4 transgenic animals derived by dna...

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4Transgenic Animals Derived by DNA MicroinjectionMarianne Bruggemann, Michael J. Osborn, Biao Ma, Suzanne Avis, Ignacio Anegon,and Roland Buelow

4.1Introduction

Antibodies are produced in all jawed vertebrates with diverse repertoires beinggenerated by DNA rearrangement, first for the immunoglobulin heavy (IgH) V(variable), D (diversity), and J (joining) segments, then by IgL (light) V and Jsegments [1, 2]. In mammals, this rearrangement is initiated at the pre B-cell stagein bone marrow cells which subsequently express surface IgM and migrate viathe cardiovascular system to other lymphatic organs such as spleen and lymphnodes. Upon antigen encounter, low affinity binders can be edited by somatichypermutation and this may be followed by cellular expansion [3]. For therapeuticapplications, monoclonal antibodies have been derived from rodents using spleencells from immunized animals fused to myeloma cells and from human bloodlymphocytes using phage and ribosome display. In addition, rodent antibodies havebeen ‘‘humanized’’ and transgenic animals have been generated to produce humanantibody repertoires [4]. Figure 4.1 provides a list of FDA-approved monoclonalantibodies produced in the last ∼20 years, which shows a recent increase of fullyhuman antigen binders derived from transgenic mice.

Here, we summarize the production of fully human monoclonal antibodies inmice and rats generated by DNA microinjection and compare these strains withlines derived from manipulated embryonic stem (ES) cells. In the previous edition,we provided extensive details of the various transgenic constructs to express fullyhuman Ig [5]. In this review, particular emphasis is on the efficiency of innovativetransgenic constructs, which, in combination with new KO approaches, yield highexpression levels and diverse monoclonal antibodies of sub-nanomolar affinity aseffectively as endogenous loci in normal animals.

Handbook of Therapeutic Antibodies, Second Edition. Edited by Stefan Dubel and Janice M. Reichert.c© 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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2 4 Transgenic Animals Derived by DNA Microinjection

2013

Fully human TG mouse derived

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Figure 4.1 FDA-approved therapeutic monoclonal antibodies from 1994 to 2013. (Under-lined products have been withdrawn (www.immunologylink.com/FDA-APP-Abs.html andhttp://en.wikipedia.org/wiki/Monoclonal antibody therapy).)

4.2Construction of Human Ig Transloci

The human Ig loci have been cloned in bacterial artificial chromosome (BAC) andyeast artificial chromosome (YAC) libraries and all genes have been sequenced [6].For the IgH locus 38–46 functional VHs have been identified followed by ∼23 Dsegments, 6 JH segments, and 9 constant (C)-region genes on a region of ∼1.3 Mb[7, 8]. The Igκ locus accommodates up to 36 functional Vκs, 5 Jκs, and 1 Cκ, allon ∼2.6 Mb, but with Vκ genes in both transcriptional orientation and clusteredon two well-separated regions [9, 10]. For the Igλ locus 29–33 functional Vλs havebeen identified, followed by 4–5 Jλ-Cλ genes on an ∼1 Mb region [11, 12].

Sizes and contents of fully human Ig loci introduced and expressed in transgenicanimals have been summarized [5, 13] with many regions being quite small andincomplete. Here we focus on a new strategy to assemble, integrate, and expressIg loci on cointegrated BACs of several hundred kilobase pairs with extensive Vand C regions.

4.2.1IgH

The first germline configured construct to rearrange and express chimeric humanH-chains in mice was a plasmid minilocus of ∼25 kb [14], which was followed

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4.2 Construction of Human Ig Transloci 3

by fully human regions of ∼100 kb on cointegrated cosmids [15] or a YAC [16].These constructs on plasmids, cosmids, and YACs contained human V-, D-, J-, andC-region genes, which after rearrangement would provide fully human antibodyH-chains. In general, larger constructs with more genes allowed better expressionin a transgenic animal when the natural configuration was maintained [17]. Forexample, XenoMouse animals [18] carry a substantially larger and more diverseIgH translocus and this led to a better expression when compared to smaller andless diverse transloci [19–21]. Nevertheless, it became clear that fully human IgHtransloci were suboptimal regarding their efficiency in human antibody productionand it has been suggested that this may be caused by the imperfect interaction ofmembrane-expressed human C regions with rodent cellular signaling components[21, 22]. It was also reasoned that inclusion of large parts of cis-acting controlsequences might improve affinity maturation as sequences downstream of Cα inrat and mouse may play an important role in class-switch recombination as well ashypermutation [21, 23, 24].

To overcome the shortcomings in expressing fully human H-chains from trans-genes, a rat strain was generated that carries a translocus with human VH, D,and JH genes in natural configuration but linked to the rat C-region locus [22].Figure 4.2 shows the inserted IgH locus made up from three modified BACs,which provided the sequence, and homology regions for overlapping integrationof human VHs, and human D and JH segments linked to rat C-region genes withthe complete 3′ regulatory region. The three parts of the chimeric region wereassembled from several BACs containing human and, separately, rat genomicsequences by purification of the relevant fragments, design of large oligos forjoining up regions, and transformation into yeast to obtain circular yeast artificialchromosomes (cYACs). A shuttle (cYAC/BAC) vector allowed recombination in

Color Fig.: 4.2D1-1 ----D7-27 J

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Figure 4.2 IgH BACs with human VH, D, JH segments and rat C-region locus [22].(a) Homologous integration of three BACs (all near 200 kb) by overlapping regions of ∼11 kb.(b) Head-to-tail tandem integration.

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Figure 4.3 Igκ BACs containing human Vκ, Jκ, and Cκ genes [22]. (a) Homologousintegration by overlapping regions of ∼14 and ∼40 kb on two IgKV BACs of ∼150 kb and theKDE (kappa deleting element) on a ∼55 kb BAC. (b) Head-to-tail tandem integration.

yeast and transfer to Escherichia coli for BAC purification. Mixtures of linearizedBAC DNA were subsequently used for microinjection [22].

4.2.2Ig𝛋

The expression of human L-chain in transgenic mice was initially achieved withminigene constructs containing one or two Vκ genes. Later cosmids and BACsprovided many different Vκ genes, which were well expressed and providedextensive VκJκ diversity [5]. Figure 4.3 illustrates the integration of human Igκsequences from three different BACs obtained by digests using rare cutting restric-tion enzymes, which produced overlapping fragments. A performance comparisonof naturally spaced and closely assembled Vκ genes revealed poor expression oftightly spaced Vκ sequences on small DNA constructs [17], probably independentof the integrated copy number. Using larger regions on YACs and from multipleBACs showed impressive results with extensive transgene rearrangement and goodexpression even in a wildtype (Wt) background and thus successful competitionwith the endogenous locus.

4.2.3Ig𝛌

There are few reports on the expression of transgenic human Igλ constructsand perhaps the most successful approach was the use of a YAC extended bycosmids and containing the authentic region of the human Igλ locus with over

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4.3 BAC Integration 5

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Figure 4.4 Human Igλ YAC [25] containing 17 Vλ and all J-Cλs followed by the 3′ enhancerregion on ∼300 kb.

half of all Vλ genes (Figure 4.4) [25, 26]. As diverse use and good expressioneven in a Wt background was obtained, the human Igλ translocus must provideregulatory sequences to allow broad utilization in mice and rats despite theseanimals producing little endogenous Igλ [20, 22, 26].

4.3BAC Integration

When purified YAC or BAC DNA was microinjected into fertilized eggs, a recurringproblem was the instability of the large linear DNA with strand breakage occurringduring the purification process or when forcing the DNA through the injectionneedle [5]. For the purification of large fragments obtained from restrictiondigests separated on agarose gels via conventional or pulsed field electrophoresis,electroelution using Elutrap™ worked efficiently [27]. DNA recovery was usually20–50% of the starting material. For fragments up to 200 kb, the DNA could beprecipitated and redissolved in microinjection buffer at the desired concentration.For the separation of ∼10 kb vector DNA from >150 kb BAC insert, sepharose4B-CL filtration columns using a microinjection buffer gave very good results [28]and avoided DNA precipitation [22].

Cointegration of several large fragments proved beneficial for the reconstitutionof an authentic Ig locus. The purification of 400+ kb YAC fragments, which areprone to degradation and difficult to obtain at the required concentration, wasavoided. For DNA injection, equal molar amounts of the different purified BACfragments at concentrations of 0.5–3 ng μl−1 were mixed. In many cases, three largefragments were coinjected, which resulted in transgenic integration of at least 1%of injected eggs. Tandem insertion was frequently observed; either by homologousintegration or head-to-tail integration as shown in Figures 4.2 and 4.3. Integrationsuccess was initially identified by an extensive genomic polymerase chain reaction(PCR) and then confirmed by the analysis of rearranged V(D)J transcripts whererecombinations of the most 5′ to the most 3′ gene segments were readily found[22]. Germline transmission was frequently seen, with mosaicism being less of aproblem compared with ES cell technology.

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4.4Designer Zinc Finger Endonucleases to Silence Endogenous Ig Loci

In the mouse, gene targeting in ES cells allows the generation of heritablemutations or knockout animals. As suitable stem cells were lacking for the rat,an alternative method using zinc finger (endo)nucleases (ZFNs) was attemptedrecently producing several knockout lines with fully silenced Ig loci [22, 29, 30].The targeting sites of a ZFN pair contain a unique DNA-homology region of2× (9–18) nucleotides on opposite DNA strands. Three to six zinc fingers bind oneach strand with high specificity and a 6 bp spacer in between the targeting sitespermits FokI cleavage (Figure 4.5) [31]. ZFN constructs are assembled on plasmidsand the targeting efficiency can be assessed by transfection into cell lines beforemicroinjection of the purified expression cassettes into one-cell embryos [29]. Aninteresting observation was that cleavage and DNA repair could lead to variousmutations and sizable deletions. For example, disruption of the rat Cμ gene, whichsilenced the IgH locus, was achieved in a number of rats carrying deletions rangingfrom a few bases to hundreds of nucleotides [29]. Targeting of the JH locus produceda deletion of about 2.5 kb resulting in removal of all JH segments [30] and it may bepossible to use mixtures of ZFN pairs to remove larger regions by design.

Silencing of the IgH locus was also achieved in the rabbit using two ZFN pairsdesigned to target and replace Cμ exons 1 and 2 [32]. It appears that gene targetingwith designer ZFNs induces double-strand breaks at the desired target sites

Color Fig.: 4.5ZFN target sequence

DNA repair and non homologous

end-joining

ZFN induced cleavage

Deletion Insertion

Fokl

Fokl

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Figure 4.5 Sequence-recognition and cleav-age function of FokI, a two-domain zincfinger nuclease, allows target specific DNAcleavage for gene disruption or insertion(www.biochem.utah.edu/carroll/public html/

research/backgr.html). Targeted genedisruptions and germline modifications ofall rat Ig loci using ZFNs have been accom-plished [22, 29, 30].

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4.5 Expression Comparison of Fully Human and Chimeric IgH Loci 7

and subsequently nonhomologous end-joining repair, which results in targetedreplacement or deletion in a relatively high frequency of embryos born. In manycases, the breeding efficiency was little affected, which led to the conclusion thattargeting of unique sequences may avoid detrimental deletions.

4.5Expression Comparison of Fully Human and Chimeric IgH Loci

In many transgenic strains, mainly mice-derived in the last 20 years, improvedcloning strategies allowed the addition of a large number of human V genes to Dand/or J regions followed by one or more human C-region genes [5]. Such translocicontained only human genes, with expression control sometimes provided by shortrat or mouse enhancer sequences. In these strains, human Ig loci are integrated inthe germline and endogenous mouse Ig loci have been rendered nonfunctional bygene targeting [20, 33, 34]. Although fully human therapeutic antibodies have beenderived from these transgenic mouse lines (e.g., from XenoMouse [35]), frequentlydifficulties have been encountered with eliciting diverse human antibody responses[21]. A possible reason for this observation is a compromised immune responsebecause of the imperfect interaction of the human constant region with the mousecellular signaling machinery.

A reduction in the numbers of surface IgM+ cells in fully human IgH transgenicmice compared to Wt animals is illustrated in Figure 4.6a, which shows reducedlevels, from 14% to 3% for bone marrow cells and 30% to 19% for spleen cells[20]. A similar reduction in spleen cells has been observed in other transgenicstrains [35].

For transgenic rats carrying the constructs shown in Figure 4.2, no significantB-cell reduction was observed when Wt and transgenic lines where compared(Figure 4.6b) [22]. B-cell numbers found in normal rats were very similar orindistinguishable when compared to rats carrying a chimeric human IgH locusbred with Ig KO lines (termed OmniRat when carrying a chimeric IgH locus, ahuman IgL [κ and/or λ] locus, and endogenous KOs of IgH, IgK, and IgL). In IgH(and IgH+ IgL) KO strains obtained by ZFN targeting, no Ig+ cells were identifiedand B-cell development was abrogated (Figure 4.6c). Importantly, the OmniRatstrain with human IgVH, D, and JH segments linked to germline-configured ratIgCH regions produces chimeric antibodies and immune responses in a highlyefficient manner [22].

Previous analyses by us and other laboratories showed much reduced IgM andIgG titers in transgenic human antibody mice [5, 20, 21, 33, 36–38]. In theseanimals, the 3′ regulatory region was incomplete and contained only one of thefour enhancer elements. A problem was that class-switch recombination, changingCμ to Cγ, was much less efficient than observed in normal animals, hence relativelylittle IgG was produced. Sometimes, a significant amount of trans-switching fromhuman Cμ to endogenous mouse Cγ was found along with transgene switching[39]. For a rearranged transgenic IgH locus that lacks the 3′ enhancer region, a

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Figure 4.6 Flow cytometry analysis of bonemarrow (bm) and spleen lymphocytes stainedwith anti-IgM and anti-B220 (CD45R) usinghuman, mouse, or rat-specific antibodies. (a)Comparison of a wild type (Wt) mouse lineand a fully human transgenic mouse line car-rying human VHs, Ds, JHs, and human Cμ,expressed in a KO background with silencedendogenous IgH and Igκ loci [20]. (b) A wildtype rat and a translocus (chimeric) rat linecarrying human VHs, Ds, JHs, part of the ratC region with several C genes, and disabled

endogenous Ig loci [22]. Please note that fixedarea settings (boxed cells) for Wt and trans-genic lines stained with different reagents canslightly increase or decrease relevant popula-tions. (c) Ig deficient rat-line (KO) obtainedby ZFN technology. In bone marrow, A refersto pro/pre B cells (CD45R+IgM−) and Brefers to immature B cells (CD45R+IgM+).In spleen, A refers to lymphocyte precur-sors (CD45R+IgM−), B to follicular B cells(CD45R+IgM+), and C to marginal zone Bcells (CD45RlowIgM+).

reduction in transgenic but an increase in interchromosomal class-switch has beenidentified [40]. In OmniRat, the levels of chimeric IgM and IgG in serum with fullyhuman idiotypes but rat C regions and entirely human Igκ and Igλ loci were similarto that in Wt or normal rats (Figure 4.7a,b). Interestingly the level of IgG was notreduced in OmniRat despite the lack of Cγ2a in the translocus construct, whichsuggests that class-switching is similarly efficient but is using different C genes.

Purification of Ig by capturing with either anti-human κ or anti-human λ L-chainaffinity matrix (Figure 4.7c,d) also demonstrated that normal amounts are readilyexpressed; chimeric IgM and IgG levels with human L-chain from ∼3-month-oldtransgenic rats were very similar compared to the Ig levels found in older children[22]. It is also interesting to note that the fully human L-chain transloci are generallywell expressed in many transgenic lines with or without particular KO background[17, 26]. This suggests that expression of fully human L-chain is not biased orreduced by imperfect interaction with the rodent cellular machinery or impeded byassociation with chimeric H-chains.

The benefit of finding normal B-cell development and differentiation in atransgenic line, accompanied by high IgG expression, led to several essentialquestions: do class-switch and hypermutation function effectively in OmniRatand will immunization generate high affinity antibodies, ideally monoclonals by

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References 9

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Figure 4.7 Purified Ig from OmniRat ani-mals, Wt rats, and human serum. (a) IgMlevels were very similar in OmniRat and Wt.(b) IgG levels were a few milligrams permilliliter in both OmniRat and Wt but no

IgG2a was produced in OmniRat as Cγ2a isnot present on the translocus. (c,d) Purifica-tion of human Igκ and human Igλ, respec-tively [22].

cell fusion? In fully human transgenic IgH locus lines, reduced hypermutationand clonal expansion of particular V genes has been identified [21], which we donot see in OmniRat [22]. Indeed transcripts from nearly all human V, D, and Jsegments present in OmniRat have been identified in lymphoid tissue with upto 80% of VH genes linked to D-JH-rat Cγ being hypermutated, many extensively.Successful immunization using various antigens resulted in a large number ofhigh affinity IgG monoclonals and similar numbers of hybridomas were obtainedfrom OmniRat and Wt control animals compared side by side [22].

4.6Outlook

Significant improvements in the production of human antibody repertoires havebeen achieved by the expression of a chimeric IgH locus with human VH, D, andJH segments and rodent C genes. Extensive diversity and hypermutation was foundand many high affinity antibodies were identified by cell fusion. It appears thatoptimal interaction with the rodent cellular-signaling machinery can be achievedwhen species-specific CH regions and control sequences are preserved. As foundpreviously, extensive diversity was also seen for the introduced human Igκ and Igλtransloci. For therapeutic applications, a desired human CH region can easily replacethe rat CH region in a monoclonal antibody without compromising antigen binding.

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dubel c04.tex V2 - 04/09/2014 2:56pm

‘‘keywords/abstract

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Abstract

Human antibody expression in transgenic rodents has been significantly improvedby the integration of large chimeric DNA constructs linking human VH, D,and JH segments to rat constant (C) regions and regulatory sequences. Light-chain constructs were fully human. The Ig loci were cloned and modified inartificial minichromosomes and added to the rat germline by DNA microinjectioninto fertilized oocytes. Silencing of all endogenous Ig loci was achieved usingzinc finger nuclease constructs, which accomplished targeted gene disruption ofsometimes several kilobases. DNA rearrangements provided extensive diversity,and near-normal or wild-type levels of transgenic IgM and IgG were produced.Hypermutation was readily found and many high affinity antibodies could beproduced by cell fusion. For therapeutic applications, the rat C-region could beeasily replaced by a desired human C without the loss of antigen binding.

Keywords

transgenic rodents; ZFN knockout; chimeric antibody repertoires; human epitopes;class-switch recombination; lymphocyte development

Affiliation for the Author: Marianne Bruggemann1, Michael J. Osborn1, Biao Ma1,Suzanne Avis1, Ignacio Anegon2, and Roland Buelow1,3

1Recombinant Antibody Technology Ltd., Babraham Research Campus, Babraham,Cambridge, CB22 3AT, UK2INSERM Unit 1064, Transgenic Rats Nantes Platform, ITUN CHU de Nantes,Nantes F44093, France3Open Monoclonal Technology, Inc., 2747 Ross Road, Suite A, Palo Alto, CA,94303, USA

4 transgenic animals derived by dna...

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