EP0453554A1 - Anticorps murins humains chimeriques a specificite aux antigenes du vih - Google Patents

Anticorps murins humains chimeriques a specificite aux antigenes du vih

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Publication number
EP0453554A1
EP0453554A1 EP91900088A EP91900088A EP0453554A1 EP 0453554 A1 EP0453554 A1 EP 0453554A1 EP 91900088 A EP91900088 A EP 91900088A EP 91900088 A EP91900088 A EP 91900088A EP 0453554 A1 EP0453554 A1 EP 0453554A1
Authority
EP
European Patent Office
Prior art keywords
immunoglobulin
antigen
fragment
chain
derivative
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Withdrawn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
EP91900088A
Other languages
German (de)
English (en)
Other versions
EP0453554A4 (en
Inventor
Marc D. Better
Arnold H. Horwitz
Pradip Ghosh-Dastidar
Randy R. Robinson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mitsubishi Tanabe Pharma Corp
Xoma Corp
Original Assignee
Green Cross Corp Japan
Xoma Corp
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Green Cross Corp Japan, Xoma Corp filed Critical Green Cross Corp Japan
Publication of EP0453554A1 publication Critical patent/EP0453554A1/fr
Publication of EP0453554A4 publication Critical patent/EP0453554A4/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Withdrawn legal-status Critical Current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
    • C07K16/1072—Regulatory proteins, e.g. tat, rev, vpt
    • 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
    • C07K16/1054—Lentiviridae, e.g. HIV, FIV, SIV gag-pol, e.g. p17, p24
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion

Definitions

  • This invention relates to genetically-engineered antibodies with specificity for the Human Immunodeficiency Virus (HIV) and their derivatives, nucleotide and protein sequences coding therefor, as well as methods of obtaining and manipulating such sequences.
  • HIV Human Immunodeficiency Virus
  • mAb Monoclonal antibody
  • mAb Monoclonal antibody
  • MAbs produced from hybridomas are already widely used in diagnostic and basic scientific studies. Their efficacy in the treatment of human diseases, including viral and microbial infections, to a large extent remains to be demonstrated.
  • mouse mAbs Although they display extraordinarily specificity and can influence the progression of human disease, mouse mAbs, by their very nature, have limitations in their applicability to human medicine. Most obviously, since they are derived from mouse cells, they are recognized as foreign protein when introduced into humans and elicit immune responses. Similarly, since they are distinguished from human proteins, they are cleared rapidly from circulation. Finally, mouse antibodies may not be recognized as effectively as human antibodies by human effector cells or molecules.
  • human mAb-producing cell lines are obtained from Epstein Barr Virus (EBV) immortalized cells derived from human blood and may therefore not be useful for scale-up and production of human pharmaceuticals.
  • Immortalized human antibody-producing cell lines may also carry human viral sequences including the Human Immunodeficiency Virus.
  • mice since human viruses are thought to have evolved in the face of the human immune response, key antigens may not be recognized by the human immune system. Such antigens would not be expected to elicit useful immune responses in man. In contrast, those viral antigens that are immunogenic in mice can be used for the production of mouse mAbs which may have therapeutic utility in humans.
  • novel chimeric antibodies of this invention have been created using both the hybridoma and genetic engineering technologies to provide reagents.
  • the chimeric antibodies and products derived therefrom should have significant utility for the treatment and diagnosis of human disease.
  • the invention provides engineered chimeric mouse-human antibodies of desired variable (V) region specificity able to recognize certain antigens encoded by HIV and selected human constant (C) region properties, produced after gene cloning and expression of light (L) and heavy (H) chains.
  • V variable
  • C constant
  • the chimeric antibodies and their derivatives may have applicability in the treatment and diagnosis of individuals infected with the Human Immunodeficiency Virus (HIV).
  • HIV Human Immunodeficiency Virus
  • the cloned immunoglobulin gene products and their derivatives can be produced in mammal i an or microbial cel l s .
  • the invention provides cDNA sequences coding for immunoglobulin chains comprising a human C region and a non-human V region.
  • the immunoglobulin chains are both heavy and light.
  • the invention provides sequences as above, present in recombinant DNA molecules, in vehicles such as plasmid vectors, capable of expression in desired prokaryotic or eukaryotic hosts.
  • the invention provides hosts capable of producing, by culture, the chimeric antibodies and methods of using these hosts.
  • the invention also provides individual chimeric immunoglobulin chains, as well as complete assembled molecules having human C regions and mouse V regions with specificity for HIV antigens, wherein both V regions have the same binding specificity.
  • immunoglobulin chains and/or molecules provided by the invention are:
  • An antibody with monovalent specificity for an HIV antigen i.e., a complete, functional immunoglobulin molecule comprising:
  • Antibody fragments such as Fab, Fab', and F(ab') 2 .
  • the invention also provides for a genetic sequence, especially a cDNA sequence, coding for the V region of desired specificity of an antibody molecule H and/or L chain with or without human C regions, linked to a sequence coding for a polypeptide different than an immunoglobulin chain (e.g., an enzyme).
  • a genetic sequence especially a cDNA sequence, coding for the V region of desired specificity of an antibody molecule H and/or L chain with or without human C regions, linked to a sequence coding for a polypeptide different than an immunoglobulin chain (e.g., an enzyme).
  • cDNA sequences are particularly advantageous over genomic sequences (which contain introns), in that cDNA sequences can be expressed in bacteria or other hosts which lack appropriate RNA splicing systems.
  • FIGURE 1 Nucleotide sequence of the coding strand for the 2E12 mouse L chain V region (V L ). Shown is the nucleotide sequence from the end of the oligo-dC tail to the J k 2 - C k junction. Also shown is the amino acid sequence deduced from the nucleotide sequence. Shown in bold are the oligonucleotides used for site directed mutagenesis and the sites at which restriction site modifications were made.
  • FIGURE 2 Nucleotide sequence of the coding strand for the 2E12 mouse H chain V region (V H ). Shown is the nucleotide sequence from the end of the oligo-dC tail to the J H 2 - C H 1 junction. Also shown is the amino acid sequence deduced from the nucleotide sequence. Shown in bold are the oligonucleotides used for site directed mutagenesis and the sites at which restriction site modifications were made.
  • FIGURE 3 Construction scheme for the chimeric mouse-human Chimer1 H chain mammalian expression plasmid, pING3112.
  • the variable region from the cDNA clone pClG-12 was engineered to be compatible with the eucaryotic expression plasmid pING2227.
  • Plasmid pING2227 contains the following gene regulatory elements useful for expression in mammalian cells: 1) the IgG H chain enhancer element, 2) an Abelson LTR promoter, 3) the SV40 16S splice site, and 4) the IgG H chain polyadenylation signal sequence. It also contains the entire human IgGl C region from pGMH-6 (Liu, A. Y., et al., Proc. Natl. Acad. Sci., USA 84:3439-3443. 1987).
  • pING3112 contains the neomycin phosphotransferase gene which allows for G418 selection in transfected cells.
  • FIGURE 4 Construction scheme for the chimeric mouse-human Chimer1 L chain mammalian expression plasmid pING3110.
  • the V region from the cDNA clone pClK-7 was engineered to be compatible with the eucaryotic expression plasmid pING1712.
  • Plasmid pING1712 contains the following gene regulatory elements useful for expression in mammalian cells: 1) the IgH enhancer element, 2) the Abelson LTR promoter, 3) the SV40 16S splice site, and 4) a human kappa (k) polyadenylation signal sequence. It also contains the entire human C k region, Liu A.Y., et al., supra, and the GPT gene which allows for mycophenolic acid resistance in transfected cells.
  • FIGURE 5 Construction scheme for the bacterial Chimer1 Fab expression plasmid pING3127.
  • Plasmid pING3127 contains the following elements useful for expression in E. coli: 1) the araC gene, 2) the inducible araB promoter, 3) the dicistronic Fd and K Chimer1 genes fused to the pelB leader sequence, 4) the trpA transcription termination sequence, and 5) the tetR gene, useful for selection in E. coli.
  • the specific cloning steps are described in detail in Example 3.
  • FIGURE 6 Yeast expression plasmids for Fab expression. Shown are the yeast expression plasmid pING3114 containing the Chimer1 L chain gene fused to the yeast PGK promoter, invertase signal sequence and PGK polyadenylation signal (a); the similar yeast plasmids pING3117 and pING3137 containing the Fd gene (b); the yeast expression plasmid pING3118 containing the L chain promoter/leader fusion with PGK transcription termination signal (c); similar yeast plasmid pING3138 containing the Fd gene (d); and the final 2 gene yeast expression plasmid pING3142 (e).
  • FIGURE 7 Nucleotide sequence of the coding strand for the 2G12 mouse V H region. Shown is the nucleotide sequence from the end of the oligo-dC tail to the J H 3 - C H 1 junction. Also shown is the amino acid sequence deduced from the nucleotide sequence. Shown in bold are the oligonucleotides used for site directed mutagenesis and the sites at which restriction site modifications were made.
  • FIGURE 8 Nucleotide sequence of the coding strand for the 2G12 L chain mouse V region. Shown is the nucleotide sequence from the end of the oligo-dC tail to the J K 1 - C K junction. Also shown is the amino acid sequence deduced from the nucleotide sequence. Shown in bold are the oligonucleotides used for site directed mutagenesis and the sites at which restriction site modifications were made.
  • FIGURE 9 Construction scheme for the chimeric mouse-human Chimer2 H chain mammalian expression plasmid, pING3004.
  • the variable region from the cDNA clone pC2G-6 was engineered to be compatible with the eukaryotic expression plasmid pING2227.
  • Plasmid pING2227 contains the gene regulatory elements described in the legend to Figure 3.
  • FIGURE 10 Construction scheme for the chimeric mouse-human Chimer2 L chain mammalian expression plasmid pING3005.
  • the V region from the cDNA clone pC2K-14 was engineered to be compatible with the eucaryotic expression plasmid pING1712.
  • Plasmid pING1712 contains the gene regulatory elements described in the legend to Figure 4.
  • FIGURE 11 Construction scheme for the bacterial Chimer2 Fab expression plasmid pING3211. Plasmid pING3211 contains the elements useful for expression in E. coli described in the legend to Figure 5. The specific cloning steps are described in detail in Example 5.
  • FIGURE 12 Nucleotide sequence of the coding strand for the 1C11 mouse V H region. Shown is the nucleotide sequence from the end of the oligo-dC tail to the J H 3 - C H 1 junction. Also shown is the amino acid sequence deduced from the nucleotide sequence. Shown in bold is the oligonucleotide used for site directed mutagenesis and the sites at which restriction site modifications were made.
  • FIGURE 13 Nucleotide sequence of the coding strand for the 1C11 mouse V L region. Shown is the nucleotide sequence from the end of the oligo-dC tail to the J k 1 - C k junction. Also shown is the amino acid sequence deduced from the nucleotide sequence. Shown in bold is the oligonucleotide used for site directed mutagenesis and the sites at which restriction site modifications were made.
  • FIGURE 14 Construction scheme for the chimeric mouse-human Chimer4 H chain mammalian expression plasmid, pING2255.
  • the variable region from the cDNA clone pC4M-8 was engineered to be compatible with the eucaryotic expression plasmid pING2227.
  • Plasmid pING2227 contains the gene regulatory elements described in the legend to Figure 3.
  • FIGURE 15 Construction scheme for the chimeric mouse-human Chimer4 L chain mammalian expression plasmid pING2258.
  • the V region from the cDNA clone pC4K-16 was engineered to be compatible with the eucaryotic expression plasmid pING1712.
  • Plasmid pING1712 contains the gene regulatory elements described in the legend to Figure 4.
  • FIGURE 16 Construction scheme for the bacterial Chimer4 Fab expression plasmid. This plasmid contains the elements useful for expression in E. coli described in the legend to Figure 5. The specific cloning steps are described in detail in Example 8.
  • FIGURE 17. Nucleotide sequence of the coding strand for the 4D12 mouse V H region. Shown is the nucleotide sequence from the end of the oligo-dC tail to the J H 4 - C H 1 junction. Also shown is the amino acid sequence deduced from the nucleotide sequence. Shown in bold are the oligonucleotides used for site directed mutagenesis and the sites at which restriction site modifications were made.
  • FIGURE 18 Nucleotide sequence of the coding strand for the 4D12 mouse V L region. Shown is the nucleotide sequence from the end of the oligo-dC tail to the J k 5 - C k junction. Also shown is the amino acid sequence deduced from the nucleotide sequence. Shown in bold are the oligonucleotides used for site directed mutagenesis and the sites at which restriction site modifications were made.
  • FIGURE 19 Construction scheme for the chimeric mouse-human Chimer5 H chain mammalian expression plasmid, pING3126.
  • the variable region from the cDNA clone pC5G-30 was engineered to be compatible with the eucaryotic expression plasmid pING2227.
  • Plasmid pING2227 contains the gene regulatory elements described in the legend to Figure 3.
  • FIGURE 20 Construction scheme for the chimeric mouse-human Chimer5 L chain mammalian expression plasmid pING3132.
  • the V region from the cDNA clone pC5K-4 was engineered to be compatible with the eucaryotic expression plasmid pING1712.
  • Plasmid pING1712 contains the gene regulatory elements described in the legend to Figure 4.
  • FIGURE 21 Construction scheme for the bacterial Chimer5 Fab expression plasmid pING3139. Plasmid pING3139 contains the elements useful for expression in E. coli as described in the legend to Figure 5.
  • the invention provides chimeric antibodies that can be used for the treatment and diagnosis of individuals infected with HIV, either alone or in combination with other reagents.
  • the chimeric antibodies contain mouse V regions which recognize certain antigens encoded by the HIV genome. In several of the examples described herein, the chimeric antibodies recognize those antigens recognized by the mouse mAbs 2E12, 2G12, 1C11, and 4D12.
  • the method of production combines five elements:
  • mRNA messenger RNA
  • H and L chain J regions have different sequences, but a high degree of sequence homology exists (greater than 80%) among each group, especially near the C region. This homology is exploited in this invention and consensus sequences of H and L chain J regions were used to design oligonucleotides for use as primers for introducing useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments.
  • C region cDNA module vectors prepared from human cells and modified by site-directed mutagenesis to place a restriction site at the analogous position in the human sequence were used. For example, one can clone the complete human C K region and the complete human C ⁇ 1 region. An alternative method utilizing genomic C region clones as the source for C region module vectors would not allow these genes to be expressed in systems such as bacteria where enzymes needed to remove intervening sequences are absent. Cloned V region segments are excised and ligated to L or H chain C region module vectors. In addition, the human C ⁇ 1 region can be modified by introducing a termination codon thereby generating a gene sequence which encodes the H chain portion of an Fab molecule.
  • the coding sequences with linked V and C regions are then transferred into appropriate expression vehicles for expression in appropriate hosts, prokaryotic or eukaryotic.
  • Two coding DNA sequences are said to be "operably linked” if the linkage results in a continuously translatable sequence without alteration or interruption of the triplet reading frame.
  • a DNA coding sequence is operably linked to a gene expression element if the linkage results in the proper function of that gene expression element to result in expression of the coding sequence.
  • Expression vehicles include plasmids or other vectors. Preferred among these are vehicles carrying a functionally complete human C H or C L chain sequence having appropriate restriction sites engineered so that any V H or V L chain sequence with appropriate cohesive ends can be easily inserted therein. Human C H or C L chain sequence-containing vehicles are thus an important embodiment of the invention. These vehicles can be used as intermediates for the expression of any desired complete H or L chain in any appropriate host.
  • yeast provides substantial advantages for the production of immunoglobulin H and L chains. Yeasts carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies now exist which utilize strong promoter sequences and high copy number plasmids which can be used for production of the desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., prepeptides) (Hitzman, et al., 11th International Conference on Yeast, Genetics and Molecular Biology, adjoin, France, September 13-17, 1982).
  • Yeast gene expression systems can be routinely evaluated for the levels of production, secretion and the stability of chimeric H and L chain proteins and assembled chimeric antibodies. Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeasts are grown in media rich in glucose can be utilized. Known glycolytic genes can also provide very efficient transcription control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. A number of approaches may be taken for evaluating optimal expression plasmids for the expression of cloned immunoglobulin cDNAs in yeast (see Glover, D.M., ed., DNA Cloning, Vol. II, pp45-66, IRL Press, 1985).
  • PGK phosphoglycerate kinase
  • Bacterial strains may also be utilized as hosts for the production of antibody molecules or antibody fragments described by this invention, E. coli K12 strains such as E. coli W3110 (ATCC 27325), and other enterobacteria such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species may be used.
  • E. coli K12 strains such as E. coli W3110 (ATCC 27325)
  • enterobacteria such as Salmonella typhimurium or Serratia marcescens
  • various Pseudomonas species may be used.
  • Plasmid vectors containing replicon and control sequences which are derived from species compatible with a host cell are used in connection with these bacterial hosts.
  • the vector carries a replication site, as well as specific genes which are capable of providing phenotypic selection in transformed cells.
  • a number of approaches may be taken for evaluating the expression plasmids for the production of chimeric antibodies or antibody chains encoded by the cloned immunoglobulin cDNAs in bacteria (see Glover, D.M., ed., DNA Cloning, Vol. I, IRL Press, 1985).
  • Mammalian cells are grown in vitro or in vivo.
  • Mammalian cells provide post-trans! ational modifications to immunoglobulin protein molecules including leader peptide removal, folding and assembly of H and L chains, glycosylation of the antibody molecules, and secretion of functional antibody protein.
  • Mammalian cells which may be useful as hosts for the production of antibody proteins include cells of lymphoid origin, such as the hybridoma Sp2/0-Ag14 (ATCC CRL 1581) or the myeloma P3X63Ag8 (ATCC TIB 9), and its derivatives. Others include cells of fibroblast origin, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61). Many vector systems are available for the expression of cloned H and L chain genes in mammalian cells (see Glover, D.M., ed., DNA Cloning. Vol. II, pp143-238, IRL Press, 1985). Different approaches can be followed to obtain complete H 2 L 2 antibodies.
  • H and L chains in the same cells to achieve intracellular association and linkage of H and L chains into complete tetrameric H 2 L 2 antibodies.
  • the co-expression can occur by using either the same or different plasmids in the same host. Genes for both H and L chains can be placed into the same plasmid, which is then transfected into cells, thereby selecting directly for cells that express both chains. Alternatively, cells may be transfected first with a plasmid encoding one chain, for example the L chain, followed by transfection of the resulting cell line with an H chain plasmid containing a second selectable marker.
  • H 2 L 2 molecules via either route could be transfected with plasmids encoding additional copies of H, L, or H plus L chains in conjunction with additional selectable markers to generate cell lines with enhanced properties, such as higher production of assembled H 2 L 2 antibody molecules or enhanced stability of the transfected cell lines.
  • the invention provides chimeric immunoglobulin chains, either H or L, with specificity toward viral antigens of the human immunodeficiency virus, HIV.
  • a chimeric chain contains a C region substantially similar to that present in a natural human immunoglobulin, and a non-human V region having the desired anti-viral specificity of the invention.
  • the invention also provides immunoglobulin molecules having H and L chains associated so that the overall molecule exhibits the desired binding and recognition properties.
  • immunoglobulin molecules monovalent, divalent, or molecules with the invention's V region binding domains attached to moieties carrying desired functions.
  • This invention also provides for "fragments" of chimeric immunoglobulin molecules, which include Fab, F(ab'), and F(ab') 2 molecules.
  • the invention also provides for "derivatives" of the chimeric immunoglobulins, which term includes those proteins encoded by truncated or modifed genes to yield molecular species functionally resembling the immunoglobulin fragments.
  • the modifications include, but are not limited to, addition of genetic sequences coding for cytotoxic proteins such as plant and bacterial toxins and tumor necrosis factor (TNF).
  • the fragments and derivatives can be produced from any of the hosts of this invention.
  • Antibodies, fragments or derivatives having chimeric H chains and L chains of the same or different V region binding specificity can be prepared by appropriate association of the individual polypeptide chains, as taught, for example by Sears et al. (Proc. Natl. Acad. Sci. USA 72:353-357 (1975)). With this approach, hosts expressing chimeric H chains (or their derivatives) are separately cultured from hosts expressing chimeric L chains (or their derivatives), and the immunoglobulin chains are separately recovered and then associated. Alternatively, the hosts can be co-cultured and the chains allowed to associate spontaneously in the culture medium, followed by recovery of the assembled immunoglobulin, fragment or derivative.
  • the chimeric antibodies of this invention recognize epitopes of antigens encoded by the HIV genome, including core antigens, reverse transcriptase, and envelope glycoproteins.
  • These HIV antigens include the glycoproteins encoded by the viral env gene (gp160, gp120, gp41), and proteins encoded by the viral gag gene (p55, p45, p39, p24, and p18), and pol gene (p65, p51) (Musing, M.A. et al., Nature 313:450-458 (1985)).
  • the Chimer1, Chimer2, Chimer4, and ChimerS antibodies recognize various of these antigens (see Examples, below) and can be used alone or in combination with antibodies that recognize other viral components.
  • the chimeric antibodies react with viruses, subviral particles, viral proteins, viral peptides, and HIV-infected cells expressing viral antigens and should exhibit therapeutic activity in infected individuals via normal immune and host defense mechanisms.
  • the antibodies of this invention should be useful for treatment of asymptomatic HIV-infected individuals to prevent progression of the infectious process, as well as individuals with clinical symptoms of HIV infection (Acquired Immune Deficiency Syndrome (AIDS) and AIDS Related Complex (ARC)), by reducing viral burden and alleviating symptoms related to circulating viral antigens or virus-infected cells.
  • Circulating HIV proteins including gp120 and p24 are thought to be associated with immunological dysfunction.
  • Administration of Chimer1, Chimer2, Chimer4, and Chimer5, either singly or in combination therapy, should benefit individuals with low titers of anti-HIV antibodies.
  • Treatment of an individual infected with HIV using the antibodies, fragments or derivatives of this invention comprises parenterally admininstering a single or multiple doses of the antibody, fragment or derivative.
  • the effective dose is a function of the individual chimeric antibody, the presence and nature of a conjugated therapeutic agent (see below), the patient and his clinical status, and can vary from about 10 ng/kg body weight to about 100 mg/kg body weight.
  • the route of administration may include intravenous, subcutaneous, intramuscular, intrapulmonary, intraperi- toneal, intranasal, intrathecal, transdermal or other known routes.
  • the antibodies of this invention can be adapted for therapeutic efficacy by virtue of their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against HIV-infected cells.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • an endogenous source or an exogenous source of effector cells (for ADCC) or complement components (for CDC) can be utilized.
  • Antibodies that effectively mediate ADCC or otherwise mark infected cells for destruction by host effector cells should be especially useful after acute HIV infection when relatively few host cells carry the virus.
  • the chimeric antibodies of this invention, their fragments, and derivatives can be used therapeutically as immunoconjugates (see for review: Dillman, R.O., Ann. Int. Med. 111:592-603 (1989)). They can be coupled to cytotoxic proteins, including, but not limited to Ricin- A, Pseudomonas toxin, Diptheria toxin, and TNF. Toxins conjugated to antibodies or other ligands, are known in the art (see, for example, Olsnes, S. et al., Immunol. Today 10:291-295 (1989)). Plant and bacterial toxins typically kill cells by disrupting the protein synthetic machinery.
  • the chimeric antibodies of this invention can be conjugated to additional types of therapeutic moieties including, but not limited to, radionuclides, cytotoxic agents and anti-viral drugs, to treat individuals infected with HIV.
  • therapeutic moieties including, but not limited to, radionuclides, cytotoxic agents and anti-viral drugs, to treat individuals infected with HIV.
  • radionuclides which can be coupled to antibodies and delivered in vivo to sites of antigen include 212 Bi, 131 I, 186 Re, and 90 Y, which list is not intended to be exhaustive.
  • the radionuclides exert their cytotoxic effect by locally irradiating the cells, leading to various intracellular lesions, as is known in the art of radiotherapy.
  • Cytotoxic drugs which can be conjugated to antibodies and subsequently used for in vivo therapy include, but are not limited to, daunorubicin, doxorubicin, methotrexate, and Mitomycin C. Cytotoxic drugs interfere with critical cellular processes including DNA, RNA, and protein synthesis.
  • Antiviral drugs which may be conjugated to antibodies and subsequently used for in vivo therapy include, but are not limited to, acyclovir, azidothymidine, adenine arabinoside, dideoxyinosine, and protease inhibitors. Antiviral drugs act by interfering with virus-specific enzymes such as reverse transcriptase, with viral proteases, and with the metabolism and incorporation of nucleotides into nucleic acids.
  • the chimeric antibodies, fragments or derivatives of this invention may be advantageously utilized in combination with other chimeric antibodies, or with lymphokines or hemopoietic growth factors , etc . , whi ch serve to increase the number or acti vity of effector cells which interact with the antibodies.
  • Antibodies specific for HIV-infected cells should play an active role in limiting HIV spread within an individual. Earlier intervention in the infectious process may delay or prevent the later emergence of more pathogenic HIV strains or variants within an infected individual.
  • the antibodies, fragments or derivatives of this invention may permit the establishment and maintenance of a longer asymptomatic course which would improve the life of infected individuals.
  • Interventive therapy with the chimeric antibodies of this invention may also be useful either alone or in conjunction with other anti-viral therapies to prevent accidental viral exposure from developing into symptomatic disease.
  • Prophylactic immunization with the antibodies of this invention may effectively reduce the risk of a successful viral infection upon challenge, much as other antibody preparations are used in the management of hepatitis and cytomegalo- virus infections.
  • the chimeric antibodies, fragments, or derivatives of this invention, attached to a solid support can be used to remove the virus, viral antigens, or virus-infected cells from fluids or tissue or cell extracts. In a preferred embodiment, they are used to remove HIV from blood or blood plasma products. In another preferred embodiment, the chimeric antibodies are advantageously used in extracorporeal immunoadsorbent devices, which are known in the art (see, for example, Seminars in Hematology, Vol. 26 (2 Suppl. 1) (1989)). Patient blood or other body fluid is exposed to the attached antibody, resulting in partial or complete removal of circulating HIV virions, viral antigens (free or in immune complexes), or virus- infected cells, following which the fluid is returned to the body.
  • This immunoadsorption can be implemented in a continuous flow arrangement, with or without interposing a cell centrifugation step. See, for example, Terman, D.S. et al., J. Immunol. 117:1971-1975 (1976).
  • the chimeric antibodies of this invention can be used for any and all uses in which the murine mAbs can be used, with the clear advantage that mouse-human chimeric antibodies are more compatible with the human body.
  • the mouse mAb 2E12 (described as anti-gp120,160 mAb by Yoshihara, P. et al., Proc. 4th Int'l. Conf. on AIDS. June, 1988, p237) was derived from a hybridoma fusion utilizing spleen cells from a BALB/c mouse immunized with an HIV viral lysate, and was then cloned in the presence of purified env gene product. The Sp2/0 myeloma line was used as the fusion partner. Clone 2E12 produces immunoglobulin of the IgGl subclass. MAb 2E12 was reactive against cloned env gene product by ELISA analysis.
  • 2E12 was capable of immunofluorescent staining of HIV-infected cells.
  • Western blot analysis of the 2E12 mAb against viral lysates demonstrated a predominant 120 kD band as well as a weaker 160 kD band, depending upon the lysate used.
  • Oligo-dG tailed pBR322, pUC18, pUC19, M13mp18, and M13mp19 were purchased from BRL (Gaithersburg, MD).
  • DNA manipulations involving purification of plasmid DNA by buoyant density centrifugation, restriction endonuclease digestion, purification of DNA fragments by agarose gel electrophoresis, ligation and transformation of E. coli were as described by Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, (1982), or other standard procedures. Restriction endonucleases and other DNA/RNA modifying enzymes were purchased from Boehringer-Mannheim (Indianapolis, IN), BRL, and New England Biolabs (Beverly, MA). 2. RNA Purification and cDNA Library Construction
  • Oligo-dT primed cDNA libraries were prepared from poly(A)+ RNA by the methods of Gubler, V., et al. (Gene 25:263 (1983)). The cDNA was dC-tailed with terminal deoxynucleotide transferase and annealed to dG-tailed pBR322. cDNA libraries were screened by hybridization (Maniatis, T., et al., supra) with 32 P-labeled, nick-translated DNA fragments, i.e., for K clones with a mouse C ⁇ region probe and for H chain clones with a mouse IgGl C region probe
  • nucleotide sequences of the cDNA clones show that they are immunoglobulin V region clones as they contain amino acid residues diagnostic of V domains (Kabat, et al., Sequences of Proteins of
  • the L chain vector pING1712 was made by first making a plasmid DNA containing a test chimeric L chain gene (pING2122) and adding a mouse Abelson LTR promoter, a splice region, and a mouse genomic kappa region 3' to the polyadenylation signal.
  • the H chain mouse enhancer 0.7 kb Xbal to EcoRI fragment from M13 M8alphaRX12 (Robinson, R.R., et al., suoral was inserted into Xbal plus EcoRI cut M13mp19.
  • the enhancer-containing Hindlll to Bglll fragment was inserted into the BollI to Hindlll region of pSH6, an E. coli recombinant plasmid DNA that contains unique Xhol. Bglll, and Hindlll sites, in that order.
  • the enhancer-containing Xbal to Xhol fragment was then inserted into the enhancer Xbal to Xhol region of pING2121b, an expression plasmid identical to pING2108b (Liu, A.Y., et al., J. Immunology 139:3521 (1987)) except that the L6 V L region (Liu, A.Y., et al., Proc. Natl. Acad. Sci. USA 84:3439 (1987)) was used in its construction instead of the 2H7 V L region.
  • the resulting plasmid was pING2122.
  • the mouse Abel son virus LTR was obtained from pelin2 (provided by Dr. Owen Witte, UCLA).
  • pelin2 contains the p120 viral 3' LTR (Reddy, E.P., et al., Proc. Natl. Acad. Sci. USA 80: 3623 (1983)) except that the Bglll site at viral position 4623 has been modified by insertion of the EcoRI oligonucleotide linker GGATTCC.
  • the 0.8 kb EcoRI to Konl fragment of pelin2 containing the p120 3' LTR promoter was inserted into Kpnl plus EcoRI cut pUC18.
  • the LTR was excised as an EcoRI to Sall fragment and ligated to EcoRI plus Sall cut pING2122, resulting in a plasmid where the LTR promoter is adjacent to the L6 L chain gene (pING2126).
  • An Xhol to Sall fragment containing SV40 16S splice donor and acceptor sites was excised from pUC12/pL1 (Robinson, R.R. et al., supra) and inserted into the Sall site of pING2126, screening for the orientation where the splice donor was between the LTR and the L chain gene (pING2133) .
  • the polyadenylation/transcription termination region of the kappa expression vector was also modified.
  • the first step was the Hindlll digestion and religation of plasmid pING2121a, which is identical to pING2108a (Liu, A.Y., et al., supra) except that the L6 V L was used in its construction instead of the 2H7 V L , to form pING2121a-deltaH.
  • the 1.1 kb Bglll to BamHI fragment of mouse genomic DNA distal to the polyadenylation site (Xu, M., et al., J. Biol. Chem. 261:3838 (1986) was isolated from pS107A (provided by Dr.
  • the Abelson LTR promoter was also used in the chimeric H chain expression vector pING1714.
  • pING2111 Robot, R.R., et al., supra
  • the Aatll to Sall fragment containing the Abelson LTR promoter was excised from pING2133 and ligated to the large Aatll to Sall fragment of pING1707 to form pING1711.
  • the H chain enhancer was deleted from pING1711 by EcoRI digestion, T4 polymerase treatment, ligation to Aatll oligonucleotide linker, and cleavage and religation with Aatll to form the 7.7kb expression vector pING1714.
  • a similar plasmid, pING2227 contains two additional regulatory elements, the IgH enhancer and the human genomic IgG polyadenylation sequence.
  • pING2227 is identical to pING1712 in the region from Bglll to Sall containing the IgH enhancer the Abelson LTR promoter, and the 16S slice donor and acceptor sites.
  • the human genomic IgG 3' end sequence was ligated as an 1185 bp Xmalll DNA fragment into an Xmalll site located 6 bp past the termination codon for the H chain gene in pING1714.
  • the 1300 bp Xmalll fragment containing the genomic gamma 3' end was isolated from a derivative of pHG3A (Ellison, et al., Nucl. Acids Res. 10:4071 (1982)).
  • the J region mutagenesis primer 5'-GGCTGAGGAGACGGTGACCGTGG-3' was used to insert a BstEII site into the M13 subclone pClGSDK, and the oligonucleotide 5'-GAGGTCCTGTCGACTTAGTAACTGT-3' was used to insert a Sall restriction site into pClGBstEII upstream of the initiation codon ATG.
  • the restriction fragment containing the 2E12 V H region bounded by Sall and BstEII was then cloned into the expression vector PING2227.
  • the J region mutagenesis primer 5'-GTTTTATTTCAAGCTTGGTCC-S' was used to insert a Hindlll site into the M13 subclone pClKSDM, and the oligonucleotide 5'-TGAGAACTTGGTCGACAGAGTCCGGCG-3' was used to insert a Sail restriction site into pClKHIII upstream of the initiation codon ATG.
  • the restriction fragment containing the 2E12 V L region bounded by Salland Hindlll was then cloned into the expression vector pING1712.
  • the cell line Sp2/0 (American Type Culture Collection #CRL1581) was grown in Dulbecco's Modified Eagle Medium plus 4.5 g/l glucose (DMEM, Gibco) plus 10% fetal bovine serum. Media were supplemented with glutamine/penicillin/streptomycin (Irvine Scientific, Irvine, California). The electroporation method of Potter, H., et al., (Proc. Natl. Acad. Sci.. USA, 81:7161 (1984)) was used. After transfection, cells were allowed to recover in complete DMEM for 24 hours, and then seeded at 1-3 x 10 4 cells per well in 96-well culture plates in the presence of selective medium.
  • DMEM Dulbecco's Modified Eagle Medium plus 4.5 g/l glucose (DMEM, Gibco) plus 10% fetal bovine serum. Media were supplemented with glutamine/penicillin/streptomycin (Irvine Scientific, Irvine, California). The electroporation method of Potter, H.,
  • G418 (GIBCO) selection was at 0.8 mg/ml, and mycophenolic acid (Cal biochem) was at 6 ⁇ g/ml plus 0.25 mg/ml xanthine.
  • the electroporation technique gave a transfection frequency of 1-10 x 10 -5 for the Sp2/0 cells.
  • the Chimer1 L chain expression plasmid pING3110 was linearized by digestion with Pvul restriction endonuclease and transfected into Sp2/0 cells, giving mycophenolic acid resistant clones which were screened for L chain synthesis. The best producer after outgrowth and subsequent subcloning, was transfected with Pvul-linearized pING3112, the expression plasmid containing the Chimer1 H chain gene. After selection with G418, the clone producing the most L plus H chain, Sp2/0-3310 11E10 + 3312 5C11.4C9, secreted antibody at approximately 5 ⁇ g/ml.
  • Sp2/0-3110 11E10.1B2 + 3112 5C11.4C9 cells were grown in culture medium HB101 (Hana Biologies) + 1% Fetal Bovine Serum, supplemented with 10mM HEPES, 1x Glutamine-Pen-Strep (Irvine Scientific #9316). The spent medium was centrifuged at about 5,000 xg for 20 minutes. The antibody level was measured by ELISA. Approximately 23L of cell culture supernatant was concentrated 14-fold over a S10Y30 cartridge using DC-10 concentrator (Amicon Corp).
  • E. coli Bacteria are suited for production of chimeric antibodies expressed from mammalian cDNA since entire coding sequences can be expressed from well characterized promoters.
  • Escherichia coli is one of many useful bacterial species for production of foreign proteins since a wealth of genetic information is available for optimization of its gene expression.
  • E. coli can be used for production of foreign proteins internally or for secretion of proteins out of the cytoplasm, where they most often accumulate in the periplasmic space (Gray, et al., Gene 39:247 (1985); Oka, et al., Proc. Natl. Acad. Sci. USA 82:7212 (1985)).
  • Secretion from the E. coli cytoplasm has been observed for many proteins and requires a signal sequence.
  • Proteins produced internally in bacteria are often not folded properly (Schoner, et al., BioTechnology 3:151 (1985)), Protein secreted from bacteria, however, is often folded properly and assumes native secondary and tertiary structures (Hsiung, et al., BioTechnology 4:991 (1986).
  • a Fab molecule consists of two nonidentical protein chains linked by a single disulfide bridge. These two chains are the intact antibody L chain and the V, J, and C H 1 portions of the antibody heavy chain, Fd.
  • the proper cDNA clones for the Chimer1 L and Fd genes have already been identified.
  • these cDNA clones were organized into a single bacterial operon (a dicistronic message) as gene fusions to the pectate lyase (pelB) gene leader sequence from Erwinia carotovora (Lei, et al., J. Bacteriol. 169:4379 (1987) and expressed from a strong regulated promoter.
  • the result is a system for the simultaneous expression of two protein chains in E. coli, and the secretion of immunologically active, properly assembled Fab of Chimer1 antibody.
  • the following sections detail the secretion of Chimer1 Fab from E. coli. 1. Assembly of the pelB Leader Seouence Cassette
  • Erwinia carotovora codes for several pectate lyases (poly- galacturonic acid trans-eliminase) (Lei, et al., Gene 35:63 (1985)). Three pectate lyase genes have been cloned, and the DNA sequence of these genes has been determined. When cloned into E. coli under the control of a strong promoter, the pelB gene is expressed and large quantities of pectate lyase accumulate in the periplasmic space and culture supernatant. The pelB signal sequence functions efficiently in E. coli and was used as a secretion signal for antibody genes in this example. (Other signal sequences would also be useful for this application.) The nucleotide sequence surrounding the signal sequence of the pelB gene is published (Lei, et al., supra).
  • the pelB signal sequence contains a Haelll restriction site at amino acid 22, adjacent to the signal peptidase cleavage site: ala-ala.
  • Plasmid pSS1004 (Lei, et al., supra) containing the pelB gene in pUC8 (Vierra et al., Gene 19:259 (1982)) was digested with Haelll and EcoRI. This DNA was ligated with an eight base pair Sstl linker to Sspl and EcoRI cut pBR322.
  • the resulting plasmid contained a 300 bp fragment which included the 22 amino acid leader sequence of pelB and about 230 bp of upstream E. caratovora DNA.
  • This plasmid, pING173, contains an insert that upon digestion with Sstl and treatment with T4 DNA polymerase can be ligated directly to a DNA fragment flanked by the first amino acid of a mature coding sequence for any gene to generate a protein fusion containing a functional bacterial leader sequence in frame with the incoming gene.
  • the Sstl to EcoRI restriction fragment in pING173 was cloned into pUC18 (Yanich-Perron, et al., Gene 33:103 (1985)) to generate pRR175, which contains the pelB leader and adjacent upstream non-coding sequence (including a ribosome binding site) downstream of the lac promoter.
  • Plasmid pING1500 derived from pRR175, contains only the region from the -48 of the pelB gene to an Xhol site downstream of the pelB leader, and includes the Sstl site at the junction.
  • a Kpnl site was similarly introduced at the junction of the leader peptide and mature coding region of the 2E12 H chain with the oligonucleotide primer 5'-GTTGCACCTGGTACCGGACACCTGTAG-3' ( Figure 2).
  • the Chimer1 V L region containing a Bsml restriction site at the signal sequence processing site and a unique Hindlll site in the J region of pClKHB served as the starting point for bacterial expression.
  • the plasmid pClKHB was cut with Bsml, treated with T4 polymerase, and digested with Hindlll ( Figure 5A),
  • the 320 bp fragment containing the V L region was purified and ligated to pING1500 that was cut with Sstl, treated with T4 polymerase, and cut with Xhol ( Figure SB) along with a Hindlll to Xhol restriction fragment containing the human C k plus 15 nucleotides of 3' genomic DNA from pING3102 ( Figure 5C).
  • the resulting plasmid that contained a pelB::Chimer1 L chain fusion was sequenced to determine that the proper in-frame fusion was formed. This plasmid was called pING3111.
  • the intact Chimer1 chimeric Fd gene containing a Kpnl restriction site at the signal sequence processing site and a BstEII restriction site in the J region in pClGBK served as the starting point for bacterial expression.
  • the plasmid pClGBK was cut with Kpnl, treated with T4 polymerase, and digested with BstEII ( Figure 5D).
  • the approximately 335 bp fragment containing the Fd V region was purified and ligated to pING1500 that was cut with Sstl, treated with T4 polymerase, and cut with Xhol (Figure 5E) along with the human CH1 region (which had been previously generated by introducing a stop codon in hinge, Robinson, R.R., et al., supra) from pF3D ( Figure 5F).
  • the resulting plasmid that contained a pelB::Chimer1 Fd fusion was sequenced to determine that the proper in-frame fusion was formed. This plasmid was called pING3115.
  • both L chain and Fd need to be produced simultaneously within the cell.
  • plasmids constructed with each of these genes separately, a series of expression vectors were constructed that contain both genes aligned so that transcription from a single promoter will specify both genes. This was done in a way that minimized the noncoding DNA between the two genes.
  • Each gene has a ribosome binding site needed for translation initiation and the identical DNA sequence from -48 to the pelB leader::antibody gene junction.
  • Plasmid pING3111 was cut with Sphl, treated with T4 polymerase, cut with EcoRI, and the vector fragment was purified ( Figure 5G).
  • pING3115 was cut with Xhol, treated with T4 polymerase, cut with EcoRI and the fragment containing the Fd gene was purified ( Figure 5H). These two purified DNA fragments were ligated to produce pING3116, which contained the two Chimer1 gene fusions linked in close proximity. The two-gene cistron was placed under the control of the araB promoter in pING3104.
  • Plasmid pING3116 was cut with Sphl, treated with T4 polymerase, cut with Xhol, and the fragment containing the Fd and k genes was purified (Figure 51). This DNA fragment was ligated to the vector fragment from pING3104 that had been cut with EcoRI, treated with T4 polymerase, and cut with Xhol ( Figure 5J), generating pING3119. The unique BstEII fragment in the Fd C region was shown by restriction analysis to be in the incorrect orientation; when the Fd gene was assembled, it assembled improperly. To correct this, pING3119 was cut with BstEII and religated to generate the final Chimer1 Fab vector pING3127 ( Figure 5K). This vector contains all the necessary features for expression of Chimer1 Fab in E. coli. 6. Production of Chimer1 Fab in Bacteria
  • Chimer1 Fab from pING3127 in E. coli is under the inducible control of the araB promoter. Upon arabinose induction, Fab secreted into the growth medium increases more than 10-fold.
  • Fab was detected in the fermentation broth by antibody sandwich ELISA using rabbit anti-human K chain antiserum as the solid phase agent to bind Fab, followed by detection with monoclonal anti-human Fd and goat anti-mouse IgG peroxidase conjugate.
  • the total amount of protein was estimated by a colorimetric assay, and the sample was loaded onto a carboxymethylcellulose type (CM52, Whatman) column at a ratio of 10 mg total protein per lg CM52 (pre-equilibrated with 10mM sodium phosphate buffer at pH 6.8).
  • the CM52, column was eluted with a linear gradient of increasing NaCl concentration (0-0.1N) in the same phosphate buffer.
  • the fractions containing Fab were further analyzed by SDS-PAGE and then pooled.
  • the combined Fab fractions were concentrated over a YM10 membrane to an Fab concentration of about 1 mg/ml, and stored frozen.
  • the Chimer1 Fab purified from E. coli has identical molecular weight properties as other Fab molecules produced from E. coli, as assessed by SDS gel electrophoresis.
  • the bacteri ally-produced Chimer1 Fab is correctly assembled as a kappa plus Fd chain dimer because of its positive reaction in the ELISA detecting molecules with both Kappa and Fd determinants, and both chains are properly folded because the Fab reacts with gp120 on Western immunoblot test strips. 7. Study Performed on Chimer1 Fab
  • Chimer1 Fab was tested along with 2E12 mouse antibody and Chimer1 antibody for binding to antigen fixed to commercially available HIV Western immunoblot test strips (Dupont or equivalent). Chimer1 Fab was incubated with the test strip, followed by interaction with goat anti-human Fab antibody, and then with rabbit anti-goat IgG. Detection of bound antibody was with Protein A-gold followed by silver enhancement (BioRad). Chimer1 Fab specifically recognized the same size antigen on the test strip as did Chimer1 whole antibody, specifically recognizing an antigen of about 120 kD.
  • yeast cells are capable of expressing and secreting foreign proteins.
  • yeast served as the host for the production of mouse-human chimeric Fab.
  • Saccharomyces cerevisiae strain PS6 (ura3 leu2 MATa) was developed at INGENE and used as a host for yeast transformations performed as described by Ito, et al., J. Bacteriol. 153:163-168 (1983). Yeast transformants were selected on SD agar (2% glucose, 0.67% yeast nitrogen base, 2% agar) and grown in SD broth buffered with 50mM sodium succinate, pH 5.5.
  • the gene sequences encoding the mature form of the V L region of 2E12 and containing a Hindlll site in the 0 region (as described in Example 1) and a Bsml site introduced at the signal sequence processing site was fused to the human C k region.
  • Plasmid pCIKHB was cut with Bsml, treated with T4 polymerase, and cut with HindIII.
  • the V region fragment was ligated along with the human C L region prepared from pING3102 as a Hindlll to Xhol fragment into pING1149.
  • the plasmid pING1149 contains the gene sequence encoding the yeast invertase signal sequence (Taussig, R. et al., Nucl.Acids Res.
  • the resultant plasmid pING3114 contains the PGK promoter (P), along with the invertase signal sequence (S), fused to the Chimer1 coding sequence.
  • P PGK promoter
  • S invertase signal sequence
  • the PGK promoter - invertase signal sequence - chimeric L chain (V,C K ) fusion was cloned into a partial 2 micron circle (2 ⁇ ), ura3 yeast expression vector containing the PGK polyadenylation signal (Tm) to generate pING3118 ( Figure 6C).
  • the gene sequence encoding the mature form of the V H region of Chimer1 containing a BstEII site in the J region (as described in Example 1) and a Kpnl site introduced at the signal sequence processing site from pCIGBK was fused along with the human C H 1 region (which had been previously generated by introducing a stop codon in hinge, Robinson, R.R., et al., supra) from pF3D into pING1149 that had been cut with Pstl, treated with T4 polymerase and cut with Xhol. This generated plasmids pING3117 and pING3137.
  • Plasmid pING3117 had the correct fusion junction between the invertase leader sequence and the mature V region coding sequence, but a BstEII fragment from the C H 1 region of pF3D was in the incorrect orientation. Plasmid pING3137 had improperly fused the invertase leader to the Chimer1 V H region, but had the C region BstEII fragment in the correct orientation.
  • a BamHI to Pstl fragment from pING3117 containing the PGK promoter, invertase leader and a portion of the Chimer1 V region was ligated along with a Pstl to Xhol fragment from pING3137 which contained the remainder of the Chimer1 V region and the J-C H 1 portion into a partial 2 micron circle (2 ⁇ ) expression vector containing the PGK polyadenylation signal (Tm) to generate pING3138, Figure 6D.
  • a single yeast expression vector containing both the chimeric L chain and Fd chain genes and their respective expression signals was constructed from pING3118 and pING3138.
  • This final vector, pING3142, Figure 6E contains a portion of 2 micron circle (oriY, REP3) and the two selectable markers leu2d and ura3.
  • the plasmid pING3142 was transformed into S. cerevisiae PS6 and the transformants were grown in broth under selective conditions as described above.
  • the culture supernatants were assayed by ELISA and contained Fab levels of approximately 900 ng/ml.
  • This material can be purified from fermentation broth and is expected to have properties identical to those of Fab produced as described in Example 2; yeast Chimer1 Fab is expected to have the same binding specificity as Fab produced in E. coli.
  • the mouse mAb 2G12 (described as anti-p24 mAb in: Yoshihara, P. et al., Proc. 4th Int'l. Conf. on AIDS, June, 1988, p237; and Marcus- Sekura, C.J. et al., Biochim. Biophys. Acta 949:213-223 (1988)) was derived from a hybridoma fusion utilizing spleen cells from a BALB/c mouse immunized with purified gag gene product. The Sp2/0 myeloma line was used as the fusion partner. Clone 2G12 produced immunoglobulin of the IgGl subclass. MAb 2G12 was reactive against cloned gag gene product by ELISA analysis. In addition, 2G12 was capable of immunofluorescent staining HIV-infected cells. Western blot analysis of the 2G12 mAb against viral lysates demonstrated predominant bands developed at 55, 45, 39, and 24 kD.
  • the nucleotide sequences of the cDNA clones show that they are immunoglobulin V region clones as they contain amino acid residues diagnostic of V domains (Kabat, et al., supra).
  • the J region mutagenesis primer 5'-GAGACGGTGACCAGAGTCCCT-3' was used to insert a BstEII site into the M13 subclone pSWO.
  • the restriction fragment containing the 2G12 V H region bounded by Sall and BstEII was then cloned into the expression vector pING2240 (pING2240 is identical to pING2227 except it contains a different antibody V region).
  • the J region mutagenesis primer 5'-GTTTGATTTCAAGCTTGGTGC-3' was used to insert a Hindlll site into the M13 subclone pC2-K, and the oligonucleotide 5'-TGTCTGTGTGTCGACAGTGTGATGC-3' was used to insert a Sall restriction site into pSW6 upstream of the initiation codon ATG.
  • the restriction fragment containing the 2G12 V L region bounded by Salland Hindlll in pSW7 was then cloned into the expression vector pING2216 (pING2240 is identical to pING1712 except it contains a different antibody V region).
  • the cell line Sp2/0 (American Type Culture Collection #CRL1581) was grown, transfected, and selected as described in Example 1, above.
  • the Chimer2 L chain expression plasmid pING3005 was linearized as described in Example 1. The best producer after outgrowth and subsequent subcloning, was transfected with Pvul-linearized pING3004, the expression plasmid containing the Chimer2 H chain gene. After selection with G418, the clone producing the most L plus H chain, Sp2/0-3005 + 30043E9, secreted antibody at approximately 5 ⁇ g/ml.
  • the Chimer2 antibody was eluted with various pH gradients (pH 7-2) and eluted between pH 3.6-4.0. Fractions containing antibody (46% yield) were combined and concentrated 85-fold by ultrafiltration (YM30 membrane, stirred cell, Amicon Corp.) diluted 25-fold with PBS, reconcentrated 10-fold, diluted 10-fold with PBS, and finally reconcentrated 4-fold. The antibody was stored in 1.0 ml aliquots at -20°C. 5. Studies Performed on the Chimer2 Antibody
  • Bacteria are suited for production of chimeric antibodies expressed from mammalian cDNA since entire coding sequences can be expressed from well characterized promoters.
  • the following sections detail the secretion of Chimer2 Fab from E. coli.
  • the Chimer2 V L region containing a Bsml restriction site at the signal sequence processing site and a unique Hindlll site in the J region of pSW8 served as the starting point for bacterial expression.
  • the plasmid pSW8 was cut with Bsml, treated with T4 polymerase, and digested with Hindlll ( Figure 11A).
  • the approximately 320 bp fragment containing the V L region was purified and ligated to pING 1500 that was cut with Sstl, treated with T4 polymerase, and cut with Xhol (Figure 11B) along with a Hindlll to Xhol restriction fragment containing the human C k plus 15 nucleotides of 3' genomic DNA from p3Q2 ( Figure 11C).
  • the resulting plasmid that contained apelB::Chimer2 L chain fusion was sequenced to determine that the proper in-frame fusion was formed. This plasmid was called pSW10-B.
  • the intact Chimer2 chimeric Fd gene containing a Bsml restriction site at the signal sequence processing site and a BstEII restriction site in the J region in pSW2 served as the starting point for bacterial expression.
  • the plasmid pSW2 was cut with Bsml, treated with T4 polymerase, and digested with BstEII ( Figure 11D).
  • the approximately 336 bp fragment containing the Fd V region was purified and ligated to pING1500 that was cut with Sstl, treated with T4 polymerase, and cut with Xhol ( Figure HE) along with the human CH1 region (which had been previously generated by introducing a stop codon in hinge, Robinson, R.R., et al., supra) from pF3D ( Figure 11F).
  • the resulting plasmid that contained a pelB::Chimer2 Fd fusion was sequenced to determine that the proper in-frame fusion was formed. This plasmid was called pSW4-B.
  • both L chain and Fd need to be produced simultaneously within the cell.
  • plasmids constructed with each of these genes separately, a series of expression vectors were constructed that contain both genes aligned so that transcription from a single promoter will specify both genes. This was done in a way that minimized the noncoding DNA between the two genes.
  • Each gene has a ribosome binding site needed for translation initiation and the identical DNA sequence from -48 to the pelB leader::antibody gene junction.
  • Plasmid pSW10-B was cut with Sphl, treated with T4 polymerase, cut with EcoRI, and the vector fragment was purified ( Figure 11G).
  • pSW4-B was cut with Xhol. treated with T4 polymerase, cut with EcoRI and the fragment containing the Fd gene was purified ( Figure 11H).
  • These two purified DNA fragments were ligated to produce pMB1, which contained the two Chimer2 gene fusions linked in close proximity.
  • the two gene cistron was placed under the control of the araB promoter in pING3107.
  • Plasmid pMB1 was cut with Sphl, treated with T4 polymerase, cut with Xhol, and the fragment containing the Fd and K genes was purified ( Figure 111).
  • This DNA fragment was ligated to the vector fragment from pING3107 that had been cut with EcoRI, treated with T4 polymerase, and cut with Xhol ( Figure 11J), generating pING3211.
  • This vector contains all the necessary features for expression of Chimer2 Fab in E. coli.
  • Chimer2 Fab from pING3211 in E. coli is under the inducible control of the araB promoter. Upon arabinose induction, Fab secreted into the growth medium increases more than 10-fold.
  • Example 2 About 7 liters of a culture supernatant were concentrated, analyzed, and stored as in Example 2.
  • the Chimer2 Fab purified from E. coli has identical molecular weight properties as other Fab molecules produced from E. coli. as assessed by SDS gel electrophoresis.
  • the bacterially-produced Chimer2 Fab is correctly assembled as a K plus Fd chain dimer because of its positive reaction in the enzyme immunoassays detecting molecules with both K and Fd determinants, and because it reacts with core protein on commercially available test strips.
  • Chimer2 Fab was tested along with 2G12 mouse antibody and Chimer2 antibody for binding to antigen fixed to commercially available HIV Western test strips (Dupont or equivalent).
  • Chimer2 Fab was incubated with the test strip, followed by interaction with goat anti-human Fab antibody, and then with rabbit anti-goat IgG. Detection of bound antibody was with Protein A-gold (BioRad) followed by silver enhancement.
  • Chimer2 Fab specifically recognized the same size antigen on the test strip as did Chimer2 whole antibody, specifically recognizing antigen of about 24 kD.
  • Yeast cells are capable of expressing and secreting foreign proteins.
  • yeast serve as a host for the production of mouse-human chimeric Fab.
  • Yeast strains and growth conditions are as in Example 3.
  • the gene sequences encoding the mature form of the V L region of 2G12 and containing a Hindlll site in the J region (as described in Example 4) and a Bsml site introduced at the signal sequence processing sitp was fused to the human C k region. Plasmid pSW8 was cut with Bsml, treated with T4 polymerase, and cut with Hindlll. The V region fragment was ligated along with the human C L region prepared from p3Q2 as a Hindlll to Xhol fragment into pING1149.
  • the plasmid pING1149 contains the gene sequence encoding the yeast invertase signal sequence (Taussig, R., et al., supra) under control of the yeast PGK promoter (Hitzeman, R.A., et al., supra).
  • the resultant plasmid pSWll-Y (similar to Figure 6A) contains the PGK promoter (P) along with the invertase signal sequence (S) fused to the Chimer2 coding sequence.
  • the gene sequence encoding the mature form of the Chimer2 L chain was fused in frame to the gene sequence encoding the yeast invertase signal sequence.
  • the PGK promoter - invertase signal sequence - chimeric L chain (V,C k ) fusion was cloned into a partial 2 micron circle (2 ⁇ ), ura3 yeast expression vector containing the PGK polyadenylation signal (Tm) to generate pSW13-Y (similar to Figure 6C).
  • the PGK promoter - invertase signal sequence - chimeric Fd chain (V,CH1) fusion was cloned into a partial 2 micron circle (2 ⁇ ), ura3 yeast expression vector containing the PGK polyadenylation signal (Tm) to generate pSW12-Y (similar to Figure 6D).
  • a single yeast expression vector containing both the chimeric L chain and Fd chain genes and their respective expression signals was constructed from pSW13-Y and pSW12-Y.
  • This final vector, pING3208 (similar to Figure 6E), contains a portion of 2 micron circle (oriY, REP3) and the two selectable markers leu2d and ura3.
  • the plasmid pING3208 was transformed into S. cerevisiae PS6 and the trar.sformants were grown in broth under selective conditions as described above.
  • the culture supernatants were assayed by ELISA and contained Fab levels of approximately 100 ng/ml.
  • This material can be purified from fermentation broth and is expected to have properties identical to those of Fab produced as described in Example 3; yeast Chimer2 Fab is expected to have the same binding specificity as Fab produced in E. coli.
  • the mouse mAb 1C11 (developed by and commercially available from Epitope Inc., Beaverton OR) was derived from a hybridoma fusion utilizing spleen cells from a BALB/c mouse immunized with an HIV viral lysate.
  • the Sp2/0 myeloma line was used as the fusion partner.
  • Clone 1C11 produced immunoglobulin of the IgM subclass.
  • MAb 1C11 was reactive with the cloned pol gene product by Western blot analysis.
  • 1C11 was capable of immunofluorescent staining of HIV- infected cells.
  • Western blot analysis of the 1C11 mAb against viral lysates demonstrated predominant bands developed at 51 and 65 kD.
  • RNA was prepared and a cDNA library was constructed in pBR322 as described in Example 1. cDNA libraries were screened by hybridization (Maniatis, T., supra) with 32 P-labeled, nick translated DNA fragments, i.e., for kappa clones with a mouse C k region probe and for H chain clones with a mouse IgM C region probe.
  • the nucleotide sequences of the cDNA clones show that they are immunoglobulin V region clones as they contain amino acid residues diagnostic of V domains (Kabat, et al., supra).
  • the DNA sequence of the 1C11 H chain gene is very similar to the previously cloned and chimerized anti-cancer A10 antibody gene.
  • the region from at least 46 bp upstream of the initiation codon ATG, through the antibody leader sequence and to a BamHI restriction site in the V region at amino acids 16 and 17 contains identical DNA sequence.
  • a Sall restriction site was introduced into the A10 H chain cDNA sequence upstream of the ATG initiation codon by site-directed mutagenesis with the primer 5 ⁇ TGTCTGTGTCGACCACTGAAGA-3'.
  • the 1C11 H chain contains the J H 3 sequence which contains a Pstl site at its 3' end.
  • the Hindlll site (amino acids 3 and 4) and Pstl site (J region) occurring in pC4M-8 (underlined and bold in Figure 12) were used to mobilize the the unique Chimer4 V region sequences adjacent to a Sall to Hindlll DNA fragment from pTK7, which shared the 5' untranslated region, leader sequence, and the shared V region amino acid sequence.
  • the resulting plasmid, pYZ124 contained the complete Chimer4 V H region, leader sequence and accompanying 5' untranslated sequence.
  • the Sall to Pstl fragment from pYZ124 was cloned into the expression vector pING2227 to generate the Chimer4 H chain vector PING2255 ( Figure 14).
  • the cDNA clone containing the 1C11 L chain, pC4K-16 was adapted for mammalian expression taking advantage of site directed mutagenesis using the polymerase chain reaction (Horton, R.M., et al., 1989, Gene 77:61).
  • a DNA fragment was amplified, using pC4K-16 as a substrate, which contained a Sall restriction site upstream of the initiation codon ATG and a Bglll site in the J region with the oligonucleotides 5'-GGCCGTCGACTCACCTGGACATGAT-3' and 5' -AGCGCAGATCTCCAGCTTGGTGCC-3' .
  • the DNA fragment was cut with Sal l and BglII and cloned into pING2016Egpt (Robinson, R.R., et al., supra) to generate an in-frame V-J-C k chimeric gene fusion. Subsequent cloning to generate the final L chain vector, pING2258 is as described in Figure 16.
  • the cell line Sp2/0 (American Type Culture Collection #CRL1581) was grown, transfected, and selected as described in Example 1, above.
  • the Chimer4 L chain expression plasmid, pING2258, and the H chain expression plasmid, pING2255, were each linearized by digestion with Pvul restriction endonuclease and co-transfected into Sp2/0 cells, giving mycophenolic acid resistant clones which were screened for L + H chain synthesis and G418 resistance.
  • DMEM fetal bovine serum
  • HEPES HEPES buffer
  • glutamine glutamine
  • penicillin penicillin
  • streptomycin Irvine Scientific, Irvine
  • Chimer4 was purified in a similar manner to that described for Chimer1 and Chimer2 in Examples 1 and 4 respectively. Purified
  • Chimer4 antibody was tested to demonstrate that it retained the antigen binding characteristics of the mouse 1C11 antibody using commercially available HIV Western strips (Dupont or equivalent), as described above for Chimer1 and Chimer2.
  • Chimer4 and mouse 1C11 antibodies both recognized the same HIV reverse transcriptase antigen.
  • Bacteria are suited for production of chimeric antibodies expressed from mammalian cDNA since entire coding sequences can be expressed from well characterized promoters.
  • the following sections detail the secretion of Chimer4 Fab from E. coli.
  • V-J region (containing 10 bp of CH1) from pING2255 was purified as a BamHI to Apal fragment and the CH1 fragment (which had been previously generated by introducing a stop codon in hinge, Robinson, R.R., et al., supra) from pQ16D was purified as an Apal to Xhol fragment.
  • a three-way ligation of these fragments generated pYZ117, Figure 16.
  • the Chimer4 V L region was adapted from pC4K-16 for bacterial expression as shown in Figure 16.
  • Two primers were used to obtain an ol igonucleotide by polymerase chain reaction, 5'-GATATCCAGATGACACAGACTACATCC-3' and 5' -AGCGCAGATCTCCAGCTTGGTGCC-3 ' which generated a DNA fragment that was blunt-ended at one end and contained a Bglll site in the J region at the other end.
  • the DNA fragment was then cloned into pING1500 that was digested with Sstl, treated with T4 polymerase and cut with BamHI, generating pING3314.
  • Plasmid pING3314 contains the pelB leader fused in-frame with the Chimer4 V k -J k region.
  • both L chain and Fd need to be produced simultaneously within the cell.
  • plasmids constructed with each of these genes separately, a series of expression vectors were constructed that contain both genes aligned so that transcription from a single promoter will specify both genes. This was done in a way that minimized the noncoding DNA between the two genes.
  • Each gene has a ribosome binding site needed for translation initiation and the identical DNA sequence from -48 to the pel B leader::antibody gene junction.
  • a two-gene vector containing the entire pelB::Fd gene and the pelB-V k -J k region was constructed by cutting pING3314 with Sphl, treating it with T4 polymerase and cutting it with EcoRI and cloning into it the Fd gene from pYZ117 as an Xhol. T4 polymerase treated, EcoRI cut fragment, generating pING331S.
  • Plasmid pING3315 was cut with Sphl, treated with T4 polymerase and cut with Xhol, and the fragment containing the Fd and K genes was purified. The DNA fragment was ligated into the vector fragment of pING3303 that had been cut with Pstl, treated with T4 polymerase and cut with Xhol, generating pING3316.
  • the final two-gene expression vector, pING3405, was constructed in a three way ligation with two fragments from pING3316 [Apal to Xhol (vector) and the Apal to Hindlll (Fd/ ⁇ ) and the Hindlll to Xhol fragment of pYZ125. [pYZ125 contains the V k region of Chimer4 fused to the C k region derived from pING2016Egpt]. These steps are outlined in Figure 16.
  • Chimer4 Fab is produced and purified as described in Examples 2 and 5 for Chimer1 and Chimer2 Fab.
  • the purified Chimer4 Fab is tested for antigen binding as described in Examples 2 and 5.
  • the Chimer4 Fab is expected to have the same binding specificity as the Fab fragment of the mouse 1C11 antibody.
  • Yeast cells are capable of expressing and secreting foreign proteins.
  • yeast serve as a host for the production of mouse-human chimeric Fab.
  • This Fab molecule is identical to the Fab produced in E. coli outlined in Example 8, except that a single amino acid change occurred at position 23 (from an isoleucine to a leucine). This was an unexpected result of DNA polymerization during site directed mutagenesis. This amino acid change is not expected to alter antigen binding ability.
  • Yeast strains and growth conditions are as in Example 3.
  • the gene sequences encoding the mature form of the V L region of 1C11 was adapted for yeast expression as follows. Sequential site directed mutagenesis of an M13 subclone of pC4K-16 with the J region primers 5'-CATCAGCCCGTTAGATCTCCAGCTTGG-3' and the leader mature primer 5'-CATCTGGATATCTGCAGTGGTACCTTGAA-3' generated pYZ122, which contained a Pstl site at the leader mature junction and a Bglll site in the J region. Plasmid pYZ122 was cut with Pstl, treated with T4 polymerase and cut with Hindlll (a site located in the V region; see Figure 13).
  • the V region fragment was ligated along with the VJCK region prepared from pYZ125 (see Example 8) as a Hindlll to Xhol fragment into pING1149.
  • the plasmid pING1149 contains the gene sequence encoding the yeast invertase signal sequence (Taussig, R. et al., supra) under control of the yeast PGK promoter (Hitzeman, R.A., et al., supra).
  • the resultant plasmid pING3157 contains the PGK promoter (P), along with the invertase signal sequence (S), fused to the Chimer4 coding sequence.
  • P PGK promoter
  • S invertase signal sequence
  • the gene sequence encoding the mature form of the Chimer4 L chain was fused in-frame to the gene sequence encoding the yeast invertase signal sequence.
  • the PGK promoter - invertase signal sequence - chimeric L chain (V,C k ) fusion was cloned into a partial 2 micron circle (2 ⁇ ), ura3 yeast expression vector containing the PGK polyadenylation signal (Tm) to generate pING3158 (similar to Figure 6C).
  • pING2225 was cut with BamHI, incubated with calf intestinal alkaline phosphatase (CIAP), and cut with Apal.
  • the plasmid pTK14, which contains the PGK promoter - invertase signal sequence-chimeric A10 Fd chain fusion was cut with BamHI, treated with CIAP and cut with Apal; in a separate reaction, pTK14 was cut with BamHI.
  • the vector fragment from the former reaction and the PGK-invertase-V-region fragment from the latter were ligated to the BamHI to Apal V-J region fragment from pING2255 to generate pYZ116.
  • the PGK promoter - invertase signal sequence - chimeric Fd chain (V-J-C H 1) fusion (similar to Figure 6B) was cloned into a partial 2 micron circle (2 ⁇ ), ura3 yeast expression vector containing the PGK polyadenylation signal (Tm) to generate pYZ118 (similar to Figure 6D).
  • a single yeast expression vector containing both the chimeric L chain and Fd chain genes and their respective expression signals was constructed from pYZ118 and pING3158.
  • This final vector, pING3169 (similar to Figure 6E), contains a portion of 2 micron circle (oriY, REP3) and the two selectable markers leu2d and ura3.
  • the plasmid pING3169 is transformed into S. cerevisiae PS6 and the transformants are grown in broth under selective conditions.
  • the culture supernatants contain Fab.
  • This material is purified from fermentation broth and is expected to have properties identical to those of Fab produced as described in Example 8; yeast Chimer4 Fab is expected to have the same binding specificity as Fab produced in E . coli.
  • the mouse mAb 4D12 (described as anti-p18 mAb in Yoshihara, P. et al., Proc. 4th Int'l. Conf. on AIDS, June, 1988, p237) was derived from a hybridoma fusion utilizing spleen cells from a BALB/c mouse immunized with purified gag gene product. The Sp2/0 myeloma line was used as the fusion partner. Clone 4D12 produces immunoglobulin of the IgGl subclass. MAb 4D12 is reactive against the cloned gag gene product by ELISA analysis. In addition, 4D12 is capable of immunofluorescent staining of HIV-infected cells. Western blot analysis of the 4D12 mAb against viral lysates demonstrates predominant bands developed at 55, 45, and 39 kD. An additional weak band is developed at 18 kD depending upon the lysate used.
  • RNA was prepared, a cDNA library was constructed in pBR322, and L and H chain cDNA clones were identified as described in Example I.
  • the complete nucleotide sequences of the V region of these clones were determined ( Figures 17 and 18) by the dideoxy chain termination method. These sequences predict V region amino acid compositions that agree well with the observed compositions, and predict peptide sequences which have been verified by direct amino acid sequencing of portions of the V regions.
  • nucleotide sequences of the cDNA clones show that they are immunoglobulin V region clones as they contain amino acid residues diagnostic of V domains (Kabat, et al., supra).
  • the cDNA clone containing the 4D12 H chain, pC5G-30 was adapted for mammalian expression by introducing convenient restriction endonucleases sites by convenient cloning and site directed mutagenesis (Kramer, W., et al., supra) of appropriate M13 subclones, Figure 19. Oligonucleotides were synthesized on a Cyclone DNA synthesizer, New Brunswick Scientific Co., and purified by acrylamide gel electrophoresis. A Bell site, Figure 17, located approximately 45 bp upstream of the initiation codon ATG was used for introduction of a Sail restriction site 5' of the antibody V region.
  • the J region mutagenesis primer 5'-GAGACGGTGACCGAGGTTCCT-3' was used to insert a BstEII site into the M13 subclone pING3122.
  • the restriction fragment containing the 4D12 V H region bounded by Sall and BstEII was then cloned into the expression vector pING2240 (pING2240 is identical to pING2227 except it contains a different antibody V region).
  • a Bcll site, Figure 18, located approximately 30 bp upstream of the initiation codon ATG was used to introduce a Sall restriction site into pING312S upstream of the initiation codon ATG.
  • restriction fragment containing the 4D12 L chain V region bounded by Sall and Hindlll in pING3130 was then cloned into the expression vector pING1712 (pING2216 is identical to pING1712 except it contains a different antibody V region).
  • Sp2/0-3132 + 3126 1G4 cells were grown in culture medium DMEM
  • Chimer4 in Example 7 Purified Chimer5 was tested to demonstrate that it retained the antigen binding characteristics of the mouse 4D12 antibody using commercially available HIV Western strips (Dupont or equivalent) as described for Chimer1, Chimer2, and Chimer4, above.
  • Bacteria are suited for production of chimeric antibodies expressed from mammalian cDNA since entire coding sequences can be expressed from well characterized promoters.
  • the following sections detail the secretion of Chimer5 Fab from E. coli.
  • a Apal site was similarly introduced at the junction of the leader peptide and mature coding region of the 4D12 H chain with the oligonucleotide primer 5'-TGGACCTGGGCCCGAACACCTGC-3' ( Figure 17).
  • the Chimer5 L chain V region containing a Pstl restriction site at the signal sequence processing site and a unique Hindlll site in the J region of pING3128 served as the starting point for bacterial expression.
  • the plasmid pING3128 was cut with Pstl. treated with T4 polymerase, and digested with Hindlll ( Figure 21A).
  • the 320 bp fragment containing the V L region was purified and ligated to pING1500 that was cut with Sstl, treated with T4 polymerase, and cut with Xhol (Figure 21B) along with a Hindlll to Xhol restriction fragment containing the human C k plus 15 nucleotides of 3' genomic DNA from pING3102 ( Figure 21C).
  • the resulting plasmid that contained a pelB::Chimer5 L chain fusion was sequenced to determine that the proper in-frame fusion was formed. This plasmid was called pING3133.
  • the intact Chimer5 chimeric Fd gene containing a Apal restriction site at the signal sequence processing site and a BstEII restriction site in the J region in pING3129 served as the starting point for bacterial expression.
  • the plasmid pING3129 was cut with Apal, treated with T4 polymerase, and digested with BstEII ( Figure 21D).
  • the approximately 336 bp fragment containing the Fd V region was purified and ligated to pING1500 that was cut with Sstl, treated with T4 polymerase, and cut with Xhol (Figure 21E) along with the human CH1 region (which had been previously generated by introducing a stop codon in hinge, Robinson, R.R., et al., supra) from pF3D ( Figure 21F).
  • the resulting plasmid that contained a pelB::Chimer5 Fd fusion was sequenced to determine that the proper in-frame fusion was formed. This plasmid was called pING3131.
  • both L chain and Fd need to be produced simultaneously within the cell.
  • plasmids constructed with each of these genes separately, a series of expression vectors were constructed that contain both genes aligned so that transcription from a single promoter will specify both genes. This was done in a way that minimized the noncoding DNA between the two genes.
  • Each gene has a ribosome binding site needed for translation initiation and the identical DNA sequence from -48 to the pelB leader::antibody gene junction.
  • Plasmid pING3133 was cut with SphI, treated with T4 polymerase, cut with EcoRI. and the vector fragment was purified ( Figure 21G). Similarly, pING3131 was cut with Xhol. treated with T4 polymerase, cut with EcoRI and the fragment containing the Fd gene was purified ( Figure 21H). These two purified DNA fragments were ligated to produce pING3136, which contained the two Chimer5 gene fusions linked in close proximity. The two gene cistron was placed under the control of the araB promoter in pING3107. Plasmid pING3136 was cut with Sphl, treated with T4 polymerase, cut with Xhol, and the fragment containing the Fd and K genes was purified ( Figure 21I).
  • This DNA fragment was ligated to the vector fragment from pING3107 that had been cut with EcoRI. treated with T4 polymerase, and cut with Xhol ( Figure 21J), generati ng pING3139.
  • This vector contains all the necessary features for expression of Chimer5 Fab in E. coli.
  • Chimer5 Fab from pING3139 in E. coli is under the inducible control of the araB promoter. Upon arabinose induction, Fab secreted into the growth medium increased more than 10-fold.
  • the E . coli strain harboring pING3139 was was cultured at 32oC in 10L of minimal medium, supplemented with 1.7% glycerol as the carbon source. Dissolved oxygen was maintained at 20% of saturation by adition of a 5X concentrate of glycerol-supplemented minimal medium.
  • the induction solution was added over a period of about 20 hours, then 5X concentrate without minimal medium was added for about 4 hours.
  • Fab was detected in the fermentation broth by ELISA.
  • Example 2 About 7 liters of a culture supernatant was concentrated, analyzed, and stored as in Example 2.
  • the Chimer5 Fab purified from E. coli had identical molecular weight properties as other Fab molecules produced from E. coli, as assessed by SDS gel electrophoresis.
  • the bacterially-produced Chimer5 Fab was correctly assembled as a K plus Fd chain dimer because of its positive reaction in the enzyme immunoassays detecting molecules with both K and Fd determinants, and because it reacted with core protein on commercially available test strips.
  • Chimer5 Fab was tested along with 4D12 mouse antibody and Chimer5 antibody for binding to antigen fixed to commercially available HIV Western test strips (Dupont or equivalent).
  • Chimer5 Fab was incubated with the test strip, followed by interaction with goat anti-human Fab antibody, and then with rabbit anti-goat IgG. Detection of bound antibody was with Protein A-gold (BioRad) followed by silver enhancement.
  • Chimer5 Fab specifically recognized the same size antigens of 65, 45, and 39 kD on the test strip as did Chimer5 whole antibody.
  • Yeast cells are capable of expressing and secreting foreign proteins.
  • yeast serve as a host for the production of mouse-human chimeric Fab.
  • Yeast strains and growth conditions are as in Example 3.
  • the gene sequences encoding the mature form of the V L region of 4D12 and containing a Hindlll site in the J region (as described in Example 11) and a Pstl site introduced at the signal sequence processing site was fused to the human C k region.
  • Plasmid pING3128 was cut with Pstl, treated with T4 polymerase, and cut with Hindlll.
  • the V region fragment was ligated along with the human C L region prepared from pING3102 as a Hindlll to Xhol fragment into pING1149.
  • the plasmid pING1149 contains the gene sequence encoding the yeast invertase signal sequence (Taussig, R.
  • the resultant plasmid pING3134 (similar to Figure 6A), contained the PGK promoter (P), along with the invertase signal sequence (S), fused to the Chimer5 coding sequence.
  • P PGK promoter
  • S invertase signal sequence
  • the PGK promoter - invertase signal sequence - chimeric L chain (V,C K ) fusion was cloned into a partial 2 micron circle (2 ⁇ ), ura3 yeast expression vector containing the PGK polyadenylation signal (Tm) to generate pING3140 (similar to Figure 6C).
  • the gene sequence encoding the mature form of the V H region of Chimer5 containing a BstEII site in the J region (as described in Example 11) and a Apal site introduced at the signal sequence processing site from pING3129 was fused along with the human C H 1 region (which had been previously generated by introducing a stop codon in hinge, Robinson, R.R., et al., supra) from pF3D into pING1149 that had been cut with Pstl, treated with T4 polymerase and cut with Xhol. This generated plasmid pING3135.
  • the PGK promoter - invertase signal sequence - chimeric Fd chain (V,C H 1) fusion was cloned into a partial 2 micron circle (2 ⁇ ), ura3 yeast expression vector containing the PGK polyadenylation signal (Tm) to generate pING3141 (similar to Figure D).
  • a single yeast expression vector containing both the chimeric L chain and Fd chain genes and their respective expression signals was constructed from pING3140 and pING3141.
  • This final vector, pING3143 similar to Figure 6E, contains a portion of 2 micron circle (oriY, REP3) and the two selectable markers leu2d and ura3.
  • the plasmid pING3143 is transformed into S. cerevisiae PS6 and the transformants are grown in broth under selective conditions as described above.
  • Transformed yeast cultures secrete Chimer4 Fab into the culture medium, and this material is purified from fermentation broth and is expected to have properties identical to those of Fab produced as described in Example 3; yeast Chimer5 Fab is expected to have the same binding specificity as Fab produced in E. coli.
  • the examples presented above demonstrate a number of important qualities of the Chimer1, Chimer2, Chimer4, and Chimer5 antibodies and the genetically engineered fragments and derivatives of the invention.
  • the chimeric antibodies, fragments and derivatives bind to the same antigens as the parent mouse mAbs. This is evidenced by direct binding of chimeric antibodies, fragments and derivatives to Western immunoblot test strips prepared with lysates of HIV.
  • chimeric antibodies of this invention represent potentially important agents for treatment of HIV infection.
  • Antibodies such as Chimer1, Chimer2, Chimer 4, and Chimer 5 or their corresponding fragments and derivatives, either alone, as immunoconjugates, or in combination with other agents may be advantageously used for HIV treatment.
  • the chimeric antibodies of this invention may also be used for any diagnostic purpose for which the similar mouse antibodies or their derivatives can be used. Furthermore, the chimeric antibodies, fragments and derivatives may be used as immobilized reagents for ex vivo adsorption of viruses, viral antigens, or virus-infected cells.
  • Chimeric antibody molecules such as those of the present invention, and their derivatives, embody a combination of the advantageous characteristics of mouse and human mAbs. Like mouse mAbs, they can recognize and bind to viral antigens; however, unlike mouse mAbs, the "human specific" properties of chimeric antibodies lower the likelihood of an immune response to the antibodies, and result in prolonged survival in the circulation through reduced clearance. These properties have been observed when a chimeric antibody directed against a tumor marker was introduced in patients, LoBuglio, et al., 1989, Proc. Natl. Acad. Sci. USA 86:4220-4224.
  • the human component of a chimeric antibody may enhance its ability to mediate target destruction, for example, of virally infected cells, in combination with human effector cells or complement.
  • any desired antibody isotype can be combined with any particular antigen combining site. This invention also enables the direct production of one or more domains of the antibody molecule in functionally active form.
  • the E. coli strains deposited are as follows:
  • E. coli MC1061 transformed with pING3127 was granted ATCC Accession #68146.
  • E. coli MC1061 transformed with pING3211 was granted ATCC Accession #68147.
  • E. coli MC1061 transformed with pING3405 (G271) was granted ATCC Accession #68148.
  • E. coli MC1061 transformed with pING3139 was granted ATCC Accession #68149.
  • the transfected hypbridoma cell lines are as follows:
  • S p /0 (pING3110 + pING3112) (C863) was granted ATCC Accession # HB10277.
  • S p /0 (pING3005 + pING3004) (C850) was granted ATCC Accession # HB10276.

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Abstract

Sont décrits des immunoglobulines, des fragments ou des dérivés chimériques présentant toute ou partie d'une région constante d'immunoglobuline humaine et d'une région variable murine, ayant une spécificité à l'égard d'antigènes du virus-1 d'immudéficience humaine (VIH-1), leurs procédés de production et leurs emplois.
EP19910900088 1989-11-13 1990-11-13 Chimeric mouse human antibodies with specificity to hiv antigens Withdrawn EP0453554A4 (en)

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JP2005522192A (ja) 2001-07-19 2005-07-28 パーラン セラピューティクス， インコーポレイテッド マルチマータンパク質およびマルチマータンパク質を作製および使用する方法
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EP0327000A2 (fr) * 1988-01-30 1989-08-09 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Fragments de gènes codant pour la région variable d'un anticorps anti-HIV, anticorps chimériques anti-HIV exprimés lors de leur utilisation et procédé pour leur préparation

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WO1991007493A1 (fr) 1991-05-30

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