Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1991-03-26
1991-12-24
Rollins, John W.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
435 691, 435 701, 435 703, 435 91, C07H 1700, C12P 2106, C12P 2102, C12P 1934
Patent
active
050754319
DESCRIPTION:
BRIEF SUMMARY
Carcinoembryonic antigen (CEA) is extensively used in the in vitro immunodiagnosis of human colon adenocarcinomas. This invention relates to a chimeric anti-CEA antibody.
BACKGROUND OF THE INVENTION
CEA is the best characterized human tumor-associated antigen and the most widely used tumor marker for the in vitro diagnosis of human colon cancers. CEA, however, is one of a family of closely related genes including normal cross-reacting antigen (NCA) and biliary glycoprotein (BGPI).
Many antibodies to tumor markers cross-react with related antigens. Accordingly, the development of antigen-specific monoclonal antibodies (MABs) for in vitro and in vivo diagnosis and therapy requires a good knowledge of the number, quality and biodistribution of related cross-reactive antigens. This requirement has restricted the number of acceptable tumor markers which, in the case of colon cancer, includes CEA, CA 17-1A and TAG-72.
Careful immunochemical characterization of the MAB to be used is required with respect to its specificity and affinity for the target antigen and for related antigens. Systematic application of a MAB that is cross-reactive with a related antigen must be avoided to foreclose risk of potentially severe side effects.
Murine MAB T84.66 (ATCC Accession No. BH 8747) IgG1,k shows a high affinity constant (2.6.times.10.sup.10 M.sup.- 1) and no cross reactivity to other members of the CEA gene family. T84.66 is therefore ideally suited for immunodetection and immunotherapy studies.
Use of murine MABs including T84.66 for detection and therapy of human tumors is constrained by patient immune response against the heterologous immunoglobulin. The production of human anti-mouse antibodies (HAMA) leads to reduced efficiency of the MAB and to potentially serious manifestations of acute and chronic allergic complications for the patient. See Levy, et al. Ann. Rev. Med. 34:107-116 (1983); Houghton, et al. Proc. Natl. Acad. Sci. USA, 82:1242-1246 (1985) and Sears, et al. J. Biol. Resp. Modifiers 3:138-150 (1984).
Recombinant DNA technology provides attractive methods to generate MABs useful to circumvent such problems from chimeric human
on-human genes. In addition, recombinant antibody genes provide a renewable source of antibodies which can be further engineered to alter affinity constants and effector functions. Using different approaches, a number of antibody genes and derivatives thereof have been constructed which code for antibody chimeras directed against tumor-associated antigens. Sahagen, et al, J.Immunol. 137:1066-1074 (1986); Sun, et al., Proc.Natl.Acad.Sci.USA 84:214-218 (1987); Nishimura, et al., Cancer Res. 47:999-1005 (1987); Liu, et al. Proc.Natl.Acad. Sci.USA 84:3439-3443 (1987).
Beidler, et al, J. Immunology 141:4053-4060 (1988) describe a murine/human chimeric antibody constructed by using variable light and variable heavy regions from a murine hybridoma CEM231.6.7 specific for CEA. The parental hybridoma bound antigen with an affinity of 5.times.10.sup.9 M.sup.-1, chimeric subclones bound antigen at 2.times.10.sup.10 M.sup.-1, and 1.times.10.sup.10 M.sup.-1. Clinical utility of the chimera, including lack of cross-reactivity was not demonstrated.
SUMMARY OF THE INVENTION
This invention includes the cloning and sequencing of the genes coding for MAB T84.66, the amino acid sequence of the variable regions for the light and heavy chains, and the construction of mouse/human chimeric IgG-1 antibody genes using T84.66 variable region genes and human constant region genes. The gene constructs were transfected into murine myeloma cells (Sp2/0) by electrophoration and into CHO cells by lipofection. The chimeric antibodies exhibited a specificity and affinity for CEA similar to that of the T84.66 immunoglobulin produced by the murine hybridoma cell line.
DESCRIPTION OF THE FIGURES
FIG. 1A is the sequence of the kappa light chain of the T84.66 monoclonal antibodies.
FIG. 1B is the sequence of the gamma heavy chain of the T84.66 monoclonal antibodies.
FIG. 2A shows the construction of a plasmid fo
REFERENCES:
A. Liu et al., P.N.A.S., 84:3439-3443, 1987.
C. Beidler et al., J. Imm. 141(11):4053-4060, 1988.
L. Sun et al., P.N.A.S., 84:214-218, 1987.
Sahagan et al., J. Imm. 137(3):1066-1074, 1986.
Chen et al., Mol. Cell. Biol. 7(8):2745-2752, 1987.
Bergman et al., Proc. Nat. Acad. Sci. 81:7041-7045, 1984.
Neumaier Michael
Riggs Arthur D.
Shively John E.
City of Hope
Irons Edward S.
Rollins John W.
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