同时、 分开或连续给药。 其它抗病毒试剂包括利巴韦林，金刚烷， 脲， IL-2, IL-12 和五羧 链胞酸， 但不仅限于这些。
此领域技术人员应不难了解， 本发明抗体同源物或其活性片段 的免疫治疗有效量将视施用程序、 所施用的抗体同源物的单位剂量、 抗体同源物是否与其他药剂一同使用、 病人的免疫及健康情况、 所 施用的特定抗体同源物的抗病毒活性而定。 实施例
下面结合具体实施例与附图， 对本发明进一步加以描述， 但这 些描述并不构成对本发明的限制。 实施例 1. 可抑制 HIV感染 CD4+细胞的抗 CD4单克隆抗体的 制备
小鼠： 6周龄雌性 BALB/c鼠为厦门大学生命科学院实验动物中 心提供。
杂交瘤的制备： 我们使用标准的体内免疫方式和 PEG融合方法 获得单克隆抗体， 详细方法参见 Ed Harlow 等人， Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory 1988. 简要过 程如下：
小鼠免疫： 将溶于 PBS的重组 sCD4抗原溶液与弗氏完全佐剂 ( CFA )等体积混合乳化， 经四肢肌肉多点注射， 每只每次注射实 施例 2中制备的纯化重组 sCD4抗原 5 g (总体积 50μί ) 。 首次免 疫后 15天和 29天， 分别用同样剂量的重组抗原溶液加弗氏不完全 佐剂 （IFA )进行加强免疫。 融合前 72小时， 尾静脉加强注射 5 g 不加佐剂的抗原 1次进行加强免疫。
融合： 取血清中和效价最高的小鼠脾脏细胞与小鼠骨髓瘤细胞 相融合， 先把脾脏研磨得到脾细胞悬液， 细胞计数。按 1/6于脾细胞 的数量取培养的 SP2/0小鼠骨髓瘤细胞， 混合后离心， 经 50%聚乙 二醇（PEG2000 )作用 1 分钟， 使脾细胞与小鼠骨髓瘤细胞 SP2/0 融合。加入 50mL含有 20%FBS的 RPMI1640培养基重悬融合细胞， 将细胞悬液与等体积的饲养细胞混合后， 分置于 96孔细胞培养板中 ( 200μΙ孔 )于 5 %二氧化碳培养箱（ ESPEC BNA-311 ) 37*€培养。
3天后，用含有 20%FBS的 HT培养基（ 1.361mg黄嘌呤 (hypoxanthn, H)、 0.388mg胸腺嘧 核苷（ thymidine, T ) ,加 RPMI 1640( GIBCO 公司）培养基至 lOOmLo 45 ~ 50 条件下溶解后， 过滤除菌。 半保 留换液。
杂交瘤的筛选： 融合后细胞在 96孔细胞板上培养 10天后， 吸 取细胞上清利用下述 HIV感染 TZM-bl细胞中和检测方法 96孔细胞 培养板中所得杂交瘤细胞上清培养液。 对于中和效果为阳性的细胞 克隆， 利用有限稀释法进行克隆化， 经 3次克隆化后， 得到所分泌 的抗体能够稳定抑制 HIV感染 TZM-bl细胞的单克隆抗体细胞株 (见 实施例 3 ) 。
筛选结果： 获得两株单克隆抗体， 分别为： 15A7和 14G7。
抗体的类型及亚类鉴定： 以实施例 2中制备的纯化 sCD4包被 ELISA板。每孔加入单克隆抗体的细胞上清 ΙΟΟμΙ^, 37*€温育 30分 钟；在 TECAN全自动洗板机上用 PBST洗涤 5次，每次间隔 20秒， 扣干， 加入合适稀释度的 HRP-山羊抗小鼠 IgM、 IgGl、 IgG2a、 IgG2b、 IgG3抗体（ Serotec公司）酶标二抗， 于 37"C温育 30分钟； 在 TEC AN全自动洗板机上用 PBST洗涤 5次， 每次间隔 20秒， 扣 干， 加入显色液 A ( H202 )和 B ( TMB )各 1滴， 于 37*€显色 10 分钟， 加入 1 滴终止液（2M H2S04 ) ； 在 TECAN酶标仪上测量 OD45()nm (参比波长为 620nm ) , 阈值为 2倍的阴性均值， OD值大 于阈值为阳性， OD值小于阈值为阴性。 结果单克隆抗体 15A7 为 IgGl型 , 14G7为 IgG2b型。
杂交瘤的培养： 稳定的杂交瘤单克隆抗体细胞株先在二氧化碳 培养箱中扩增培养， 经 96孔培养后转移至 24孔， 再转移至 50mL 细胞瓶中扩增培养。取 10周龄的健康 BALB/c小鼠，腹腔注射 0.5mL/ 只液状石蜡， 1-2周后，每只小鼠腹腔注射 lxlO6个杂交瘤细胞， 7-10 天后收集腹水。 3000rpm离心 15分钟， 吸取中间澄清部分的液体， 0.45μιη的微孔滤膜过滤除菌， 分装后 -20 保存。
单克隆抗体的纯化：将腹水用 0.02Μ、ρΗ7.4的 PBS( 81mL 0.2M Na2HP04, 19mL 0.2M NaH2P04, 加生理盐水至 1L )对倍稀释， 搅 拌下逐滴緩慢加入硫酸铵（浓度达到 50%饱和度）， 4*€过夜。
12000rpm离心 15分钟，弃上清，将沉淀溶于原腹水体积 2倍的 PBS 中。搅拌下逐滴緩慢加入硫酸铵，使硫酸铵浓度达到 33%饱和度， 4*€ 静置过夜。 12000rpm离心 15分钟， 弃上清， 将沉淀溶于原腹 水体积 2倍的 PBS中。 搅摔下逐滴緩慢加入硫酸铵 (浓度达到 50% 饱和度） ， 4*€过夜。 4*C , 12，000rpm离心 15分钟， 弃上清。 将沉 淀溶于适量的 PBS中，装入透析袋中，放入 50-100倍含 20mM NaCl, H 7.8的 120mM Tris-HCl緩冲液中 4*€搅拌下脱盐 12小时左右， 期间更换 3次以上透析液。 取出后分装 -20 保存。
实施例 2. 用做抗原的重组人 CD4片段多肽的制备
用 4故模板之人 CD4胞外区片段的制备： 以 TZM-bl细胞系
( Cat.No. 8129 National Institutes of Health AIDS Research and Reference Reagent Program )中的人 CD4 cDNA为模板， CD4(dl)F ( 5'- CGG CAT ATG AAG AAA GTG GTG CTG GGC -3' )为正向 引物， CD4(d4)R ( 5'-
GCGAATTCTTACCATGTGGGCAGAACCTT -3' )为反向引物， 在 PCR热循环仪（ Biometra T3 )按照如下条件进行 PCR反应： 94 *€ 10分钟；随后是 94 30秒， 56 30秒，， C 1分钟的 25个循环， 最后为 72 延伸 10分钟。 得到特异的 l.lkb左右大小的用做制备 各截短的重组人 CD4多肽 ^¾板 DNA片段。将上述获得的 PCR产 物与商售的 pMD 18-T载体（ TaKaRa公司生产）连接，经 Nde I/EcoR I酶切鉴定， 得到插入 sCD4基因的阳性克隆。 利用 M13(+)/(-)引物， 测序得经过上述方法制备的 CD4胞外区的 DNA片段保守序列， 并 用做制备各截短的重组人 CD4多肽的模板。
以如上获得的人 CD4 的 N 端胞外区的免疫球蛋白样结构域 D1-D4序列为模板， 利用表 1中的正向引物（其 5'端引入限制性内 切酶 Ndel )和反向引物（其 5'端引入限制型内切酶 EcoRI位点） 。 在 PCR热循环仪（ Biometra T3 )按照如下条件进行 PCR反应： 94 *€ 10分钟； 随后是 94 30秒， 56 30秒， ， C 45秒的 25个循环， 最后为 ，1/C 延伸 10 分钟。 其中， CD4(dl) 所用正向引物为 CD4(dl)F , 反向引物为和 CD4(dl)R; CD4(d2) 所用正向引物为 CD4(d2)F , 反向引物为和 CD4(d2)R; CD4(d3) 所用正向引物为 CD4(d3)F , 反向引物为和 CD4(d3)R; CD4(d4) 所用正向引物为 CD4(d4)F , 反向引物为和 CD4(d4)R; CD4(dl-2)所用正向引物为 CD4(dl)F,反向引物 CD4(d2)R得到特异的 DNA片段， 为编码本发 明所述各截短的人 CD4的多核苷酸序列，其氨基酸序列如图 1所示。
表 1. CD4各结构域截短的重组蛋白克隆引物序列
引物名称 引物序列
CD4(dl)F 5'- CGGCATATGAAGAAAGTGGTGCTGGGC-3'
CD4(dl)R 5'-GGAATTCTTAGAACACTAGCAATTGCACC-3'
CD4(d2)F 5'-CCGCATATGGGATTGACTGCCAACTCTGAC-3'
CD4(d2)R 5'- GGGGAATTCTTAGGAGGCCTTCTGGAAAG-3'
CD4(d3)F 5'-GCCCATATGAGCATAGTCTATAAGAAAGAGGG-3' CD4(d3)R 5'- GGGAATTCTTACACCACCAGGTTCAC-3' CD4(d4)F 5'- GCCCATATGATGAGAGCCACTCAGCTCC-3'
CD4(d4)R 5'- GCGAATTCTTACCATGTGGGCAGAACCTT-3'
表达载体 pTO-T7依照文献构建（罗文新等， 生物工程学报， 2000, 16:53-57 )。 用于表达本发明所述多肽的载体构建的具体步骤 包括：将如上述获得的 PCR产物与商售的 pMD 18-T载体（ TaKaRa 公司生产）连接， 经 Nde I/EcoR I酶切鉴定， 得到插入各区段 CD4 基因的阳性亚克隆； Nde I/EcoR I酶切获得各区段的 CD4基因片段， 再与经 Nde I/EcoR I酶切的 pTO-T7表达载体相连接， Nde I/EcoR I 酶切鉴定得到插入个区段 CD4 基因的多肽重组表达克隆 pTO-T7-CD4(dl) 、 pTO-T7-CD4(d2) 、 pTO-T7-CD4(d3) 、 pTO-T7-CD4(d4)、 pTO-T7-CD4(dl-2)和 pTO-T7-sCD4。
各区段截短的 CD4重组多肽的表达： Ιμί的重组表达载体质粒 ( 0.15mg/mL )转化 40μΙ 的氯化钙法制备的感受态大肠杆菌 BL21 (购自新英格兰生物实验室公司 ） ， 涂布于卡那霉素 （终浓度 25mg/mL, 下同）抗性的固体 LB培养基， 37*€静置培养 10~12小 时， 至单菌落清晰可辨。 挑取单菌落至含 4mL卡那霉素抗性的液体 LB培养基之试管， 37*€ 220转 /分振荡培养 10小时， 至菌体浓度为 OD600 * 0.8,从中取 lmL保存于 4*€ ,剩余的 3mL加入 3·0μί 0.8Μ IPTG (终浓度为 0.8 mM ) , 31V 220转 /分振荡进一步诱导培养转 化的表达菌株 4 小时。 取 1.5mL 经诱导培养的表达菌株培养液， 12,000转 /分离心 30秒， 弃去上清， 扣干， 用 ΙΟΟμί lx凝胶加样緩 冲液（ 50mM Tris Cl ( pH6.8 ) , lOOmM二疏苏糖醇（ DTT ) , 2% SDS, 0.1%溴酚蓝， 10%甘油）重悬； 沸水浴处理 10分钟； 12,000 转 /分， 离心 10分钟； 取出 ΙΟμί进行 15%丙烯酰胺 SDS-PAGE分 析。 挑选表达量较高的转化菌株进行大量表达并长期保种。 每个 1L的三角培养瓶装 500mL新鲜 LB液体培养基， 分别转 接 200μί的保存菌液， 37*€振荡培养 11小时， 至菌体浓度 OD600 « 1.0。 然后向每培养瓶加入 500μί 0.8Μ IPTG至终浓度为 0.8mM， 振荡诱导培养 4小时， 离心收集菌体。
CD4(dl)、 CD4(d2)、 CD4(d3)、 CD4(d4)、 CD4(dl-2)和 sCD4 截短多肽在大肠杆菌中重组表达的包涵体的洗涤纯化： 重组菌株经 诱导表达所得培养液 4*C 7，000rpm离心 5分钟后，弃上清； 菌体沉 淀以每 500mL培养基加 20mL裂解液（ 50mM Tris Cl, 10mM EDTA, 300mM NaCl pH7.2 )计算， 重新悬浮于裂解液中。 用超声波破碎仪 ( SONICS&MATERIALS公司的 Uilbra-Cell VCX500型）破碎细 胞， 以 500mL培养液所得菌体悬浮液 20mL为例破碎条件为： 设置 55%功率， 水浴， 每次脉冲破碎时间为 2秒， 间隔 4秒， 共破碎时 间为 4分钟； 12,000转 /分 4*€下离心 10分钟， 弃上清； 沉淀用 1/2 菌体悬浮液体积的緩冲液 I 溶液（ 200mM Tris Cl, pH8.5, 5mM EDTA, lOOmM NaCl )重悬， 加入等体积含 4%Triton-X100的緩冲 液 I， 其中 Triton-XlOO的终浓度为 2%。 37"€下 200rpm振荡 30 分钟； 10，000rpm 4*C离心 10分钟， 弃上清； 重复 2% Triton-XlOO/ 緩冲液 I处理一次。 沉淀用与菌体悬浮液等体积的緩冲液 I重悬， 37*€ 200rpm振荡 30分钟； 4*€ 8，000rpm离心 10分钟， 弃上清； 重复緩冲液 I处理两次。 沉淀用与菌体悬浮液等体积的緩冲液 I重 悬； 37*C 200rpm振荡 30分钟； 4*€ 10，000rpm离心 10分钟， 弃 上清； 沉淀用与菌体悬浮液等体积的 2M尿素 /緩冲液 I重悬； 37*€ 200rpm振荡 30分钟； 4"C 12，000rpm离心 10分钟， 留上清。 样品 分别编号为 CD4(dl) 2M、 CD4(d2) 2M、 CD4(d3) 2M、 CD4(d4) 2M、 CD4(dl-2) 2M和 sCD4 2M;沉淀用等体积的 4M尿素 /緩冲液 I重悬； 37*€ 200rpm振荡 1小时， 4*€放置过夜（10小时）； 4"C 12,000rpm 离心 10分钟，留上清，样品编号为 CD4(dl) 4M、CD4(d2) 4M、CD4(d3) 4M、 CD4(d4) 4M、 CD4(dl-2) 4M和 sCD4 4M;沉淀用等体积的 8M 尿素 /緩冲液 I重悬； 37 200rpm振荡 1小时， 放置过夜 ( 10 小时）； 4°C 12,000rpm离心 10分钟， 留上清。样品编号为 CD4(dl) 8M、 CD4(d2) 8M、 CD4(d3) 8M、 CD4(d4) 8M、 CD4(dl-2) 8M和 sCD4 8M;弃沉淀。分别取 CD4(dl) 、 CD4(d2)、 CD4(d3)、 CD4(d4)、 CD4(dl-2)和 sCD4的 2M/4M/8M上清各 ΙΟΟμί,加入凝股加样緩冲 液（50mM Tris Cl (pH6.8)， lOOmM二疏苏糖醇（ DTT ) , 2%SDS, 0.1%溴酚蓝， 10%甘油）； 沸水浴处理 10分钟； 12，000rpm离心 10 分钟； 取出 ΙΟμί进行 15%丙烯酰胺 SDS-PAGE分析， 以判断目的 蛋白在 2Μ/4Μ/8Μ尿素 /緩冲液 I中所占的比重。
各截短的 CD4重组多肽的复性： 分别将 CD4(dl)、 CD4(d2)、 CD4(d3)、 CD4(d4)、 CD4(dl-2)和 sCD4的 4M或 8M样品分装到透 析袋（美国联合碳化公司生产， 36DM, 截留分子量 8,000-10,000 ) , 按样品体积：透析液体积 =1:100的比例于 4 梯度透析至 lxPBS( 1L 中含有 Na2HP04 12H20 2.9g, KH2P04 0.24g, NaCl 8.0g, KC1 0.2g, pH7.4 ) 中 （8M尿素 /緩冲液 I-4M尿素 /緩冲液 I-2M尿素 /緩冲液 I-lxPBS ) 。 理论上， 透析结束时， 样品的尿素含量为 4xl(T6M; 透 析样品经 4*C 12,000rpm离心 10分钟； 上清液通过 0.22μιη的微孔 滤膜过滤， 可用于进一步纯化。
CD4(dl)、 CD4(d2)、 CD4(d3)、 CD4(d4)、 CD4(dl-2)和 sCD4 等截短的 CD4重组多肽的 HPLC凝胶过滤色谱纯化。
对复性样品采用如下 HPLC系统和方法进一步纯化。
仪器系统： Agilent Technologies 1200 series HPLC, 层析柱： TSK GEL G5000PWXL 7.8mmx30cm, 洗脱液： lxPBS pH7.4, 流 速： 0.5mL/min, 检测器波长： 280nm, 样品量： ΙΟΟμ 收集模式： 手动收集。 结果表明： 样品经 15%丙烯酰胺 SDS-PAGE分析目的蛋白峰的纯度， 纯度可达 70~90%。
图 2 显示了各表达质粒转化的大肠杆菌菌体经诱导后， SDS-PAGE ( A 小图为 CD4(dl)、 B 小图为 CD4(d2)、 C 小图为 CD4(d3)、 D小图为 CD4(d4)、 E小图为 CD4(dl-2)、 F小图为 sCD4 ) 显示 CD4(dl)、 CD4(d2)、 CD4(d3)、 CD4(d4)和 CD4(dl-2)、 sCD4 在相应位置有表达条带， 表达量约为 10~40%之间（图 2.A-F各小图 的第 2道） ， 主要以包涵体的形式存在。 经 2M、 4M和 8M尿素洗 涤， 可见 2M尿素上清（图 2.A~F各小图的第 3道）， 4M尿素上清 (图 2.A~F各小图的第 4道） ， 和 8M尿素上清（图 2.A~F各小图 的第 5道） ； 对 4M或 8M上清样品以梯度透析复性后， HPLC中 经分子筛柱层析纯化（图 2. A~F各小图的第 6道） 。 实施例 3. 单克隆抗体阻断 HIV感染 TZM-bl细胞活性验证 选用 5株分别属于 B、 C、 D、 D/C亚型的 HIV全基因实验株病 毒（HIVNL4.3， HIV89.6， HIV94 uGii4 » HIVMJ4， HIVWCML249 )， 通过细 胞中和实验测定单抗 15A7， 14G7对上述病毒感染 CD4+细胞的阻断 /中和效果。 TZM-bl细胞按照 1.5X104个细胞 /孔的浓度接种于 96孔 细胞培养板中， 12小时后用于检测。 按照如图 3坐标所列的稀释浓 度稀释单抗 15A7和 14G7, 在 96孔 U底板中每孔加入 ΙΟΟμί抗体 稀幹液和 50μί各 HIV病毒悬液（稀释至 100 TCID50 )， 37"€温育 1小时。 将混合液 150μί加入 96孔细胞培养板上培养好的 TZM-bl 细胞、 培养 48小时后， 显色， Elispot计数， 并计算中和效率。 对照组用 PBS代替 15A7抗体。结果如图 3和图 4，可见单抗 15A7, 14G7对上述各亚型 HIV均具有很好的阻断 /中和效果， 见表 2: 表 2.单抗 15A7, 14。7对5林111¥病毒的细胞中和效果
IC50( g/mL)
15A7 14G7
HIVNL4-3 0.465 1.126
HIV89.6 0.058 0.340
Figure imgf000048_0001
HIVMJ4 0.071 0.235
HIVwCML249 0.293 0.429 实施例 4.单克隆抗体 15A7, 14G7标记细胞表达 CD4的免疫 荧光检测
表达 CD4的细 Jf&^: Tzm-bl( Cat.No. 8129 National Institutes of Health AIDS Research and Reference Reagent Program ) ； U87.CD4.CXCR4 ( Cat.No.4036 National Institutes of Health AIDS Research and Reference Reagent Program ) ； U87.CD4.CCR5 ( Cat.No. 4035 National Institutes of Health AIDS Research and Reference Reagent Program )„上述 3株细胞是均可以稳、定表达 CD4 的贴壁细胞。
准备细胞： 实验前 1天将上述三株细胞用 0.25%的胰蛋白酶消 化制备成单细胞悬液， 浓度为 5xl05/mL， 将细胞悬液按照 100μΙ 孔接种入预先铺好无菌盖玻片的 24 孔板中， 然后每孔补充 400μΙ DMEM完全培养基 (添加 10%FBS, 2mM L-glutamine, O.lmM非 必须氨基酸及 1%双抗） 5%二氧化碳培养箱 培养 24小时。
细胞固定： 将 24孔板中的培养基吸出， 每孔加入 500μί PBS 洗一次， 然后每孔加入 500μί 4%多聚甲醛（ lOOmL PBS含有 4g多 聚甲醛）室温避光固定 5分钟， 吸出 4%多聚甲醛固定液。每孔加入 lmL PBS, 室温放置 5分钟， 然后吸出， 重复用 PBS洗 2次。 封闭： 在 24孔板盖上放一张封口膜， 24孔板上每个孔的位置滴 加 25μί山羊血清， 用眼 ϋ小心的从 24孔板中取出盖玻片， 用吸 水纸吸去盖玻片上多余的液体， 然后将盖玻片置于山羊血清的液滴 内， 确保细胞面向下。 将放有盖玻片的 24孔板盖放入湿盒（保持饱 和湿度） 室温孵育 1小时。
一抗孵育： 小心取出盖玻片， 翻动盖玻片使细胞面朝上， 再将 其放入 24孔板， 加入 lmL PBS室温放置 5分钟。 再取一张新的封 口膜覆盖在 24孔板盖上， 按照 24孔板上孔的位置， 滴加 25μί 单 克隆抗体 15A7, 14G7的 PBS稀幹液（ 1:1000稀释 ) 。 对照滴单克 隆抗体 H5F4 ( anti-P24 )的 PBS稀幹液（ 1:1000稀释 ) , 按照封闭 一样的操作， 将盖玻片放置于一抗的液滴内， 确保细胞面向下。 将 放有盖玻片的 24孔板盖放入湿盒（保持饱和湿度）室温孵育 1小时。
二抗孵育： 小心取出盖玻片， 翻动盖玻片使细胞面朝上， 再将 其放入 24孔板， 加入 lmL PBS室温放置 5分钟。 然后吸出 PBS, 重复用 PBS洗两次。 取一张新的封口膜覆盖在 24孔板盖上， 按照 24孔板上孔的位置，滴加 25μί二抗，二抗为 anti-Mouse IgG (whole molecule)-FITC ( Cat.No. F9006, Sigma )用 PBS 1:200稀释。 按照 封闭一样的操作， 将盖玻片放置于二抗的液滴内， 确保细胞面向下。 将放有盖玻片的 24孔板盖^湿盒（保持饱和湿度）室温避光孵育 30分钟。
DAPI染核： 小心取出盖玻片， 翻动盖玻片使细胞面朝上， 再将 其放入 24孔板， 加入 lmL PBS室温放置 5分钟。 然后吸出 PBS, 重复用 PBS洗两次。 取一张新的封口膜覆盖在 24孔板盖上， 按照 24孔板上孔的位置， 滴加 25μί用 PBS稀幹好的 DAPI ( 1:2000稀 释） ， 按照封闭一样的操作， 将盖玻片放置于 DAPI 的液滴内， 确 保细胞面向下。将放有盖玻片的 24孔板盖放入湿盒（保持饱和湿度 ) 室温避光孵育 5分钟。
封片： 小心取出盖玻片， 翻动盖玻片使细胞面朝上， 再将其放 入 24孔板， 加入 ImL PBS室温放置 5分钟。 然后吸出 PBS, 重复 用 PBS洗两次。标记载玻片， 并在上面滴一滴封片剂。从 24孔板中 取出盖玻片， 用吸水纸吸去盖玻片上多余的液体， 然后细胞面朝下， 将盖玻片置于封片剂液滴中。 用吸水纸轻轻除去盖玻片上的封片剂 , 再在边缘涂上指甲油， 用来封闭盖玻片边缘。 避光干燥。 用共激光 扫描共聚焦显微镜观察并拍照。
结果如图 5和图 6所示， 单克隆抗体 15A7, 14G7可以 f艮好的 结合在三株细胞的细胞膜上。 这表明单克隆抗体可以与三株细胞表 面表达的 CD4蛋白结合。
实施例 5.各截短的 CD4多肽阻断 /抑制单克隆抗体 15A7, 14G7 标记 CD4+细胞的流式细胞仪检测
Tzm-bl按照 1.5xl04个细胞 /孔铺于 96孔细胞培养板中， 5%二 氧化碳培养箱 孵育 12小时后用于实验。将初步纯化的 CD4(dl)， CD4(d2), CD4(dl-2), CD4(d3), CD4(d4), sCD4蛋白用 10%FBS 的 DMEM培养基稀释到终浓度 l(^g/mL，再分别加入按照稀释的单 克隆抗体 15A7、 14G7,使抗体的终浓度达到图 7和图 8横坐标轴中 标记的浓度， 然后 37 孵育 1小时 。将上述混合液加入预先铺好的 Tzm-bl细胞， 37"€孵育 1小时。用 0.25%的胰酶消化细胞，转入 4mL 离心管中， 2mLPBS洗一次， l，500rpm离心 5分钟弃上清，用 ΙΟΟμΙ 含有 RAM-FITC ( 1:200稀释 )的 PBS重悬细胞， 25 "C孵育 30分钟。
2mL PBS/管洗一次， l，500rpm离心 5分钟弃上清， 用 500μί PBS 重悬细胞， 流式细胞仪检测抗体结合情况。 具体结果如图 7和图 8 所示。 由图 7和图 8可知， CD4(dl-2)对单克隆抗体 15A7、 14G7与 细胞的反应有影响最显著 ( <0.01 ) ； CD4(dl)、 sCD4对单克隆抗 体 15A7, 14G7与细胞的反应有影响（ ρ<0·05 )；而 CD4(d2)、CD4(d3)、 CD4(d4)对单克隆抗体 15A7, 14G7与细胞的反应影响与 PBS差别 不大（Ρ>0·05 ) 。 实施例 6. 单克隆抗体轻链基因和重链基因可变区的克隆 半贴壁培养 107个表达抗体 15A7， 14G7的杂交瘤细胞， 吹管吹 起贴壁细胞使悬浮， 转移到新的 4mL 离心管中， l，500rpm 离心 3 分钟， 收集沉淀的细胞， 重悬于 ΙΟΟμί无菌 PBS ( ρΗ7·45 ) 中， 转 移到一新的 1.5mL离心管中。加入 800μί TrizoK Roche, Germany )， 轻轻颠倒混匀， 静置 10分钟。 加入 200μί 氯仿， 剧烈振荡 15秒， 静置 10分钟， 4°C 12，000rpm 离心 15分钟， 转移上层液体至一新的 1.5mL离心管中， 加入等体积的异丙醇， 混匀， 静置 10分钟。 4°C 12，000rpm离心 10分钟， 弃上清， 加入 600μί 75 %乙醇洗涤， 4。C 12,000rpm离心 5分钟， 弃上清， 沉淀于 60 "C真空抽干 5分钟。 透 明的沉淀溶于 70μί DEPC Η20中， 分装成两管。 每管加入 Ιμί反 转录引物， 其中一管加入的反转录引物为 MVJkR ( 5'-CCg TTT (T/g)AT (T/C)TC CAg CTT ggT (g/C)CC-3' )， 用于扩增轻链可变区 基因，另一管加入的反转录引物为 MVDJhR( 5，-C ggT gAC Cg(T/A) ggT (C/g/T)CC TTg (g/A)CC CCA-3' ) ， 用于扩增重链可变区基因。 每管再加入 luL dNTP ( 10mM, TAKARA ) ， 置 72°C 水浴 10分 钟， 立即放到冰浴中置 5分钟，加入 ΙΟμί 5x反转录緩冲液， Ιμί AMV ( lOimit/ L, Pormega ) , l L RNasin (40unit/ L， Promega), 混匀后于 42 "Ό将 RNA反转录成 cDNA。
抗体基因可变区的分离采用聚合酶链式反应 ( PCR ) 法， 使用 根据 Novagen公司的 Ig-Prime kits合成的引物组以及另外设计合成 的两条下游引物 MVJkR、 MVDJhR (上海博亚公司合成）， MVJkR 为轻链可变区基因扩增的下游引物， MVDJhR为重链可变区基因扩 增的下游引物。模板即为以上合成的两种 cDNA。 PCR条件为： 94°C 5分钟， 94°C 40秒， 53°C复性 1分钟， 72°C 50秒， 35个循环， 72°C 延伸 15分钟。 回收目的片段并克隆至 pMD 18-T载体（ TaKaRa ) , 并测序， 序列经 Blast分析比对后确定抗体可变区序列， 并推测出相 应的氨基酸序列。
依上述方法分别从单克隆抗体 15A7,14G7杂交瘤细胞株中克隆 出其抗体可变区基因， 并推算出相应的氨基^^列。 表 3所示为所 用的上游引物序列， 单克隆抗体 15A7 重链可变区核甘酸 SEQ ID ΝΟ:1; 重链氨基酸序列编号 SEQ ID NO:2; 轻链可变区核甘酸 SEQ ID NO:3; 轻链氨基酸序列编号 SEQ ID NO:4。 单克隆抗体 14G7重 链可变区核甘酸 SEQ ID NO:17; 重链氨基酸序列编号 SEQ ID NO:18; 轻链可变区核甘酸 SEQ ID NO:19; 轻链氨基酸序列编号 SEQ ID NO:20。 互补决定区 ( complementary determinant region, CDR )是通过 Kabat方法确定的 （Kabat EA， Wu TT， Perry HM, Gottesman KS, Coeller K. Sequences of proteins of immunological interest, U.S Department of Health and Human Services, PHS, NIH, Bethesda, 1991 ) 。
表 3.扩增 15A7，14G7可变区基因的引物序列
单抗林
引物名称 引物序列
可变区基因
15A7 Vh MuIgVh5'-E2 5'-GGCCCAGCCGGCCGAGGTGCAGCTG(G/T)TGGAG(A/T)C(C/
T)-3'
15A7 Vk MuIgkV15'-Dl 5'-GGTGGAGGCGGGAGTGAAACGACACTCACGCAGTCTC-3'
14G7 Vh MuIgVh5'-E2 5'-GGCCCAGCCGGCCGAGGTGCAGCTG(G/T)TGGAG(A/T)C(C/
T)-3'
14G7 Vk MuIgkV15'-Dl 5'-GGTGGAGGCGGGAGTGAAACGACACTCACGCAGTCTC-3' 将获得的可变区重链基因序列输入 VBASE2 数据库 ( http://www.vbase2.org/ )进行比对分析。 结果表明， 所克隆的 V 片段均为 Igh-VJ558 VH1族， Vh基因大小分别为 351bp,编码由 117 个氨基酸残基组成的多肽， 具有与 KABAT结构完全对应的结构特 征。 重链 CDR区如表 4所示。
将获得的可变区轻链基因序列输入 VBASE2 数据库进行比对 分析。 结果表明， 所克隆的 V片段均为 IGKV21 族， Vk基因大小 分别为 15A7 Vk: 339bp,编码由 113个氨基酸残基组成的多肽， 14G7 Vk: 324bp，编码由 108个氨基酸残基组成的多肽，具有与 KABAT结 构完全对应的结构特征。 轻链 CDR区如表 4所示。
表 4· 15A7,14G7 Vh 和 Vk CDR氨基酸序列
抗体 CDR氨基酸序列
单链抗体株
CDR1 CDR2 CDR3
GYFFTDYV IYPGSGSS ARRGTGLGFAY
15A7 Vh
(SEQ ID NO: 5) (SEQ ID NO:6) (SEQ ID NO: 7)
15A7 Vk QSVDYDGDSY VAS QQSNEDPPT
(SEQ ID NO: 8) (SEQ ID NO:9) (SEQ ID NO: 10)
GYTFTDYF IYPNNGGT ARRGYDEGFAY
14G7 Vh
(SEQ ID NO: 21) (SEQ ID NO:22) (SEQ ID NO: 23)
14G7 Vk QIIGTS YAS QQSNTTWPFT
(SEQ ID NO: 24) (SEQ ID NO:25) (SEQ ID NO: 26) 实施例 7. 单链抗体的表达及活性检测
将抗体基因的重链和轻链可变区通过（GGGGS ) 3短肽连接成 单链抗体 DNA片段。 以 15A7 VHF为正向引物， 15A7 VHR为反向 引物扩增出 15A7重链可变区片段，以 15A7 VKF为正向引物， 15A7 VKR为反向引物扩增出 15A7轻链可变区片段。 引物序列见表 5。 分别回收两个抗体基因片段。 再以重链可变区和轻链可变区片段互 为引物及模板在新的 PCR系统中进行重叠延伸，得到少量完整单链 抗体片段。然后以完整片段为模板，以 15A7 VHF为正向引物， 15A7 VKR为反向引物进行大量扩增重叠的 15A7单链抗体片段。 回收单 链抗体片段， 以 Nde l/Xho l酶切回收到单链抗体片段， 克隆到相同 酶切的 pET-30a-c(+)原核表达载体中， 载体如图 9所示。 以 ER2566 大肠杆菌作为表达菌株， 采用常规方法表达， 结果表达的蛋白质是 以不溶性的包涵体形式存在。 以常规方法洗涤纯化包涵体， 结果单 链抗体主要溶解于 8M尿素中。 将溶解于 8M尿素中的单链抗体蛋 白逐步对 lxPBS透析复性， 12，000rpm离心 10分钟去除沉淀， 将最 终得到的初步纯化的单链抗体溶液进行活性测定。
表 5. 单链抗体克隆用引物
引物名称 引物序列
15A7VHF 5'-CATATGATGCAAGTTTTACTACAGCAGTCT-3'
S'-ACTCCCGCCTCCACCGCTACCACCCCCTCCAGATCCG
15A7VHR
CCACCTCCTGCTGCAGAGACAGTGACCA-3'
S'-GGAGGTGGCGGATCTGGAGGGGGTGGTAGCGGTGGA
15A7VKF
GGCGGGAGT GACATTGTGCTGACCCAATC-3'
15A7VKR 5'-AAGCTTCTCGAG CCGTTTGATTTCCAGCTTGG-3'
14G7VHF 5'-CATATGATGGAGGTCCAGCTGCAACAG-3'
S'-ACTCCCGCCTCCACCGCTACCACCCCCTCCAGATCCG
14G7VHR
CCACCTCCAGAGACAGTGACCAGAGTCCCTT'
S'-GGAGGTGGCGGATCTGGAGGGGGTGGTAGCGGTGGA
14G7VKF
GGCGGGAGTGACATCTTGCTGACTCAGTC
14G7VKR S'-AAGCTTCTCGAGTTACCGTTTCATTTCCAGCTT -3' 采用竟争 ELISA法检测初步纯化出的 15A7单链抗体和 14G7 单链抗体的活性。在聚苯乙烯板条的孔中包被 CD4抗原，并用 lxED ( lxPBS溶液中加入 2%明胶、 0.5%酪蛋白和 2%蔗糖）封闭，待测 孔中加入 ΙΟΟμί上述单链抗体溶液，阳性对照孔中则分别加入 ΙΟΟμί 单克隆 15Α7、 单克隆抗体 14G7, 阴性对照孔中加入 ΙΟΟμΙ不相关 单抗 1F2稀释液或 10(^L PBS, 三孔重复。 各孔稍混匀后于 37 反 应 1小时后， 以 biotin标记的 15A7、 14G7再反应 1小时， 再加入 SA-HRP作为二抗反应 30分钟后，加入 A、 B显色液于 显色 15 分钟， 终止反应后用酶标仪读数。 结果见表 6和表 7, 15A7单链抗 体和 14G7 单链抗体均显示明显的阻断其对应的单克隆抗体与 CD4(D1)蛋白的结合。
表 6. 15A7单链抗体对 15A7全抗的阻断的活性检测
一抗反应物
包被抗原
15A7-scfv 15A7 PBS 1F2
CD4(dl) 1.068 0.522 1.678 1.592
CD4(dl-2) 1.159 1.080 1.209 1.095 sCD4 0.395 0.338 0.236 0.237
CD4(d2) 0.082 0.077 0.058 0.069 表 1. 14G7单链抗体对 14G7全抗的阻断的活性检测
一抗反应物
包被抗原
14G7-scfv 14G7 PBS 1F2
CD4(dl) 1.002 0.513 1.718 1.488
CD4(dl-2) 1.052 0.989 1.119 1.120 sCD4 0.412 0.323 0.225 0.253
CD4(d2) 0.076 0.058 0.049 0.058 采用 ELISA法检测初步纯化出的 15A7单链抗体、 14G7单链抗 体与 CD4蛋白反应活性。将 15A7单链抗体、 14G7单链抗体标记上 生物素 biotin,并用 HCV-ED稀幹。在聚苯乙烯板条的孔中包被 CD4 抗原 CD4(dl)、 CD4(d2)、 CD4(dl-2)、 CD4(d3)、 CD4(d4)和 sCD4， 并用 lxED封闭， 待测孔中加入 ΙΟΟμί上述标记了 biotin的单链抗 体溶液， 阳性对照孔中则加入 ΙΟΟμί标记了生物素（biotin )的单克 隆抗体 15A7、 标记了生物素（biotin )的单克隆抗体 14G7, 阴性对 照孔中加入 10(^L HCV-ED溶液， 三孔重复。各孔稍混勾后于 37*€ 反应 1小时后， 加入 SA-HRP作为二抗反应 30分钟后， 加入 A、 B 显色液于 37*€显色 15分钟， 终止反应后用酶标仪读数。 结果见表 8 和表 9, 说明初步纯化出的 15A7单链抗体蛋白对 CD4蛋白具有较 高的活性。
表 8. 15A7单链抗体与 CD4蛋白反应活性的检测
包被抗原 一抗反应物
15A7scfv-biotin 15A7-biotin 阴性对照
CD4(dl) 0.596 3.779 0.214
CD4(d2) 0.392 0.096 0.036
CD4(dl-2) 1.376 2.134 0.026
CD4(d3) 0.159 0.068 0.023
CD4(d4) 0.227 0.197 0.045 sCD4 0.438 0.428 0.091
N444(对照） 0.145 0.109 0.021
14G7单链抗体与 CD4蛋白反应活性的检测 包被抗原 一抗反应物
14G7scfv-biotin 14G7-biotin 阴性对照
CD4(dl) 0.615 3.256 0.198
CD4(d2) 0.422 0.076 0.043
CD4(dl-2) 1.469 2.348 0.031
CD4(d3) 0.148 0.076 0.025
CD4(d4) 0.212 0.232 0.051 sCD4 0.525 0.521 0.089
N444(对照） 0.160 0.115 0.023 实施例 8. 单克隆抗体 Fab片段及 F(ab')2的制备
单克隆抗体 Fab片段的制备：纯化的单克隆抗体 15A7( 2mg/mL ) 用 20mM PB ( pH 7.0 )緩冲液透析过夜。 将木瓜酶（购自 Sigma公 司） 以相同緩冲液配制成 lmg/mL， 内含 20mM EDTA, 20mM半 胱氨酸， 37"€孵育 30分钟，将激活的酶緩冲液加入透析后的抗体中， 酶:抗体重量比 = 1:1000， 37"€孵育 15小时。 加入礁乙酰胺（终浓度 30mM )避光水浴 1小时，终止反应。将酶切好的单克隆抗体对 20mM ΤΒ ( ρΗ8·0 )緩冲液透析过夜， 然后上 DEAE-HPLC纯化。用 20mM TB ( pH8.0 )平衡柱子， 用含有 0.05M NaCl的 20mM TB ( pH8.0 ) 线性洗脱 15分钟 Fab洗脱，用含有 1M NaCl的 20mM TB ( pH 8.0 ) 洗下 Fc及少量未切开的全抗。上样量 10-100mg抗体，流速 lmL/min, 检测波长 280nm。 收集的 FabSDS-PAGE检测纯度后用 10kD规格 浓缩管浓缩， 对 20mM PBS ( pH7.4 )透析后， 除菌 （ 0·22μιη微孔 滤膜）分装， 即得单克隆抗体 15A7的 Fab纯品。
单克隆抗体 F(ab')2片段的制备： 纯化的单克隆抗体 15A7 ( 2mg/mL )用 6mM Na2HP04 12H20与 7mM柠檬酸混合緩冲液透 析过夜。将胃蛋白 S (购自 Sigma公司）以相同緩冲液配制成 lmg/mL 加入透析后的抗体中， 酶:抗体重量比 = 1:100, 37 孵育 12小时。 将酶切好的单克隆抗体对 20mMTB ( H 8.0 )緩冲液透析过夜， 然 后上 DEAE-HPLC纯化。 用 20mM TB ( pH 8.0 )平衡柱子， 用含有 1M NaCl的 20mM TB ( pH 8.0 )线性洗脱 30分钟，分别洗下 F(ab')2 和少量未切开的全抗。 上样量 10-100mg抗体， 流速 lmL/min,检测 波长 280nm。 收集的 F(ab')2 经 SDS-PAGE检测纯度后用 10kD规 格浓缩管浓缩， 对 20mM PBS ( pH7.4 )透析后， 除菌 （ 0·22μιη微 孔滤膜）分装， 即得单克隆抗体 15A7的 F(ab')2纯品。
纯化后的单克隆抗体 15A7, 单克隆抗体 15A7 Fab片段和单克 隆抗体 F(ab')2片段 SDS-PAGE结果如图 10所示： 其中第 1道是蛋 白分子量 marker; 第 2道是经过还原剂 β-巯基乙醇后沸水浴 10分 钟处理的单克隆抗体 15A7; 第 3道是经过还原剂 β-巯基乙醇后沸水 浴 10分钟处理的单克隆抗体 15A7 Fab片段； 第 4道是经过还原剂 β-巯基乙醇后沸水浴 10分钟处理的单克隆抗体 15A7 F(ab')2片段； 第 5道是单克隆抗体 15A7; 第 6道是 15A7 Fab片段； 第 7道是单 克隆抗体 15A7 F(ab')2片段。 实施例 9. 抗体 15A7及其活性片段中和活性 IC50的测定 选用 B亚型病毒株 HIVNL4-3, 用细胞中和实验检测抗体 15A7及 其活性片段中和活性 IC50。TZM-bl细胞按照 1.5X104个细胞 /孔的浓 度接种于 96孔细胞培养板中， 12小时后用于检测。 按照 ΙΟΟμ^ϋΐΙ 的稀释浓度 2倍稀释抗体 15A7及其 Fab、 F(ab')2片段， 稀释 20个 梯度，在 96孔 U底板中每孔加入 ΙΟΟμί抗体稀释液和 50μί HIV病 毒悬液 (稀释至 100 TCID50 ) ， 37"€温育 1小时。 将混合液 150μί 加入 96孔细胞培养板上培养好的 TZM-bl细胞， 37"€培养 48小时 后， 显色后用 Elispot计数， 按照下列公式计算中和效率， 50%以上 中和活性的定为阳性， 低于 50%中和活性的为阴性。 对照组用 PBS 代替 15A7抗体。 图 11显示了单克隆抗体 15A7和其 Fab和 F(ab')2 片段中和 HIVNL4-3的效果，其中 F(ab')2中和效果与全抗体相当。 IC50 的计算方法如下：
lgIC50=Xm-I(P-(3-Pm-Pn)/4)
其中： Xm为 lg 最大剂量； I为 lg (最大剂量 /相临剂量)； P为阳 性反应率之和； Pm为最大阳性反应率； Pn为最小阳性反应率。 结 果总结见表 10。
表 10. 单抗 15A7及其片段对 5株 HIV病毒的细胞中和效果
IC50( g/mL)
15A7 15A7-Fab 15A7-F(ab')2
HIVNL4-3 0.465 10.297 0.687
HIV89.6 0.058 NT* NT
Figure imgf000059_0001
HIVMJ4 0.071 NT NT
HIVwCML249 0.293 NT NT
*NT: 未检测 实施例 10. 单克隆抗体 15A7标记 CD4+细胞的流式细胞仪检测 本实验所用的表达 CD4 的细胞系： Tzm-bl ( Cat.No. 8129 National Institutes of Health AIDS Research and Reference Reagent Program ) ； U87.CD4.CXCR4 ( Cat.No.4036 National Institutes of Health AIDS Research and Reference Reagent Program ) ； U87.CD4.CCR5 ( Cat.No. 4035 National Institutes of Health AIDS Research and Reference Reagent Program ) ； MT4(Cat.No. 120 National Institutes of Health AIDS Research and Reference Reagent Program)； H9 ( Cat.No. 87 National Institutes of Health AIDS Research and Reference Reagent Program ) 。 上述 5林细胞均可以 稳定表达 CD4, 其中 Tzm-bl U87.CD4.CXCR4, U87.CD4.CCR5 3 株为贴壁细胞， MT4、 H9为悬浮细胞。
PBMC抽取健康志愿者的静脉血分离， 步骤如下： 抽取静脉血 20mL加到肝素（ 5-10IU/mL )抗凝管中，加入 20mL PBS悬浮细胞。 将 20mL 细胞悬液小心加在 lOmL Ficoll-Paque PLUS ( Cat.No. 17-1440-03 GE Healthcare Bio-Sciences AB )上， 室温水平离心 500g 20分钟。 此时离心管中形成分层， 最上面是血浆， 血浆层和淋巴细 胞分离液之间是 PBMC, 吸去部分最上层的血浆， 收集血浆层和淋 巴细胞分离液交界面的 PBMC到 50mL离心管中。加入 40mL PBS, 混匀后 200g离心 10分钟，弃掉上清，收集细胞沉淀，加入 40mL PBS 重悬， 混匀后 500g离心 10分钟， 再重复 1次。 用 1 %台盼蓝染色检 测细胞活力 （应 >95 % ) 并计数细胞， 用 AIM-V Medium ( Cat.No.12055 Invitrogen )培养基培养。
实验所用抗体： FITC标记的 anti-CD4 ( Cat.No. 555346, BD Biosciences ) , PE标记 CXCR4 ( Cat.No. 555974, BD Biosciences ) , PE-Cy5标记 CCR5 ( Cat.No. 556889, BD Biosciences )以上抗体全 部用 PBS 1:200稀释用。
单克隆抗体 15A7 ( 1:1000稀释）作为一抗， Anti-Mouse IgG (whole molecule)-FITC ( Cat.No. F9006 Sigma )用 PBS 1:200稀释 作为二抗。
准备细胞： 将 Tzm-bl、 U87.CD4.CXCR4, U87.CD4.CCR5 等 3林贴壁细胞用 0.25%的胰蛋白酶消化制备成单细胞悬液， MT4、 H9和 PBMC为悬浮细胞， 用移液管吹打成单细胞悬液， 细胞浓度 均调整为 lxlo mL, 取 4mL细胞悬浮液等量分装 8管， 并随机分 成 、 B、 C、 D四组， 每组两管， 一管用于抗体标记， 一管为阴性 对照。 每管细胞加入 1.5mL PBS颠倒混匀， 1500rpm离心 5分钟， 弃掉上清， 收集细胞沉淀。
标记抗体：用稀释好的抗体溶液 200μΙ重悬细胞并混勾。 Α、 Β、 C 组 分 别 加 入 FITC-anti-CD4 、 PE-anti-CXCR4 、 PE-Cy5-anti-CCR5抗体， 室温避光孵育 30分钟。 加入 2mL PBS混 匀， l，500rpm离心 5分钟，弃掉上清，收集细胞沉淀，并用 500μί PBS 重悬。 流式细胞仪检测抗体标记情况。
D组一抗用单克隆抗体 15A7溶液重悬细胞， 室温孵育 1小时， 加入 2mL PBS混匀， 1500rpm离心 5分钟， 弃掉上清， 重复上述操 作 1 次， 收集细胞。 200 L 二抗 anti-Mouse IgG (whole molecule)-FITC溶液重悬细胞并混匀， 室温避光孵育 30分钟。 加入 2mL PBS, l，500rpm离心 5分钟， 弃掉上清， 收集细胞沉淀， 并 用 500μί PBS重悬。流式细胞仪检测抗体标记情况：图 12是 Tzm-bl 细胞标记后的流式细胞仪检测检测结果； 图 13是 MT4细胞标记后 的流式细胞仪检测检测结果； 图 14是 H9细胞标记后的流式细胞仪 检测检测结果； 图 15是 U87.CD4.CXCR4细胞标记后的流式细胞仪 检测检测结果；图 16是 U87.CD4.CCR5细胞标记后的流式细胞仪检 测检测结果； 图 17是 PBMC细胞标记后的流式细胞仪检测检测结 果。 各细胞标记后的阳性率结果统计于表 11 , 流式检测结果将阴性 对照值设定在 1%， 15A7 抗体与各细胞系表面受体的结合情况与 anti-CD4抗体具有明显的相关性。 表 11. 单抗 15A7对各细胞系表面受体流式检测结果
CD4 CXCR4 CCR5 15A7
Tzm-bl 99.7% 97.2% 99.4% 100%
MT4 84.3% 20.3% 2.2% 99.5%
H9 80.4% 96.8% 21.0% 99.4%
U87.CD4.CXCR4 18.4% 90.2% 32.1% 41.0%
U87.CD4.CCR5 97.7% 55.0% 91.3% 99.4%
PBMC 33.8% 90.7% 19.1% 51.0%
实施例 11. 单克隆抗体 15A7与表达 HIV的细胞结合情况的流 式细胞仪检测
293-FT细胞按照 5xl04个细胞 /孔的浓度接种于 24孔细胞培养 板中， 5%二氧化碳培养箱 37*€孵育 12小时，用 0.6μδ pNL4-3质粒 ( HIVNIj4_3感染性克隆全基因质粒） 转染细胞， 转染试剂为梭华 ( Sofast，厦门太阳马生物工程有限公司） ， 转染方法参见该试剂的 操作指南； 用未转染的细胞作为对照。 转染 48小时后， 将 24孔板 中的培养基吸出， 用 0.25%的胰蛋白酶消化制备单细胞悬液， 将细 胞转移到 4mL离心管中， 每孔对应一管。 每管细胞悬液加入 1.5mL PBS混匀， l，500rpm离心 5分钟，弃掉上清，收集细胞沉淀。用 200μΙ 单克隆抗体 15A7工作液（ 1:1000稀释）重悬细胞； 对照组用 PBS 重悬细胞， 用 1:100稀释的 anti-p24单抗 H5F4作为 pNL4-3转染效 果的参照， 混匀后室温孵育 1 小时， 每管加入 2mL PBS 混匀， 1500rpm离心 5分钟， 弃掉上清， 收集细胞沉淀。 用 PBS 1:200稀 释 anti-Mouse IgG (whole molecule)-FITC ( Cat.No. F9006, Sigma ) 作为二抗， 每管加入 200μί二抗重悬细胞并混勾， 室温避光孵育 30 分钟。 然后加入 2mL PBS混匀， l，500rpm离心 5分钟， 弃掉上清， 收集细胞沉淀， 用 50(^L PBS重悬。 用流式细胞仪检测抗体标记与 蛋白孵育后的细胞的情况。 结果见图 18, 阳性率结果总结于表 12 中， 可见： p24表达在 293FT-HIV+细胞中有表达， 阳性率为 13.4% , 说明 pNL4-3质粒转染细胞后在细胞内复制表达。 单克隆抗体 15A7 与 293FT-HIV结合效果效率为 1% , 可知 15A7与 HIV不结合。
表 12. 15A7与 293FT和转染了 pNL4-3的 293FT结合情况
15A7 H5F4 PBS
293FT-HIV 1.0% 13.4% 1.1%
293FT 1.1% 1.2% 0.8%
实施例 12. 单克隆抗体 15A7标记表达 HIV的细胞的免疫荧光 检测
准备细胞： 使用 0.25%的胰蛋白酶将 293FT细胞消化并制备成 单细胞悬液。 将细胞按照 5xl04个细胞 /孔的浓度孔接种入预先铺好 无菌盖玻片的 24孔板中， 然后每孔补充 400μί DMEM完全培养基 (添加 10%FBS, 2mM L-glutamine, O.lmM非必须 J ^酸及 1%双 抗） 5%二氧化碳培养箱 培养 12小时。 用 0.6 g pNL4-3质粒 ( HIVNIj4_3感染性克隆全基因质粒） 转染细胞， 转染试剂为梭华 ( Sofast，厦门太阳马生物工程有限公司） ， 转染方法参见该试剂的 操作指南；用未转染的细胞作为对照。转染 48小时后进行细胞固定。
细胞固定通透：将 24孔板中的培养基吸出，每孔加入 500μί PBS 洗一次， 然后每孔加入 500μί 4%多聚甲醛（ lOOmL PBS含有 4g多 聚甲醛）室温避光固定 5分钟， 吸出 4%多聚甲醛固定液。每孔加入 lmL PBS ,室温放置 5分钟，然后吸出，重复用 PBS洗 2次。将 PBS 吸出， 每孔加入 500μΙ 0.3% Triton-XlOO ( lOOmL PBS含有 300μί Triton-XlOO ) 室温通透 5分钟， 吸出 0.3% Triton-X100。 每孔加入 lmL PBS , 室温放置 5分钟， 然后吸出， 重复用 PBS洗 2次。 封闭： 在 24孔板盖上放一张封口膜， 24孔板上每个孔的位置滴 加 25μί山羊血清， 用眼 ϋ小心的从 24孔板中取出盖玻片， 用吸 水纸吸去盖玻片上多余的液体， 然后将盖玻片置于山羊血清的液滴 内， 确保细胞面向下。 将放有盖玻片的 24孔板盖放入湿盒（保持饱 和湿度） 室温孵育 1小时。
一抗孵育： 小心取出盖玻片， 翻动盖玻片使细胞面朝上， 再将 其放入 24孔板， 加入 lmL PBS室温放置 5分钟。 再取一张新的封 口膜覆盖在 24孔板盖上， 按照 24孔板上孔的位置， 滴加 25μί 单 克隆抗体 15A7的 PBS稀释液（ 1:1000稀幹） 。 对照滴单克隆抗体 H5F4 ( anti-P24 )的 PBS稀释液（ 1:1000稀释） ， 按照封闭一样的 操作， 将盖玻片放置于一抗的液滴内， 确保细胞面向下。 将放有盖 玻片的 24孔板盖^湿盒（保持饱和湿度） 室温孵育 1小时。
二抗孵育： 小心取出盖玻片， 翻动盖玻片使细胞面朝上， 再将 其放入 24孔板， 加入 lmL PBS室温放置 5分钟。 然后吸出 PBS, 重复用 PBS洗两次。 取一张新的封口膜覆盖在 24孔板盖上， 按照 24孔板上孔的位置，滴加 25μί二抗，二抗为 anti-Mouse IgG (whole molecule)-FITC ( Cat.No. F9006, Sigma )用 PBS 1:200稀释。 按照 封闭一样的操作， 将盖玻片放置于二抗的液滴内， 确保细胞面向下。 将放有盖玻片的 24孔板盖^湿盒（保持饱和湿度）室温避光孵育 30分钟。
DAPI染核： 小心取出盖玻片， 翻动盖玻片使细胞面朝上， 再将 其放入 24孔板， 加入 lmL PBS室温放置 5分钟。 然后吸出 PBS, 重复用 PBS洗两次。 取一张新的封口膜覆盖在 24孔板盖上， 按照 24孔板上孔的位置， 滴加 25μί用 PBS稀幹好的 DAPI ( 1:2000稀 释） ， 按照封闭一样的操作， 将盖玻片放置于 DAPI 的液滴内， 确 保细胞面向下。将放有盖玻片的 24孔板盖放入湿盒（保持饱和湿度） 室温避光孵育 5分钟。
封片： 小心取出盖玻片， 翻动盖玻片使细胞面朝上， 再将其放 入 24孔板， 加入 ImL PBS室温放置 5分钟。 然后吸出 PBS, 重复 用 PBS洗两次。标记载玻片， 并在上面滴一滴封片剂。从 24孔板中 取出盖玻片， 用吸水纸吸去盖玻片上多余的液体， 然后细胞面朝下， 将盖玻片置于封片剂液滴中。 用吸水纸轻轻除去盖玻片上的封片剂 , 再在边缘涂上指甲油， 用来封闭盖玻片边缘。 避光干燥。 用激光扫 描共聚焦显微镜观察并拍照。
结果如图 19所示， 单克隆抗体 15A7没有结合在 293FT和转染 过 pNL4-3质粒的 293FT细胞上。 表明单克隆抗体与 HIV不结合。 实施例 13. 截短的 CD4多肽阻断 HIV感染细胞的竟争中和实验 Tzm-bl细胞按照 1.5X104个细胞 /孔铺与 96孔板中， 5%二氧化 碳培养箱 37 孵育 12小时后用于中和实验。将初步纯化的 CD4(dl), CD4(dl-2), CD4(d2), CD4(d3), CD4(d4)梯度稀释后按照 ΙΟΟμΙ^孔 与 100 TCID50的 HIVNL4_3混合后 37*€孵育 1小时， 将上述混合液 加入预先铺好的 Tzm-bl细胞， 5%二氧化碳培养箱 37 培养 48小时， 显色后用 Elispot计数，并计算中和效率。结果如图 20所示， CD4(dl)、 CD4(dl-2)和 sCD4具有很好的阻断 HIV病毒感染细胞的活性：其中 CD4(dl)的 IC50为 0.143 g/mL, CD4(dl-2)的 IC50为 0.25(^g/mL, sCD4的 IC50为 1.815 g/mL。 实施例 14. 单克隆抗体 15A7与各截短的 CD4多肽的反应性 间接 ELISA法检测： 将纯化的 CD4(dl)、 CD4(d2)、 CD4(d3)、 CD4(d4)、 CD4(dl-d2)和 sCD4蛋白用 IxPBS包被緩冲液分别稀释 至终浓度 0.5 g/mL, 在 96孔聚乙烯微量滴定板的各孔表面上 吸附 2小时， 再置于 4*€过夜。 用 PBST洗涤液（8.0g NaCl、 0.2g KH2P04、 2.9g Na2HP04 12H20, 0.2g KC1和 0.5mL吐温 20， 加去 离子水至 1升， pH为 7.4 ) 洗涤滴定板以除去未结合的抗原蛋白一 次。 然后用 200μΙ孔的封闭液（lxPBS溶液中加入 2%明胶、 0.5% 酪蛋白和 2%蔗糖） 37*€封闭 2小时。 甩净、 拍干后真空封闭， 4*€ 保存。
检测时， 于每孔中加入每孔加入 ΙΟΟμί稀释的 15A7抗体每次 反应设置一不相关单抗的阴性对照。 37*€温育 1小时， 用 PBST洗 涤液洗 5遍， 拍干后加入 1:5000稀释的过氧化物酶标记的羊抗鼠免 疫球蛋白 （HRP-GAM Ig, DAKO公司） ， 37"€温育 30分钟， 用 PBST洗涤液洗 5遍， 拍干后先后加入底物液 A、 B各 50μί (底物 液 Α成分为： 13.42g Na2HP04.12H20、 4.2g柠檬酸 H20和 0.3g过 氧化氢， 用去离子水中调节体积为 700mL; 显色液 B成分为： 0.2g 四甲基联苯胺、 20mL 二甲基甲酰胺用去离子水中调节体积为 700mL ) , 37*€显色 15分钟， 加入 50μί终止液（ 2Μ H2S04 )终止 反应，并于酶标仪上检测各孔的 OD450值。表 13结果可知， CD4(dl)、 CD4(dl-2)和 sCD4蛋白与抗体 15A7有反应。
表 13. CD4(dl)、 CD4(d2)、 CD4(d3)、 CD4(d4)、 CD4(dl-2)和 sCD4蛋白 ELISA活性检测 单抗浓度 (ng/mL) 10,000 5,000 2,500 1,250 625 310 155 75 37 18 9 P
15A7 2.7575 2.683 2.7345 2.0185 1.6845 1.0415 0.648 0.257 0.047 0.0785 0.0395 0.0
CD4(dl)
Control 0.134 0.062 0.038 0.009 0.0045 0.003 0.002 0.0075 0.005 0.003 0.01 0.0
15A7 0.005 0.016 0.0045 0.0065 0.0055 0.002 0.0025 0.0035 -0.0005 -0.0105 -0.0005 0.0
CD4(d2)
Control 0.0505 0.0305 0.0235 0.0125 0.013 0.0165 0.014 0.01 0.0115 0.0075 0.009 0.0
15A7 0.051 0.0115 0.005 0.0115 0.007 0.0115 0.013 0.0035 0.0025 0.0075 0.007 0.
CD4(d3)
Control 0.2695 0.0165 0.015 0.0155 0.009 0.0095 0.018 0.0045 0.004 0.0065 0.0235 0.
15A7 0.1135 0.0275 0.0155 0.016 0.0105 0.021 0.0055 0.007 0.004 0.0025 0.0035 0.0
CD4(d4)
Control 0.1115 0.023 0.017 0.0095 0.007 0.0055 0.0005 0.004 0.0055 0.0055 0.0065 0.0
15A7 1.6685 1.19 0.9905 0.934 0.505 0.369 0.292 0.1275 0.0405 0.0375 0.021 0.00
CD4(dl-2)
Control 0.0385 0.0095 0.006 0.005 0.0075 0.0055 0.005 0.003 0.0055 0.0075 0.0055 0.00
15A7 0.895 0.7406 0.6603 0.3087 0.287 0.1553 0.1216 0.066 0.038 0.016 0.017 0.0 sCD4
Control 0.01 0.011 0.013 0.007 0.018 0.0103 0.007 0.0243 0.017 0.009 0.007 0.0
Western法检测： HPLC纯化后的各 CD4重组蛋白还原样及非 还原样（不含 DTT )分别以 15 %的 SDS-PAGE电泳分离， 电转移 至硝酸纤维素膜上，加入 5 %的脱脂奶于室温封闭 1.5小时；将用 5 % 脱脂奶封闭好的膜裁剪成约 0.8cm宽的条， 分别加入单克隆抗体， 室温反应 1小时； TNT ( 8.765g NaCl、 1.21g Tris Base和 0.5mL吐 温 20, 加去离子水至 1L, 调 pH为 8.0 )洗涤三次， 每次间隔 5分 钟； 以 5 %脱脂奶 1:5000稀释的 AP标记山羊抗小鼠（ AP-GAM, H+L )酶标二抗（Protos公司产品） ， 室温反应 1小时； TNT洗涤 三次， 每次间隔 5分钟； 向 10mL AP緩冲液（ lOOmM NaCl, 5mM MgCl2, lOOmM Tris-HCl,调 pH为 9.5 )中加入 66μί NBT溶液（ 0.5g NBT溶于 10 mL 70%的二甲基甲酰胺中）和 33μί BCIP溶液（ 0.5g BCIP 溶于 lOmL 70%的二甲基甲酰胺中）， 室温反应 15 分钟。
Western印迹结果见图 21: 可见单克隆抗体 15A7仅与还原组分的 CD4(dl-2)和 sCD4有较好的反应性； 而与非还原组分的 CD4(dl-2) 和 sCD4有较好的反应性，与非还原组分的 CD4(dl)有较弱的反应性。 实施例 15. 单克隆抗体 15A7在有或无 HIV病毒存在情况下标 记 CD4+细胞的检测
准备细胞： TZM-bl细胞按照 lxlO5个细胞 /孔的浓度接种于 24 孔细胞培养板中， 12小时后用于实验。 24孔板中加入病毒（稀释至 100 TCID50 ) , 对照用 DMEM培养基代替。 孵育 2小时后， 弃含有病毒的上清液， 并用 PBS洗 3次。 加入单克隆抗体 15A7的 稀释液， 37*€孵育 1小时。 用 0.25%的胰酶消化细胞， 转入 4mL离 心管中， 2mLPBS洗一次， l，500rpm离心 5分钟 弃上清， 用 ΙΟΟμΙ 含有 RAM-FITC(1:200稀释)的 PBS重悬细胞， 25*€孵育 30分钟。
2mL PBS/管洗一次， l，500rpm离心 5分钟弃上清， 用 50(^LPBS重 悬细胞， 流式细胞仪检测抗体结合情况。 结果如图 22所示： 在 HIV 存在的情况下， 单克隆抗体 15A7仍可标记 TZM-bl , 且标记的阳性 率有明显增强 （ρ<0·05 ) 。 实施例 16. 单克隆抗体 15A7对细胞的毒性检测
TZM-bl细胞按照 1.5X104/孔的浓度接种于 96孔细胞培养板中， 5%二氧化碳培养箱 37 孵育 12小时用于检测。将 96孔板中的培养 基吸出， 加入 ΙΟΟμί无酚红的 DMEM培养基， 5%二氧化碳培养箱 37*€孵育 2小时， 按照 Cell Counting Kit-8试剂盒（碧云天生物技 术研究所）说明书每孔加入 ΙΟμί CCK-8, 5%二氧化碳培养 箱孵育， 并在孵育 20、 40、 60分钟后分别取样用酶标仪读板， 检测 其吸光值。 本次实验用 PBS代替单克隆抗体 15A7作为对照， 无酚 红的 DMEM培养基不加细胞作为背景。具体吸光值见表 14， 15Α7、 PBS与 DMEM培养基的吸光值无显著差异（ Ρ>0·05 ) , 这表明单克 隆抗体 15A7对 Tzm-bl细胞没有毒性。
表 14. 15A7单抗细胞毒性检测
20分钟 40分钟 60分钟
MOCK 0.031 0.021 0.028
15A7 0.558 0.769 0.783
PBS 0.451 0.635 0.655
DMEM 0.485 0.751 0.749 实施例 17. 15A7 与截短的 CD4 多肽孵育后阻断 HIV 感染 Tzm-bl细胞的中和实驗
选用 B亚型的 HIV全基因实验株病毒 HIVNL4_3利用细胞中和实 验检测单抗 15A7与截短的 CD4多肽孵育后对 Tzm-bl细胞的感染 情况。 Tzm-bl细胞按照 1.5xl04/孔铺与 96孔板中， 12小时后用于中 和实验。 按照 lOO g/mL的稀释浓度梯度稀释单克隆抗体 15A7, 倍 比稀释 20 个梯度， 稀幹的培养基中分别加入相应的 CD4(dl) , CD4(dl-2), sCD4多肽， 对照为 PBS, 实验按照 25pM的浓度使用 sCD4多肽，稀释液体积为 ΙΟΟμΙ^孔。 同时用 PBS代替 15A7抗体梯 度稀释抗体，稀释的培养基中也分别加入相应的 CD4(dl), CD4(dl-2) sCD4多肽，从而验证 sCD4多肽自身阻断 HIV感染 Tzm-bl的情况。
HIV用量为 100 TCID50/孔 ， 体积为 50μΙ 孔， 与上述稀释液混合 后在 37。C恒温培养箱中孵育 1小时， 将上述混合液加入预先铺好的 Tzm-bl细胞中， 再在 37 5%二氧化碳培养箱培养 48小时后固定 通透并显色。 用 Elispot计数并计算中和效率。 实验结果见附图 23: 从实验结果可以看出， CD4(dl)， CD4(dl-2), sCD4多肽对单克隆抗 体 15A7阻断 HIV感染 TZM-bl有明显的影响 （ ρ<0·01 ) 。 实施例 18. CD4(dl)、 CD4(dl-2)、 sCD4与转染了 HIV感染性克 隆质粒的细胞流式细胞仪检测
293-FT细胞按照 5xl04个细胞 /孔的浓度接种于 24孔细胞培养 板中， 5%二氧化碳培养箱 37*€孵育 12小时，用 0.6μδ pNL4-3质粒 ( HIVNIj4_3感染性克隆全基因质粒） 转染细胞， 转染试剂为梭华 ( Sofast，厦门太阳马生物工程有限公司） ， 转染方法参见该试剂的 操作指南； 用未转染的细胞作为对照。 转染 48小时后， 将 24孔板 中的培养基吸出， 用 0.25%的胰蛋白酶消化制备单细胞悬液， 将细 胞转移到 4mL离心管中， 每孔对应一管。 每管细胞悬液加入 1.5mL PBS混匀， l，500rpm离心 5分钟，弃掉上清，收集细胞沉淀。用 200μΙ PBS重悬细胞，加入终浓度为 0·5μΜ的 CD4(dl)、 CD4(dl-2)、 sCD4 蛋白；对照组用 PBS替代蛋白溶液，室温孵育 1小时。每管加入 2mL PBS混匀， l，500rpm离心 5分钟，弃掉上清，收集细胞沉淀。用 200μΙ 单克隆抗体 15A7工作液（ 1:1000稀释 )重悬细胞； 对照组用 200μΙ 1:100稀释的 anti-Human CD4 ( Clone Q4120, Sigma, 识别表位位 于 CD4 D1结构域）重悬细胞， 用 1:100稀释的 anti-p24单抗 H5F4 作为 pNL4-3转染效果的参照， 混匀后室温孵育 1 小时， 每管加入 2mL PBS混匀， 1500rpm离心 5分钟， 弃掉上清， 收集细胞沉淀。 用 PBS 1:200稀释 anti-Mouse IgG (whole molecule)-FITC ( Cat.No. F9006, Sigma )作为二抗， 每管加入 200μΙ二抗重悬细胞并混匀， 室温避光孵育 30分钟。 然后加入 2mL PBS混匀， l，500rpm离心 5 分钟， 弃掉上清， 收集细胞沉淀， 用 50(^L PBS重悬。 用流式细胞 仪检测抗体标记与蛋白孵育后的细胞的情况。 结果见图 24: 其中 A 小图是以单克隆抗体抗体 15A7 为第一抗体标记分别与 CD4(dl)、 CD4(dl-2)、 sCD4蛋白孵育的 293FT细胞、 293FT-HIV+细胞的结果； B小图是 anti-Human CD4 ( Clone Q4120 )为第一抗体标记分别与 CD4(dl)、 CD4(dl-2)、 sCD4蛋白孵育的 293FT细胞、 293FT-HIV+ 细胞的结果。 阳性率结果总结于表 15 中， 可见： p24 表达在 293FT-HIV+细胞中有表达， 阳性率为 7.2%， 说明 pNL4-3质粒转染 细胞后在细胞内复制表达， 但由于 p24蛋白主要存在于细胞内部， 因此在没有通透的情况下， 检测阳性率偏低； 通过对照抗体 anti-Human CD4 ( Clone Q4120 ) 标记细胞的阳性率结果可知 CD4(dl)、CD4(dl-2)、sCD4均与 293FT-HIV+细胞有结合，其中 sCD4 结合能力最强；单克隆抗体 15A7与结合了上述 CD4(dl)、 CD4(dl-2)、 sCD4 蛋白的 293FT-HIV+细胞的标记情况比阳性对照抗体 anti-Human CD4 ( Clone Q4120 )与相应细胞的标记效果好。 表 15. 15A7检测 CD4蛋白与转染了 pNL4-3的 293FT结合情况
CD4(dl) CD4(dl-2) sCD4 H5F4 PBS
15A7 0.6% 0.7% 6.1%
Figure imgf000072_0001
anti-CD4 1.2% 0.7% 3.4%
15A7 8.0% 13.2% 28.2%
293FT-HIV+ /.I /o 1.0%
anti-CD4 7.7% 10.2% 13.6%
实施例 19. 单克隆抗体 15A7与 CD4(dl-2)、 sCD4亲和力的测 定
将实施例 2中纯化的 CD4(dl-2)、 sCD4蛋白标记上生物素， 具 体操作如下： 将上述蛋白用 PBS稀释到 4mL终浓度为 10μΜ， 加入 ΙΟΟμί 浓度为 4mM 的生物素（EZ-Link Sulfo-NHS-LC-Biotin , Cat.No· 21335, Thermo Scientific), 混匀后室温孵育 2小时， 然后 装入透析袋中， 放入 1L PBS中 4"€搅拌下透析， 3小时更换一次透 析液， 重复 4次。
将上述透标记有生物素的 CD4(dl-2)、 sCD4 作为抗原， 结合 Streptavidin(SA) Biosensors探针 (Cat.No· 185019， ForteBio), 按照 操作手册提示， 用 ForteBio Octet RED蛋白相互作用仪分析单克隆 抗体 15A7与 CD4(dl-2)、 sCD4蛋白的亲和力： 单克隆抗体 15A7用 PBS稀释到 400nM、 100nM、 50nM三个浓度检测并计算出亲和力， 阴性对照抗体为 anti-P24单抗 H4F5。结果见表 16:单克隆抗体 15A7 与两种蛋白的亲和力接近 1.20χ10·9Μ。
表 16. 单克隆抗体 15A7与截短的 CD4蛋白的亲和力
KD (M) ka (1/Ms) kd (l/s)
CD4(dl-2) 1.18X10-9 4.22x10s S.OOxlO—4 sCD4 1.20X10-9 3.37x10s 4.04xl0 4 实施例 20. 点突变的 CD4(dl-2)多肽的表达
对区段 CD4基因多肽重组表达克隆 pTO-T7-CD4(dl-2)进行定 点突变，将目标位点氨基酸突变为丙氨酸，引物序列见表 17。用 PCR 热循环仪（ Biometra T3 )按照如下条件进行 PCR反应： 94 X： lOmin; 随后是 94 X： 2min, 56 *€ 50s, 72 X： 7min的 17个循环，最后为 72*€ 延伸 10min。 PCR结束后加入内切酶 Dpn I在 37*€反应 3h以上消 化模板质粒， 以 ER2566 大肠杆菌作为表达菌株， 采用常规方法表 达， 结果表达的蛋白是以不溶性的包涵体形式存在。 以常规方法纯 化包涵体， 目的蛋白主要溶解于 8M尿素中。 将溶解于 8M尿素中 的蛋白逐步透析到 1 X PBS中完成复性， 12，000rpm离心 10分钟去 除沉淀， 得到初步纯化的定点突变蛋白 CD4(dl-2)-F43A、 CD4(dl-2)-K46A、 CD4(dl-2)-R59A、 CD4(dl-2)-N73A。
表 17. CD4(dl-2)片段各定点突变克隆的引物序列
引物名称 引物序列
CD4(dl-2)-F43A-F 5'-GGAAATCAGGGCTCCGCATTAACTAAAGGTCCA-3'
CD4(dl-2)-F43A-R 5'-TGGACCTTTAGTTAATGCGGAGCCCTGATTTCC-3'
CD4(dl-2)-K46A-F 5'-GGCTCCTTCTTAACTGCAGGTCCATCCAAG-3'
CD4(dl-2)-K46A-R 5'-CTTGGATGGACCTGCAGTTAAGAAGGAGCC-3'
CD4(dl-2)-R59A-F 5'-GCTGACTCAAGAGCAAGCCTTTGGGAC-3'
CD4(dl-2)-R59A-R 5'-GTCCCAAAGGCTTGCTCTTGAGTCAGC-3'
CD4(dl-2)-N73A-F 5'-CCCCTGATCATCAAGGCACTTAAGATAGAAGAC-3'
CD4(dl-2)-N73A-R 5'-GTCTTCTATCTTAAGTGCCTTGATGATCAGGGG-3'
实施例 21. 间接 ELISA 法检测单克隆抗体 15A7 与点突变 CD4(dl-2)多肽的反应性
将初 步 纯化 的 CD4(dl-2)-F43A 、 CD4(dl-2)-K46A 、 CD4(dl-2)-R59A、 CD4(dl-2)-N73A和 CD4(dl-d2)蛋白用 1 x PBS 包被緩冲液分别稀释至终浓度 0.5 g/mL, 按照 ΙΟΟμΙ^孔加入 96孔 聚乙烯微量滴定板中 37 孵育 2小时， 再置于 4 过夜。 用 PBST 洗涤液（ 8.0g NaCl、 0.2g KH2PO4、 2.9g Na2HP04 12H20、 0.2g KC1 和 0.5ml吐温 20, 加去离子水至 1升， pH为 7.4 )洗涤滴定板一次 以除去未结合的抗原蛋白。 然后用 200 μ Ι孔的封闭液（ 1 X PBS溶 液中加入 2%明胶、 0.5%酪蛋白和 2%蔗糖） 37*€封闭 2小时。甩净、 拍干后真空封闭， 4 保存。
检测时， 每孔加入 ΙΟΟμί稀幹的 15A7抗体。 孵育 1小时， 用 PBST洗涤液洗 5遍， 拍干后加入 1:5000稀释的过氧化物酶标记 的羊抗鼠免疫球蛋白 （HRP-GAM Ig， DAKO公司） , 37 孵育 30 分钟，用 PBST洗涤液洗 5遍，拍干后先后加入底物液 A、 B各 50μί (底物液 Α成分为： 13.42g Na2HP04 12H20、 4.2g柠檬酸 ·Η20和 0.3g过氧化氢，用去离子水中调节体积为 700mL;显色液 B成分为： 0.2g 四甲基联苯胺、 20mL 二甲基甲酰胺用去离子水中调节体积为 700mL ) , 37*€显色 15分钟， 加入 50μί终止液（ 2Μ H2S04 )终止 反应， 并于酶标仪上检测各孔的 OD450值。 结果见表 18结果， 其 中 CD4(dl-2)的 F43、 K46、 R59、 N73 突变成丙氨酸后， 对 15A7 的结合能力显著下降。
表 18.CD4(dl-2)定点突变各蛋白与 15A7的 EIA反应活性
15A7浓度 (ng/mL) 10000 5000 2500 1250 625 310 155 75 37 18 9
CD4(dl-2)-F43A 0.962 0.514 0.295 0.191 0.111 0.064 0.043 0.03 0.029 0.016 0.041
CD4(dl-2)-K46A 0.265 0.128 0.157 0.165 0.100 0.087 0.089 0.056 0.053 0.160 0.095 0
CD4(dl-2)-R59A 0.696 0.581 0.348 0.198 0.125 0.067 0.050 0.044 0.027 0.022 0.027 0
CD4(dl-2)-N73A 1.658 0.539 0.392 0.24 0.121 0.087 0.139 0.094 0.073 0.082 0.052 0
-4 CD4(dl-2) 1.885 1.382 1.246 0.986 0.776 0.624 0.511 0.262 0.25 0.266 0.165 0
实施例 22. Western印记杂交法检测单克隆抗体 15A7与点突变 CD4(dl-2)多肽的反应性
将初 步 纯化 的 CD4(dl-2)-F43A 、 CD4(dl-2)-K46A 、 CD4(dl-2)-R59A、 CD4(dl-2)-N73A和 CD4(dl-d2)蛋白还原样及非 还原样 (不含 DTT)分别以 13.5 %的 SDS-PAGE电泳分离， 电转移至 硝酸纤维素膜上， 加入 5 %的脱脂奶于室温封闭 1.5小时； 封闭后的 膜加入一抗 15A7 (以 1: 200稀释单克隆抗体的脱脂奶） 中， 4 反 应过夜; TNT ( 8.765g NaCl, 1.21g Tris Base和 0.5ml Tween-20， 加去离子水至 1L,调 pH为 8.0 )洗涤三次，每次间隔 5分钟；以 5 % 脱脂奶 1:5000稀释的 AP标记山羊抗小鼠（ GAMH+L-AP )为酶标二 抗（Protos公司产品） ， 室温反应 1小时； TNT洗涤三次， 每次间 隔 5分钟；向 10 mL AP緩冲液（ lOOmM NaCl, 5mM MgCl2, lOOmM Tris-HCl, pH为 9.5 )中加入 66μί NBT溶液（ 0.5g NBT溶于 10 mL 70%的二甲基甲酰胺中）和 33μί BCIP溶液（ 0.5g BCIP溶于 10 mL 70%的二甲基甲酰胺中），室温反应 15分钟。 Western印迹结果参见 图 25: 其中 CD4(dl-2)的 F43、 K46、 R59、 N73突变成丙氨^， 单克隆抗体 15A7对蛋白的结合能力显著下降。
表 19. CD4(dl-2)定点突变各蛋白与 15A7的 EIA反应活性
15A7浓度 (ng/mL) 10000 5000 2500 1250 625 310 155 75 37 18 9
CD4(dl-2)-F43A 0.962 0.514 0.295 0.191 0.111 0.064 0.043 0.03 0.029 0.016 0.041
CD4(dl-2)-K46A 0.265 0.128 0.157 0.165 0.100 0.087 0.089 0.056 0.053 0.160 0.095
CD4(dl-2)-R59A 0.696 0.581 0.348 0.198 0.125 0.067 0.050 0.044 0.027 0.022 0.027
CD4(dl-2)-N73A 1.658 0.539 0.392 0.24 0.121 0.087 0.139 0.094 0.073 0.082 0.052
CD4(dl-2) 1.885 1.382 1.246 0.986 0.776 0.624 0.511 0.262 0.25 0.266 0.165

Claims

权 利 要 求
1、 一种单克隆抗体或其抗原结合部分， 其具有至少一项选自以 下的特征：
(i) 特异性结合人表面抗原分化簇 4蛋白；
(ii) 与含有人表面抗原分化簇 4蛋白 D1结构域的截短形式的人 表面抗原分化簇 4多肽特异性反应；
(iii)对人免疫缺陷病毒包膜糖蛋白 gpl20与人表面抗原分化簇 4蛋白的反应无明显影响； 和
(iv) 阻断人免疫缺陷病毒感染 CD4+细胞及再感染，和 /或阻断由 此引起的疾病， 例如艾滋病。
2、 一种单克隆抗体或其抗原结合部分， 其中所述抗体的重链的 CDR1. CDR2和 CDR3和所述抗体的轻链的 CDR1、CDR2和 CDR3 分别是 CCTCC保藏号 C201098的杂交瘤产生的抗体 15A7或抗体 14G7的重链 CDR1. CDR2和 CDR3和轻链 CDR1. CDR2和 CDR3。
3、 一种单克隆抗体或其抗原结合部分， 其中所述抗体的重链可 变区和轻链可变区分别是 CCTCC保藏号 C201098的杂交瘤产生的 抗体 15A7或抗体 14G7的重链可变区和轻链可变区。
4、 权利要求 1-3任一项蛋白的单克隆抗体或其抗原结合部分， 其中所述抗体的重链可变区的氨基酸序列和轻链可变区的氨基酸序 列分别如 SEQ ID NO:2和 SEQ ID NO:4所示；或者所述抗体的重链 可变区的氨基酸序列和轻链可变区的氨基酸序列分别如 SEQ ID NO:18和 SEQ ID NO:20所示。
5、如权利要求 1-4任一项所述的单克隆抗体或其抗原结合部分， 其中所述抗体的重链 CDR1、 CDR2 和 CDR3的 ^^^列分别为 SEQ ID NOs:5-7或由 SEQ ID NOs:ll-13的核苷酸序列编码， 所述 抗体的轻链 CDR1、 CDR2和 CDR3的氨基酸序列分别为 SEQ ID NOs:8-10或由 SEQ ID NOs:14-16的核苷酸序列编码； 或者， 所述 抗体的重链 CDR1、 CDR2 和 CDR3的氨基酸序列分别为 SEQ ID NOs:21-23或由 SEQ ID NOs:27-29的核苷酸序列编码， 所述抗体的 轻链 CDR1、CDR2和 CDR3的^ 列分别为 SEQ ID NOs:24-26 或由 SEQ ID NOs:30-32的核苷酸序列编码。
6、如权利要求 1-5任一项所述的单克隆抗体或其抗原结合部分， 其为 Fab、 Fab'、 F(ab')2、 Fv或单链抗体。
7、如权利要求 1-5任一项所述的单克隆抗体或其抗原结合部分， 其中，所述的单克隆抗体结合人表面抗原分化簇 4蛋白的 KD值小于 1x10 5 M。
8、如权利要求 1-7任一项所述的单克隆抗体或其抗原结合部分， 其中， 所述的单克隆抗体包括非 CDR区， 该非 CDR区是来自非鼠 类的物种。
9、 一种特异性结合人表面抗原分化簇 4蛋白的单克隆抗体， 其 是 CCTCC保藏号 C201098的杂交瘤产生的抗体 15A7。
10、 一种杂交瘤细胞株， 其是 CCTCC保藏号 C201098的杂交
11、 一种检测样品中人表面抗原分化簇 4蛋白的方法， 其包括 如下步骤：
a )将所述的样品与权利要求 1-9任一项的单克隆抗体或其抗原 结合部分接触；
b )检测所述的单克隆抗体与所述样品中的蛋白的反应；
c )检测所述的单克隆抗体与所述样品中的蛋白及人免疫缺陷病 毒包膜蛋白的反应；
优选地， 其中所述的单克隆抗体或其抗原结合部分附着在固相 载体上； 优选地， 其中所述的固相载体选自： 微滴定板、 磁颗粒、 胶乳颗粒和硝化纤维素膜； 更优选地， 其中所述的单克隆抗体或其 抗原结合部分是按如此方向附着在所述固相上， 该方向能够增加所 述单克隆抗体与所述样品的结合效率； 更优选地， 其中所述的单克 隆抗体或其抗原结合部分是通过其恒定区域而附着在所述固相上 的。
12、 一种治疗受试者中由人免疫缺陷病毒感染引发的疾病的方 法，其包括向所述的受试者施用治疗上有效量的、权利要求 1-9任一 项所述的单克隆抗体或其抗原结合片段。
13、权利要求 1-9任一项所述的单克隆抗体用于制备治疗或预防 以 CD4+细胞为目标细胞的疾病（如艾滋病）的药物的用途， 用于制 备阻断 HIV感染 CD4+细胞及再感染的药物的用途。
14、一种药物组合物，其包含如权利要求 1-9任一项所述的单克 隆抗体或其抗原结合部分以及药学上可接受的载体； 优选地， 其中 所述的单克隆抗体或其抗原结合部分为 Fab、 Fab'、 F(ab')2或 Fv。
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WO2023015217A1 (en) 2021-08-04 2023-02-09 Sana Biotechnology, Inc. Use of cd4-targeted viral vectors
WO2023114949A1 (en) 2021-12-16 2023-06-22 Sana Biotechnology, Inc. Methods and systems of particle production
WO2023133595A2 (en) 2022-01-10 2023-07-13 Sana Biotechnology, Inc. Methods of ex vivo dosing and administration of lipid particles or viral vectors and related systems and uses
WO2023150647A1 (en) 2022-02-02 2023-08-10 Sana Biotechnology, Inc. Methods of repeat dosing and administration of lipid particles or viral vectors and related systems and uses
WO2023193015A1 (en) 2022-04-01 2023-10-05 Sana Biotechnology, Inc. Cytokine receptor agonist and viral vector combination therapies
WO2024026377A1 (en) 2022-07-27 2024-02-01 Sana Biotechnology, Inc. Methods of transduction using a viral vector and inhibitors of antiviral restriction factors
WO2024044655A1 (en) 2022-08-24 2024-02-29 Sana Biotechnology, Inc. Delivery of heterologous proteins
WO2024064838A1 (en) 2022-09-21 2024-03-28 Sana Biotechnology, Inc. Lipid particles comprising variant paramyxovirus attachment glycoproteins and uses thereof
WO2024119157A1 (en) 2022-12-02 2024-06-06 Sana Biotechnology, Inc. Lipid particles with cofusogens and methods of producing and using the same

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