WO1992001719A1

WO1992001719A1 - Chimeric hiv-1-neutralizing immunoglobulins

Info

Publication number: WO1992001719A1
Application number: PCT/US1990/004048
Authority: WO
Inventors: Ruey S. Liov; Edward M. Rosen; Cecily R. Y. Sun; Bill N. C. Sun; Michael S. C. Fung; Tse W. Chang; Nancy T. Chang
Original assignee: Tanox Biosystems, Inc.
Priority date: 1990-07-18
Filing date: 1990-07-18
Publication date: 1992-02-06

Abstract

Chimeric HIV-1-neutralizing immunoglobulins, made up of a non-human antigen binding region and a human constant region, are discribed. One particularly good HIV-1-neutralizing antibody is designated CGP 47 439. These antibodies are useful for immunotherapy of AIDS, AIDS related complex (ARC), and passive immunization of infected but asymptomatic individuals, seronegative persons in high risk groups, or persons who have been exposed to HIV-1 and are at risk of infection.

Description

CHIMERIC HIV-1-NEUTRALIZING IMMUNOGLOBULINS

Background of the Invention

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

.al., (1984) J. Biol. Resp. Modif.1:138. 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. Furthermore, antibodies that bind to Fc receptors on the surface of phagocytic cells can cause antibody-dependent cell-mediated cytotoxicity of virus-infected cells.

Monoclonal antibodies have been developed against the exterior

glycoprotein gpl20 of the human immunodeficiency virus type 1 (HIV-1), the

causative agent of acquired immunodeficiency syndrome (AIDS). See Fung, S.C. et ,al., BioTechnology 5:940 (1987). These 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. These HIV-1-neutralizing antibodies have applications in therapy and prevention of AIDS.

Most monoclonal antibodies presently available, including antiviral monoclonal antibodies, are murine antibodies. The production of human antibodies by somatic cell fusion is generally difficult. Techniques for production of murine monoclonal antibodies, however, are well established.

Murine antibodies, however, 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

increases the likelihood that these undesirable immune-related reactions will occur in patients.

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

constant region. See, e.g., Morrison, S.L. et al. (1984) "Chimeric Human Antibody Molecules: Mouse Antigen-binding Domains with Human Constant Region Domains" Proc. Natl. Acad. Sci. USA 81:6851; Neuberger, M.S. and Rabbits, T.H. "Production of Chimeric Antibodies" PCT Application No. PCT/GB85 00392; Sun, L.K. £l.al., (19871 Proc. Natl. Acad. Sci. USA 84:214: Iiu, A.Y. £l_al„ (1987) 1 Immunol. 139:3521; Sahagan, B.G. ^.al., (1986) J. Immunol. 137:1066; Liu, A.Y.

£1^1., ⁽19871 Proc. Natl. Acad. Sci. USA 84:3439. Because 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. Furthermore, the human constant region may provide for an antibody with a longer jn vivo half life and better effector function.

Summary of the Invention

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"), or viral-inhibition during early-stage HIV-1 infection. They can also be

used for passive immunization against HIV-1 infection in individuals with a risk of infection because of actual exposure to HIV-1, or a high likelihood of such exposure.

Brief Description of the Drawings 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. (A) Plasmid pSV184ΔHneo.BAT123V_κ.hC_κ contains a chimeric light chain gene construct consisting of a 4.4 Kbp Hind III fragment of mouse V_κ gene fused with the human C_κ gene. This plasmid contains a neo selection marker. (B) Plasmid pSV2ΔHgpt.BAT123V_H. hC_γl 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_γl gene. The plasmid carriers a _ggt selection marker. B:Bam HI, E:Eco RI, H:Hind III, S:Sal I, V:variable region gene, Cxonstant region.

Detailed Description of the Invention

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,

divalent or polyvalent. 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 (H₂L-,) formed of two associated dimers. Polyvalent antibodies can be produced, for example, by employing heavy chain constant regions which aggregate (e.g.. μ- 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.

The variable regions of the chimeric immunoglobulins are derived from nonhuman (preferably murine) immunoglobulins having the desired viral specificity and viral-neutralizing properties. In preferred embodiments, 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-

-in-part of U.S. Patent Application Serial No. 057,445, filed May 29, 1987) and also in published international application PCT/US88/01797, and in Fung, £l _ai. supra. (The teachings of these references are incorporated by reference herein.) These 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 TCID₅₀ 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 α, S, e, γ or μ. 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 λ 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. In general, DNA 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. The fused gene is assembled in or inserted into an expression vector for transfection of the appropriate recipient cells.

In preferred embodiments 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. For example, 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. 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

Laboratory (1982). Screening of the genomic library for the functionally rearranged variable region can be accomplished with the use of appropriate DNA probes such as DNA segments containing the mouse germline J region DNA sequences and sequences downstream. Identification and confirmation of the correct clones are then achieved by DNA sequencing of the cloned genes and comparing the sequence to the corresponding sequence of the full length, properly

spliced mRNA. 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).

Genes encoding antibody light and heavy chains can be obtained generally from immunoglobulin-producing lymphoid cells. 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. For example, 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.

In preferred embodiments, 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. Examples of 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. In 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.

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

into the cell. See, e^, Potter, et al (1984) PNAS 81:7161. Another way to introduce DNA is by protoplast fusion. 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 presence of polyethylene glycol. After protopl-ast fusion, the transfectants are selected and isolated. (Oi, et al., (1983) 30:825). Finally, the

DEAE-dextran procedure described by Cullen, £i.ai., (1984) Nature. 307:241 can also be used.

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. Such 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

contaminated with HIV-1 positive blood. Because some HIV-1-infected blood used for transfusion is not detected and screened correctly, it is also possible to use the protective chimeric antibodies in patients receiving blood transfusions. 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. 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.

Many factors, such as GM-CSF (granulocyte monocyte-colony stimulation factor) or M-CSF (monocyte-colony stimulation factor), are known to induce the proliferation of leukocytes, including those mediating ADCC. In m vitro experiments, 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

steroids, gelonin, abrin, ricin, Pseudomonas toxin, diptheria toxin, pokeweed antiviral peptide, tricathecums, radioactive nuclides, and membrane-lytic enzymes (such as phospholipases). 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

immune-related responses.

As mentioned above, chimeric immunoglobulins of this invention (formed, for example, from HIV-1-neutralizing immunoglobulins such as BAT123) 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.

EXAMPLES

Cloning and Identification of the Functionally Rearranged V_L and V_H Genes of BAT123

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.

The sequences of these mRNA's, determined either by direct sequencing of the mRNA molecule or from the cDNA clones, 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.

The 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

sequence. The primers used for this mRNA sequencing were:

5'dTGGATGGTGGGAAGATG3' for light chain mRNA and 5'dGGCCAGTGGATAGAC3' for heavy chain mRNA (both primers were obtained from Pharmacia, Nutley, NJ). These oligonucleotides were complementary to the mRNA sequences in the constant regions at positions proximal to the junctions of J and C regions of the molecules. Primer extension was accomplished with the use of AMV reverse transcriptase and terminated by dideOaXynucleotides. The nucleotide sequence of mRNA's was determined by the gel electrophoresis and subsequent autoradiography.

A genomic DNA library for BAT123 cells was constructed in λ phage vector -2001 (Karin, J., Natthes, W.D.H., Gait, MJ., Brenner, S. (1984) Gene

32:21 -- /4). High molecule weight genomic DNA from BAT123 hybridoma cells was partially digested with restriction endonuclease Sau 3AI and size fractionated on a 10-40% sucrose density gradient. DNA fragments of 18-23 Kbp were ligated with λ-2001/BrτιHl arms (Stratagene Cloning Systems, La Jolla, CA). and packaged by using Gigapack Gold packaging extracts (Stratagene). This genomic library "'as first screened for the functionally rearranged variable region gene of BAT123 light chain (V_L). 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_κ probe; Max, E.E., Maizel, J.V., and Leder, P. (19811 J. Biol. Chem. 256:5116-5120^') and two oligonucleotide probes V^ and V_κ_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

5'dATATAACTATCACCATCA3'. The probes were synthesized by using the phosphoramidite chemistry on an Applied Biosystems DNA synthesizer model

381.

Approximately 5x10^s phage recombinants were screened initially with ³²P-labeled mouse J_κ DNA probe. Plaque hybridizations were carried out in

5xSSC with 50% (v/v) formamide at 42°C for 16 hours (lxSSC=0.15M NaCl, 0.015M sodium citrate). Final washes were in 0.2xSSC/0.1% SDS at 65°C. Two positive clones were obtained. They were subsequently screened with the use of ³²P-labeled oligonucleotide probes V^ and V_κ_₂. Hybridization with the oligo-probes was carried out in 5xSSC at 37°C for 18 hours and washes were carried out in 2xSSC/0.1% SDS at room temperature. One of these clones, V_κ123-23, was shown to hybridize with the J_κ 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.

This clone was used in the subsequent construction of the mouse/human chimeric

L chain gene. For the cloning of the functionally rearranged variable region genes for

BAT123 heavy chain (V_H), partial 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 λ vector λgtWESλB (Leder, P., Timeier, D., and Enquiest, L. (1977)

Science 196:175-177). 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

BAT123 mRNA sequence. The hybridization conditions for these probes were as described above for K chain. Screening of these two EcoRI partial libraries with J_H probe resulted in the isolation of 3 independent phage clones containing a 7.5 Kbp, 6.6 Kbp or a 4.5 Kbp DNA fragment. Subsequent hybridization using oligo- nucleotide probe V_H-1 revealed that only the phage clone containing a 4.5 Kbp insert, clone V_H123-E3, hybridized with the probe. DNA sequencing of this clone

showed that it contained a V_H sequence identical to that from V_H mRNA of BAT123. This clone was used in the construction of the mouse-human chimeric

H chain gene.

Fusion of Murine V with Human C exons and Introduction into Murine Myeloma Cells

The functionally rearranged L and H chain V genes isolated from BAT123 cells were joined to human K .and γl C region genes in expression vectors containing dominant selectable markers, .neo (Southern, P.J. & Berg, P. (1981) J.

Mol. Appl. Genet.1:327-341) and g ϊ (Mulligan, R.C. and Berg, P. (1981) Proc. Natl. Acad. Sci. USA 78:2072-2076). respectively. To construct the desired chimeric gene, the Hind III fragment of pV184ΔHneo.DNSV_L-hC_κ (Oi, V.T. and Morrison, S.L. (1986) Biotechniques 4:214-221) containing the dansyl-specific V_L

gene was replaced with the 4.4 Kbp Hind III fragment containing the L chain gene of BAT123 derived from clone Vκl23-23. The structure of the resulting plasmid ρSV184ΔHneo.BAT123.V_k.hCκ is shown in Figure IA.

The chimeric H chain gene was constructed by replacing the EcoRI fragment in the pSV2ΔHgpt. DNSV_H.hC_γl plasmid containing the dansyl-specific V_H gene with the 4.5-kbp EcoRI fragment containing the functionally rearranged

BAT123 H chain gene derived from the phage clone V_H123-E2. The structure of the resulting plasmid pSV2ΔHgpt.BAT123.V_H.hC_γl is shown in Figure IB. The L and H chain chimeric genes shown in Figure 1 were used to

transfect mouse myeloma cells. 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

,52:456) was adopted to transfer the chimeric genes into Sp2/0 cells. To facilitate the DNA transfection, Sp2/0 cells were seeded at 5 x 10⁶ 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 10⁷ Sρ2/0 cells were cotransfected with CsCl-ethidium bromide gradient purified pSV184ΔHneo.BAT123.V_k.hC_κ (150 μg) and pSV2ΔHgpt.BAT123.V_H.hC_γl (150 μg) using the calcium phosphate precipitation method. Two days after transfection, cells were subcultured in microtiter plates at a density of 1 x 10^s cells per well in culture medium containing 400 μg/ml G418 and 0.2 μg/r^,l mycophenolic acid. The frequency of transfectants resistant to both selection drugs was approwmately 2 x 10^"5. 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,

707 of the 1200 transfectants tested gave a positive signal in the ELISA, indicating

that the intact chimeric antibody was secreted, and that this antibody retained the 20 ability to bind HIV-1 gpl20. The untransfected parental cell line gave a negative

result.

TABLE 1 Level of Secretion of Chimeric Antibody bv Transfectomas

Recipient Number

Cell Transfectoma

Tested

SP2/0 1200 493 511 196 59

* Transfectomas were scored based on their OD in ELISA. "Negative" is defined as OD of 0.0 to 0.1, "Weakly Positive" denotes 0.1 to 0.2, and "Positive" indicates 0.2-3.0.

Production of Chimeric Antibody from Transfectoma Cell Lines

Seventeen 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.

To estimate the level of chimeric antibody production and to prepare the antibody for further characterization, 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 μg/ml, a level somewhat higher than the level produced by BAT123 hybridoma cells (20 μg/ml).

Biochemical Analysis of Chimeric Antibody Produced bv CAGl-51-4

Purified chimeric immunoglobulin was used to characterize the biochemical/immunological properties of the chimeric antibody produced by CAGl-51-4 transfectoma.

A) Isoelectric Points

Isoelectric focusing (IEF) 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 System™ 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.

B) Reactivity of the Chimeric Antibody Produced by CAGl-51-4 to Anti-Mouse as well as Anti-Human Antisera

When the chimeric antibody was subjected to a 10% SDS-polyacrylamide gel electrophoresis under reducing conditions (Laemmli, U.K. (1970) Nature

227:680-685) two b.ands were observed. One protein band of molecular weight of 53,000 daltons corresponded to the chimeric heavy chains, and exhibited a close similarity in size to the heavy chains of BAT123 immunoglobulin. Another protein band which was produced, with the size of approjdmately 23,000 dalton, denoted the light chains of antibodies. The slightly slower mobility observed for chimeric light chains is also seen in other chimeric antibodies and may not necessarily be attributed to the larger size of human light chain K constant region

(hC_K) than the murine counterpart. The fully assembled H₂L₂ 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.

To test whether the chimeric antibody indeed incorporated the constant regions of human immunoglobulin, 2 μg of the chimeric antibody and BAT 123 were electroblotted onto a nitrocellulose membrane (100 volts, 1 hour in a transblot buffer consisting of 25 mM Tris-HCl, 192 mM glycine, 20% (v/v) metha¬ nol, pH 8.3) after the proteins were resolved in 10% SDS-PAGE under reducing conditions in a BioRad Mini-Protein II Dual Slab Cell apparatus. One of the replica membrane filters was reacted with biotinylated anti-mouse antibody

(Vector Laboratory, Burlingame, CA) whereas the other filter was reacted with biotinylated anti-human antibody in blotto buffer consisting of 5% non-fat dry milk in phosphate buffered saline (PBS). After 1 hour of incubation at 37°C, the membrane filters were washed with 2 changes of PBS + 0.1% Tween 20 (PBST) and both membranes were reacted with horseradish perαxidase-avidin conjugate in blotto buffer at room temperature for 30 minutes. After final washes with PBST the reactive protein bands were visualized by color development using 4-chloro-l-naphthol (4-CN) and hydrogen peroxide.

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

antiserum whereas BAT123 did not show appreciable reactivity to the antiserum. This result demonstrated that portions of the chimeric antibody molecule was indeed derived from human immunoglobulin.

C) The IgG Subclass of the Chimeric Antibody Produced by CAGl-51-4

The heavy chain chimeric gene was constructed by splicing the V_H gene of

BAT123 into the coding sequence of human C_γl region. The resulting chimeric antibody was therefore expected to be of IgGl subclass. To confirm the isotype of the expressed constructed chimeric antibody the following experiment was conducted. Mouse anti-human IgGl and IgG3 antibodies (Fisher Biotech) were separately coated onto microtiter wells in series of 2-fold dilutions. Chimeric antibody at 20 μg/ml was added to these wells and incubated at 37°C for 1 hour. After washes with PBST the complexes were detected by incubation with goat anti-human-horseradish peroxidase conjugate (Vector) with a subsequent color development. The results clearly demonstrated that chimeric antibody produced by CAGl-51-4 is of IgGl subclass, as the anti-human IgGl antibodies showed a far greater affinity for the chimeric antibody.

D) Antigen Specificity of the Chimeric Antibody Produced by

CAGl-51-4

The amino acid residues within an immunoglobulin molecule that are directly involved in the formation of the antigen binding site are generally

believed to be in the complementarity determining region (CDR) which reside in the variable domain (V) of the immunoglobulin. To determine if the chimeric antibody retained the antigen specificity of the parent antibody the following experiments were performed.

In the first experiment the chimeric antibody was allowed to react with a commercially available immunoblot strip (gift of Dr. Robert Ting, Biotech

Research Labs) that contains all antigens of purified HIV-1 resolved in SDS-PAGE, with a subsequent electrotransblot on to nitrocellulose membrane filters. The reaction was carried out at room temperature for 16 hours in blotto buffer. The reactive complexes were then detected by incubations first with biotinylated-anti-human antisera (Vector) followed by avidin-horseradish peroxidase, with washes between each incubation step, and finally visualized by color development with the enzyme substrates 4-CN and hydrogen peroxide.

The antigen band that reacted with the chimeric antibody was identical to

the one detected by BAT123 antibody in a parallel reaction and corresponded to the viral envelope glycoprotein gpl20 in a viral antigen profile displayed by reaction with a reference serum from a patient with AIDS. This result showed that the chimeric antibody retained the antigen specificity of BAT123 to bind HIV-l-gpl20.

To further test whether 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

acid sequence of Arg-Ile<51n-Arg-GlyPro-GlyArg-Ala-Phe-Val-Thr-Ile-GlyLys. The antigen specificity of the murine antibody BAT123 was therefore well preserved upon conversion into a mouse/human chimeric antibody.

Biological Activity of the Chimeric Antibody Produced bv CAGl- 1-4

A) Neutralization of HIV-1 Infection to H9 Cells by Chimeric Antibodies

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

ten-fold serial dilutions. The

of the viral stock was determined as the infective dose at which half of the number of the microcultures was infected. In the neutralization assay, an infectivity dose equivalent to 20 times of the

TCID₅₀ was used. 50 μl of 60 times

of the viral stock was preincubated with 50μl 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, 50μl of growth medium without any antibodies was used. The mixtures were kept at 37°C in a 5% C0₂ incubator for 1 hour. The final concentration of the antibody was 100, 50, 25, 12.5 and 6.25μg_/ ml. Each concentration of the tested antibodies was performed in triplicate. At the end of the incubation, 50μl of 4 x 10⁶/ml H9 cells was added.

The H9 cells were harvested in log phase and pre-incubated for 1 hour at 37°C with 2 μg/ml polybrene in the RPMI-1640 growth medium containing 15% heat-inactivated fetal bovine serum before being added to the mixture. At the end of the incubation, the cells in each microculture wells were resuspended, 40 μl of the cell suspension was added to 200 μl of fresh growth medium in the corresponding wells of another microculture plates. The microculture plates were kept at 37°C in 5% C0₂ in an incubator. On day 3, day 5, day 7, day 9, day 11, and day 14, 150 μl 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 150μl fresh growth medium.

In the antigen capture assay, the HIV-1 specific antigens in the cell-free supernatant were measured by their affinity for the immunoglobulins from patients with AIDS. One hundred μl of diluted purified AIDS patient immunoglobulin (1:2000, approximately 5μg/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 μl of phosphate-buffered saline (PBS). The wells were blocked with 220 μl 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. 50 μl of the test samples (undiluted or in appropriate dilution) together with 50 μl of PBSTB (PBS containing 1% BSA and 0.1% Tween 20) were added to the well. The negative control contained 50 μl of the growth medium. The plate was incubated for 1 hour at 37°C. Then it was rinsed three times with PBST. lOOμl 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. lOOμl of a substrate solution (con- taining 20 mM sodium acetate buffer pH 6.0, 0.001% 3,3',5,5' tetramethyl-

benzidine and 0.001% hydrogen peroxide) was added to each well and incubated for 30 minutes at room temperature. Then 50μl of 2M H₂S0₄ was added to each well to stop the reaction. The absorbance was read at 490 nm. The readings from the triplicate were averaged and compared between the control and the test antibody for the percent of inhibition when neutralizing antibodies were added.

The results showed that at all dilutions the chimeric antibody completely

neutralized the HIV-1 infection to H9 cells at a 14-day assay. This neutralizing activity was identical to that of the parent murine antibody BAT123. A control murine antibody (anti-hCG) and the growth medium did not exhibit any inhibitory activity.

B) Inhibition of Svncvtium Formation bv the Chimeric Antibody Produced hv CAGl-51-4

The HIV-1-neutralizing activity of the chimeric antibody produced by

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,

Los Angeles). A culture of HeLa-CD4⁺ cells were plated onto wells of 96-well microculture plates at 20,000 cells per well. The plates were incubated for 36 hours and by this time, the monolayer epithelial cells were almost confluent. When 1 x 10⁴ infected H9 cells were added to the confluent HeLa-CD4⁺ cells, the cells formed contacts and fused, and within 18 hours multinucleated giant cells

(syncytia) formed. Those syncytia with more than five nuclei could be easily identified and enumerated, thus providing quantitative measurements of syncytium formation. When the effects of chimeric antibodies on syncytium formation were studied, 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 μl. .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. As shown in Table 2, 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 μg/ml, 10 μg/ml, -and 5 μg/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.

Table 2 Inhibition of Syncytium Formation Between HIV-1 Infected H9 Cells and HeLa-CD4⁺ Cells by the Chimeric Antibody Produced by CAGl- 51-4

* No. of syncytia from 5 randomly selected microscopical field at a magnification of 100X.

Results expressed in mean ± S.D., n=3

32 Isolation of CGP 47 439 Chimeric Antibody

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 μg/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 μg/ml when grown for four days under selection pressure with

5% FCS. 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

Pharmacia's Phast System™ and IEF was done as recommended by the

manufacturer. 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 μl peroxidase conjugated with goat anti-human IgG. The ELISA was performed three times, and the mean values were determined.

The results showed that the immunologic specificity was substantially the same as the standard, non-chimeric antibody BAT123. The potency was determined to be about 1.09 times that of the standard, non-chimeric antibody BAT123. An HIV-1-neutralizing assay, measured by the extent of syncytium

formation inhibition in the manner described above using HIV-1 infected H9 cells and CD4-expressing HeLa cells, was also performed. The IC^ μg/ml was substantially the same as the standard, non-chimeric antibody BAT123, as was the overall neutralization. Equivalents

Those skilled in the art will recognize, or be able to ascertain using no

more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to

be encompassed by the following claims.

Claims

1. A chimeric, viral-neutralizing immunoglobulin which binds to the gpl20 region of HIV-1 with a potency and immunologic specificity substantially the same as BAT123 mouse immunoglobulin, comprising a viral-specific antigen-binding region of non-human origin and a constant region of human origin.

2. The chimeric immunoglobulin of claim 1 wherein the antigen-binding region is of mouse origin.

3. The chimeric immunoglobulin CGP 47 439.

4. The transfectoma cell line CAG1-51-4-7A.6.

5. A method of treating an HIV-1 infected individual, a patient with AIDS or AIDS-related complex, an individual at risk of HIV-1 exposure, or an individual exposed to HIV-1 and at risk of infection, comprising administering to the individual a therapeutic amount of the chimeric

HIV-1-neutralizing immunoglobulin of claim 3.