- Email: [email protected]
Antibody discovery: the use of transgenic mice to generate human monoclonal antibodies for therapeutics
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Antibody discovery: the use of transgenic mice to generate human monoclonal antibodies for therapeutics Sirid-Aimée Kellermann and Larry L Green* Technical advances made in the 1980s and early 1990s resulted in monoclonal antibodies that are now approved for human therapy. Novel transgenic mouse strains provide a powerful technology platform for creating fully human monoclonal antibodies as therapeutics; ten such antibodies have entered clinical trials since 1998 and more are in preclinical testing. Improved transgenic mouse strains provide a powerful technology platform for creating human therapeutics in the future. Addresses Abgenix, Inc., 6701 Kaiser Drive, Fremont, CA 94555, USA *e-mail: [email protected] Current Opinion in Biotechnology 2002, 13:593–597
generate human antibodies (see the article in this issue by Kretzschmar and von Rüden), optimizing such antibodies for high affinity and potency can be labor intensive. The rationale behind mice transgenic for human immunoglobulin loci is to harness the natural recombination and affinity maturation machinery to generate human antibodies of wide diversity and high affinity. Furthermore, facile rodent hybridoma generation technology requires less expertise in the antibody generation stage, making the process of recovering human antibodies of wide activity efficient and accessible. In this review, we discuss historical and recent advances in the use of transgenic mice to produce fully human antibodies.
0958-1669/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved.
Development of transgenic mice producing fully human antibodies
Abbreviations HIV human immunodeficiency virus Ig immunoglobulin mAb monoclonal antibody YAC yeast artificial chromosome
The evolution of transgenic mouse technologies was first marked by the report of a transgenic mouse containing a predominantly human IgH minilocus, permitting the expression of IgM antibodies with human µ chains, but containing mouse light chains [3]. Analysis of resulting mAbs from these and other mice [4] revealed that rearrangement and hypermutation occurred, indicating that the endogenous cell signaling machinery of mice was compatible with human immunoglobulin sequence elements.
Introduction Although the first monoclonal antibody (mAb) to be approved as a human therapeutic was a mouse antibody, muromonab-CD3 (OKT3, anti-CD3), murine mAbs are generally not well tolerated as multi-dosed therapeutics in immuno-competent individuals because they can be rapidly cleared and have the potential to be highly immunogenic, putting the recipient at risk of severe anaphylaxis and even death. Broad utilization of mAbs as therapeutics requires the non-human sequence to be minimized. Chimeric antibody technology, followed by humanization, led to successes such as infliximab (Remicade, anti-tumor necrosis factor α) and trastuzumab (Herceptin, anti-human epidermal growth factor 2). However, chimeric antibodies may still be considerably immunogenic, and humanization requires sophisticated molecular biological techniques and might sacrifice the affinity and potency of the original antibody. An obvious source of fully human antibodies is human B cells; however, there has been limited published success in ‘immunizing’ human B cells in vitro or in immunizing mice engrafted with human B cells. The recovery of stable human B cell hybridomas making high-affinity IgG mAbs is rare, although recent advances have been reported [1,2]. Furthermore, in humans the circulating antibody repertoire does not generally contain specificity to self proteins, which comprise the majority of targets for human antibody therapeutics. Although display technologies using systems such as phage, ribosomes or yeast can be powerful tools to
The majority of antibodies in these early transgenic strains were still murine, because of the activity of functional endogenous immunoglobulin loci. Inactivation of the murine IgH and Igκ loci through gene targeting solved this problem. Mice with inactivated endogenous IgH loci have arrested B-cell development [5,6], whereas mice with inactivated Igκ loci lack mouse Igκ antibodies and make only mouse Igλ antibodies [7,8]. These strains, despite displaying profound defects in B-cell development, still contained the necessary trans-acting factors for immunoglobulin gene function, such as factors for transcription and immunoglobulin gene recombination. Consequently, they provided a genetic background into which human immunoglobulin transgenes could be bred and would function [9–11]. Indeed, human immunoglobulin transgenes restored B-cell development to IgH and Igκ knock-out mice. Furthermore, the percentage of B cells making human antibodies and the circulating levels of human antibodies were significantly enhanced. Others used the cre–lox recombination method to replace the murine constant region genes Cκ and Cγ1 with the human counterparts [12,13]. These resulting mice produce chimeric antibodies with murine variable regions and human constant regions which, although of limited therapeutic value, are useful as standardization reagents in diagnostic assays [14].
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Figure 1
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Schematic representations of (a) the human immunoglobulin heavy chain (IgH) locus, (b) the human immunoglobulin κ light chain (Igκ) locus, and (c) the human immunoglobulin λ light chain (Igλ) locus. Functional V (variable) genes with open reading frames (filled circles), pseudogenes (white squares) and non-rearranged genes with open reading frames (gray or white circles) are represented. The OP, AP, LP and B designations in the human Igκ locus refer to clusters of related Vκ gene families. Unlike the IgH and Igκ loci, which each have a cluster of J genes, the human Igλ locus has seven JλCλ pairs, of which there are typically four functional clusters (filled boxes) and three
pseudogenes (white rectangles). For the IgH locus, the primary immune response is mediated through recombination of VDJ genes to form a functional variable region, which is linked on a transcript to the Cµ constant region, and together comprise the µ heavy chain of the IgM antibody class. After class-switch recombination within the IgH locus, the variable region is operably linked to another constant region gene, with the consequent deletion of the genes encoding Cµ, Cδ and any other intervening C genes. The C genes Cγ3, Cγ1, Cα1, Cγ2, Cγ4, Cε and Cα2 encode the antibody classes IgG3, IgG1, IgA1, IgG2, IgG4, IgE and IgA2, respectively.
The megabase size of many human genes, including the human germline IgH, Igκ and Igλ loci, drove the development of techniques for the introduction of transgenes on yeast artificial chromosomes (YACs) into the mouse germline (see Figure 1) [15–17]. This breakthrough enabled the generation of transgenic mice carrying much larger portions of the human immunoglobulin loci than possible with miniloci [10,18–23]. Indeed, mice with YAC transgenes, compared with transgenic mice with smaller transgenes, displayed better reconstitution of B-cell development, higher antibody productivity, and greater somatic hypermutation [6,22]. The superiority of larger, germline-configured transgenes may result from the representation of more variable genes as well as the germline-configured cis regulatory elements.
were constrained to class switch from IgM to either IgG1 or IgG4 were soon produced through replacement of the Cγ2 gene on the YAC with the genes for either human Cγ1 or Cγ4, followed by introduction into the mouse germline. The constrained switch to IgG1, IgG2 or IgG4 in these three XenoMouse® strains can be a benefit when the design goals for a potential therapeutic antibody require either the presence or absence of effector function. Because the YAC transgenes are integrated into a mouse chromosome, they offer superior genetic stability. To date, five fully human antibodies from the XenoMouse® strains have been used in human clinical trials.
The XenoMouse® strains of mice were the first engineered mice to include a majority of both the human VH and Vκ repertoire. These XenoMouse® mice contained megabasesized YACs that, on the heavy chain locus, had 34 functional VH genes, the entire DH and JH regions, and the human Cγ2 gene in a functional configuration downstream of Cµ and Cδ; the κ light chain locus contained 18 functional Vκ genes, all five functional Jκ regions, as well as the Cκ gene [23]. Additional strains of XenoMouse® mice that
The increased variable gene complexity of the XenoMouse® strains improved transit through several checkpoints of B-cell development, leading to a larger B cell compartment than in transgenic mice with fewer V genes [6]. Human IgG mAbs from these mice are consistently of low nanomolar or subnanomolar affinity [23,24], and have a diverse, human adult-like repertoire with CDR3 regions of a length more like human than mouse [25•]. Further demonstration of the diversity of the immune response in the XenoMouse mice is evidenced by the multiple epitopes recognized by a panel
The use of transgenic mice to generate therapeutic antibodies Kellermann and Green
of mAbs raised against the gp120 protein of human immunodeficiency virus (HIV) [26•]. Indeed, the wide diversity and fidelity with which the naïve and immune human antibody repertoire is reproduced in these mice have made them a useful tool to investigate diseases such as primary biliary cirrhosis [27] and antiglomerular basement membrane disease (Goodpasture’s syndrome) [28]. Fishwild and colleagues [29] augmented the number of human Vκ segments in the HuMab strains of transgenic mice by introducing a 450 kb YAC. These mice could generate low nanomolar to subnanomolar affinity human antibodies to proteins such as human CD4 [29] and digoxin [30]. Other scientists introduced human chromosome fragments that were several megabases in size into mice, presumably enabling the transfer of entire IgH and Igκ loci [31]. Breeding of these mice with mice having inactivated IgH/Igκ loci generated double-transchromosomic/doubleknockout mice. These ‘TC’ mice have reported levels of circulating IgG1, IgG2, IgG3 and IgG4 reminiscent of those in human serum [32]. These mice also make human Cµ and Cα chains, but because the IgM and IgA antibodies contain a murine J chain, they cannot be considered fully human. The transmission efficiency of the human extrachromosomal fragments was moderate to low [32]. The instability of the transchromosome carrying the Igκ locus was particularly detrimental, as hybridoma production was less than 10% of that seen with normal mice; the majority of hybridomas lost the Igκ TC fragment and instead expressed mouse λ chain. Breeding of the HuMab™ strains of mice with the TC mice produced the KM mouse, which possesses the IgH transchromosome of the TC mouse and the Igκ transgene (with its approximately 50% of the Vκ repertoire) of the HuMab™ mouse.
Where are we now? Future innovations Improvements continue to be made to these transgenic mice. The primary aim is to further enhance the ability to generate a large panel of high-affinity antibodies to a desired antigen, from which can be selected the best therapeutic candidates. The immune repertoire can be increased (e.g. by introducing the human Igλ locus), which represents 40% of the human antibody repertoire. Nicholson et al. [33] recently generated such a ‘five-feature’ mouse in which human IgH, Igκ and Igλ transloci were introduced into a murine IgH/Igκinactivated background. These animals made both human IgMκ and IgMλ antibodies. Because the IgH transgene lacks a downstream human Cγ gene, they cannot classswitch to human IgG, obviating the direct isolation of high-affinity fully human IgG antibodies from these mice. Ribosome display libraries were generated from hyperimmunized ‘five-feature’ mice as an alternative strategy to recover monoclonal antibodies [34]. Similarly, the XenoMouse strains of mice were augmented through the introduction of the entire human Igλ locus on a YAC transgene. These XMG1-KL, XMG2-KL and XMG4-KL
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strains produce both human IgGκ and IgGλ antibodies, and the human κ:λ ratio of 60:40 is recapitulated. These mice are capable of mounting robust immune responses to generate a diverse repertoire of IgGκ and IgGλ mAbs with high affinity. Further improvements in the immune response of humanantibody-producing mice may be obtained by crossing them with inbred or autoimmunity-prone genetic backgrounds, such as lpr, or an FcγRIIb-deficient background [35]. This could improve antigen presentation or promote breaking tolerance against targets for which the mouse ortholog is highly conserved. Such backcrossing to enhance the immune response might be obviated by improving immunization parameters such as improved adjuvants and the route of antigen administration. Because the transgenic mice produce a set of diverse, high-affinity human antibodies, a new challenge is the rapid identification and recovery of a set of candidate mAbs that meet the required affinity and potency characteristics for a human therapeutic. Improved production of hybridomas, coupling more efficient fusion procedures with high-throughput screening methods, accelerates the antibody discovery phase. The direct culture of B cells from hyperimmunized mice [36] followed by functional screening of antibodies can sample the immune repertoire efficiently. Finally, antibody display libraries from hyperimmunized mice may enable the in vitro isolation of high-affinity antibodies, eliminating the need for repeated rounds of molecular engineering that are typically required to enhance the activity of mAbs from naïve human display libraries. Gazing farther into the future, advances in the cloning of livestock coupled with the genetic engineering techniques used to generate transgenic mice may allow the generation of fully human polyclonal antibodies in amounts sufficient for clinical testing and even commercialization. For example, it may now be feasible to inactivate endogenous immunoglobulin loci and introduce the human immunoglobulin loci by fusion technologies [15,31,37•].
Conclusions: fully human antibodies from transgenic mice come of age The first transgenic-mouse-derived fully human antibody entered human clinical trials in 1998 [38]. Since that time, other mAbs have followed including antibodies against the epidermal growth factor receptor [39], CD4 [40], CTLA-4 (cytotoxic T lymphocyte-associated antigen 4), and several cancer-specific target antigens. A plethora of human antibodies from transgenic mice is in preclinical and early clinical testing, and the data accumulated from patients to date suggests that these antibodies are meeting expectations for lack of immunogenicity and pharmacokinetic characteristics. Efficacy must be evaluated on a case-by-case basis, being a function of both antibody affinity/potency as well as the disease relevance of the target antigen.
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Fully human antibodies will be useful for chronic indications such as rheumatoid arthritis and other inflammatory diseases where repeated antibody administration is needed. The technology may also be applicable to passive immunotherapy of infectious diseases. For example, mAbs from XenoMouse mice specific for Pneumococcus purified capsular polysaccharides as well as Pseudomonas aeruginosa lipopolysaccharide have been shown to have protective activity [41,42]. More recently, investigators have found that XenoMouse® mice immunized with recombinant HIV gp120 generated a robust response and a panel of diverse mAbs was isolated, including those with potent neutralizing activity against HIV [26•]. HuMab™ mice have been used to generate antibodies to Shiga toxin that were protective in animals [43] and, thus, may prove useful for the treatment of hemolytic-uremic syndrome in humans. These advances portend the successful application of transgenic mouse technology for the production of human mAbs against a wide array of clinically relevant targets.
Acknowledgements We thank our colleagues Michael Gallo and Chris Hare for critical reading of the manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest
10. Green LL, Hardy MC, Maynard-Currie CE, Tsuda H, Louie DM, Mendez MJ, Abderrahim H, Noguchi M, Smith DH, Zeng Y: Antigenspecific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nat Genet 1994, 7:13-21. 11. Wagner SD, Williams GT, Larson T, Neuberger MS, Kitamura D, Rajewsky K, Xian J, Brüggemann M: Antibodies generated from human immunoglobulin miniloci in transgenic mice. Nucleic Acids Res 1994, 22:1389-1393. 12. Zou YR, Gu H, Rajewsky K: Generation of a mouse strain that produces immunoglobulin kappa chains with human constant regions. Science 1993, 262:1271-1274. 13. Zou YR, Muller W, Gu H, Rajewsky K: Cre-loxP-mediated gene replacement: a mouse strain producing humanized antibodies. Curr Biol 1994, 4:1099-1103. 14. Simon T, Kneusel R, Haubruck H, Liedvogel B, Diarect AG: New ways for the standardization of autoantibody assays: chimeric monoclonal antibodies. Scand J Clin Lab Invest Suppl 2001, 235:91-97. 15. Jakobovits A, Moore AL, Green LL, Vergara GJ, Maynard-Currie CE, Austin HA, Klapholz S: Germ-line transmission and expression of a human-derived yeast artificial chromosome. Nature 1993, 362:255-258. 16. Schedl A, Montoliu L, Kelsey G, Schutz G: A yeast artificial chromosome covering the tyrosinase gene confers copy-numberdependent expression in transgenic mice. Nature 1993, 362:258-261. 17.
Strauss WM, Dausman J, Beard C, Johnson C, Lawrence JB, Jaenisch R: Germ line transmission of a yeast artificial chromosome spanning the murine α1(I) collagen locus. Science 1993, 259:1904-1907.
18. Choi TK, Hollenbach PW, Pearson BE, Ueda RM, Weddell GN, Kurahara CG, Woodhouse CS, Kay RM, Loring JF: Transgenic mice containing a human heavy chain immunoglobulin gene fragment cloned in a yeast artificial chromosome. Nat Genet 1993, 4:117-123.
1.
Karpas A, Dremucheva A, Czepulkowski BH: A human myeloma cell line suitable for the generation of human monoclonal antibodies. Proc Natl Acad Sci USA 2001, 98:1799-1804.
19. Davies NP, Brüggemann M: Extension of yeast artificial chromosomes by cosmid multimers. Nucleic Acids Res 1993, 21:767-768.
2.
Vaisbourd M, Ignatovich O, Dremucheva A, Karpas A, Winter G: Molecular characterization of human monoclonal antibodies derived from fusions of tonsil lymphocytes with a human myeloma cell line. Hybrid Hybridomics 2001, 20:287-292.
20. Popov AV, Butzler C, Frippiat JP, Lefranc MP, Brüggemann M: Assembly and extension of yeast artificial chromosomes to build up a large locus. Gene 1996, 177:195-201.
3.
Brüggemann M, Caskey HM, Teale C, Waldmann H, Williams GT, Surani MA, Neuberger MS: A repertoire of monoclonal antibodies with human heavy chains from transgenic mice. Proc Natl Acad Sci USA 1989, 86:6709-6713.
4.
Taylor LD, Carmack CE, Schramm SR, Mashayekh R, Higgins KM, Kuo CC, Woodhouse C, Kay RM, Lonberg N: A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins. Nucleic Acids Res 1992, 20:6287-6295.
5.
Jakobovits A, Vergara GJ, Kennedy JL, Hales JF, McGuinness RP, Casentini-Borocz DE, Brenner DG, Otten GR: Analysis of homozygous mutant chimeric mice: deletion of the immunoglobulin heavy-chain joining region blocks B-cell development and antibody production. Proc Natl Acad Sci USA 1993, 90:2551-2555.
6.
Green LL, Jakobovits A: Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes. J Exp Med 1998, 188:483-495.
7.
κ locus: Zou YR, Takeda S, Rajewsky K: Gene targeting in the Igκ efficient generation of λ chain-expressing B cells, independent of κ. EMBO J 1993, 12:811-820. gene rearrangements in Igκ
8.
Chen J, Trounstine M, Kurahara C, Young F, Kuo CC, Xu Y, Loring JF, Alt FW, Huszar D: B cell development in mice that lack one or both immunoglobulin kappa light chain genes. EMBO J 1993, 12:821-830.
9.
Lonberg N, Taylor LD, Harding FA, Trounstine M, Higgins KM, Schramm SR, Kuo CC, Mashayekh R, Wymore K, McCabe JG: Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 1994, 368:856-859.
21. Zou X, Xian J, Davies NP, Popov AV, Brüggemann M: Dominant κ locus replacing mouse light expression of a 1.3 Mb human Igκ chain production. FASEB J 1996, 10:1227-1232. 22. Xian J, Zou X, Popov AV, Mundt CA, Miller N, Williams GT, Davis CG, Neuberger MS, Brüggemann M: Comparison of the performance of κ minilocus and Yac-based human Igκ κ a plasmid-based human Igκ transloci for the production of human antibody repertoires in transgenic mice. Transgenics 1998, 2:333-343. 23. Mendez MJ, Green LL, Corvalan JR, Jia XC, Maynard-Currie CE, Yang XD, Gallo ML, Louie DM, Lee DV, Erickson KL et al.: Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice. Nat Genet 1997, 15:146-156. 24. Green LL: Antibody engineering via genetic engineering of the mouse: XenoMouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies. J Immunol Methods 1999, 231:11-23. 25. Gallo ML, Ivanov VE, Jakobovits A, Davis CG: The human • immunoglobulin loci introduced into mice: V (D) and J gene segment usage similar to that of adult humans. Eur J Immunol 2000, 30:534-540. This article showed that the antibody variable gene usage in human immunoglobulin loci-transgenic mice was strikingly consistent with that observed in adult humans, suggesting faithful recapitulation of human humoral immunity and a ‘human’ antibody repertoire. 26. He Y, Honnen WJ, Krachmarov CP, Burkhart M, Kayman SC, • Corvalan J, Pinter A: Efficient isolation of novel human monoclonal antibodies with neutralizing activity against HIV-1 from transgenic mice expressing human Ig loci. J Immunol 2002, 169:595-605. This report describes the level of diversity that the humoral immune response can achieve in the XenoMouse® strain of transgenic mice, and suggests that developing a passive immunotherapy for HIV may be possible using this approach.
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27.
Sasaki M, van De WJ, Kenny TP, Gallo ML, Leung PS, Nakanuma Y, Ansari AA, Coppel RL, Neuberger J, Gershwin ME: Immunoglobulin gene usage and immunohistochemical characteristics of human monoclonal antibodies to the mitochondrial autoantigens of primary biliary cirrhosis induced in the XenoMouse. Hepatology 2001, 34:631-637.
28. Meyers KE, Allen J, Gehret J, Jacobovits A, Gallo M, Neilson EG, Hopfer H, Kalluri R, Madaio MP: Human antiglomerular basement membrane autoantibody disease in XenoMouse II. Kidney Int 2002, 61:1666-1673. 29. Fishwild DM, O’Donnell SL, Bengoechea T, Hudson DV, Harding F, Bernhard SL, Jones D, Kay RM, Higgins KM, Schramm SR, Lonberg N: High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice. Nat Biotechnol 1996, 14:845-851.
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35. Ishida I: The KM mouse: for efficient and rapid production of high affinity human monoclonal antibodies. IBC 12th Annual International Conference on Antibody Engineering. December 2001. 36. Babcook JS, Leslie KB, Olsen OA, Salmon RA, Schrader JW: A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Proc Natl Acad Sci USA 1996, 93:7843-7848. 37. •
Ishida I, Tomizuka K, Yoshida H, Tahara T, Takahashi N, Ohguma A, Tanaka S, Umehashi M, Maeda H, Nozaki C et al.: Production of human monoclonal and polyclonal antibodies in TransChromo animals. Cloning Stem Cells 2002, 4:91-102. This report suggests that the technology for generating fully human antibodies in transgenic animals may transcend mice and be applicable to polyclonal antibody generation in livestock.
30. Ball WJ Jr, Kasturi R, Dey P, Tabet M, O’Donnell S, Hudson D, Fishwild D: Isolation and characterization of human monoclonal antibodies to digoxin. J Immunol 1999, 163:2291-2298.
38. Yang XD, Corvalan JR, Wang P, Roy CM, Davis CG: Fully human anti-interleukin-8 monoclonal antibodies: potential therapeutics for the treatment of inflammatory disease states. J Leukoc Biol 1999, 66:401-410.
31. Tomizuka K, Yoshida H, Uejima H, Kugoh H, Sato K, Ohguma A, Hayasaka M, Hanaoka K, Oshimura M, Ishida I: Functional expression and germline transmission of a human chromosome fragment in chimaeric mice. Nat Genet 1997, 16:133-143.
39. Yang XD, Jia XC, Corvalan JR, Wang P, Davis CG, Jakobovits A: Eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy. Cancer Res 1999, 59:1236-1243.
32. Tomizuka K, Shinohara T, Yoshida H, Uejima H, Ohguma A, Tanaka S, Sato K, Oshimura M, Ishida I: Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies. Proc Natl Acad Sci USA 2000, 97:722-727.
40. Fishwild DM, Hudson DV, Deshpande U, Kung AH: Differential effects of administration of a human anti-CD4 monoclonal antibody, HM6G, in nonhuman primates. Clin Immunol 1999, 92:138-152.
33. Nicholson IC, Zou X, Popov AV, Cook GP, Corps EM, Humphries S, Ayling C, Goyenechea B, Xian J, Taussig MJ et al.: Antibody repertoires of four- and five-feature translocus mice carrying human immunoglobulin heavy chain and kappa and lambda light chain yeast artificial chromosomes. J Immunol 1999, 163:6898-6906. 34. He M, Menges M, Groves MA, Corps E, Liu H, Brüggemann M, Taussig MJ: Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display. J Immunol Methods 1999, 231:105-117.
41. Russell ND, Corvalan JR, Gallo ML, Davis CG, Pirofski L: Production of protective human antipneumococcal antibodies by transgenic mice with human immunoglobulin loci. Infect Immun 2000, 68:1820-1826. 42. Hemachandra S, Kamboj K, Copfer J, Pier G, Green LL, Schreiber JR: Human monoclonal antibodies against Pseudomonas aeruginosa lipopolysaccharide derived from transgenic mice containing megabase human immunoglobulin loci are opsonic and protective against fatal pseudomonas sepsis. Infect Immun 2001, 69:2223-2229. 43. Mukherjee J, Chios K, Fishwild D, Hudson D, O’Donnell S, Rich SM, Donohue-Rolfe A, Tzipori S: Human Stx2-specific monoclonal antibodies prevent systemic complications of Escherichia coli O157:H7 infection. Infect Immun 2002, 70:612-619.
Antibody discovery: the use of transgenic mice to generate human monoclonal antibodies for therapeutics Sirid-Aimée Kellermann and Larry L Green* Technical advances made in the 1980s and early 1990s resulted in monoclonal antibodies that are now approved for human therapy. Novel transgenic mouse strains provide a powerful technology platform for creating fully human monoclonal antibodies as therapeutics; ten such antibodies have entered clinical trials since 1998 and more are in preclinical testing. Improved transgenic mouse strains provide a powerful technology platform for creating human therapeutics in the future. Addresses Abgenix, Inc., 6701 Kaiser Drive, Fremont, CA 94555, USA *e-mail: [email protected] Current Opinion in Biotechnology 2002, 13:593–597
generate human antibodies (see the article in this issue by Kretzschmar and von Rüden), optimizing such antibodies for high affinity and potency can be labor intensive. The rationale behind mice transgenic for human immunoglobulin loci is to harness the natural recombination and affinity maturation machinery to generate human antibodies of wide diversity and high affinity. Furthermore, facile rodent hybridoma generation technology requires less expertise in the antibody generation stage, making the process of recovering human antibodies of wide activity efficient and accessible. In this review, we discuss historical and recent advances in the use of transgenic mice to produce fully human antibodies.
0958-1669/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved.
Development of transgenic mice producing fully human antibodies
Abbreviations HIV human immunodeficiency virus Ig immunoglobulin mAb monoclonal antibody YAC yeast artificial chromosome
The evolution of transgenic mouse technologies was first marked by the report of a transgenic mouse containing a predominantly human IgH minilocus, permitting the expression of IgM antibodies with human µ chains, but containing mouse light chains [3]. Analysis of resulting mAbs from these and other mice [4] revealed that rearrangement and hypermutation occurred, indicating that the endogenous cell signaling machinery of mice was compatible with human immunoglobulin sequence elements.
Introduction Although the first monoclonal antibody (mAb) to be approved as a human therapeutic was a mouse antibody, muromonab-CD3 (OKT3, anti-CD3), murine mAbs are generally not well tolerated as multi-dosed therapeutics in immuno-competent individuals because they can be rapidly cleared and have the potential to be highly immunogenic, putting the recipient at risk of severe anaphylaxis and even death. Broad utilization of mAbs as therapeutics requires the non-human sequence to be minimized. Chimeric antibody technology, followed by humanization, led to successes such as infliximab (Remicade, anti-tumor necrosis factor α) and trastuzumab (Herceptin, anti-human epidermal growth factor 2). However, chimeric antibodies may still be considerably immunogenic, and humanization requires sophisticated molecular biological techniques and might sacrifice the affinity and potency of the original antibody. An obvious source of fully human antibodies is human B cells; however, there has been limited published success in ‘immunizing’ human B cells in vitro or in immunizing mice engrafted with human B cells. The recovery of stable human B cell hybridomas making high-affinity IgG mAbs is rare, although recent advances have been reported [1,2]. Furthermore, in humans the circulating antibody repertoire does not generally contain specificity to self proteins, which comprise the majority of targets for human antibody therapeutics. Although display technologies using systems such as phage, ribosomes or yeast can be powerful tools to
The majority of antibodies in these early transgenic strains were still murine, because of the activity of functional endogenous immunoglobulin loci. Inactivation of the murine IgH and Igκ loci through gene targeting solved this problem. Mice with inactivated endogenous IgH loci have arrested B-cell development [5,6], whereas mice with inactivated Igκ loci lack mouse Igκ antibodies and make only mouse Igλ antibodies [7,8]. These strains, despite displaying profound defects in B-cell development, still contained the necessary trans-acting factors for immunoglobulin gene function, such as factors for transcription and immunoglobulin gene recombination. Consequently, they provided a genetic background into which human immunoglobulin transgenes could be bred and would function [9–11]. Indeed, human immunoglobulin transgenes restored B-cell development to IgH and Igκ knock-out mice. Furthermore, the percentage of B cells making human antibodies and the circulating levels of human antibodies were significantly enhanced. Others used the cre–lox recombination method to replace the murine constant region genes Cκ and Cγ1 with the human counterparts [12,13]. These resulting mice produce chimeric antibodies with murine variable regions and human constant regions which, although of limited therapeutic value, are useful as standardization reagents in diagnostic assays [14].
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Figure 1
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Current Opinion in Biotechnology
Schematic representations of (a) the human immunoglobulin heavy chain (IgH) locus, (b) the human immunoglobulin κ light chain (Igκ) locus, and (c) the human immunoglobulin λ light chain (Igλ) locus. Functional V (variable) genes with open reading frames (filled circles), pseudogenes (white squares) and non-rearranged genes with open reading frames (gray or white circles) are represented. The OP, AP, LP and B designations in the human Igκ locus refer to clusters of related Vκ gene families. Unlike the IgH and Igκ loci, which each have a cluster of J genes, the human Igλ locus has seven JλCλ pairs, of which there are typically four functional clusters (filled boxes) and three
pseudogenes (white rectangles). For the IgH locus, the primary immune response is mediated through recombination of VDJ genes to form a functional variable region, which is linked on a transcript to the Cµ constant region, and together comprise the µ heavy chain of the IgM antibody class. After class-switch recombination within the IgH locus, the variable region is operably linked to another constant region gene, with the consequent deletion of the genes encoding Cµ, Cδ and any other intervening C genes. The C genes Cγ3, Cγ1, Cα1, Cγ2, Cγ4, Cε and Cα2 encode the antibody classes IgG3, IgG1, IgA1, IgG2, IgG4, IgE and IgA2, respectively.
The megabase size of many human genes, including the human germline IgH, Igκ and Igλ loci, drove the development of techniques for the introduction of transgenes on yeast artificial chromosomes (YACs) into the mouse germline (see Figure 1) [15–17]. This breakthrough enabled the generation of transgenic mice carrying much larger portions of the human immunoglobulin loci than possible with miniloci [10,18–23]. Indeed, mice with YAC transgenes, compared with transgenic mice with smaller transgenes, displayed better reconstitution of B-cell development, higher antibody productivity, and greater somatic hypermutation [6,22]. The superiority of larger, germline-configured transgenes may result from the representation of more variable genes as well as the germline-configured cis regulatory elements.
were constrained to class switch from IgM to either IgG1 or IgG4 were soon produced through replacement of the Cγ2 gene on the YAC with the genes for either human Cγ1 or Cγ4, followed by introduction into the mouse germline. The constrained switch to IgG1, IgG2 or IgG4 in these three XenoMouse® strains can be a benefit when the design goals for a potential therapeutic antibody require either the presence or absence of effector function. Because the YAC transgenes are integrated into a mouse chromosome, they offer superior genetic stability. To date, five fully human antibodies from the XenoMouse® strains have been used in human clinical trials.
The XenoMouse® strains of mice were the first engineered mice to include a majority of both the human VH and Vκ repertoire. These XenoMouse® mice contained megabasesized YACs that, on the heavy chain locus, had 34 functional VH genes, the entire DH and JH regions, and the human Cγ2 gene in a functional configuration downstream of Cµ and Cδ; the κ light chain locus contained 18 functional Vκ genes, all five functional Jκ regions, as well as the Cκ gene [23]. Additional strains of XenoMouse® mice that
The increased variable gene complexity of the XenoMouse® strains improved transit through several checkpoints of B-cell development, leading to a larger B cell compartment than in transgenic mice with fewer V genes [6]. Human IgG mAbs from these mice are consistently of low nanomolar or subnanomolar affinity [23,24], and have a diverse, human adult-like repertoire with CDR3 regions of a length more like human than mouse [25•]. Further demonstration of the diversity of the immune response in the XenoMouse mice is evidenced by the multiple epitopes recognized by a panel
The use of transgenic mice to generate therapeutic antibodies Kellermann and Green
of mAbs raised against the gp120 protein of human immunodeficiency virus (HIV) [26•]. Indeed, the wide diversity and fidelity with which the naïve and immune human antibody repertoire is reproduced in these mice have made them a useful tool to investigate diseases such as primary biliary cirrhosis [27] and antiglomerular basement membrane disease (Goodpasture’s syndrome) [28]. Fishwild and colleagues [29] augmented the number of human Vκ segments in the HuMab strains of transgenic mice by introducing a 450 kb YAC. These mice could generate low nanomolar to subnanomolar affinity human antibodies to proteins such as human CD4 [29] and digoxin [30]. Other scientists introduced human chromosome fragments that were several megabases in size into mice, presumably enabling the transfer of entire IgH and Igκ loci [31]. Breeding of these mice with mice having inactivated IgH/Igκ loci generated double-transchromosomic/doubleknockout mice. These ‘TC’ mice have reported levels of circulating IgG1, IgG2, IgG3 and IgG4 reminiscent of those in human serum [32]. These mice also make human Cµ and Cα chains, but because the IgM and IgA antibodies contain a murine J chain, they cannot be considered fully human. The transmission efficiency of the human extrachromosomal fragments was moderate to low [32]. The instability of the transchromosome carrying the Igκ locus was particularly detrimental, as hybridoma production was less than 10% of that seen with normal mice; the majority of hybridomas lost the Igκ TC fragment and instead expressed mouse λ chain. Breeding of the HuMab™ strains of mice with the TC mice produced the KM mouse, which possesses the IgH transchromosome of the TC mouse and the Igκ transgene (with its approximately 50% of the Vκ repertoire) of the HuMab™ mouse.
Where are we now? Future innovations Improvements continue to be made to these transgenic mice. The primary aim is to further enhance the ability to generate a large panel of high-affinity antibodies to a desired antigen, from which can be selected the best therapeutic candidates. The immune repertoire can be increased (e.g. by introducing the human Igλ locus), which represents 40% of the human antibody repertoire. Nicholson et al. [33] recently generated such a ‘five-feature’ mouse in which human IgH, Igκ and Igλ transloci were introduced into a murine IgH/Igκinactivated background. These animals made both human IgMκ and IgMλ antibodies. Because the IgH transgene lacks a downstream human Cγ gene, they cannot classswitch to human IgG, obviating the direct isolation of high-affinity fully human IgG antibodies from these mice. Ribosome display libraries were generated from hyperimmunized ‘five-feature’ mice as an alternative strategy to recover monoclonal antibodies [34]. Similarly, the XenoMouse strains of mice were augmented through the introduction of the entire human Igλ locus on a YAC transgene. These XMG1-KL, XMG2-KL and XMG4-KL
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strains produce both human IgGκ and IgGλ antibodies, and the human κ:λ ratio of 60:40 is recapitulated. These mice are capable of mounting robust immune responses to generate a diverse repertoire of IgGκ and IgGλ mAbs with high affinity. Further improvements in the immune response of humanantibody-producing mice may be obtained by crossing them with inbred or autoimmunity-prone genetic backgrounds, such as lpr, or an FcγRIIb-deficient background [35]. This could improve antigen presentation or promote breaking tolerance against targets for which the mouse ortholog is highly conserved. Such backcrossing to enhance the immune response might be obviated by improving immunization parameters such as improved adjuvants and the route of antigen administration. Because the transgenic mice produce a set of diverse, high-affinity human antibodies, a new challenge is the rapid identification and recovery of a set of candidate mAbs that meet the required affinity and potency characteristics for a human therapeutic. Improved production of hybridomas, coupling more efficient fusion procedures with high-throughput screening methods, accelerates the antibody discovery phase. The direct culture of B cells from hyperimmunized mice [36] followed by functional screening of antibodies can sample the immune repertoire efficiently. Finally, antibody display libraries from hyperimmunized mice may enable the in vitro isolation of high-affinity antibodies, eliminating the need for repeated rounds of molecular engineering that are typically required to enhance the activity of mAbs from naïve human display libraries. Gazing farther into the future, advances in the cloning of livestock coupled with the genetic engineering techniques used to generate transgenic mice may allow the generation of fully human polyclonal antibodies in amounts sufficient for clinical testing and even commercialization. For example, it may now be feasible to inactivate endogenous immunoglobulin loci and introduce the human immunoglobulin loci by fusion technologies [15,31,37•].
Conclusions: fully human antibodies from transgenic mice come of age The first transgenic-mouse-derived fully human antibody entered human clinical trials in 1998 [38]. Since that time, other mAbs have followed including antibodies against the epidermal growth factor receptor [39], CD4 [40], CTLA-4 (cytotoxic T lymphocyte-associated antigen 4), and several cancer-specific target antigens. A plethora of human antibodies from transgenic mice is in preclinical and early clinical testing, and the data accumulated from patients to date suggests that these antibodies are meeting expectations for lack of immunogenicity and pharmacokinetic characteristics. Efficacy must be evaluated on a case-by-case basis, being a function of both antibody affinity/potency as well as the disease relevance of the target antigen.
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Fully human antibodies will be useful for chronic indications such as rheumatoid arthritis and other inflammatory diseases where repeated antibody administration is needed. The technology may also be applicable to passive immunotherapy of infectious diseases. For example, mAbs from XenoMouse mice specific for Pneumococcus purified capsular polysaccharides as well as Pseudomonas aeruginosa lipopolysaccharide have been shown to have protective activity [41,42]. More recently, investigators have found that XenoMouse® mice immunized with recombinant HIV gp120 generated a robust response and a panel of diverse mAbs was isolated, including those with potent neutralizing activity against HIV [26•]. HuMab™ mice have been used to generate antibodies to Shiga toxin that were protective in animals [43] and, thus, may prove useful for the treatment of hemolytic-uremic syndrome in humans. These advances portend the successful application of transgenic mouse technology for the production of human mAbs against a wide array of clinically relevant targets.
Acknowledgements We thank our colleagues Michael Gallo and Chris Hare for critical reading of the manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest
10. Green LL, Hardy MC, Maynard-Currie CE, Tsuda H, Louie DM, Mendez MJ, Abderrahim H, Noguchi M, Smith DH, Zeng Y: Antigenspecific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nat Genet 1994, 7:13-21. 11. Wagner SD, Williams GT, Larson T, Neuberger MS, Kitamura D, Rajewsky K, Xian J, Brüggemann M: Antibodies generated from human immunoglobulin miniloci in transgenic mice. Nucleic Acids Res 1994, 22:1389-1393. 12. Zou YR, Gu H, Rajewsky K: Generation of a mouse strain that produces immunoglobulin kappa chains with human constant regions. Science 1993, 262:1271-1274. 13. Zou YR, Muller W, Gu H, Rajewsky K: Cre-loxP-mediated gene replacement: a mouse strain producing humanized antibodies. Curr Biol 1994, 4:1099-1103. 14. Simon T, Kneusel R, Haubruck H, Liedvogel B, Diarect AG: New ways for the standardization of autoantibody assays: chimeric monoclonal antibodies. Scand J Clin Lab Invest Suppl 2001, 235:91-97. 15. Jakobovits A, Moore AL, Green LL, Vergara GJ, Maynard-Currie CE, Austin HA, Klapholz S: Germ-line transmission and expression of a human-derived yeast artificial chromosome. Nature 1993, 362:255-258. 16. Schedl A, Montoliu L, Kelsey G, Schutz G: A yeast artificial chromosome covering the tyrosinase gene confers copy-numberdependent expression in transgenic mice. Nature 1993, 362:258-261. 17.
Strauss WM, Dausman J, Beard C, Johnson C, Lawrence JB, Jaenisch R: Germ line transmission of a yeast artificial chromosome spanning the murine α1(I) collagen locus. Science 1993, 259:1904-1907.
18. Choi TK, Hollenbach PW, Pearson BE, Ueda RM, Weddell GN, Kurahara CG, Woodhouse CS, Kay RM, Loring JF: Transgenic mice containing a human heavy chain immunoglobulin gene fragment cloned in a yeast artificial chromosome. Nat Genet 1993, 4:117-123.
1.
Karpas A, Dremucheva A, Czepulkowski BH: A human myeloma cell line suitable for the generation of human monoclonal antibodies. Proc Natl Acad Sci USA 2001, 98:1799-1804.
19. Davies NP, Brüggemann M: Extension of yeast artificial chromosomes by cosmid multimers. Nucleic Acids Res 1993, 21:767-768.
2.
Vaisbourd M, Ignatovich O, Dremucheva A, Karpas A, Winter G: Molecular characterization of human monoclonal antibodies derived from fusions of tonsil lymphocytes with a human myeloma cell line. Hybrid Hybridomics 2001, 20:287-292.
20. Popov AV, Butzler C, Frippiat JP, Lefranc MP, Brüggemann M: Assembly and extension of yeast artificial chromosomes to build up a large locus. Gene 1996, 177:195-201.
3.
Brüggemann M, Caskey HM, Teale C, Waldmann H, Williams GT, Surani MA, Neuberger MS: A repertoire of monoclonal antibodies with human heavy chains from transgenic mice. Proc Natl Acad Sci USA 1989, 86:6709-6713.
4.
Taylor LD, Carmack CE, Schramm SR, Mashayekh R, Higgins KM, Kuo CC, Woodhouse C, Kay RM, Lonberg N: A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins. Nucleic Acids Res 1992, 20:6287-6295.
5.
Jakobovits A, Vergara GJ, Kennedy JL, Hales JF, McGuinness RP, Casentini-Borocz DE, Brenner DG, Otten GR: Analysis of homozygous mutant chimeric mice: deletion of the immunoglobulin heavy-chain joining region blocks B-cell development and antibody production. Proc Natl Acad Sci USA 1993, 90:2551-2555.
6.
Green LL, Jakobovits A: Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes. J Exp Med 1998, 188:483-495.
7.
κ locus: Zou YR, Takeda S, Rajewsky K: Gene targeting in the Igκ efficient generation of λ chain-expressing B cells, independent of κ. EMBO J 1993, 12:811-820. gene rearrangements in Igκ
8.
Chen J, Trounstine M, Kurahara C, Young F, Kuo CC, Xu Y, Loring JF, Alt FW, Huszar D: B cell development in mice that lack one or both immunoglobulin kappa light chain genes. EMBO J 1993, 12:821-830.
9.
Lonberg N, Taylor LD, Harding FA, Trounstine M, Higgins KM, Schramm SR, Kuo CC, Mashayekh R, Wymore K, McCabe JG: Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 1994, 368:856-859.
21. Zou X, Xian J, Davies NP, Popov AV, Brüggemann M: Dominant κ locus replacing mouse light expression of a 1.3 Mb human Igκ chain production. FASEB J 1996, 10:1227-1232. 22. Xian J, Zou X, Popov AV, Mundt CA, Miller N, Williams GT, Davis CG, Neuberger MS, Brüggemann M: Comparison of the performance of κ minilocus and Yac-based human Igκ κ a plasmid-based human Igκ transloci for the production of human antibody repertoires in transgenic mice. Transgenics 1998, 2:333-343. 23. Mendez MJ, Green LL, Corvalan JR, Jia XC, Maynard-Currie CE, Yang XD, Gallo ML, Louie DM, Lee DV, Erickson KL et al.: Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice. Nat Genet 1997, 15:146-156. 24. Green LL: Antibody engineering via genetic engineering of the mouse: XenoMouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies. J Immunol Methods 1999, 231:11-23. 25. Gallo ML, Ivanov VE, Jakobovits A, Davis CG: The human • immunoglobulin loci introduced into mice: V (D) and J gene segment usage similar to that of adult humans. Eur J Immunol 2000, 30:534-540. This article showed that the antibody variable gene usage in human immunoglobulin loci-transgenic mice was strikingly consistent with that observed in adult humans, suggesting faithful recapitulation of human humoral immunity and a ‘human’ antibody repertoire. 26. He Y, Honnen WJ, Krachmarov CP, Burkhart M, Kayman SC, • Corvalan J, Pinter A: Efficient isolation of novel human monoclonal antibodies with neutralizing activity against HIV-1 from transgenic mice expressing human Ig loci. J Immunol 2002, 169:595-605. This report describes the level of diversity that the humoral immune response can achieve in the XenoMouse® strain of transgenic mice, and suggests that developing a passive immunotherapy for HIV may be possible using this approach.
The use of transgenic mice to generate therapeutic antibodies Kellermann and Green
27.
Sasaki M, van De WJ, Kenny TP, Gallo ML, Leung PS, Nakanuma Y, Ansari AA, Coppel RL, Neuberger J, Gershwin ME: Immunoglobulin gene usage and immunohistochemical characteristics of human monoclonal antibodies to the mitochondrial autoantigens of primary biliary cirrhosis induced in the XenoMouse. Hepatology 2001, 34:631-637.
28. Meyers KE, Allen J, Gehret J, Jacobovits A, Gallo M, Neilson EG, Hopfer H, Kalluri R, Madaio MP: Human antiglomerular basement membrane autoantibody disease in XenoMouse II. Kidney Int 2002, 61:1666-1673. 29. Fishwild DM, O’Donnell SL, Bengoechea T, Hudson DV, Harding F, Bernhard SL, Jones D, Kay RM, Higgins KM, Schramm SR, Lonberg N: High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice. Nat Biotechnol 1996, 14:845-851.
597
35. Ishida I: The KM mouse: for efficient and rapid production of high affinity human monoclonal antibodies. IBC 12th Annual International Conference on Antibody Engineering. December 2001. 36. Babcook JS, Leslie KB, Olsen OA, Salmon RA, Schrader JW: A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Proc Natl Acad Sci USA 1996, 93:7843-7848. 37. •
Ishida I, Tomizuka K, Yoshida H, Tahara T, Takahashi N, Ohguma A, Tanaka S, Umehashi M, Maeda H, Nozaki C et al.: Production of human monoclonal and polyclonal antibodies in TransChromo animals. Cloning Stem Cells 2002, 4:91-102. This report suggests that the technology for generating fully human antibodies in transgenic animals may transcend mice and be applicable to polyclonal antibody generation in livestock.
30. Ball WJ Jr, Kasturi R, Dey P, Tabet M, O’Donnell S, Hudson D, Fishwild D: Isolation and characterization of human monoclonal antibodies to digoxin. J Immunol 1999, 163:2291-2298.
38. Yang XD, Corvalan JR, Wang P, Roy CM, Davis CG: Fully human anti-interleukin-8 monoclonal antibodies: potential therapeutics for the treatment of inflammatory disease states. J Leukoc Biol 1999, 66:401-410.
31. Tomizuka K, Yoshida H, Uejima H, Kugoh H, Sato K, Ohguma A, Hayasaka M, Hanaoka K, Oshimura M, Ishida I: Functional expression and germline transmission of a human chromosome fragment in chimaeric mice. Nat Genet 1997, 16:133-143.
39. Yang XD, Jia XC, Corvalan JR, Wang P, Davis CG, Jakobovits A: Eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy. Cancer Res 1999, 59:1236-1243.
32. Tomizuka K, Shinohara T, Yoshida H, Uejima H, Ohguma A, Tanaka S, Sato K, Oshimura M, Ishida I: Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies. Proc Natl Acad Sci USA 2000, 97:722-727.
40. Fishwild DM, Hudson DV, Deshpande U, Kung AH: Differential effects of administration of a human anti-CD4 monoclonal antibody, HM6G, in nonhuman primates. Clin Immunol 1999, 92:138-152.
33. Nicholson IC, Zou X, Popov AV, Cook GP, Corps EM, Humphries S, Ayling C, Goyenechea B, Xian J, Taussig MJ et al.: Antibody repertoires of four- and five-feature translocus mice carrying human immunoglobulin heavy chain and kappa and lambda light chain yeast artificial chromosomes. J Immunol 1999, 163:6898-6906. 34. He M, Menges M, Groves MA, Corps E, Liu H, Brüggemann M, Taussig MJ: Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display. J Immunol Methods 1999, 231:105-117.
41. Russell ND, Corvalan JR, Gallo ML, Davis CG, Pirofski L: Production of protective human antipneumococcal antibodies by transgenic mice with human immunoglobulin loci. Infect Immun 2000, 68:1820-1826. 42. Hemachandra S, Kamboj K, Copfer J, Pier G, Green LL, Schreiber JR: Human monoclonal antibodies against Pseudomonas aeruginosa lipopolysaccharide derived from transgenic mice containing megabase human immunoglobulin loci are opsonic and protective against fatal pseudomonas sepsis. Infect Immun 2001, 69:2223-2229. 43. Mukherjee J, Chios K, Fishwild D, Hudson D, O’Donnell S, Rich SM, Donohue-Rolfe A, Tzipori S: Human Stx2-specific monoclonal antibodies prevent systemic complications of Escherichia coli O157:H7 infection. Infect Immun 2002, 70:612-619.