Proceedings of the National Academy of Sciences of the United States of America. Annual subject and author indexes.

We have created mouse-human antibody molecules of defined antigen-binding specificity by taking the variable region genes of a mouse antibody-producing myeloma cell line with known antigen-binding specificity and joining them to human immunoglobulin constant region genes using recombinant DNA techniques. Chimeric genes were constructed that utilized the rearranged and expressed antigen-binding variable region exons from the myeloma cell line SI07, which produces an lgA (K) anti-phosphocholine antibody. The heavy chain variable region ex on was joined to human IgG I or lgG2 heavy chain constant region genes, and the light chain variable region ex on from the same myeloma was joined to the human K light chain gene. These genes were transfected into mouse myeloma cell lines, generating transformed cells that produce chimeric mouse-human IgG (K) or IgG (K) anti-phosphocholine antibodies. The transformed cell lines remained tumorigenic in mice and the chimeric molecules were present in the ascitic fluids and sera of tumor-bearing mice. The capability to transfer immunoglobulin genes into lymphoid cells where they produce protein in quantities sufficient for structural studies Cl-3) provides us with the opportunity to generate and characterize novel immunoglobulin molecules. Cloned variable (V) region genes from mouse or rat hybridoma cell lines can be ligated to human constant (C) region genes and we would expect that these chimeric genes can be transfected into mouse myeloma cells, which then will produce novel human antibody molecules. We would thus produce antibodies that are largely human but which have antigen-binding specificities generated in mice. The additional potential for in vitro manipulation and alteration of both the antigen-binding site and the structures correlated with biological effector functions of these antibody molecules using recombinant DNA techniques would introduce a powerful approach for further understanding antibody structure, function, and immunogenetics. As we show here, both chimeric mouse heavy chain V region exon (VH)-human heavy chain C region genes and chimeric mouse light chain V region cxon (VK)-human K light chain gene constructs are expressed when transfected into mouse myeloma cell lines. When both chimeric heavy and light chain genes are transfected into the same myeloma cell, an intact tetrameric (H2L2) chimeric antibody is produced. In this study we used V 11 and VK exons from the mouse phosphocholine (PCho)-binding antibody-producing S107 myeloma cell line (4, 5). Chimeric mouse-human anti-PCho antibodies were produced in culture by appropriate transfectcd cell lines or by "transfcctomas" obtained when such cell lines arc injected into mice. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with lH U.S.C. §1734 solely to indicate this fact. 6851 MATERIALS AND METHODS Chimeric Genes. The cloned S107 VH and S107 VK genes were gifts from Matthew Scharff (Albert Einstein College of Medicine, Bronx, NY). The S107 VH gene was spliced to human IgGl and IgG2 C region genes by using Sal I linkers as shown in Fig. IA. Both constructs were inserted into the vector pSV2~H-gpt (1, 6). The S107 VK gene was spliced to the human K gene at a unique f/indlll site located in the large intron between the K light chain joining and C (JK and CK) region exons as shown in Fig. lB. This chimeric light chain gene construct was inserted into both PSV2~H-gpt and pSV2-neo plasmid vectors (7). Transfection. Protoplast fusion and calcium phosphate precipitation techniques (1, 8, 9) were used to transfect these chimeric immunoglobulin genes into the J558L myeloma cell line (a A. light chain-producing mouse myeloma cell line) and an immunoglobulin nonproducing derivative of the P3 myeloma cell line. Mycophenolic acid (GIBCO) was used for selection of cells transfected with pSV2~-gpt vectors as described (1, 3). G418 (GIBCO) at 1.0 mg/ml was used for selection of cells transfected ,with pSV2-neo vectors (7). Light and heavy chain chimeric immunoglobulin genes were transfected sequentially by protoplast fusion using G418 selection for the chimeric light chain gene vector and mycophenolic acid for the chimeric heavy chain gene ve'ctor. The protoplast fusion transfection procedure used was as described (1). Transfection using the calcium phosphate precipitation procedure was done by transfecting a mixture of 40 11-g each of the chimeric light and chimeric heavy chain pSV2~H-gpt vectors into 5 x 106 cells. Mycophenolic acid was used to select for transformed cell lines as described (1). Antigen Binding. PCho binding of antibody secreted into the culture supernates of transfected cell lines w~s analyze? by using a solid-phase radioimmunoassay d~scnb~d previously (10). PCho-keyhole limpet hemocyamn ~ntlg~n w~s bound to 96-well polyvinyl plates; bindin~ of chimenc an~I­ PCho antibodies was detected by using LSI-labeled protem A or 1251-labeled-anti-human lgG. PCho-binding antibodies in biosynthetically labeled culture supernates and c~ll .lYsates of transfected cell lines also were analyzed by bmdmg the biosynthetically labeled antibody to PCho-coupled ~eph­ arose 4B (Pharmacia) and then eluting the bound antibody with PCho-haptcn. The bound and eluted antibody was e~­ amined by NaDodS04/polyacrylamide gel electrophoresis (NaDodS04/PAGE). Biosynthetic-labeling procedures were as described (11). Idiotope Analysis. Three hybridoma anti-idiotope antibodies, also kindly provided by Matthew Scharff and Angela Abbreviations: V, variable; C, constant; J, joining; H• heavy chain; L• light chain; PCho, phosphocholine; kb,. kilobase(s); NEPHGE, nonequilibrium pH gradient electrophoresiS. This material was copied PFIZER EX. 1031 Page 7 6852 Immunology : Morri son e t a/. Proc. N at/. A cad. Sci. USA 81 (1 984) A 1am. HI (~I) Sal I (Hind Ill) lll.m HI pSV2AH-S 107 HuG 1 t--1:H::ID---·-~~.--tH~B"i~---· Mouse VDJ Human constant region