transgenic mouse; deactivated endogenous immunoglobulin heavy

(12) United States Patent Kucherlapati et al.

US006673986B1

US 6,673,986 B1 *J an. 6, 2004

(10) Patent N0.: (45) Date of Patent:

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GENERATION OF XENOGENEIC ANTIBODIES

Inventors: Raju Kucherlapati, Darien, CT (US);

Assignee:

Notice:

Appl. No.:

Filed:

Aya Jakobovits, Menlo Park, CA (US); Sue Klapholz, Stanford, CA (US); Daniel G. Brenner, Redwood City, CA (US); Daniel J. Capon, Hillsborough, CA (US)

Abgenix, Inc., Fremont, CA (US)

This patent issued on a continued pros ecution application ?led under 37 CFR 1.53(d), and is subject to the twenty year patent term provisions of 35 U.S.C. 154(a)(2).

Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days.

This patent is subject to a terminal dis claimer.

08/031,801 Mar. 15, 1993

Related US. Application Data

Continuation-in-part of application No. 07/919,297, ?led on Jul. 24, 1992, now abandoned, which is a continuation-in part of application No. 07/610,515, ?led on Nov. 8, 1990, now abandoned, which is a continuation-in-part of applica tion No. 07/466,008, ?led on Jan. 12, 1990, now abandoned.

Int. Cl.7 .................. .. A01K 67/033; A01K 67/027;

C12N 15/87; C12N 15/63 US. Cl. ......................... .. 800/18; 800/13; 435/325;

435/455 Field of Search .............................. .. 800/2, 13, 18;

4,950,599 4,959,313 5,175,384 5,204,244 5,416,260 5,545,806 5,545,807 5,569,825 5,591,669 6,114,598

424/88; 435/455, 325

References Cited

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A A A A A A A A A A

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* 1/1997

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Krimpenfort . 820/2

Fell et al. .... .. 435/696

Koller ..... .. 820/2

Lonberg ..... .. 800/2

Surani et al. 800/2 Lonberg et al. 800/2 Krimpenfort ...... .. 800/2

Kucherlapati et al. ...... .. 800/18

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WO 94/00569 1/1994 WO 94/02602 2/ 1994

OTHER PUBLICATIONS

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(List continued on neXt page.)

Primary Examiner—Anne M. Wehbe’ (74) Attorney, Agent, or Firm—Fish & Neave; James F. Haley, Jr.; Jane T. Gunnison

(57) ABSTRACT

The subject invention provides non-human mammalian hosts characterized by inactivated endogenous Ig loci and functional human Ig loci for response to an immunogen to produce human antibodies or analogs thereof. The hosts are produced by multiple genetic modi?cations of embryonic cells in conjunction with breeding. Different strategies are employed for recombination of the human loci randomly or at analogous host loci. Chimeric and transgenic mammals, particularly mice, are provided, having stably integrated large, Xenogeneic DNA segments. The segments are intro duced by fusion with yeast spheroplasts comprising yeast arti?cial chromosomes (YACs) which include the Xenoge neic DNA segments and a selective marker such as HPRT, and embryonic stem cells.

3 Claims, 24 Drawing Sheets

US 6,673,986 B1

OTHER PUBLICATIONS

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Huxley et al., “The Human HPRT Gene on a Yeast Articial Chromosome Is Functional When Transferred to Mouse Cells by Cell Fusion” (1991) Genomics 9:742—750. Mortensen et al., “Production of HomoZygous Mutant ES Cells With a Single Targeting Construct” (1992) Mol. and Cell. Biol. 12(5):2391—2395. Davies et al., “Targeted alterations in yeast arti?cial chro mosomes for inter—species gene transfer” (1992) Nucl. Acids Res. 20:2693—2698.

Zachau, “The Human Immunoglobulin k Locus and Some of its Acrobatics” (1990) Biol. Chem. 371:1—6. Matsuda et al., “Structure and physical map of 64 variable segments in the 3’0.8—megabase region of the human immu noglobulin heavy—chain locus” (1993) Nature Genetics 3:88—94. Shin et al., “Physical map of the 3’ region of the human immunoglobulin heavy chain locus: clustering of autoanti body—related variable segments in one haplotype” (1991) EMBO 10:3641—3645.

Ayares, et al., “Sequence Homology Requirements for Inter molecular Recombination in Mammalian Cells,” Proc. Natl. Acad. Sci. USA 83:5199—5203 (1986). Berman, et al., Content and Organization of the Human Ig VH Locus: De?nition of Three NeW VH Families and Linkage to the Ig CH Locus, EMBO J. 7:727—738 (1988). Brinster, et al., “Introns Increase Transcriptional Ef?ciency in Transgenic Mice,” Proc. Natl. Acad. Sci., USA, 85:836—840 (1988). Buttin, et al., “Exogenous Ig Gene Rearrangement in Trans genic Mice: A NeW Strategy for Human Monoclonal Anti body Production,” Trends in Genetics 3(8):205—206 (1987). Capecchi, et al., “Altering The Genome By Homologous Recombination,” Science 244:1288—1292 (1989). Choi et al., “RNA Splicing Generates a Variant Light Chain from an Aberrantly Rearranged k Gene,” Nature 286:776—779 (1980). Doetschman, et al., “Targeted Mutation of the Hprt Gene in Mouse Embryonic Stem Cells,”Proc. Natl. Acad. Sci. USA 85:8583—8587 (1988). Green, et al., “Antigen—Speci?c Human Mnonoclonal Anti bodies from Mice Engineered With Human Ig Heavy and Light Chain YACs,” Nature Genetics 7:13—21 (1994). Hooper et al., “HPRT—De?cient (Lesch—Nyhan) Mouse Embryos Derived from Gemline ColoniZation by Cultured Cells,” Nature 326:292—295 (1987). J akobovits, et al., “Germ—Line Transmission and Expression of a Human—Derived Yeast Arti?cial Chromosome,” Nature 362:255—258 (1993). Johnson, et al., “Targeting of Nonexpressed Genes in Embryonic Stem Cells Via Homologous Recombination,” 245:1234—1236 (1989). Kucherlapati, R., “Homologous Recombination in Mamma lian Somatic Cells,” Prog. Nucleic Acid Res. Mol. Biol. 36:301—310 (1989). Mansour, et al., “Disruption of the Proto—Oncogene Int—2 In Mouse Embryo—Derived Stem Cells: A General Strategy for Targeting Mutations to Non—Selectable Genes,” Nature 336—352 (1988). Max, et al., “Sequences of Five Potential Recombination Sites Encoded Close to an Immunoglobulin k Constat Region Gene,”Proc. Natl.Acad. Sci., USA 76(7):3450—3454 (1979).

US 6,673,986 B1

Miller, et al., “Structural Alterations in J Regions of Mouse Immunoglobulin )» Genes are Associated With Differential Gene Expression,” Nature 295:428—430 (1982). Morrison, “Success in Speci?cation,” Nature, 368:812—813 (1994). Orkin, et al., “Mutation in an Intervening Seguence Splice Junction in Man,” Proc. Natl. Acad. Sci. USA 78(8):5041—5045 (1981). RajeWsky, et al., “Evolutionary and Somatic Selection of the Antibody Repertoire in the Mouse,” Science 238:1088—1094 (1987). RamireZ—Solis, et al., “Chromosome Engineering in Mice,” Nature 378:720—724 (1995). Sakano, et al., “Sequences at the Somatic Recombination Sites of Immunoglobulin Light—Chain Genes,” Nature 280:288—294 (1979). Sakano, et al., “TWo Types of Somatic Recombination are Necessary for the Generation of Complete Immunoglobulin Heavy—Chain Genes,” Nature 286:676—683 (1980). Schedl, et al., “Transgenic Mice Generated By Pronuclear Injection of aYeast Arti?cal Chromosome,” Nucl. Acids Res. 20:3073—3077 (1992). Schedl, et al., “A Method for the Generation of YAC Transgenic Mice by Pronuclear Microinjection,” Nucleic Acids Research 21(20):4783—4787 (1993). SchWartZberg et al., “Germ—Line Transmission of a c—abl Mutation Produced by Targeted Gene Disruption in ES Cells,” Science 246:799—803 (1989). Seidman et al., “A Mutant Immunoglobulin light Chain is Formed by Aberrant DNA—and RNA—Splicing Events,” Nature 286:779—783 (1980).

ShimiZu, et al., “Immunoglobulin Double—Isotype Expres sion by Trans—mRNA in a Human Immunoglobulin Trans genic Mouse,” Proc. Natl. Acad. Sci., USA 86:8020—8023 (1989). Straus, et al., “Germ Line Transmission of a Yeast Arti?cial Chromosome Spanning the Murine (x1(1) Collagen Locus,” Science 259:1904—1907 (1993).

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* cited by examiner

U.S. Patent Jan. 6, 2004 Sheet 1 0f 24 US 6,673,986 B1

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