Bispecific human IgG by design

Journal of Immunological Methods 248 (2001) 7–15www.elsevier.nl / locate / jim

Review

Bispecific human IgG by design

*Paul CarterDepartment of Molecular Oncology, Genentech Inc, 1 DNA Way, South San Francisco, CA 94080-4990, USA

Abstract

A major obstacle facing the development of bispecific antibodies as therapeutics has been the formidable task of producingthese complex molecules in sufficient quantity and purity for clinical trials. These production difficulties have been largelyovercome with the advent of efficient methods for the secretion of designer bispecific antibody fragments such as diabodiesand miniantibodies from Escherichia coli. In contrast, the creation of bispecific IgG by the coexpression of two different IgGis highly inefficient due to unwanted pairings of the component heavy and light chains. A robust technology for the creationof bispecific IgG has recently been developed that virtually precludes IgG contaminants, as reviewed here. This technology isanticipated to spur the clinical development of bispecific IgG and other bifunctional Fc-containing molecules such asantibody/ immunoadhesin hybrids and bispecific immunoadhesins. 2001 Elsevier Science B.V. All rights reserved.

Keywords: Bispecific antibodies; Antibody engineering; Immunotherapy

1. Introduction to bispecific antibodies BsAb have also been used to target viruses, virally-infected cells and bacterial pathogens as well as to

As their name implies, bispecific antibodies deliver thrombolytic agents to blood clots (Cao and(BsAb) bind to two different epitopes usually on Suresh, 1998; Koelemij et al., 1999; Segal et al.,distinct antigens. BsAb have potential clinical utility 1999).in targeting tumor cells or tumor vasculature with A great challenge facing the development of BsAbcytotoxic machinery including, immune effector as therapeutics has been the difficulty and expense ofcells, radionuclides, drugs and toxins. In addition, generating clinical grade material using hybrid hy-

bridoma technology (Milstein and Cuello, 1983) tocreate bispecific IgG (BsIgG) or directed-chemical

Abbreviations: Ab/ IA, antibody/ immunoadhesin hybrid; coupling to generate bispecific F(ab9) (BsF(ab9) )2 2ADCC, antibody-dependant cellular cytotoxicity; BsAb, bispecific fragments (Brennan et al., 1985; Glennie et al.,antibody; BsF(ab9) , bispecific F(ab9) fragment; BsIgG, bispecific2 2 1987). This production problem has been largelyIgG; CDC, complement-dependant cytotoxicity; c-Mpl, human

overcome, for BsAb fragments at least, with thethrombopoietin receptor; H chain, heavy chain; HER2, humanepidermal growth factor receptor 2; HER3, human epidermal advent of a plethora of formats for recombinantgrowth factor receptor 3; L chain, light chain; scFv, single-chain production by secretion from E. coli (reviewed byFv fragment ¨Carter et al. (1995); Pluckthun and Pack (1997); and

*Current address: Department of Protein Engineering, ImmunexHudson (1999)).Corporation, 51 University Street, Seattle, WA 98101-2936, USA.

In contrast to the rapid progress with BsAbTel.: 11-206-389-4037; fax: 11-206-624-7496.E-mail address: [email protected] (P. Carter). fragments, the development of a robust and generic

0022-1759/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0022-1759( 00 )00339-2

8 P. Carter / Journal of Immunological Methods 248 (2001) 7 –15

route to BsIgG has proved exceedingly difficult. the IgG products of a hybrid hybridoma (Suresh etNevertheless, as reviewed here, technology is now al., 1986b). Moreover, these contaminants includeavailable for the facile production of BsIgG and monospecific IgG that can compete with the activityother related bispecific molecules. In addition, fac- of the BsIgG and thus must be removed. In sometors influencing the choice between BsIgG and BsAb cases it has been possible to meet the great challengefragments for clinical applications are discussed. of purifying BsIgG away from the very closely

related IgG contaminants (Cao and Suresh, 1998).Indeed, BsIgG from hybrid hybridomas, used direct-

2. Traditional route to bispecific IgG ly or proteolysed to BsF(ab9) , have been evaluated2

in at least five small-scale clinical trials (Carter et al.,BsIgG are commonly produced by the coexpres- 1995).

sion of two different IgG in hybrid hybridomas(Milstein and Cuello, 1983), also known as quad-romas. Unfortunately, such IgG coexpression yields 3. Generic and efficient route to bispecificup to nine unwanted heavy (H) and light (L) chain human IgGpairings (Suresh et al., 1986a) (Fig. 1A). This reflectsthat IgG H chains can form homodimers, as well as We reasoned that the clinical potential of BsIgGthe desired heterodimers, and that many L chains would be substantially enhanced by the advent of awill pair with non-cognate as well as cognate H general and efficient route to these molecules. Such achains. Thus, BsIgG likely represent only 10–50% of technology should utilize humanized or human anti-

Fig. 1. Technology for creating BsIgG using H chains engineered for heterodimerization and a common L chain (Merchant et al., 1998). (A)Coexpression of two different IgG (black and white) yields up to nine unwanted chain pairings in addition to the desired BsIgG (Suresh etal., 1986a). (B) Engineering IgG H chains for heterodimerization virtually eliminates six of these unwanted chain pairings. (C) Usage of IgGwith a common L chain (gray) avoids the remaining three unwanted chain pairings.

P. Carter / Journal of Immunological Methods 248 (2001) 7 –15 9

bodies, rather than rodent antibodies, to minimize therisk of immunogenicity of BsIgG in patients. Inaddition, human and humanized antibodies permitefficient secondary immune functions if desired, andlonger plasma half-lives (Vaughan et al., 1998; Quanand Carter, 2000). Such considerations motivated usto develop a robust BsIgG technology that virtuallyprecludes IgG chain-mispairing contaminants (Mer-chant et al., 1998). Six out of nine unwanted IgGchain combinations were greatly reduced by proteinengineering of antibody H chains so that theypreferentially form heterodimers over homodimers(Fig. 1A,B). The L chain mispairing problem wascircumvented entirely by constructing a BsIgG fromantibodies that utilize identical L chains (Fig. 1B,C).

3.1. Engineering antibody heavy chains forheterodimerization

The most extensive site of protein–protein inter-action between the H chains of human IgG mole-cules is between their C 3 domains (Deisenhofer,H

1981). In contrast, C 2 domains interact primarilyH

via their attached carbohydrate. Two inter H chaindisulfide bonds are formed during antibody expres-sion in mammalian cells, although this is dependentupon the presence of C 2 and C 3 domains (King etH H Fig. 2. Engineering of C 3 domains from IgG for heterodimeriza-H

al., 1992). Thus H chain assembly apparently pro- tion of corresponding H chains. (A) Rational design strategies forengineering C 3 domains. (B) Optimization of C 3 knobs-into-motes the formation of hinge disulfide bonds rather H H

holes mutants using phage display technology. A knob mutationthan vice versa. This led us to hypothesize that C 3Hwas fixed whereas three residues in the partner C 3 domain wereHdomains direct the association of antibody H chainsrandomized (*) to create a hole mutant library. (C) Ab/ IA

and that the interface between C 3 domains mightH cotransfection assay used to study heterodimer formation. (D)be engineered to promote the formation of hetero- Summary of best yields of Ab/ IA hybrid using five different

engineering strategies (Ridgway et al., 1996; Atwell et al., 1997;dimers over homodimers.Merchant et al., 1998). ’Knobs-into-holes1phage’ refers to theSeveral rational design strategies have been usedstrategy depicted in (B) in which rationally designed stericallyto engineer antibody H chains for heterodimeriza-complementary mutations were optimized by selection from a

tion, namely, sterically complementary, ‘knobs-into- phage display library (Atwell et al., 1997). Mutations are shownholes’, mutations, disulfide bonds, and salt bridges using the one letter code for the wild-type residue followed by the

residue number and then the mutant residue. Mutations in the(Fig. 2A). For the knobs-into-holes approach a knobsecond subunit follow the slash (/ ) and are indicated with a primevariant was obtained by replacing a small amino acidsign (9) on the residue position. W/SAV is an abbreviation for thewith a larger one in the first C 3 domain, e.g.H multiple mutant, T366W/T3669S:L3689A:Y4079V.

T366Y. The knob was designed to insert into a holein the second C 3 created by judicious replacementH

of a large residue with a smaller one, e.g. Y407T (Merchant et al., 1998). Attempts to form salt(Ridgway et al., 1996). In order to create inter-C 3 bridges were made by juxtaposing oppositelyH

domain disulfide bonds, six different pairs of cys- charged residues on either side of the C 3 domainH

teine mutants were constructed in which a disulfide interface (P. Carter and M. Merchant, unpublishedbond could be modeled with favorable geometry data).


Knobs-into-holes mutations were optimized using antibody H and L chains. Indeed, we have en-phage display technology (Atwell et al., 1997). gineered the interface between V and V domains toL H

Briefly, a C 3 knob mutant, T366W, was first facilitate the formation of a bispecific diabody (ZhuH

generated. A library of C 3 hole mutants was then et al., 1997), albeit at the expense of slightly reducedH

created by randomizing residues 366, 368 and 407 antigen binding affinities.that are in proximity to the knob on the partner C 3 A much simpler solution was to completely cir-H

domain. The C 3 knob mutant was genetically fused cumvent the L chain mispairing problem. This wasH

to a peptide flag, whereas the C 3 hole library was accomplished using antibodies with identical LH

fused to M13 gene III. Phage displaying stable C 3 chains that bind to different antigens by virtue ofH

heterodimers were then selectively recovered by their distinct H chains. Such antibodies are verypanning using an anti-flag antibody (Fig. 2B). readily isolated from phage libraries that have vast H

The propensity of rationally designed and phage- chain repertoires and a unique (Nissim et al., 1994)optimized C 3 variants to heterodimerize was as- or very few (Vaughan et al., 1996) different LH

sessed by their ability to direct the formation of an chains. For example, we assessed the frequency ofanti-CD3/CD4-IgG antibody/ immunoadhesin hybrid identical L chains from the library of Vaughan et al.(Ab/ IA) (Chamow et al., 1994). Briefly, the CD4- (1996) by comparing amino acid sequences for 117IgG and anti-CD3 H chain variants were coexpressed V domains from scFv binding to eleven differentL

in 293 cells, together with the anti-CD3 L chain. The antigens. ScFv sharing identical L chains wereantibody and immunoadhesin products were quan- identified for the majority (50 out of 55) of thetified by scanning laser densitometry following pro- possible pairwise combinations of two differenttein A affinity chromatography and SDS–PAGE antigen specificities (Merchant et al., 1998). Indeed,(Ridgway et al., 1996; Atwell et al., 1997; Merchant identical L chains were found in all cases exceptet al., 1998) (Fig. 2C). where five or fewer sequences were available for one

As expected, chains containing wild-type C 3 or both of the antigen specificities.H

domains gave yields of |50% Ab/IA hybrid. Incontrast, the preferred disulfide bond (D399C/K3929C) gave a yield of 73% Ab/IA hybrid and the 3.3. Test of human bispecific IgG technologymost successful ‘salt bridge’ (charge pair mutantL368D:Y407F/T3669K) resulted in |86% Ab/IA Our next step was to create a human BsIgG byyield. This is very similar to the Ab/IA yield using antibodies sharing identical L chains in con-achieved with the designed knobs-into-holes variant junction with H chains engineered for heterodimeri-(T366W/Y4079A), and the corrresponding phage- zation. We chose to create a BsIgG binding to theoptimised variant (T366W/T3669S:L3689A: human thrombopoietin receptor (c-Mpl) and toY4079V). The most successful strategy of five tested human epidermal growth factor receptor 3, (HER3)combined knobs-into-holes mutations with an en- from a pragmatic consideration of reagent availabili-gineered disulfide bond, to yield 95% Ab/IA hybrid ty. The common L chain was cotransfected with the(Fig. 2D). This latter combination of mutants, name- two H chains containing the preferred C 3 mutationsH

ly S354C:T366W/Y3499C:T3669S:L3689A:Y4079V, (Fig. 2C). The IgG products were purified by proteinwas chosen for the construction of a BsIgG. A chromatography and then analyzed by SDS–

PAGE. The BsIgG preparation gave rise to a singlemajor band of greater electrophoretic mobility than

3.2. Circumventing mispairing of antibody light IgG containing wild-type C 3 domains (Merchant etH

chains al., 1998). This increased mobility reflects the forma-tion of the engineered disulfide bond. As anticipated,

The protein engineering strategies used to create the BsIgG, but not the parental anti-c-Mpl and anti-antibody H chain heterodimers could, in principle, HER3 IgG, could bind simultaneously to c-Mpl andhave been applied to remodel the interface between HER3 antigens (Merchant et al., 1998).


3.4. IgG with heavy chain heterodimerizing hybrids (Merchant et al., 1998), engineered H chainsmutations efficiently supports ADCC are anticipated to be useful in creating bispecific

immunoadhesins (Dietsch et al., 1993).Mutations created for H chain heterodimerization

are fully buried and anticipated not to propagate 3.6. Potential risk of immunogenicity of bispecificmajor structural changes to the surface of the corre- IgGsponding C 3 domains. Nevertheless, it was im-H

portant to study the effects of these C 3 mutations A potential risk of treating patients with en-H

upon Fc-mediated functions such as antibody-depen- gineered proteins is that of eliciting a neutralizingdent cell-mediated cytotoxicity (ADCC) and com- antibody response or a T cell response. Unfortuna-plement-dependent cytotoxicity (CDC) as well as tely, such immunogenicity issues can only be defini-effects upon plasma half-life. Thus far we have tively addressed through costly and time-consuminginvestigated the impact of H chain mutations clinical trials. Nevertheless, the risk of immuno-(S354C:T366W and Y349C:T366S:L368A:Y407V) genicity for human BsIgG appears small as the C 3H

on the ability of an IgG to support ADCC. This mutations are fully buried and few in number (six).comparison was made using huMAb4D5-5, a mono- Encouragingly, humanized antibodies containingspecific humanized IgG that binds to human epider- several dozen foreign and mainly exposed residues inmal growth factor receptor 2 (HER2), (Carter et al., their variable domains have elicited undetectable1992b), rather than a BsIgG. This reflects that the (Caron et al., 1994; Sharkey et al., 1995; Baselga etnecessary control molecule containing a wild-type Fc al., 1996; Hale and Waldmann, 1996) or only minorregion is easily prepared for the monospecific IgG (Isaacs et al., 1992; Vincenti et al., 1997) humanbut not for the BsIgG. Anti-HER2 antibodies con- anti-human antibody responses.taining engineered C 3 domains had similar potencyH

in ADCC with breast cancer cell line, SK-BR-3, that 3.7. Binding affinity of bispecific IgGoverexpresses HER2. Both antibodies showed com-parable, low activity against the non-tumor breast Antigen-binding affinities in the low nanomolar orepithelial cell line, HBL100, which expresses 33-fold picomolar range are desirable and perhaps essentialless HER2 than SK-BR-3 cells (Lewis et al., 1993). for each arm of a BsIgG destined for human therapy.

In some cases it will likely be possible to construct3.5. Broad applicability of bispecific IgG such high affinity human BsIgG using scFv isolated

¨technology directly from a large naıve phage library. Indeed,Vaughan et al. (1996) have previously reported many

Our BsIgG technology is applicable to antibodies (nine) high affinity scFv (K 50.3–8 nM) from thed

recognizing virtually any pair of antigens, since it is same scFv phage library panned by us to identifyroutinely possible to identify antibodies with differ- antibodies utilizing the same L chains. These highent antigen-binding specificities that share identical L affinity scFv include several with clinically signifi-chains (Nissim et al., 1994; Vaughan et al., 1996; cant binding specificities: the tumor-associated an-Merchant et al., 1998). In addition, the C 3 muta- tigen, carcinoembyronic antigen; the radionuclideH

tions required for heterodimerization can be incorpo- chelator, diethylene triamine penta-acetate; and therated into H chains of any antigen-binding spe- anti-neoplastic drug, doxorubicin. Thus, high affinitycificity. Engineered H chains will likely be useful in antigen binding is possible even starting from phagedirecting the assembly of other immunoglobulins libraries with very limited L chain repertoires. Thesesince the mutated residues are fully conserved across high affinities presumably reflect the fact that the Hhuman IgG isotypes and the majority of the C 3 chain predominates in making major contributions toH

interface residues are highly conserved (Deisenhofer, the energetics of antigen binding.1981; Miller, 1990; Kabat et al., 1991). In addition Sometimes it may be necessary to increase theto facilitating the construction of BsIgG and Ab/IA binding affinity of a BsIgG for one or both cognate


antigens. Affinity maturation of antibodies from thenanomolar to picomolar range has been accom-plished by mutagenesis of H and L chains inconjunction with selection using phage display li-braries (Yang et al., 1995; Schier et al., 1996). Theemerging technology of ribosome display holdssignificant promise for accelerating antibody affinitymaturation and reducing the effort required (Hanes etal., 1998; Knappik et al., 2000). Any L chainmutation that enhances binding to one antigen wouldof course have to be evaluated for maintainingbinding to the second antigen, prior to incorporationinto a BsIgG.

3.8. Constructing bispecific IgG from existingantibodies

It may be desirable to construct a BsIgG usingexisting human or humanized antibodies that havedifferent L chains. In such a case, engineered H

Fig. 3. Bispecific antibody formats that are particularly wellchains can still be used and are expected to reducesuited for clinical development (see Section 4). The two differentthe number of significant IgG contaminants fromantigen-binding specifities are indicated by black and whitenine or less to three or less (Fig. 1A,B). If necessary,shading, whereas the common Lchain of the BsIgG is colored

it should additionally be possible to identify a grey. A linker connecting the carboxy terminus of V to the aminoL

common L chain for these antibodies. In this instant terminus of V is indicated as a bold white or black line for theH

miniantibodies. Additional BsAb formats have been reviewed byone starts from an existing antibody and identifies a¨Carter et al. (1995), Pluckthun and Pack (1997) and Hudsonnew H chain which pairs with the original L chain

(1999).and binds to the second antigen of interest. The Fabphage library and the panning strategy of Figini et al.(1994) are particularly well-suited to the identifica- Miniantibodies are notable in that they can betion of such a H chain. created to be bispecific and also bivalent for each

¨antigen specificity (Muller et al., 1998) (Fig. 3).The format of a BsAb should be carefully tailored

4. Choice of bispecific antibody format for to the intended clinical application in terms of theclinical applications required pharmacokinetic properties, and the de-

sirability of supporting secondary immune functions,In addition to BsIgG reviewed here, three recom- CDC and ADCC. Additionally, for oncologic indica-

binant technologies seem particularly well suited to tions, the ability to penetrate tumor tissue should bethe production of BsAb for clinical applications: considered. For example, BsIgG likely represent thechemically-coupled BsF(ab9) using Fab9 fragments preferred BsAb format where a multi-day or multi-2

from E. coli (Rodrigues et al., 1992; Shalaby et al., week plasma half-live is desired and/or secondary1992), diabodies (Holliger et al., 1993) and minian- immune functions are required. However bispecific

¨tibodies (Pack and Pluckthun, 1992; Pack et al., diabodies have also been conferred with the ability to1993, 1995) (Fig. 3). Indeed, E. coli fermentation support CDC and ADCC, by designing one arm totiters approaching, or even exceeding, a gram per bind plasma IgG (Holliger et al., 1997). Bispecificliter have been achieved for Fab9 fragments (Carter diabodies have also been created to support CDC byet al., 1992a), diabodies (Zhu et al., 1996) and targeting one specificity to the complement com-miniantibodies (Pack et al., 1993; Horn et al., 1996). ponent, C1q (Kontermann et al., 1997). Bispecific


diabodies binding to plasma IgG have extended nologies, in this case chimerization and humaniza-terminal half-lives (t b|10 h) compared to simi- tion, and their application to judiciously chosen1 / 2

larly sized fragments, but are still cleared much target antigens (Quan and Carter, 2000). I anticipatefaster than IgG in mice (Holliger et al., 1997), and that recently devised BsAb technologies will fulfill apresumably man. similar role in the development of BsAb thera-

BsAb fragments seem preferable to BsIgG for peutics.clinical applications where rapid clearance is desir-able or small size is potentially advantageous tofacilitate tumor penetration. BsAb fragments also Referencescircumvent Fc interactions that can sometimes leadto toxicity problems. Alternatively, effector functions Atwell, S., Ridgway, J.B., Wells, J.A., Carter, P., 1997. Stablecan be avoided by constructing BsIgG using IgG or heterodimers from remodeling the domain interface of a2

IgG isotypes or by engineering the Fc to remove homodimer using a phage display library. J. Mol. Biol. 270,426–35.these functions.

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Caron, P.C., Jurcic, J.G., Scott, A.M., Finn, R.D., Divgi, C.R.,Despite the arduous task of making clinical gradeGraham, M.C., Jureidini, I.M., Sgouros, G., Tyson, D., Old,

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Bispecific human IgG by design

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