EP0540512A1 - Immunoglobulines chimeriques netralisant le hiv-1 - Google Patents

Immunoglobulines chimeriques netralisant le hiv-1

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Publication number
EP0540512A1
EP0540512A1 EP90912204A EP90912204A EP0540512A1 EP 0540512 A1 EP0540512 A1 EP 0540512A1 EP 90912204 A EP90912204 A EP 90912204A EP 90912204 A EP90912204 A EP 90912204A EP 0540512 A1 EP0540512 A1 EP 0540512A1
Authority
EP
European Patent Office
Prior art keywords
chimeric
hiv
antibody
cells
bat123
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP90912204A
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German (de)
English (en)
Other versions
EP0540512A4 (en
Inventor
Ruey S. Liov
Edward M. Rosen
Cecily R. Y. Sun
Bill N. C. Sun
Michael S. C. Fung
Tse W. Chang
Nancy T. Chang
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Tanox Inc
Original Assignee
Tanox Inc
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Publication date
Application filed by Tanox Inc filed Critical Tanox Inc
Priority claimed from PCT/US1990/004048 external-priority patent/WO1992001719A1/fr
Publication of EP0540512A1 publication Critical patent/EP0540512A1/fr
Publication of EP0540512A4 publication Critical patent/EP0540512A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
    • C07K16/462—Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Monoclonal antibodies for therapy have gained acceptance in recent years.
  • An individual's immune state can be influenced by administering immunoglobulin of appropriate specificity, and this has been considered a valid approach to disease control and prevention.
  • the therapeutic efficacy of certain monoclonal antibodies in anti-tumor treatment has been documented. See, e.g.. Sears, H.F. _ei
  • Viral specific antibodies can be therapeutically useful for treatment of viral infections.
  • Antibodies directed against some viral epitopes can neutralize the virus.
  • Antibodies which fix complement (C1-C9) can cause lysis of cells carrying viral antigens or directly damage enveloped viruses.
  • antibodies that bind to Fc receptors on the surface of phagocytic cells can cause antibody-dependent cell-mediated cytotoxicity of virus-infected cells.
  • glycoprotein gpl20 of the human immunodeficiency virus type 1 HIV-1
  • AIDS acquired immunodeficiency syndrome
  • monoclonal antibodies can inhibit the infection of susceptible T cells by free virions. They can also inhibit the fusion between HIV-1-infected cells and uninfected cells which results in the formation of multinucleated giant cells (syncytia), such fusion having been implicated as a major route of viral transmission and T cell death.
  • syncytia multinucleated giant cells
  • Murine antibodies have several drawbacks in human therapy. As foreign proteins, murine antibodies often evoke an endogenous in vivo immune response which may reduce or destroy their therapeutic effectiveness. In addition, murine antibodies can cause an allergic or hypersensitivity reaction in patients. In therapy, there is a need to readminister the antibody, and this readministration
  • One way to ameliorate the problems associated with the jn vivo use of a murine antibody is to convert the murine antibody to a "chimeric" antibody, consisting of the variable region of the murine antibody joined to a human
  • the chimeric antibody has a human constant region, and the constant region is the larger region which is probably responsible for inducing immune or allergic responses against antibody, the chimeric antibody is less likely to evoke an undesirable immune-related response in humans.
  • the human constant region may provide for an antibody with a longer jn vivo half life and better effector function.
  • This invention includes chimeric HIV-1 neutralizing immunoglobulins which bind to the gpl20 portion of the HIV-1 envelope glycoprotein, and which have an antigen binding (variable) region of nonhuman origin and a constant region of human origin.
  • the chimeric immunoglobulins are prepared by genetic engineering techniques and retain the viral neutralizing activity of the parent, nonhuman immunoglobulin from which they are derived.
  • the chimeric immunoglobulins are useful for immunotherapy of .AIDS, AIDS related complex
  • ARC ARC
  • ARC viral-inhibition during early-stage HIV-1 infection
  • Figure 1 is a schematic depiction of the structure of chimeric genes encoding a light and a heavy chain for a chimeric HIV-neutralizing antibody.
  • Plasmid pSV184 ⁇ Hneo.BAT123V ⁇ .hC ⁇ contains a chimeric light chain gene construct consisting of a 4.4 Kbp Hind III fragment of mouse V ⁇ gene fused with the human C ⁇ gene. This plasmid contains a neo selection marker.
  • B Plasmid pSV2 ⁇ Hgpt.BAT123V H .
  • hC ⁇ l contains a chimeric heavy chain gene construct consisting of a 4.5 Kbp Eco RI fragment of mouse V H gene fused with the human C ⁇ l gene.
  • the plasmid carriers a _ggt selection marker.
  • the chimeric immunoglobulins of this invention are made up of chimeric heavy and light immunoglobulin chains.
  • Each chimeric chain is a contiguous polypeptide that has a nonhuman variable region and a human constant region.
  • the chimeric heavy and light chains are associated to form a molecule with a functional antigen binding region.
  • the chimeric immunoglobulins of this invention can be monovalent,
  • Monovalent immunoglobulins are dimers (HL) formed of a chimeric heavy chain (H) associated (through disulfide bridges) with a chimeric light chain (L).
  • Divalent immunoglobulins are tetramers (H 2 L-,) formed of two associated dimers.
  • Polyvalent antibodies can be produced, for example, by employing heavy chain constant regions which aggregate (e.g.. ⁇ - type constant regions).
  • the chimeric immunoglobulins can be produced as antigen binding fragments. Fragments such as Fv, Fab, Fab' or F(ab') 2 can be produced.
  • variable regions of the chimeric immunoglobulins are derived from nonhuman (preferably murine) immunoglobulins having the desired viral specificity and viral-neutralizing properties.
  • the parent nonhuman antiviral immunoglobulin can neutralize broad strains and isolates of the HIV-1 virus.
  • the chimeric immunoglobulin therefore, will have those same cross-neutralizing characteristics.
  • HIV-1-neutralizing immunoglobulins are described in U.S. Patent Application Serial No. 07/137,861, filed December 24, 1987 (a continuation-
  • HIV-1-neutralizing antibodies specifically react with the glycoprotein gpl20 of HIV-1; they inhibit the infection of T cells by free virions and inhibit infection of T cells by fusion with HIV-1-infected cells. These antibodies also exhibit some degree of cross-neutralizing activity, le., they can neutralize many different strains 6 and isolates of HIV-1. Antibody BAT123 is especially preferred because of its effective neutralizing activity and significant cross-strain reactivity.
  • the BAT123 antibody inhibits with an IC of less than 10 ng/ml, the infection of a susceptible human T cell line, H9, by HTLV-III B strain at 20 times TCID 50 in a nine day assay.
  • the BAT123 antibody also inhibits some other cloned HIV-1 strains and it inhibits the in vitro replication of broad, freshly isolated field HIV-1 samples from patients.
  • BAT123 antibody is described in pending U.S. Application Serial No. 07/137,861, filed December 24, 1987, and is on deposit at the American Type Culture Collection (ATCC), Rockville, Maryland, under Accession No. HB 10438.
  • the heavy chain constant region for the chimeric immunoglobulins can be selected from any of the five isotypes ⁇ , S, e, ⁇ or ⁇ . Heavy chains of various subclasses (such as the IgG subclasses 1-4) can be used. The different classes and subclasses of heavy chains are involved in different effector functions and thus, by selecting the appropriate heavy chain constant region, chimeric antibodies with desired effector function can be produced.
  • the light chains can have either a K or ⁇ constant chain.
  • the chimeric immunoglobulins of this invention are produced by genetic engineering techniques. Appropriate recipient cells are transfected with nucleic acid constructs, preferably DNA, encoding the desired chimeric light or heavy chain.
  • nucleic acid constructs for each of the light and heavy chain components of the chimeric immunoglobulin include a fused gene having a first
  • DNA segment which encodes at least the functional portion of the variable region linked to a second DNA segment encoding at least a part of a constant region is assembled in or inserted into an expression vector for transfection of the appropriate recipient cells.
  • the fused gene construct will comprise a functionally rearranged gene encoding a variable region of a chain of a HIV-1- neutralizing immunoglobulin linked to a gene encoding a constant region of an immunoglobulin chain.
  • the construct will also include the endogenous promoter and enhancer for the variable region encoding gene.
  • the variable region encoding genes can be obtained as DNA fragments comprising the leader peptide-encoding segment, the VJ gene (functionally rearranged variable (V) regions with joining (J) segment) for the light chain or VDJ gene for the heavy chain, and the endogenous promoter and enhancer for these genes.
  • VJ functionally rearranged variable
  • J variable
  • These variable region genes can be obtained from antibody-producing cells that produce the desired viral-neutralizing antibody, by standard DNA cloning procedures. See Molecular Cloning: Laboratory Manual. T. Maniatis £j _al. Cold Spring Harbor
  • the DNA fragment containing the functionally rearranged variable region gene is linked to a DNA fragment containing the gene encoding for the desired constant region (or a portion thereof).
  • Hybridoma cell lines producing antibody against HIV-1 can be made by standard procedures. See Koprowski e£ al., U.S. Patent No. 4,196,265. In general, these procedures entail immunizing a animal with HIV-1 or a purified or partially purified viral antigen, fusing antibody- producing cells taken from the immunized animal with compatible myeloma cells to form hybridoma cells, cloning the resulting hybridoma cells and selecting clones which produce antibody against the virus. The hybridoma clones can be screened for the production of viral-neutralizing antibody by tests such as those described in U.S. Patent Application Serial No. 07/137,861, and international application PCT/US88/01797 supra, and in the Examples below.
  • Human constant regions can be obtained from antibody-producing cells by standard gene cloning techniques. Genes for the two classes of human light chains and the five classes of human heavy chains have been cloned, and thus, constant regions of human origin are readily available from these clones.
  • Chimeric immunoglobulin fragments such as the monovalent Fv, Fab or Fab' fragments or the divalent F(ab') 2 fragment can be prepared by designing a chimeric heavy chain gene in truncated form.
  • a chimeric gene encoding a F(ab'), heavy chain would include DNA sequences encoding the CH j domain and at least the sulfhydryl-containing part of the hinge region of the heavy chain.
  • the fused genes encoding either the light or heavy chains are assembled or inserted into expression vectors for incorporation into a recipient cell.
  • Suitable vectors for the chimeric gene constructs include plasmids of the types pBR322, pEMBL, and pUC.
  • the introduction of gene constructs into plasmid vectors can be accomplished by standard procedures.
  • the expression vector is designed to contain two genetic selection markers, one for selection in a prokaiyotic (bacterial) system and the other for selection in a eukaryotic system.
  • the fused genes can be produced and amplified in a bacterial system and subsequently incorporated and selected for in eukaryotic cells.
  • suitable genes for a prokaryotic system are those which confer ampicillin resistance and those which confer chloramphenicol resistance.
  • Two preferred genes for selection of eukaryotic transfectants are: (i) the xanthine-guanine phosphoribosyl-transferase gene (designated gpt). and (ii) the phosphotransferase gene from Tn5 (designated neo).
  • Selection with ggt is based on the ability of the enzyme encoded by this gene to use xanthine as a substrate for purine nucleotide synthesis; the analogous endogenous enzyme cannot.
  • a medium containing xanthine and mycophenolic acid which blocks the conversion of inosine monophosphate to xanthine monophosphate, only cells expressing the ggt gene can survive.
  • the product of the neo gene blocks the inhibition of protein synthesis in eukaryotic cells caused by the antibiotic G418 and other antibiotics in the same class.
  • the chimeric light and heavy chain genes can be placed in two different expression vectors which can be used to cotransfect a recipient cell. In this case, each vector is designed to have a different selection gene for eukarytoic
  • transfectants This -allows cotransfection of the recipient cell and selection of cotransfected cells (Le., cells that have received both vectors). Selection of co- transfected cells is accomplished by selection for both selectable markers, which can be done simultaneously or sequentially.
  • Recipient cell lines are generally lymphoid cells, for example, B lymphocytes or hybridomas.
  • the preferred recipient cell is a myeloma cell line. Myelomas can synthesize, assemble and secrete immunoglobulins encoded by transfected genes and they can glycosylate protein.
  • a particularly preferred recipient cell is the myeloma Sp2/0 which normally does not produce endogenous immunoglobulin. When transfected, the cell will produce only immunoglobulin encoded by the transfected gene constructs. Transfected myelomas can be grown in culture or in the peritoneum of mice where secreted immunoglobulin can be recovered from ascites fluid.
  • transfecting lymphoid cells with vectors containing chimeric L and H chain genes There are several methods for transfecting lymphoid cells with vectors containing chimeric L and H chain genes.
  • a preferred method is the calcium phosphate precipitation procedure described by Graham and van der Eb, (1973) Virology 52:456.
  • Another method is by electroporation, wherein recipient cells are subjected to an electric pulse in the presence of the DNA to be incorporated
  • a lysozyme is used to digest cell walls from bacteria which contain the recombinant vector with the chimeric chain gene to produce spheroplasts.
  • the spheroplasts are fused with the lymphoid cells in
  • the chimeric viral-neutralizing immunoglobulins of this invention are useful for antiviral therapy of HIV-1 infected symptomatic patients and passive immunization of persons who are seronegative but at high risk of infection, or who are infected but asymptomatic.
  • high risk candidates include fetuses carried by or babies born to HIV-1-carrier mothers and health professionals working with infected patients, or with blood products, such as dentists and nurses.
  • the antibodies may also be used in individuals shortly after (e.g., within one hour) sexual contact with HIV-1 infected persons, or after puncture by needles
  • the chimeric antibodies of the invention can be used to reduce or eliminate the free HIV-1 virions or the infected T cells.
  • the infected T cells can be eliminated by antibody dependent cellular cytotoxicity ("ADCC"), complement- mediated cytolysis, or other cytolytic or regulatory immune mechanisms.
  • ADCC antibody dependent cellular cytotoxicity
  • the chimeric antibodies can also be used as effector agents mediating an immune function or as targeting agents for cytotoxic cells. They are administered systemically, preferably by intravenous injection in a pharmaceutically acceptable vehicle, such as sterile saline.
  • the antibodies can also be administered in ' conjunction with other anti-viral agents, such as AZT, or several different chimeric HIV-1-neutralizing immunoglobulins can be administered together. These antibodies of the invention can also be used with other drugs which combat secondary AIDS-related diseases, such as pneumonia.
  • GM-CSF granulocyte monocyte-colony stimulation factor
  • M-CSF monocyte-colony stimulation factor
  • GM-CSF and M-CSF have been shown to augment the ADCC activity of monoclonal antibodies specific for surface antigens expressed on tumor cells.
  • the therapeutic effect of the chimeric antibodies of the invention should be enhanced by combining antibody therapy with factors that augment ADCC activities, as it would help to eliminate infected T cells.
  • the antibodies of the invention can also be combined with immunotoxins, thereby forming an antibody-immunotoxin conjugate which specifically targets HIV-1 infected T cells.
  • the conjugates include cytolytic or cytotoxic agents conjugated to the chimeric antibodies of the invention.
  • the cytolytic/cytotoxic agents can be selected from any of the available substances including cytotoxic
  • the antibody and the cytotoxin can be conjugated by chemical or by genetic engineering techniques.
  • the immunotoxin conjugates may be used alone or in combination with free chimeric antibodies, or with factors which augment ADCC.
  • the antibodies of the invention can be used directly for jn vivo therapy, or they may be used in extra-corporeal ex-vivo therapy.
  • the free HIV-1 virions or the infected T cells can be removed by an affinity matrix (antibody immobilized on a solid phase) that is conjugated with the chimeric antibodies of this invention. Because antibodies may leak out from the affinity column and enter into the circulation of the patient, the chimeric antibodies of the invention are preferable to other antibodies that are more immunogenic and can induce undesirable
  • chimeric immunoglobulins of this invention are capable of neutralizing many different strains and isolates of HIV-1. Further, these immunoglobulins can inhibit transmission of the virus by syncytia formation.
  • the invention is illustrated further by the following examples.
  • the cloning of the functionally rearranged V L and V H genes of BAT123 was accomplished by the screening of BAT123 genomic libraries using appropriate molecular probes in a strategy similar to that described by Oi and Morrison (Biotechniques.4:214-221). The identification and final verification of the cloned gene segments was achieved by the aid of the nucleotide sequences of mRNA's for BAT123 immunoglobulin molecules. The rationale is based on the fact that only when a variable region gene segment is appropriately joined to the J region gene in the case of K chain rearrangement or when appropriate VDJ joining occurs in the case of heavy chain rearrangement, is the full length and properly spliced immunoglobulin mRNA synthesized in the antibody producing cells.
  • sequences of these mRNA's are therefore most suitable to serve as a guide for the selection and verification of the functionally rearranged variable region genes. Any gene segment containing sequences identical to the mRNA sequence can be considered functionally rearranged.
  • sequences of the mRNA molecules corresponding to the variable regions were determined by a primer extension/dideoxynucleotide termination method with the use of mRNA prepared from the polysomes of the BAT123 hybridoma cells.
  • This direct mRNA sequencing approach eliminates the intermediate cDNA cloning step in the conventional approach to derive the mRNA sequence; hence, it provides a relatively fast way to determine the mRNA
  • the primers used for this mRNA sequencing were:
  • a genomic DNA library for BAT123 cells was constructed in ⁇ phage vector -2001 (Karin, J., Natthes, W.D.H., Gait, MJ., Brenner, S. (1984) Gene
  • the probes used for this screening included a 2.7 Kbp Hind III DNA fragment containing all of the mouse germline K chain joining regions J1-J5 (J ⁇ probe; Max, E.E., Maizel, J.V., and Leder, P. (19811 J. Biol. Chem. 256:5116-5120 ' ) and two oligonucleotide probes V ⁇ and V ⁇ _ 2 derived from the nucleotide sequence of BAT123 light chain mRNA. The sequences of these oligonucleotide probes are V ⁇ : 5'dTTTGCTGACAGTAATAGG3' and V x _ 2
  • the probes were synthesized by using the phosphoramidite chemistry on an Applied Biosystems DNA synthesizer model
  • V ⁇ 123-23 was shown to hybridize with the J ⁇ DNA probe and both oligonucleotide probes.
  • DNA sequence determination of this clone by the dideoxynucleotide termination method showed that it carried a V L gene segment with a sequence identical to that determined from BAT123 light chain mRNA.
  • V H BAT123 heavy chain
  • genomic libraries were prepared. Genomic Southern blots of the EcoRI digest with the J H probe (see below) had previously revealed 2 potentially functionally rearranged V H genes in BAT123, one being 7.5 Kbp and the other 4.5 Kbp, in addition to the 6.6 Kbp fragment which was presumably derived from the fusion parent of BAT123, i.e. NS-1 cells. Two partial libraries containing these DNA bands were prepared. High molecular weight DNA was digested with EcoRI to completion and fractionated on a 0.7% agarose gel. DNA fragments of the size 4-6 Kbp and 6-9 Kbp were isolated and ligated with ⁇ vector ⁇ gtWES ⁇ B (Leder, P., Timeier, D., and Enquiest, L. (1977)
  • the ligated DNAs were packaged and recombinant plaques were screened.
  • the probes used included a 2 Kbp BamHI-EcoRI DNA fragment containing the mouse H chain joining regions J3 and J4 (J H probe; Gough, N.M. and Bernard, O. (1981) Proc. Natl. Acad. Sci. USA 78:509-513) and an oligonucleotide probe V H -1, 5'dAGTGTGGCTGTGTCCTC3' derived from
  • the chimeric H chain gene was constructed by replacing the EcoRI fragment in the pSV2 ⁇ Hgpt. DNSV H .hC ⁇ l plasmid containing the dansyl-specific V H gene with the 4.5-kbp EcoRI fragment containing the functionally rearranged
  • the structure of the resulting plasmid pSV2 ⁇ Hgpt.BAT123.V H .hC ⁇ l is shown in Figure IB.
  • the L and H chain chimeric genes shown in Figure 1 were used to
  • the myeloma cells chosen, Sp2/0 is a non-secretory cell line (Shulman, M., Wilde, C, and Kohler, G. (1978) Nature 276:269-270) that does not produce immunoglobulin molecules of its own.
  • the calcium phosphate precipitation method (Graham and van der Eb (1973) Virology
  • Sp2/0 cells were seeded at 5 x 10 6 cells per 100-mm Petri dish which had been previously treated with histone (Sigma Chemical Co., St. Louis, Mo) and incubated for 16 hours at 37°C. Approximately 7.5 x 10 7 S ⁇ 2/0 cells were cotransfected with CsCl-ethidium bromide gradient purified pSV184 ⁇ Hneo.BAT123.V k .hC ⁇ (150 ⁇ g) and pSV2 ⁇ Hgpt.BAT123.V H .hC ⁇ l (150 ⁇ g) using the calcium phosphate precipitation method.
  • transfectoma The stable transfectants (transfectoma) were screened for the production of secreted, functional chimeric antibodies by virtue of their affinity for purified HIV-1 gpl20, a distinct characteristic of BAT123. Purified gpl20 was immobilized on microtiter plates, and allowed to react with culture supernatants from the transfectants. The antigen-antibody complexes were detected with alkaline phosphatase-conjugated antibody specific for human IgG. As shown in Table 1,
  • transfectoma lines exhibiting OD greater than 1.0 in ELISA were selected and the chimeric antibody producing cells were cloned by a single cell cloning technique from which twelve stable cell lines were established. These transfectoma cell lines were then tested for stability of chimeric antibody production in the absence of selection drugs G418 and mycophenolic acid. The cells were cultured in the medium with stepwise reduction in the two selection drugs at 2-week intervals which resulted in the complete elimination of the drugs. During each reduction of drugs the production of the chimeric antibody in these cell lines was monitored by ELISA. Three of these cell lines lost their ability to secrete chimeric .antibody upon removal of selection pressure. The remaining 9 cells lines maintain stable production of chimeric antibody at 5 weeks after complete elimination of selection drugs in the culture medium.
  • transfectoma line CAGl-51-4 (one of the 9 stable cell lines) was expanded and grown in tissue culture medium. Approximately 600 ml of culture medium was collected, from which 14.4 mg (estimated by BCA protein assay, Pierce, Rockford, IL) of the chimeric antibody was purified by utilizing r-protein A-Sepharose affinity column (Repligen Corporation, Cambridge, MA). The IgG concentration in the culture supernatant of this transfectoma cell line was therefore estimated to be 24 ⁇ g/ml, a level somewhat higher than the level produced by BAT123 hybridoma cells (20 ⁇ g/ml).
  • Purified chimeric immunoglobulin was used to characterize the biochemical/immunological properties of the chimeric antibody produced by CAGl-51-4 transfectoma.
  • IEF Isoelectric focusing gel of the purified chimeric antibody produced by CAGl-51-4 along with that of BAT123 was performed.
  • the IEF pattern was obtained by application of purified antibody samples onto Pharmacia's Phast SystemTM and IEF was carried out according to the procedure recommended by the manufacturer.
  • the IEF pattern indicated that the chimeric antibody contained two major species of molecules with pi in the range of pH 6.8 to 7.2 * whereas the corresponding molecules of BAT123 exhibited pi in the range of pH 5.6 to 5.8.
  • the replacement of constant regions of the immunoglobulin molecule from mouse to human therefore greatly altered the composition of the antibody,as reflected in the IEF pattern.
  • H 2 L 2 molecule of the chimeric antibody showed identical mobility to that of BAT123 (mw 146,000 dalton) when the immunoglobulins were resolved in the 10% SDS-PAGE under non-reducing conditions.
  • BAT123 immunoglobulin was shown to be extensively reactive with anti- mouse antiserum whereas chimeric antibody was only barely reactive to the same serum. On the other hand, chimeric antibody reacted strongly with anti-human
  • the heavy chain chimeric gene was constructed by splicing the V H gene of
  • amino acid residues within an immunoglobulin molecule that are directly involved in the formation of the antigen binding site are generally
  • CDR complementarity determining region
  • V variable domain
  • the antigen band that reacted with the chimeric antibody was identical to
  • the chimeric antibody recognizes the same antigenic determinant (epitope) within gpl20 as does BAT123
  • the antibody was allowed to react with a membrane strip containing a series of 32 overlapping oligopeptides that represent the potential antigenic determinants in gpl20 (gift of S. Petteway, Du Pont).
  • the incubation procedure was essentially the same as that for the immunoblot strip of viral antigens.
  • the result indicated that the chimeric antibody binds to the same oligopeptide as does BAT123 which has the amino
  • the ability of the chimeric antibody produced by CAGl-51-4 to neutralize the HIV-1 infection of H9 cells was measured using the similar procedure described earlier (international application PCT/US88/01797, U.S. Patent Application Serial No. 07/137,861 filed December 24, 1987, which is a continuation-in-part of U.S. Application Serial No. 07/057,445, filed May 29, 1987).
  • the virus used was prepared from culture supernatants of HTLV-IIIB-infected H9 cells. 40 ml of cell-free supernatant was centrifuged at 35,000 x g for 3 hours. The pellet was resuspended in 3 ml of growth medium. The titer of the virus was measured by infecting H9 cells with the viral stock in
  • the of the viral stock was determined as the infective dose at which half of the number of the microcultures was infected.
  • TCID 50 was used. 50 ⁇ l of 60 times of the viral stock was preincubated with 50 ⁇ l the antibodies tested in a microculture well of a 96-well plate. The antibodies tested were the chimeric antibody produced by CAGl-51-4, the murine monoclonal antibody BAT123 and a murine monoclonal antibody to human chorionic gonadotropin (anti-hCG). In the control, 50 ⁇ l of growth medium without any antibodies was used. The mixtures were kept at 37°C in a 5% C0 2 incubator for 1 hour. The final concentration of the antibody was 100, 50, 25, 12.5 and 6.25 ⁇ g / ml. Each concentration of the tested antibodies was performed in triplicate. At the end of the incubation, 50 ⁇ l of 4 x 10 6 /ml H9 cells was added.
  • the H9 cells were harvested in log phase and pre-incubated for 1 hour at 37°C with 2 ⁇ g/ml polybrene in the RPMI-1640 growth medium containing 15% heat-inactivated fetal bovine serum before being added to the mixture.
  • the cells in each microculture wells were resuspended, 40 ⁇ l of the cell suspension was added to 200 ⁇ l of fresh growth medium in the corresponding wells of another microculture plates.
  • the microculture plates were kept at 37°C in 5% C0 2 in an incubator. On day 3, day 5, day 7, day 9, day 11, and day 14, 150 ⁇ l of cell suspension from each microculture was removed and placed into a U-bottomed well of another 96-well plate. The plate was centrifuged at 200xg for 5 minutes. The supernatants were collected for HIV-1 viral antigen capture assays.
  • the wells were fed with 150 ⁇ l fresh growth medium.
  • the HIV-1 specific antigens in the cell-free supernatant were measured by their affinity for the immunoglobulins from patients with AIDS.
  • One hundred ⁇ l of diluted purified AIDS patient immunoglobulin (1:2000, approximately 5 ⁇ g/ml) was added to each well of a Cobind plate and incubated for 2 hours at 37°C. Then the wells were rinsed two times with 200 ⁇ l of phosphate-buffered saline (PBS). The wells were blocked with 220 ⁇ l of 1% bovine serum albumin (BSA) in PBS for 1 hour at 37°C. Then it was rinsed three times with PBST (PBS containing 0.1% Tween 20). The wells were then emptied.
  • PBS phosphate-buffered saline
  • test samples 50 ⁇ l of the test samples (undiluted or in appropriate dilution) together with 50 ⁇ l of PBSTB (PBS containing 1% BSA and 0.1% Tween 20) were added to the well.
  • the negative control contained 50 ⁇ l of the growth medium.
  • the plate was incubated for 1 hour at 37°C. Then it was rinsed three times with PBST. lOO ⁇ l of diluted peroxidase conjugated AIDS patients immunoglobulins was added to each well for 1 hour at room temperature. The plate was then rinsed three times with PBST. lOO ⁇ l of a substrate solution (con- taining 20 mM sodium acetate buffer pH 6.0, 0.001% 3,3',5,5' tetramethyl-
  • CAGl-51-4 was also assessed by its ability to inhibit syncytium formation.
  • the effects of chimeric antibody on HIV-1 transmission via cell fusion were studied using HIV-1 infected H9 cells and CD4-expressing HeLa cells (HeLa-CD4 + ), which fuse upon contact and form syncytia in culture.
  • HeLa is a human carcinoma cell line.
  • HeLa-CD4 + contains in its genome, CD4 encoding DNA introduced by transfection and thus, it expresses CD4 antigen on its cell surface.
  • the HeLa-CD4 + cell line was a gift from David. D. Ho (University of California,
  • syncytia formed. Those syncytia with more than five nuclei could be easily identified and enumerated, thus providing quantitative measurements of syncytium formation.
  • antibodies at different concentrations were mixed with infected H9 cells and added to the HeLa-CD4 + cells. The final total volume of the culture medium per well was 200 ⁇ l. .After 18 hours of incubation at 37°C, the wells were washed with PBS, fixed with methanol, air-dried, and stained with methylene blue.
  • the cells were examined at 100X magnification and the number of syncytia (of more than 5 nuclei) were determined in four randomly chosen fields and averaged.
  • the chimeric antibody produced by CAGl-51-4 gave 89.1%, 65.7%, and 58.6% reduction in the number of syncytia formed between H9 cells and Hela-CD4 + cells at levels of 20 ⁇ g/ml, 10 ⁇ g/ml, -and 5 ⁇ g/ml, respectively.
  • This effect is essentially identical to that caused by BAT123 and it indicates that the chimeric antibodies retained the activity to inhibit the fusion between the HIV-infected cells and uninfected cells, one of the major routes for HIV transmission.
  • the control murine antibody anti-hCG gave no effect on the syncytium formation.
  • Clones of the CAGl-51-4 cell line were carried in the hybridoma laboratory for four months, and then re-tested for monoclonal antibody production. Production was stable after four months. When about 100,000 cells were grown for five days, production was determined to be about 19 ⁇ g/ml.
  • the .antibody produced by CAGl-51-4 was then re-cloned in D-15 medium and 1% Fetal Calf Serum ("FCS"). It was determined that 90% of the cells produced a monoclonal antibody to gpl20, using an ELISA. Based on the ELISA, six subclones (CAG1-51-4-1 to 51-4-6) were selected for further growth and testing. These six subclones were then recloned in serum free medium. The subclone (CAG 1-51-4-7 A) which produced the largest amount of antibody was then selected and recloned again in serum free media. This procedure produced 16 subclones (CAG 1-51-4-7 A.1 to 51-4-7A.16). Subclone CAG1-51-4-7A.6 was found to be a high antibody producer, with 100,000 cells/ml yielding an antibody concentration of 17 ⁇ g/ml when grown for four days under selection pressure with
  • the antibody produced by this subclone was named CGP 47 439, and the subclone CAG 1-51-4-7 A.6 was deposited at the ATCC, Rockville, Maryland, on July 10, 1990, under Accession No. ATCC CRL 10499.
  • Isoelectric focusing was performed on the CGP 47 439 antibodies.
  • the IEF pattern was obtained by application of purified antibody samples onto
  • the IEF pattern indicated that the CGP 47 439 antibody had two major bands at pH 6.8 ⁇ 0.5 pH units, which was comparable to the antibody produced by the CAGl-51-4 transfectoma.
  • a peptide binding ELISA was then performed for the CGP 47 439 antibody, to determine the immunologic specificity and the potency.
  • the ELISA was performed in 96-well microtiter plates, coated with a synthetic peptide which is known to contain the same amino acid sequence as the segment of gpl20 which binds to BAT123. The plates were blocked with an ammonium chloride blocking solution. After antibody binding, labelling was with 20 ⁇ l peroxidase conjugated with goat anti-human IgG. The ELISA was performed three times, and the mean values were determined.

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Abstract

L'invention concerne des immunoglobulines chimériques neutralisant le HIV-1-, constituées d'une région de liaison d'un antigène non humain et d'une région constante humaine. Un anticorps, particulièrement efficace, neutralisant le HIV-1- prend la désignation CGP 47 439. Les anticorps sont utiles pour l'immunothérapie du SIDA, le complexe apparenté au SIDA (ARC), et l'immunisation passive des individus contaminés mais asymptomatiques, des personnes séronégatives dans les groupes à hauts risques, ou des personnes qui ont été exposées au HIV-1- et qui risquent d'être contaminées.
EP19900912204 1990-07-18 1990-07-18 Chimeric hiv-1-neutralizing immunoglobulins Withdrawn EP0540512A4 (en)

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PCT/US1990/004048 WO1992001719A1 (fr) 1990-07-18 1990-07-18 Immunoglobulines chimeriques netralisant le hiv-1
CA002087528A CA2087528A1 (fr) 1990-07-18 1990-07-18 Immunoglobulines chimeriques neutralisant le vih-1

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EP0295803B1 (fr) * 1987-05-29 1993-11-03 Shuzo Matsushita Anticorps monoclonaux

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF IMMUNOLOGY. vol. 143, no. 12, 15 December 1989, BALTIMORE US pages 3967 - 3975 Liou RS;Rosen EM;Fung MS;Sun WN;Sun C;Gordon W;Chang NT;Chang TW 'A chimeric mouse-human antibody that retains specificity for HIV gp120 and mediates the lysis of HIV-infected cells.' *
See also references of WO9201719A1 *

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