Advertisement

Human Antibody Production in Transgenic Animals

  • Marianne BrüggemannEmail author
  • Michael J. Osborn
  • Biao Ma
  • Jasvinder Hayre
  • Suzanne Avis
  • Brian Lundstrom
  • Roland BuelowEmail author
Open Access
Review

Abstract

Fully human antibodies from transgenic animals account for an increasing number of new therapeutics. After immunization, diverse human monoclonal antibodies of high affinity can be obtained from transgenic rodents, while large animals, such as transchromosomic cattle, have produced respectable amounts of specific human immunoglobulin (Ig) in serum. Several strategies to derive animals expressing human antibody repertoires have been successful. In rodents, gene loci on bacterial artificial chromosomes or yeast artificial chromosomes were integrated by oocyte microinjection or transfection of embryonic stem (ES) cells, while ruminants were derived from manipulated fibroblasts with integrated human chromosome fragments or human artificial chromosomes. In all strains, the endogenous Ig loci have been silenced by gene targeting, either in ES or fibroblast cells, or by zinc finger technology via DNA microinjection; this was essential for optimal production. However, comparisons showed that fully human antibodies were not as efficiently produced as wild-type Ig. This suboptimal performance, with respect to immune response and antibody yield, was attributed to imperfect interaction of the human constant region with endogenous signaling components such as the Igα/β in mouse, rat or cattle. Significant improvements were obtained when the human V-region genes were linked to the endogenous CH-region, either on large constructs or, separately, by site-specific integration, which could also silence the endogenous Ig locus by gene replacement or inversion. In animals with knocked-out endogenous Ig loci and integrated large IgH loci, containing many human Vs, all D and all J segments linked to endogenous C genes, highly diverse human antibody production similar to normal animals was obtained.

Keywords

Transgenic animals Antibody repertoires Human epitopes Class-switch recombination B cell development Locus silencing 

Abbreviations

BAC

Bacterial artificial chromosome

C-region

Constant-region

D

Diversity segment

EGFR

Epidermal growth factor receptor

H

Heavy chain

Ig

Immunoglobulin

J

Joining segment

L

Light chain

mAb

Monoclonal antibody

V

Variable gene

WT

Wild type

YAC

Yeast artificial chromosome

ZFN

Zinc-finger nuclease

Introduction

The first fully human monoclonal antibodies (mAbs) have been produced over 25 years ago by two parallel technologies: phage display, with selection of antigen-specific binders from blood lymphocyte libraries, and transgenic mice, with integrated human immunoglobulin (Ig) loci (Bruggemann et al. 1989a; Bruggemann and Neuberger 1996; McCafferty et al. 1990; Neuberger and Bruggemann 1997). In the transgenic approach, natural diversification and selection are being exploited as integrated loci are under the control of the animal’s immune system where they can undergo normal processes of DNA rearrangement and hypermutation. Interestingly, human or mouse, and probably all mammalian Ig loci work in very similar ways, which was confirmed in early experiments using a germline-configured chimeric construct (Bruggemann et al. 1989a).

A challenge to the transgenic antibody technology was the large size of the human Ig loci (Cook and Tomlinson 1995; Frippiat et al. 1995; Matsuda et al. 1993; Pallares et al. 1999; Weichhold et al. 1993). Emphasis was on inclusion of V genes, as many and as diverse as possible, rather than, for example, all D segments or several C genes for the H chain (reviewed in Bruggemann et al. 2007). Early approaches used minigenes and even with a small number of genes mAbs with good binding characteristics could be obtained (Green et al. 1994; Taylor et al. 1994; Wagner et al. 1994). Improvements in oocytes microinjection and the use of embryonic stem (ES) cells permitted the integration of larger regions; first on plasmids and cosmids (Bruggemann et al. 1991; Taylor et al. 1994), and then on bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) (Davies et al. 1993; Mendez et al. 1997; Nicholson et al. 1999; Popov et al. 1999; Wagner et al. 1996).

At the same time, various Ig knock-out (KO) lines were derived, with µMT being the earliest mouse line with a silenced IgH locus (Kitamura et al. 1991). Apart from studying lymphocyte development and antibody expression in mice without fully functional H- and/or L-chain loci, these approaches aided human antibody expression from introduced transgenic Ig loci significantly. Many silencing strategies were successful: removal of the JH segments, abolished DNA rearrangement (Chen et al. 1993a), deleting all CH genes eliminated H chain expression and trans-switching (Ren et al. 2004), the various Cκ or Jκ KOs and a Cλ KO silenced IgL expression (Chen et al. 1993b; Sanchez et al. 1994; Zou et al. 2003). Breeding regimes to combine H- and L-chain KOs with human IgH and IgL transloci produced multi-feature animals expressing considerable levels of diverse fully human Ig.

The production of transgenic antibody repertoires, similar or comparable to those in humans, requires diverse rearrangement combined with high expression of human V, (D) and J segments, ideally from a large gene pool. This has been achieved only recently in rodent lines, not with fully human constructs but chimeric Ig loci, where immune responses similar to wild-type (WT) animals and diversity comparable to recorded human antibody sequences were found (Lee et al. 2014; Ma et al. 2013; Osborn et al. 2013). In Fig. 1a, the human IgH locus with all VHs, Ds and JH genes/segments is illustrated. Features of the various fully human and chimeric IgH transloci, large and small, are presented in Fig. 1b.
Fig. 1

Human IgH loci. a The complete VH, D and JH region from the most 5′ VH, IgHV3–74, to the most 3′ JH segment, JH6, is accommodated on ~950 kb (Lefranc and Lefranc 2001). b Transgenic constructs and features. Animals were derived from manipulated fibroblasts (cattle) or ES cells (mouse) and by DNA microinjection (mouse and rat). In several lines, human Ig expression appeared to be reduced. Endogenous VDJ use or H chain products with non-human segments, such as mouse or cattle Vs, were obtained in strains with leaky or incomplete endogenous KO

Fully Human Ig Transloci

Fully human transloci contain exclusively human V, (D), J and C genes, while in chimeric human transloci human V, (D) and J genes or regions have been linked to host (rat, mouse or cattle) C genes. In many cases, the size of the fully human Ig constructs assembled, for example, in plasmids, cosmids or phages, limited the number of genes that could be included as these vectors could only accommodate regions below ~100 kb. This meant that V genes had to be gathered in close proximity, D and J segments were tightly linked and C genes had to be carefully chosen. The result was poor expression from such mini-gene constructs although several useful human antibodies have been derived (Lonberg et al. 1994). The assembly of larger constructs in YACs by homologous overlaps (Davies and Bruggemann 1993; Davies et al. 1992; Popov et al. 1996), and subsequent introduction of YACs into ES cells by spheroplast fusion or lipofection enabled the integration of several hundred kb up to ~1 Mb large human IgH and IgL regions into the mouse genome (Choi et al. 1993; Davies et al. 1993; Mendez et al. 1997; Nicholson et al. 1999; Popov et al. 1999). Introduction of human mini-chromosomes by microcell-mediated chromosome transfer established ES cell-derived mouse lines producing fully human mAbs (Kuroiwa et al. 2002; Tomizuka et al. 2000) and cattle, derived from manipulated fibroblasts, which expressed considerable amounts of human polyclonal Ig (Matsushita et al. 2014; Sano et al. 2013). However, the production of fully human mAbs in transchromosomic mice appeared to be less than 1 % (Tomizuka et al. 2000) and only about 50 % of fully human IgG was identified in transchromosomic cattle, often at quite reduced levels (Matsushita et al. 2014). Although problems concerned with trans-chromosome truncation, chimerism and transmission stability have been addressed, these could not be fully resolved (Kakeda et al. 2005). A solution, which considerably improved expression and efficiency of hybridoma production, was cross-breeding of IgH trans-chromo mice with Igκ XenoMouse (Ishida et al. 2002; Mendez et al. 1997).

Despite the improvement and breeding with endogenous KO lines, human antibody titers were still suboptimal or at best variable and despite diverse V, D and J usage, class-switching was frequently poor and hypermutation was relatively low (Bruggemann et al. 2007). A problem for many academic researchers was that supposedly superior transgenic animals, produced by companies, were not generally available and little support was provided to improve available lines or constructs once they had been published or shown to work in principle. Nevertheless, useful fully human IgM antibodies of reasonable affinity against HIV (when converted to human IgG1) could be retrieved even from lines carrying few V genes and lacking class-switching (Pruzina et al. 2011).

Rearrangement and expression of human IgL loci was achieved with mini-gene constructs assembled on plasmids and also larger regions on BACs and YACs; these earlier constructs have been extensively reviewed (Bruggemann et al. 2007, 2014). A comparison of human Igκ L-chain transloci showed that various germline-configured regions, from 15 kb to over 1 Mb, readily express serum Ig but that larger regions, with naturally spaced genes, produce much higher antibody yields (Xian et al. 1998). Several larger fully human IgL loci were highly expressed even in a WT background, which showed successful competition with the endogenous L-chain loci. For example, an authentic 380 kb human Igλ YAC with 15 functional Vλ genes produced high antibody titers from diverse and hypermutated transcripts (Nicholson et al. 1999; Osborn et al. 2013; Popov et al. 1999). Thus, it can be concluded that large fully human L-chain constructs can be expressed similar to WT L-chain levels in transgenic animals (Ma et al. 2013; Osborn et al. 2013).

Chimeric Human Ig Transloci

To ensure optimal physiological interaction of translocus-encoded membrane Ig with endogenous components of antigen-receptor associated polypeptides, it has been speculated and later shown that chimeric constructs with human VDJ linked to rodent CH are most efficient (Green 2014; Ma et al. 2013; Osborn et al. 2013; Pruzina et al. 2011). Strategies to link human IgH V genes, D and J segments with mouse or rat CH genes made use of BAC and YAC technology for (1) sub-cloning and joining large regions to secure overlapping integration by DNA microinjection into oocytes (Ma et al. 2013; Osborn et al. 2013) and (2) sequential site-specific integration of BACs with human V and V-D-J regions upstream of mouse Cµ by gene targeting or recombinase-mediated exchange (Lee et al. 2014; Murphy 2009). Human VHs are efficiently rearranged in transgenic mammals and their linkage to endogenous CH genes provides a flawless interplay with the cellular signaling machinery. Combined interactions of the many components that aid B cell receptor (BCR) signaling (see: http://www.biocarta.com/genes/PathwayGeneSearch.asp) secure efficient B cell activation with normal immune responses. Endogenous CH genes will also allow optimal physiological interactions necessary in the regulation of immune responses by the various host Fc receptors (Nimmerjahn and Ravetch 2007; Rowland et al. 2013).

Conventional transgenic technologies with DNA microinjection into oocytes enable the integration of multiple BACs and large YACs. This allowed the insertion of three linear and overlapping BAC regions with homologous end sequences 5′ and 3′, which provided about half of all human V genes, all D and all J segments (Osborn et al. 2013). A comparison of independent founders carrying a continuous region of over 500 kb randomly integrated found high expression levels independent of the site of integration. The conclusion derived from many chimeric (human V D J, rat C) IgH, as well as fully human Igκ and Igλ lines, is that antibodies obtained from multiple large integrations at one position can be as efficiently expressed as WT Ig if the constructs contain the necessary regulatory sequences (Ma et al. 2013). Furthermore, multiple integration of microinjected DNA, i.e., several copies of the same BAC or YAC, may well be advantageous as larger gene pools with an increased number of available genes could facilitate more extensive receptor editing (Liu et al. 2013; Nemazee and Weigert 2000; Ouled-Haddou et al. 2014; Zhang et al. 2003).

Considerable efforts have been made to integrate large numbers and possibly all human VH, D and JH segments upstream of mouse Cµ (Green 2014; Lee et al. 2014; Murphy 2009; Murphy et al. 2014). Lee et al. (2014) showed that with site-specifically integrated WT and mutated loxP sites, it was possible to provide a BAC landing pad for adding human V-regions upstream of the first endogenous C gene. This approach employed sequential integration of modified BACs, which reconstituted or replaced the mouse VH-region with equivalent human genes. The result was to reconstitute a near authentic human VH-D-JH as well as Vκ-Jκ and Vλ-Jλ region (Green 2014; Lee et al. 2014; Murphy 2009). With these insertions, duplications or multimeric integration of the same BAC are barred, which ensures a genuine V gene order and content. However, there has been no indication that non-selected or random integration of large transloci was disadvantageous and choosing particular founders (or combination of founders for IgH and IgL) secured high expression and breeding to homozygosity.

Integration of human L-chain V-J regions adjacent to the mouse CL gene has also been shown to yield extensive chimeric antibody repertoires, but reports explained that endogenous L-chain rearrangements were not entirely prohibited (Green 2014; Lee et al. 2014). In contrast, randomly integrated fully human Igκ or Igλ transloci could be cleanly expressed—without residual rodent Ig in KO lines—and extensive levels and diversity similar to WT have been obtained (Osborn et al. 2013). This means that there are no apparent advantages employing locus replacement strategies in this instance.

Ig KO Strains

Ig loci have been knocked out or disabled in mouse, rat and cattle (Bruggemann et al. 2007; Matsushita et al. 2014; Osborn et al. 2013; Tomizuka et al. 2000). Strategies involved gene targeting in ES cells via insertion or deletion using, for example, Cre/loxP which allowed the removal of >100 kb regions (Ren et al. 2004; Zou et al. 2003). Recently, the use of zinc-finger (endo)nuclease (ZFN) constructs for DNA microinjections into oocytes produced several IgH and IgL KO lines in the rat (Geurts et al. 2009; Menoret et al. 2010; Osborn et al. 2013) and silenced the IgH locus in rabbits (Flisikowska et al. 2011). Advantages of the ZFN technology are that non-homologous end joining to silence a gene or locus via deletions up to several kb can also provide a target site for homologous integration (Cui et al. 2011).

More recently combined gene targeting and locus extension have been taken to the next level. In one approach where Cre recombinase was directed to opposite loxP sites, integration was followed by inversion of the mouse VH-D-JH region and, separately, Vκ-Jκ region (Lee et al. 2014). This would not necessarily prevent DNA rearrangement but with sufficient separation from C genes splicing resulting in V(D)J-C products appeared to be minimal (Lee et al. 2014). In another approach, deletion of the whole V-region from both mouse IgH and Igκ loci was obtained (Green 2014).

A considerable advantage in expressing a human antibody repertoire in transgenic animals is the lack of all endogenous Ig as this greatly simplifies the production of polyclonal Ig, as shown in cattle (Matsushita et al. 2014), and monoclonal Ig in rats (Osborn et al. 2013) and mice (see: http://www.crescendobiologics.com/rnd/crescendo-mouse). In these animals, no IgH, Igκ and Igλ with endogenous V(D)J is being produced. The absence of endogenous Ig prevents a bias in the immune response, for example, selecting more compatible mouse idiotypes, and means that selection and purification can be avoided.

Applications

mAb therapy has been developed to provide reagents that specifically bind to cellular receptors or interact with circulating targets for their removal or neutralization. Target cell depletion can be achieved by Fc receptor interaction and antibody-dependent cellular cytotoxicity has been identified as an important mechanism to eliminate tumor cells (Hjelm et al. 2006; Kim and Ashkenazi 2013; Mellor et al. 2013). Binding of mAbs to particular cell-surface receptors via the variable domain and simultaneously engaging with natural killer cells, monocytes or macrophages via Fc receptor interaction provided by the constant region (C region) initiates a cascade of events important for removal or reduction of abnormal or malignant cells and pathogens. In many epithelial tumors, epidermal growth factor receptor is expressed at high levels and the tumor cell load of many patients could be reduced with Vectibix (see: http://www.vectibix.com). Tumor inhibition was also achieved with anti-CD20 mAbs, which were effective in initiating the destruction or killing of B cells from patients with chronic lymphocytic leukemia, another very aggressive disease (Zhang 2009).

Important validation for using transgenic lines includes how well they compare with WT animals with respect to serum titer, diversity and B cell development and if they can be bred to homozygosity, which was achieved with various rat and mouse strains. Success in DNA rearrangement and antibody expression of the first construct assembled in germline configuration (Bruggemann et al. 1989a) was followed by important progress regarding gene targeting, to knock-out the mouse Ig loci, and genetic engineering, to assemble large regions on artificial chromosomes such as YACs (reviewed in Bruggemann 2001). These advances were crucial to obtain specific, fully human antibodies from the introduced human IgH (mini)loci expressed in mice in the 1990s (Fishwild et al. 1996; Lonberg et al. 1994; Mendez et al. 1997). However, none of these early strains could match WT animals with regard to B cell numbers, antibody titers, diversity and hypermutation (Bruggemann et al. 2014; Green and Jakobovits 1998). Nevertheless, despite reduced expression levels, antibodies from these early transgenic animals, in clinical trials and on the market, have been obtained using hybridoma technology (Green 2014). To overcome the limitations of these first-generation strains, new transgenic animals have been designed (Lee et al. 2014; Ma et al. 2013; Osborn et al. 2013).

FDA approved and marketed fully human mAbs produced from the early mouse strains are listed in Table 1. The success of these mouse lines accelerated the shift from using murine and chimeric antibodies to fully human antibodies (reviewed in Bruggemann et al. 2014). Initially, conventional immunization regimes and myeloma fusion with spleen cells were adopted from procedures using WT mice or rats (Kohler and Milstein 1975; Lachmann et al. 1980). However, in recently derived strains, mAb production was significantly increased by the optimization of immunization protocols. For example, fusions of mouse myeloma cells with rat iliac lymph node cells collected after rapid immunization schemes and, separately, spleen cells obtained after multiple or booster immunizations, frequently resulted in many thousand hybridomas from one animal with sometimes over 100 diverse and high-affinity IgGs (Osborn et al. 2013).
Table 1

FDA approved fully human therapeutic monoclonal antibodies

Year

Name

Target

Treatment

Transgenic line (references)

2011

Yervoy (ipilimumab)

CTLA-4/CD152

Melanoma

Lonberg et al. (1994)

2010

Prolia (denosumab)

RANKL

Osteoporosis

Mendez et al. (1997)

2009

Simponi (golimumab)

TNF-α

Rheumatoid arthritis

Lonberg et al. (1994)

2009

Arzerra (ofatumumab)

CD20

Lymphocytic leukemia

Fishwild et al. (1996)

2009

Stelara (ustekinumab)

IL-12/IL-23

Psoriasis

Lonberg et al. (1994)

2009

Ilaris (canakinumab)

IL-1β

Auto-inflammation

Lonberg et al. (1994)

2006

Vectibix (panitumumab)

EGFR

Colorectal cancer

Mendez et al. (1997)

In 2014, a large number of defined (see: http://en.wikipedia.org/wiki/List_of_monoclonal_antibodies) and non-disclosed (see: http://biz.yahoo.com/e/130503/regn10-q.html) fully human antibodies were being evaluated in clinical trials. Close to 500 mAbs have been reported to be in active clinical development during the past 3 years, including ~25 % in phase I, ~40 % in phase II, ~20 % in phase III and ~15 % at approval stage (BioPharm Insight database). A large majority, ~60 %, are indicated primarily for treatment of cancer, e.g., reduction of solid tumors possibly by blocking growth or inducing apoptosis. Other therapeutic categories include immunological (10 %), musculoskeletal (7 %) and infectious diseases (4 %). Therapeutic antibodies are also being developed for respiratory, dermatologic, central nervous system, hematologic, eye/ear, cardiovascular, HIV, liver and other diseases. Clinical-stage antibodies derived from transgenic animals are shown in Table 2. Four distinct mouse lines have been used to generate these: HuMAb antibody or UltiMAb antibody Mouse (Lonberg et al. 1994), XenoMouse (Mendez et al. 1997), TransChromo Mouse (Ishida et al. 2002) and VelocImmune Mouse (Murphy et al. 2014).
Table 2

Promising fully human antibodies in clinical trials in 2014

Name

Target

Treatment

Transgenic line (references)

Actoxumab/bezlotoxumab

Clostridium difficile

Clostridium difficile infection

Lonberg et al. (1994)

Alirocumab

Neural apoptosis-regulated proteinase 1

Hypercholesterolemia

Murphy et al. (2014)

Anifrolumab

IFN-α/β receptor

Systemic lupus erythematosus

Lonberg et al. (1994)

Brodalumab

IL-17

Inflammatory diseases

Mendez et al. (1997)

Conatumumab

TNF-related apoptosis-inducing ligand

Solid tumors

Cancers of hematopoietic origin

Mendez et al. (1997)

Daratumumab

CD38

Multiple myeloma

Lonberg et al. (1994)

Dupilumab

IL-4Rα

Allergic disease

Murphy et al. (2014)

Eldelumab

IFN-γ-induced protein

Crohn’s disease ulcerative colitis

Lonberg et al. (1994)

Enoticumab

DLL4

Solid tumors

Murphy et al. (2014)

Evolocumab

PCSK9

Hyperlipidemia

Mendez et al. (1997)

Fulranumab

NGF

Pain

Mendez et al. (1997)

Fasinumab

Nerve growth factor

Pain

Murphy et al. (2014)

Ganitumab

IGF-1

Cancer

Mendez et al. (1997)

Guselkumab

IL-13

Psoriasis

Mendez et al. (1997)

Inclacumab

Selectin P

Cardiovascular disease

Lonberg et al. (1994)

Intetumumab

CD51

Solid tumors

Murphy et al. (2014)

Iratumumab

CD30

Hodgkin’s disease

Lonberg et al. (1994)

Lirilumab

KIR2D

Solid tumors

Lonberg et al. (1994)

Lucatumumab

CD40

Lymphoma

Mendez et al. (1997)

Necitumumab

EGFR

Non-small cell lung carcinoma

Lonberg et al. (1994)

Nesvacumab

Angiopoietin 2

Cancer

Murphy et al. (2014)

Nivolumab

PD-1

Cancer

Lonberg et al. (1994)

Patritumab

HER3

Non-small cell lung cancer

Tomizuka et al. (2000)

REGN1033

Myostatin (GDF8)

Metabolic disorders

Murphy et al. (2014)

Rilotumumab

HGF

Solid tumors

Mendez et al. (1997)

Sarilumab

IL-6

Rheumatoid arthritis

Murphy et al. (2014)

Secukinumab

IL-17A

Uveitis

Rheumatoid arthritis

Psoriasis

Mendez et al. (1997)

Teprotumumab

IGF1R/CD221

Hematologic tumors

Lonberg et al. (1994)

Ticilimumab/tremelimumab

CTLA-4

Cancer

Mendez et al. (1997)

Urelumab

4-1BB/CD137

Anti-tumor

Lonberg et al. (1994)

Vantictumab

Frizzled 7 receptor

Solid tumors

Breast cancer

Murphy et al. (2014)

Zalutumumab

EGFR

Solid tumors

Lonberg et al. (1994)

Zanolimumab

CD4

T cell lymphoma

Lonberg et al. (1994)

Different transgenic species are vitally important in providing human antibodies because of the trans-species gene similarities. Thus, sequence comparison of rat, mouse and human demonstrates that some genes are strikingly similar and that the less similar animal provides the better immune response. The other advantage of using different transgenic species (e.g. rats and mice) is that their transgene usage varies. As a result, many more genes from different families yield antibodies of high affinity for the same antigen, which may prove valuable for future immune therapy with mAb mixtures. For example, high-affinity human antibodies have recently been obtained from several other rodent lines; OmniRat (Ma et al. 2013; Osborn et al. 2013) and OmniMouse (http://www.omtinc.net), Kymouse (Lee et al. 2014) and Harbour Mouse (http://harbourantibodies.com/science-technology/h2l2/#.UyxGntzVvwI). Rabbits have also been used to produce human antibodies (Flisikowska et al. 2011). Novel antibodies with a common light chain have been obtained from the MeMo mouse (http://www.merus.nl/technology/memo.html) and OmniFlic (http://www.openmonoclonaltechnology.com/antibody_technology.html) lines. For therapeutic applications and minimizing immunogenicity, endogenous C regions can be easily replaced by the desired isotype without compromising antigen specificity (Bruggemann et al. 1989b; Presta 2006).

Conclusions

Human antibodies produced in transgenic animals represent an important, broad and rapidly growing therapeutic class with a predicted global market reaching many billion US dollars (http://www.marketresearch.com). The success is based on a now proven technology with DNA insertion of Ig loci into the germline followed by expression of human antibody repertoires. Transgenic animals are increasingly used to produce antibodies with fully human idiotypes, a trend that is expected to continue in years to come as described in reports by The Times and BBC news (both June 3, 2014) as “trials show they are fulfilling their promise fast”.

Significant improvements have been made recently with the derivation of transgenic rat and mouse lines that, after immunization, readily produce diverse high-affinity antibodies as efficiently as WT animals (Osborn et al. 2013). World-leading biopharmaceutical companies are now using these animals to generate future human therapeutic antibodies for a wide range of conditions.

References

  1. Bruggemann M, Caskey HM, Teale C et al (1989a) A repertoire of monoclonal antibodies with human heavy chains from transgenic mice. Proc Natl Acad Sci USA 86:6709–6713CrossRefPubMedCentralPubMedGoogle Scholar
  2. Bruggemann M, Winter G, Waldmann H et al (1989b) The immunogenicity of chimeric antibodies. J Exp Med 170:2153–2157CrossRefPubMedGoogle Scholar
  3. Bruggemann M, Spicer C, Buluwela L et al (1991) Human antibody production in transgenic mice: expression from 100 kb of the human IgH locus. Eur J Immunol 21:1323–1326CrossRefPubMedGoogle Scholar
  4. Bruggemann M, Neuberger MS (1996) Strategies for expressing human antibody repertoires in transgenic mice. Immunol Today 17:391–397CrossRefPubMedGoogle Scholar
  5. Bruggemann M (2001) Human antibody expression in transgenic mice. Arch Immunol Ther Exp 49:203–208Google Scholar
  6. Bruggemann M, Smith JA, Osborn MJ et al (2007) Part I: Selecting and shaping the antibody molecule, selection strategies III: transgenic mice. In: Dübel S (ed) Handbook of therapeutic antibodies. Wiley-VCH Verlag, Weinheim, pp 69–93CrossRefGoogle Scholar
  7. Bruggemann M, Osborn MJ, Ma B et al (2014) Transgenic animals derived by DNA microinjection. In: Dübel S, Reichert JM (eds) Handbook of therapeutic antibodies, 2nd edn. Wiley-VCH Verlag, WeinheimGoogle Scholar
  8. Chen J, Trounstine M, Alt FW et al (1993a) Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus. Int Immunol 5:647–656CrossRefPubMedGoogle Scholar
  9. Chen J, Trounstine M, Kurahara C et al (1993b) B cell development in mice that lack one or both immunoglobulin kappa light chain genes. EMBO J 12:821–830PubMedCentralPubMedGoogle Scholar
  10. Choi TK, Hollenbach PW, Pearson BE et al (1993) Transgenic mice containing a human heavy chain immunoglobulin gene fragment cloned in a yeast artificial chromosome. Nat Genet 4:117–123CrossRefPubMedGoogle Scholar
  11. Cook GP, Tomlinson IM (1995) The human immunoglobulin VH repertoire. Immunol Today 16:237–242CrossRefPubMedGoogle Scholar
  12. Cui X, Ji D, Fisher DA et al (2011) Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat Biotechnol 29:64–67CrossRefPubMedGoogle Scholar
  13. Davies NP, Rosewell IR, Bruggemann M (1992) Targeted alterations in yeast artificial chromosomes for inter-species gene transfer. Nucleic Acids Res 20:2693–2698CrossRefPubMedCentralPubMedGoogle Scholar
  14. Davies NP, Bruggemann M (1993) Extension of yeast artificial chromosomes by cosmid multimers. Nucleic Acids Res 21:767–768CrossRefPubMedCentralPubMedGoogle Scholar
  15. Davies NP, Rosewell IR, Richardson JC et al (1993) Creation of mice expressing human antibody light chains by introduction of a yeast artificial chromosome containing the core region of the human immunoglobulin kappa locus. Biotechnology 11:911–914CrossRefPubMedGoogle Scholar
  16. Fishwild DM, O’Donnell SL, Bengoechea T et al (1996) High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice. Nat Biotechnol 14:845–851CrossRefPubMedGoogle Scholar
  17. Flisikowska T, Thorey IS, Offner S et al (2011) Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases. PLoS One 6:e21045CrossRefPubMedCentralPubMedGoogle Scholar
  18. Frippiat JP, Williams SC, Tomlinson IM et al (1995) Organization of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2. Hum Mol Genet 4:983–991CrossRefPubMedGoogle Scholar
  19. Geurts AM, Cost GJ, Freyvert Y et al (2009) Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325:433CrossRefPubMedCentralPubMedGoogle Scholar
  20. Green LL, Hardy MC, Maynard-Currie CE et al (1994) Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nat Genet 7:13–21CrossRefPubMedGoogle Scholar
  21. Green LL, Jakobovits A (1998) Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes. J Exp Med 188:483–495CrossRefPubMedCentralPubMedGoogle Scholar
  22. Green LL (2014) Transgenic mouse strains as platforms for the successful discovery and development of human therapeutic monoclonal antibodies. Curr Drug Discov Technol 11:74–84CrossRefPubMedGoogle Scholar
  23. Hjelm F, Carlsson F, Getahun A et al (2006) Antibody-mediated regulation of the immune response. Scand J Immunol 64:177–184CrossRefPubMedGoogle Scholar
  24. Ishida I, Tomizuka K, Yoshida H et al (2002) Production of human monoclonal and polyclonal antibodies in TransChromo animals. Cloning Stem Cells 4:91–102CrossRefPubMedGoogle Scholar
  25. Kakeda M, Hiratsuka M, Nagata K et al (2005) Human artificial chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human primary fibroblasts. Gene Ther 12:852–856CrossRefPubMedGoogle Scholar
  26. Kim JM, Ashkenazi A (2013) Fcgamma receptors enable anticancer action of proapoptotic and immune-modulatory antibodies. J Exp Med 210:1647–1651CrossRefPubMedCentralPubMedGoogle Scholar
  27. Kitamura D, Roes J, Kuhn R et al (1991) A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature 350:423–426CrossRefPubMedGoogle Scholar
  28. Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497CrossRefPubMedGoogle Scholar
  29. Kuroiwa Y, Yoshida H, Ohshima T et al (2002) The use of chromosome-based vectors for animal transgenesis. Gene Ther 9:708–712CrossRefPubMedGoogle Scholar
  30. Lachmann PJ, Oldroyd RG, Milstein C et al (1980) Three rat monoclonal antibodies to human C3. Immunology 41:503–515PubMedCentralPubMedGoogle Scholar
  31. Lee EC, Liang Q, Ali H et al (2014) Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery. Nat Biotechnol 32:356–363CrossRefPubMedGoogle Scholar
  32. Lefranc MP, Lefranc G (2001) The immunoglobulin factsbook. Academic Press, London, pp 45–68Google Scholar
  33. Liu J, Lange MD, Hong SY et al (2013) Regulation of VH replacement by B cell receptor-mediated signaling in human immature B cells. J Immunol 190:5559–5566CrossRefPubMedCentralPubMedGoogle Scholar
  34. Lonberg N, Taylor LD, Harding FA et al (1994) Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 368:856–859CrossRefPubMedGoogle Scholar
  35. Ma B, Osborn MJ, Avis S et al (2013) Human antibody expression in transgenic rats: comparison of chimeric IgH loci with human VH, D and JH but bearing different rat C-gene regions. J Immunol Methods 400–401:78–86CrossRefPubMedGoogle Scholar
  36. Matsuda F, Shin EK, Nagaoka H et al (1993) Structure and physical map of 64 variable segments in the 3′0.8-megabase region of the human immunoglobulin heavy-chain locus. Nat Genet 3:88–94CrossRefPubMedGoogle Scholar
  37. Matsushita H, Sano A, Wu H et al (2014) Triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production. PLoS One 9:e90383CrossRefPubMedCentralPubMedGoogle Scholar
  38. McCafferty J, Griffiths AD, Winter G et al (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554CrossRefPubMedGoogle Scholar
  39. Mellor JD, Brown MP, Irving HR et al (2013) A critical review of the role of Fc gamma receptor polymorphisms in the response to monoclonal antibodies in cancer. J Hematol Oncol 6:1CrossRefPubMedCentralPubMedGoogle Scholar
  40. Mendez MJ, Green LL, Corvalan JR et al (1997) Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice. Nat Genet 15:146–156CrossRefPubMedGoogle Scholar
  41. Menoret S, Iscache AL, Tesson L et al (2010) Characterization of immunoglobulin heavy chain knockout rats. Eur J Immunol 40:2932–2941CrossRefPubMedGoogle Scholar
  42. Murphy A (2009) Recombinant antibodies for immunotherapy. Cambridge University Press, Cambridge, pp 100–108CrossRefGoogle Scholar
  43. Murphy AJ, Macdonald LE, Stevens S et al (2014) Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice. Proc Natl Acad Sci USA 111:5153–5158CrossRefPubMedCentralPubMedGoogle Scholar
  44. Nemazee D, Weigert M (2000) Revising B cell receptors. J Exp Med 191:1813–1817CrossRefPubMedCentralPubMedGoogle Scholar
  45. Neuberger M, Bruggemann M (1997) Monoclonal antibodies. Mice perform a human repertoire. Nature 386:25–26CrossRefPubMedGoogle Scholar
  46. Nicholson IC, Zou X, Popov AV et al (1999) 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 163:6898–6906PubMedGoogle Scholar
  47. Nimmerjahn F, Ravetch JV (2007) Fc-receptors as regulators of immunity. Adv Immunol 96:179–204CrossRefPubMedGoogle Scholar
  48. Osborn MJ, Ma B, Avis S et al (2013) High-affinity IgG antibodies develop naturally in Ig-knockout rats carrying germline human IgH/Igkappa/Iglambda loci bearing the rat CH region. J Immunol 190:1481–1490CrossRefPubMedCentralPubMedGoogle Scholar
  49. Ouled-Haddou H, Ghamlouch H, Regnier A et al (2014) Characterization of a new V gene replacement in the absence of activation-induced cytidine deaminase and its contribution to human B-cell receptor diversity. Immunology 141:268–275CrossRefPubMedCentralPubMedGoogle Scholar
  50. Pallares N, Lefebvre S, Contet V et al (1999) The human immunoglobulin heavy variable genes. Exp Clin Immunogenet 16:36–60CrossRefPubMedGoogle Scholar
  51. Popov AV, Butzler C, Frippiat JP et al (1996) Assembly and extension of yeast artificial chromosomes to build up a large locus. Gene 177:195–201CrossRefPubMedGoogle Scholar
  52. Popov AV, Zou X, Xian J et al (1999) A human immunoglobulin lambda locus is similarly well expressed in mice and humans. J Exp Med 189:1611–1620CrossRefPubMedCentralPubMedGoogle Scholar
  53. Presta LG (2006) Engineering of therapeutic antibodies to minimize immunogenicity and optimize function. Adv Drug Deliv Rev 58:640–656CrossRefPubMedGoogle Scholar
  54. Pruzina S, Williams GT, Kaneva G et al (2011) Human monoclonal antibodies to HIV-1 gp140 from mice bearing YAC-based human immunoglobulin transloci. Protein Eng Des Sel 24:791–799CrossRefPubMedGoogle Scholar
  55. Ren L, Zou X, Smith JA, Bruggemann M (2004) Silencing of the immunoglobulin heavy chain locus by removal of all eight constant-region genes in a 200-kb region. Genomics 84:686–695CrossRefPubMedGoogle Scholar
  56. Rowland SL, Tuttle K, Torres RM et al (2013) Antigen and cytokine receptor signals guide the development of the naive mature B cell repertoire. Immunol Res 55:231–240CrossRefPubMedCentralPubMedGoogle Scholar
  57. Sanchez P, Drapier AM, Cohen-Tannoudji M et al (1994) Compartmentalization of lambda subtype expression in the B cell repertoire of mice with a disrupted or normal C kappa gene segment. Int Immunol 6:711–719CrossRefPubMedGoogle Scholar
  58. Sano A, Matsushita H, Wu H et al (2013) Physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle. PLoS One 8:e78119CrossRefPubMedCentralPubMedGoogle Scholar
  59. Taylor LD, Carmack CE, Huszar D et al (1994) Human immunoglobulin transgenes undergo rearrangement, somatic mutation and class switching in mice that lack endogenous IgM. Int Immunol 6:579–591CrossRefPubMedGoogle Scholar
  60. Tomizuka K, Shinohara T, Yoshida H et al (2000) 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 97:722–727CrossRefPubMedCentralPubMedGoogle Scholar
  61. Wagner SD, Popov AV, Davies SL et al (1994) The diversity of antigen-specific monoclonal antibodies from transgenic mice bearing human immunoglobulin gene miniloci. Eur J Immunol 24:2672–2681CrossRefPubMedGoogle Scholar
  62. Wagner SD, Gross G, Cook GP et al (1996) Antibody expression from the core region of the human IgH locus reconstructed in transgenic mice using bacteriophage P1 clones. Genomics 35:405–414CrossRefPubMedGoogle Scholar
  63. Weichhold GM, Ohnheiser R, Zachau HG (1993) The human immunoglobulin kappa locus consists of two copies that are organized in opposite polarity. Genomics 16:503–511CrossRefPubMedGoogle Scholar
  64. Xian J, Zou X, Popov AV et al (1998) Comparison of the performance of a plasmid-based human Igk minilocus and YAC-based human Igk transloci for the production of human antibody repertoires in transgenic mice. Transgenics 2:333–343Google Scholar
  65. Zhang Z, Zemlin M, Wang YH et al (2003) Contribution of Vh gene replacement to the primary B cell repertoire. Immunity 19:21–31CrossRefPubMedGoogle Scholar
  66. Zhang B (2009) Ofatumumab. MAbs 1:326–331CrossRefPubMedCentralPubMedGoogle Scholar
  67. Zou X, Piper TA, Smith JA et al (2003) Block in development at the pre-B-II to immature B cell stage in mice without Ig kappa and Ig lambda light chain. J Immunol 170:1354–1361CrossRefPubMedGoogle Scholar

Copyright information

© The Author(s) 2014

Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

Authors and Affiliations

  • Marianne Brüggemann
    • 1
    • 2
    Email author
  • Michael J. Osborn
    • 1
  • Biao Ma
    • 1
  • Jasvinder Hayre
    • 1
  • Suzanne Avis
    • 1
  • Brian Lundstrom
    • 2
  • Roland Buelow
    • 2
    Email author
  1. 1.Recombinant Antibody Technology Ltd.BabrahamUK
  2. 2.Open Monoclonal Technology, Inc.Palo AltoUSA

Personalised recommendations