Synthetic antibodies as therapeutics

Review

10.1517/14712598.7.1.73 © 2007 Informa UK Ltd ISSN 1471-2598 73

Vaccines & Antibodies

Synthetic antibodies as therapeuticsGermaine FuhGenentech, Inc., Department of Protein Engineering, 1 DNA Way, South San Francisco, CA 94080, USA

Synthetic antibody libraries, whose repertoires are designed, have advancedin the last decade to rival natural repertoire-based libraries. Many types ofdiversity design have been shown to generate highly functional libraries.Defined template and defined diversity in synthetic antibody librariesimprove the process of discovering and optimizing new antibodies. Syntheticlibraries with different diversity design have targeted different epitopes onantigens, including epitopes that are unlikely to be targeted byimmunization and hybridoma. Cross-species binding antibodies are primeexamples of products generated by synthetic antibody libraries, and they arebecoming the tools of choice to validate the selection of targeted moleculesin therapeutic development. Synthetic antibody libraries complement theexisting natural repertoire-based antibody libraries and hybridoma approachto maximize the potentials of antibodies as therapeutics.

Keywords: antibody libraries, diversity design, hybridoma, synthetic antibodies

Expert Opin. Biol. Ther. (2007) 7(1):73-87

1. Introduction

Over the past decade, there have been an increasing number of examplesdemonstrating the effectiveness of antibodies as therapeutics [1]. Antibodies offersmany advantages, such as an exquisite target specificity and a reasonably predictablepharmacokinetic/pharmacodynamic profile [2]. These two desirable characteristics,among others, have contributed to the high success rate of regulatory approval forthe antibody therapeutics. The increase in our understanding of how antibodieswork, and new developments of the processes to generate and optimize them, havefueled the rapid rise in the number of antibodies on the way to clinical trials [3,4].

Almost all of the first-generation antibody therapeutics were identified frommouse hybridoma [5]. Several early methods were developed to reduce theimmunogenicity of these mouse antibodies for use in humans. One way toaccomplish this reduction is by grafting the mouse variable domain to the humanconstant domain of immunoglobulin (Ig) (mouse/human chimaera) [6,7]. Anothermethod subjects the mouse antibody to a humanization process, such as grafting theantigen binding loops (that is, the six complementarity-determining regions[CDRs]) onto the human framework region (FR) of the variable domain (seeFigure 1) [8,9]. Recently, two new approaches for generating antibody have come ontothe horizon. The first is transgenic mice carrying human antibody genes(xenomice) [10], which, like mouse hydridoma, involves in vivo immunization. Thesecond is combinatorial human antibody libraries [11-13], which bypasses theimmunization and isolates antibodies from engineered libraries in vitro.

The main technology for combinatorial antibody libraries includes four types ofdisplay: phage [14], ribosome [15], yeast [16] and bacterial display [17]. The commontheme of these four approaches is that they link phenotype (antibody) withgenotype (the corresponding encoding sequence). This is a robust process in which

1. Introduction

2. Evolution of synthetic antibody

libraries with designed diversity

3. Targeting specific epitope by

library design

4. Different diversity targets

different epitopes

5. Epitopes of

antagonistic antibodies

6. Epitopes of

agonistic antibodies

7. Epitopes and Fc-mediated

effector function

8. Epitopes of cross-species

antibodies from synthetic

antibody libraries

9. Expert opinion and conclusion

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Synthetic antibodies as therapeutics

74 Expert Opin. Biol. Ther. (2007) 7(1)

Figure 1. The antibody molecule. (A) Crystal structure of a full-length IgG (PDB ID code 1IGT) [118] is shown with carton representationgenerated using Pymol (DeLano Scientific, South San Francisco, CA). IgG is a heterotetramer of two light chain (in white) and two heavychain (in black) and is folded into two Fabs (antigen binding domain) and one Fc (crystallisable fragment). Fv is where antigen bindingoccurs and is consisted of variable domains from the light (VL) and heavy chain (VH). Antigen binding sites (paratope) mostly reside in thesix CDRs, which are presented and scaffolded on the FR. Constant domains of light chain (CL) and heavy chain (CH1, CH2 and CH3), asdenoted, are constant in sequence and bring about effector-mediated immune function by interaction with complement and Fcreceptors. (B) A detailed view of Fv (VH and VL) (Trastuzamab, PDB code 1FVC) is shown in tubes. The six CDRs are highlighted withcolours. The border residue numbers (Kabat numbering system) of each CDR are shown. Both Kabat and Chothia definition of CDRs areused [41,119]. The figure is courtesy of J Bostrom.CDR: Complementarity-determining region; FR: Framework region; Fv: Variable fragment.

CDR-H1

CDR-H2

CDR-H3

CDR-L1

CDR-L2CDR-L3

VH

VL

95

10226

35

5065

24

34

50

58

8997

B.

Fab

Fc

Fab

FR CDR

Antigen binding

Fv

Fc receptors andcomplement binding

A.

VH

VL CL

CH1

CH2

CH3

billions of variants can be simultaneously sorted based on theirbinding function. The clones with desired characteristics canthen be identified quickly by their DNA sequences. Phagedisplay is the most advanced and widely used. Ribosomedisplay offers the advantage of large library size. Yeast displayand bacterial display, when combined with cell detectiontechniques, such as fluorescence-activated cell sorting, can

rapidly compare the binding properties of large number ofclones semiquantitatively.

Initially, the approach using combinatorial antibodylibraries was primarily employed to optimize the affinity andstability of therapeutic antibodies [13]; however, in the lastdecade, combinatorial libraries have been used, not only foroptimization, but also for discovery of new antibody

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Expert Opin. Biol. Ther. (2007) 7(1) 75

therapeutics. To discover antibodies from a combinatoriallibrary, the library must contain sufficient chemical diversityso that novel antibodies to any target antigen of interest canbe generated.

Libraries can contain two types of repertoires: natural orsynthetic, or some combination of both (Figure 2). Naturalrepertoire derives from the antibody genes that exist in theB lymphocytes (∼ 109 – 1010 for human); each B cell expressesone gene recombined from the germline gene segments of Ig.The sources of B cells, as well as the immunological status ofthe donors, cloning methodologies and efficiencies, allcontribute to the differences in the library diversity [18-22] (forreview see [23]). Most libraries utilize peripheralB lymphocytes as the source repertoire, as they contain naiveB cells with less bias from immunogenic stimulations. Inaddition, peripheral B cells contain memory B cells expressingrearranged antibody gene segments with somatic mutations,which can increase the diversity of the libraries [24]. Thevariable domains (VH and VL) of immunoglobulin from thenatural sources contain multiple FRs and the hypervariableCDR loops, both of which contribute to the diversity of thenatural repertoire [25,26]. The six CDR loops are responsiblefor most of the direct interaction with antigens (Figure 1). FRsare less variable and play a role in scaffolding CDRs in their

canonical conformations [27], and occasionally make contactwith antigens.

In constructing the natural repertoire-based in vitrolibraries, the random pairing of the light chain and heavychain can also contribute to the diversity of these libraries.The theoretical diversity of the combinatorial library can belarger (up to 109 × 109) than that existing in nature at thetime of cloning (∼ 109). The practical limit on library size dueto transformation efficiency (109 – 1010), however, restrictsthe actual diversity of libraries. Advances in making largerlibraries of this type have helped to increase the representationof diversity space in the libraries and, consequently, havehelped to generate antibodies with higher affinity [20,28]. Onemain advantage of antibody libraries of this kind is that thederived antibodies can be considered fully human. However,technically, the libraries based on naturally derived diversitycan be difficult to quantitatively define and reproduce. Owingto the unique composition of each individual clone, theprocess to re-engineer the derived antibody (e.g., for affinityimprovement) is less robust. In addition, large-scale proteinproduction could be problematic. Many reported the effortsto improve antibody stability and reduce aggregationproblems [29,30]. The first combinatorial library-derivedtherapeutic antibody to reach the marketplace is adalimumab

Figure 2. Two main types of repertoires in combinatorial antibody libraries for the identification of novel antibodies.(A) A natural repertoire-based library captures its repertoire of Ig genes from immunized or non-immunized individuals. Multiple FR andhypervariable CDRs contribute to the library diversity, and the library is constructed by PCR cloning the variable domain gene into a libraryvector (B). The repertoire of a synthetic library, in contrast, is by design. CDR positions are randomized by oligonucleotide-directedmutagenesis based on a template that can contain a single or multiple FRs (C). The semisynthetic antibody libraries contain part naturaland part synthetic repertoires.CDR: Complementarity-determining region; FR: Framework region.

Repertoire Immunised Non-immunisedFR Multiple Multiple Multiple Single

Source ofdiversity

CDR + FR CDR

Constructionmethod

Oligonucleotide-directedmutagenesis

PCR cloning

Natural SyntheticDesigned

CDR + FR

CDR1 CDR2 CDR3FR1 FR2 FR3 FR4

B.

A.

CDR1 CDR2 CDR3FR1 FR2 FR3 FR4

C.

VH or VL

VH or VL

VH or VH + VL VH or VH + VL VH or VH + VL

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(a TNF-specific antibody for the treatment of rheumatoidand psoriatic arthritis; Abbot/Cambridge AntibodyTechnology), which was identified from a naturalrepertoire-based library [31].

Antibody libraries based on synthetic repertoire, on theother hand, incorporate diversity by synthetic DNAoligonucleotide-directed mutagenesis on selected templatescaffold(s) [32]. By introducing degeneracy into a syntheticoligonucleotide, the position and degree of randomization canbe precisely controlled. There are also many examples ofsemisynthetic antibody libraries, which incorporate partnatural and part synthetic repertoires.

This review discusses the evolution of different designs ofsynthetic and semisynthetic libraries, and focusses on theadvantages that synthetic antibody libraries have for targetingdifferent epitopes. It also discusses why finding the rightepitope is important for therapeutic development.

2. Evolution of synthetic antibody libraries with designed diversity

Building diversity from scratch synthetically is challengingand requires understanding of the elements within the naturalrepertoire that renders its unlimited specificities. In addition,a good grasp of combinatorial library engineering is essential.Over the last decade, the synthetic antibody libraries haveevolved and improved to a state that they can now rivalnatural libraries in productivity.

The first synthetic libraries made use of a single Fab(tetanus toxoid binding Fab) as a phage-displayedtemplate [33]. In the libraries, each of the 16-positions inCDR-H3 (95 – 102, according to Kabat numbering with100a-h between 100 and 101) have the diversity of all20 amino acids using codons (NNS)16. The choice oftargeting CDR-H3 is consistent with the observations thatCDR-H3, situated at the centre of the antibody binding site,is the most diverse CDR in amino acid sequences andstructural conformations. CDR-H3 contributesdisproportionately to direct interaction with the antigen andplays a central role for antibody diversity [34-37]. The size ofthe library is small (107) considering the DNA degeneracy is> 1020. To test the library the author used only one antigen(fluorescein), but the affinity of the best clone (∼ 0.1 µM) wasimpressively close to what would be expected from the naturalimmune response to a hapten.

The same group expanded their work by comparingthree libraries:

• libraries with randomized CDR-H3 alone with lengths of5, 10, 16 amino acids (NNK5, 10, 16) to represent the short,average and long CDR-H3 in nature, respectively

• libraries that only randomized CDR-L3 with lengths of 8, 9and 10 amino acids (Gln89-Gln90-Tyr91-NNK(4, 5, 6)-Thr97)to represent the natural range of length

• libraries that randomized both CDR-H3 and CDR-L3 [38]

They described a selection of binding clones for three haptensand observed that most binding clones came from the librariesthat randomized both the CDR-H3 and CDR-L3. Thisdespite the fact that the diversity in the library (size = 108) isonly a small portion of the full diversity of DNA(3216+6 = ∼ 1033) and amino acids (2016+6 = ∼ 1028). In naturalantibodies the most frequent residues for antigen bindingreside in both CDR-H3 and CDR-L3 [39]. No clones werederived from CDR-H3-libraries with the shortest length(NNK5) or from CDR-L3-only libraries, suggesting theselibraries had insufficient chemical diversity. Clones with lownanomolar binding affinities for the hapten antigens wereobtained. Protein antigen was not tested.

Other early work on synthetic antibody libraries using asingle Fab template incorporated synthetic diversity in fourCDRs (CDR-L1, -L3, - H2 and -H3). They randomized aselected set of positions in the three non-H3 CDR (twopositions in L1, three in L3 and four in H2) with a limited setof amino acids, and four or five positions (96 – 100) inCDR-H3 of one length (11 amino acids as the template) withfull or limited diversity [40]. This work describes an approachto build the combinatorial antibody libraries where thediversity of CDR is restricted to amino acid types present innatural antibodies. It recognizes the fact that there is limiteddiversity and biased amino acid usage in the hypervariableloops (i.e., CDRs) in nature [41]; however, the authors did notdescribe the process of how they determined the restricteddiversity for their libraries. The libraries were modestlysuccessful and produced antibodies to only one of threeprotein antigens, insulin-like growth factor-1 (a small-sizeprotein), with low micromolar affinity.

There were also a series of semisynthetic antibody librarieswhere diversity came from synthetically randomized CDR-H3within 49 or 50 natural VH germline gene segments. Thesegene segments encode the various germline CDR-H1 and -H2and the frameworks (FR1, 2 and 3). The first set ofsemisynthetic libraries consisted of a fully randomizedCDR-H3 with either five or eight residues (codon NNK(5) orNNK(8)) and contained a single light chain. The library sizewas 107 and performed modestly, generating antibodies forhapten in the micromolar range. However, it was lesssuccessful for protein antigens as antibody was generatedagainst only one out of four proteins [42].

The next set of libraries expanded the range of length ofCDR-H3 to include 4 – 12 residues for position 95 – 102,and library size was increased to 108 [43]. This resulted inhighly successful libraries for 18 antigens, including haptensand proteins. This indicates the importance of diversity in thelength of CDR-H3. The libraries were next improved bymaking large libraries using the strategy of lox-Cre mediatedrecombination [44]. They combine their semisynthetic heavychain repertoire with a diverse light chain repertoire; the latterconsisted of 47 light chain germline VL segments andsynthetically randomized CDR-L3 [45]. The library size wasdramatically increased to ∼ 1010 – 1011 and was very successful

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for the 36 test antigens (haptens and proteins); antibodies oflow nanomolar affinity were generated. The authors thenanalyzed primarily the hapten binding clones; they examinedthe 137 unique binding clones (out of the total 215 clones)and observed an uneven appearance of the VH and VL

germline segments. Some of the most frequently occurringgermline segments in these binding clones are also the mostfrequently occurring germline segments in natural antibodies.

This strategy for semisynthetic library construction wasalso adopted by de Kruif et al. [46] to generate a library thathas the 49 VH germline segments and seven light chains withCDR-H3 randomized synthetically to lengths 6 – 15. TheirCDR-H3 was designed to have ∼ 3 – 5 fully randomizedpositions (codon NNK) flanked by limitedly randomizedpositions. They did this to mimic the natural antibodysequences. Considering their moderate library size (108), theyproduced specific antibodies with reasonably high affinity inthe range of micromolar and high nanomolar antibodies.Neri and collegues use a similar approach, but strategicallychose only one germline segment for heavy and light chain,DP47 and DPK22, respectively [47]. They reasoned that thesetwo segments frequently occur in natural antibodies [48,49].Selected positions in CDR-H3 (95 – 98) and CDR-L3 (91,93, 94 and 96) were targeted for full randomization (codonNNK) due to their role as common antigen contacts. Thislibrary worked well; antibodies to six test antigens and otherantigens in a later report [50] were generated with affinity upto low nanomolar range. To improve affinity theyrandomized positions in CDR1 and 2, which werestructurally adjacent to the sites in the original scheme ofrandomization. Clones with picomolar affinity were derivedfrom a low-nanomolar parent after two rounds of affinityimprovements by first targeting six heavy chain residues, nextto light chain residues. The strategies of stepwise generationof high affinity antibodies can reasonably be extended toother clones derived from the same library. The authorsfurther extended their strategies and made another version oflibraries (ETH-2 Gold libraries) by including a VL germlinesegment DPL-16 in addition to DPK22 and DP47 as atemplate [51]. The use of their single-framework libraries,which randomizes only eight residues in the centrally locatedtwo CDR3, demonstrated that combinatorial mutagenesis inthe few positions most important for antigen binding wassufficient to generate functional libraries. Thesesemisynthetic libraries appear to have evolved and improvedwell enough to suit therapeutic development.

The first commercial venture based on the syntheticantibody library technology for generating therapeuticantibodies was Morphosys; the libraries originated from theresearch of A Pluckthun’s group. The library constructionfirst started with a careful selection of library templates byanalysing the structure, sequence diversity and usage ofgermline genes and rearranged natural antibodies [52]. Byhomology, germline genes (48, 26, 43 for VLκ, VLλ, and VH,respectively) were grouped into seven VH families (H1A,

H1B, H2, H3, H4, H5 and H6) and seven VL germlinefamilies (κ1, κ2, κ3, κ5, λ1 and λ2, λ3). A consensussequence of the rearranged natural antibodies belonging toeach of the seven germline families was chosen. CDR1 and 2of each consensus sequence were replaced with the aminoacid sequence of the germline gene of the correspondingfamily in order to remove bias from any particular antigenselection. The gene for each template was synthesized withoptimized codons for Escherichia coli expression. Uniquerestriction endonucleases sites flanking each CDR wereincorporated for the benefit of modularity. One purpose ofthe modularity is so that affinity improvement can beperformed easily by swapping libraries of different CDRsinto parent clones for selection. In total there are49 different templates from the combination of the sevenlight chains and heavy chains, which were examined for theirstability and robustness of protein expression in E. coli [30].Selected positions in CDR-L3 and CDR-H3 wererandomized in the first version of libraries (HumanCombinatorial Antibody Libraries [HuCAL]) [52], thenpositions in all six CDRs were randomized in the secondversion (HuCAL GOLD) [53]. The randomization schemeused trinucleotide-based oligonucleotides [54,55] to tailordesign the composition of natural antibody repertoire ateach position and devote higher diversity to positions thatare more likely to contact the antigen. Thus, the diversity ofHuCAL is consisted of the synthetically randomized CDRpositions and the multiple natural frameworks in the 49templates. The libraries worked well with test antigens,including human fibroblast growth factor receptor 3(FGFR3), which was recalcitrant to hybridoma approach,probably due to the high homology between human andmurine FGFR3 [56]. There are several antibodies generatedfrom the Morphosys libraries presently in various stages ofclinical development, for example, antibody 1D09C3 toMHC class II antigen on B cells for non-Hodgkin’slymphoma and antibody MOR102 to intracellular adhesionmolecule 1 for treating autoimmune diseases [57].

The synthetic antibody phage libraries at Genentech chosethe approach of using trastuzumab (humanized 4D5, h4D5)as a template to build the libraries [58,59]. There are fourrationales for using h4D5 as a phage libraries template:

• it has the framework based on a consensus sequence ofhuman VH3 and VLκ1 families for heavy and light chain,respectively [60]

• it is well expressed in E. coli and phage [61]

• the structure information is available [62]

• it is already proven in the clinic as a humanantibody therapeutic

Using structural information, residues in the three heavy chainCDRs that are surface-exposed were selected for randomizationin a manner that mimics the natural repertoire [26,41].

To improve the libraries, CDR-H1 and -H2 was fine-tunedto reduced DNA degeneracy in the oligonucleotides, but still

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covered ∼ 70 – 90% of natural repertoire in amino aciddiversity. CDR-H3 in this library was expanded to containmore length variations (7 – 19) and mimic the amino acidcomposition of natural CDR-H3 [59]. The display formatmoved from scFv to Fab, mono- or bivalently [63]. Theimprovement increased the affinity of the acquired antibodiesto low nanomolar range. The performance appears to besimilar to natural repertoire-based libraries and has beengenerating clinical candidates. The affinity can be furtherimproved to picomolar range by randomizing the light-chainCDR residues by mimicking the natural diversity [59].Another affinity improvement strategy reported later is toco-evolve selected residues from heavy chain and light chainCDR, and was shown not only to improve affinity, but alsoimprove cross-species binding [64].

Further studies on restricted diversity was explored bygenerating libraries that randomized the same set ofpositions (with CDH-H3 length variations) as above, butincluded only four amino acids (YADS library; Tyr, Ala,Asp, and Ser) or even only two amino acids (YS library; Tyrand Ser) [65,66]. This demonstrates the minimal amino acidtypes that are sufficient to present a synthetic repertoirecapable of generating antibody for a target of interest.Detailed analysis of the interaction between antibody fromeither YADS or YS libraries showed that tyrosine dominatesthe direct interactions with antigens mostly through itsaromatic ring structure. Small residues, such as serine oralanine, appear to help scaffold the tyrosine in its bindingconformation [67]. The striking results illustrate thechemical diversity contained within as few as twowell-chosen amino acids, and explain the overabundance oftyrosine and serine in natural antibody repertoire and in theantigen-binding interface [68,69].

Ladner’s group at Dyax recently reported a new set ofsemisynthetic libraries [70]. The light chain library contains anatural repertoire of λ and κ light chains cloned fromindividuals with autoimmune diseases. The heavy chain isbuilt on a single-framework of VH3 – 23, which is the sameas DP47. The randomization scheme is based on analysis ofantibody structures and germline VH genes. Selectedresidues in CDR-H1 and -H2 are fully randomized(positions 31, 33, 35, 56 and 58), which is more diversethan natural antibodies. They reasoned that high diversitybeyond the scope of natural diversity in these positions isbeneficial because these residues are poised to contact theantigens. On the other hand some positions (position 50, 52and 52a) are restrictively randomized to mimic the naturalgermline diversity. They have a unique semisyntheticapproach in that they clone CDR-H3 from donors withautoimmune disease (a natural repertoire). By combiningthe natural diversity in light chain and CDR-H3, and thesynthetically designed diversity in CDR-H1 and -H2, theselibraries were able to identify high-affinity antibodies to fourtest antigens. This set of libraries is ready to generateantibody to therapeutic targets.

3. Targeting specific epitope by library design

One advantage of synthetic antibody libraries is that librarieswith custom-designed diversity can be made to target aparticular epitope or type of epitopes. One example is thelibraries made specifically for finding antibody that blockintegrin by imbedding the integrin binding motif(Arg-Gly-Asp) in CDR-H3 of the synthetic antibodylibraries [71]. Another example is work of G Winter’s group [72]

that can be applied to nonlinear epitope. They used thestructural information of a ligand (barstar) to model the threekey residues of the ligand for binding its partner (barnase).Synthetic libraries were designed to position these three keyresidues in a similar three-dimensional relationship on theantibody surface. With seven other fully randomizedpositions, they were subjected to selection for an antibody thatbinds to the partner in the same manner as its ligand. Blockingantibodies were found. Whether the antibody–receptorbinding is mediated exactly through the designed interactionis not known. However, the novel concept shows the potentialfor synthetic libraries to target specific epitope for therapeuticantibody development. Another example is designingantibody libraries to have different antigen combining sitetopography; this work was based on analysis of the naturalantibodies that bind each type of antigen (protein, peptide orhapten) [73,74]. Synthetic antibody libraries using theseprinciples demonstrate the advantage of antibody librarieswith either long CDR-L1 or with high diversity along the wallof a proposed binding cavity when targeting small peptide orhapten [75], respectively.

4. Different diversity targets different epitopes

Antigen molecules have different chemical properties on theirmolecular surface. It is reasonable to predict that someepitopes be more favourably targeted by certain types ofsynthetic antibody libraries. One example is the set of fourdominant antibodies to vascular endothelial growth factor(VEGF) identified from three synthetic antibody libraries. Allthree libraries are constructed on identical single-frameworkand all three randomize an identical set of heavy chain CDRresidues, leaving the light chain unrandomized [65,76]. Thedifference among these libraries is solely due to designeddiversity. The four dominant clones from these three sets oflibraries have significantly different epitopes (Figure 3). Theyare: G6 from nNNS libraries, B20 from nNVT library andtwo antibodies from the YADS library, YADS1 and 2.

In nNNS and nNVT libraries, the randomization inCDR-H3 was allowed to be either all 20 amino acids or 11amino acids, respectively (the 11 amino acids, Pro, His, Thr,Ala, Tyr, His, Gln, Asn, Asp, Cys, Arg, Gly and Ser, wereencoded with NVT.) The diversity in CDR-H1 and -H2positions was restricted to 3 – 15 amino acids mimicking thenatural diversity of human antibodies [59]. In YADS libraries,

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the diversity of all selected heavy chain CDR positions wasallowed for four amino acids: Tyr, Ala, Asp and Ser [65].

G6 and B20 initially bind at ∼ 1 or 300 nM (Kd) and wereaffinity improved by randomizing a select set of CDR residuesto mimic natural diversity to 20 pM and 12 nM, respectively.YADS1 and 2, initially binding at ∼ 5 – 10 µM (Kd) wereaffinity improved by randomizing the same set of light chainCDR residues with the four amino acids (YADS) to 2 and10 nM, respectively. Crystal structures showed that thestructural contacts (structural epitopes) on VEGF for the fourantibodies are clearly distinct, but with some overlaps(Figure 3). Interestingly, G6 and B20-4 (affinity-improvedB20) resemble the binding of VEGF receptor (VEGFR)1more than YADS1 and 2. In fact, G6 nearly superimposesVEGFR1 binding exactly. Functional mapping using alaninescanning mutagenesis of the VEGF surface further showedthat the binding of these two antibodies and VEGF receptorshare a set of common hotspot residues in the N-terminalhelix of VEGF [76]. In contrast, the epitopes of YADS1 andYADS2 resemble the epitope of the hyrbridoma-derivedbevacizumab [65]. YADS1 is especially similar to bevacizumabas they both centre the epitopes on the C terminal, theso-called 80’s loops of VEGF (Figure 3).

On the antibody side, the heavy chain CDRs dominate theinteraction for all four antibodies in a similar fashion. Theresidues at the randomized positions account for most of theburied surface [67,76]. The antibodies with the epitopes thatclosely mirror the receptor epitope are derived from nNNSand nNVT libraries, which have a more complex andnatural-mimicking repertoire. The designed diversity (up to3 × 1016) of nNNS and nNVT libraries significantly exceedsthe actual library size (1010), suggesting that there are manysolutions for the receptor-like epitopes on VEGF in thesetwo libraries.

Another example of how differently designed diversitytargets different epitope is found in two synthetic antibodiesfor DR5. One antibody, BDF1, is derived from librariessimilar to the nNNS library [77] and the second, YSd1, isderived from a set of binary libraries which contain onlytyrosine or serine in the diversity in the three heavy-chainCDR and light-chain CDR3 (YS libraries) [66]. Structuralstudies showed that two high-affinity antibodies from the twolibraries bind different epitopes with overlaps betweenthemselves and with the ligand for DR5, Apo2L [77]. All ofthese examples illustrate the point that different types ofchemical diversity in the synthetic antibody libraries willtarget different epitopes.

5. Epitopes of antagonistic antibodies

As combinatorial libraries prefer to target natural binding sitesof biomolecules, it is not difficult to find antibody that canblock the binding of natural ligand. However, blockingfunction is sometimes not sufficient to make the antibody anantagonist, and antagonist development is not always

straightforward. An antibody is bivalent, such that it bringstwo target molecules to close proximity, which is a commonmechanism to trigger a signaling action. If the antigen is a cellsurface receptor, it is possible in some cases that antibodiescould directly dimerize and activate signaling activity. On theother hand, if the target antigen is a ligand, either soluble orcell-surface-bound, it is possible in some cases that theantibody could indirectly dimerize its receptor and activatethe signaling.

Some antibodies to the TNF receptor family were shown toactivate receptor signaling, but the mechanism is notclear [78-82]. If an antagonistic antibody of this type ofmolecule is desired, it is important to be aware of the

Figure 3. Different types of diversity generate antibodieswith different epitopes on human VEGF. VEGF from thecrystal structures in complex with VEGFR1 (domain 2) and variousFabs are aligned and shown as a surface (in white). Residues thatare in contact with various binding receptors (< 4.5 Å) are shownin green. VEGF residues that are not conserved in mice arecoloured in magenta on the surface for VEGFR1. The structuralepitopes of VEGF for antibodies isolated from one type ofsynthetic libraries is similar to the epitope of VEGF receptor 1(domain 2) (VEGFR1-d2) (left box) [76], although the epitopes forthe antibodies from the other type of synthetic libraries is similarto the epitope for Fab of bevacizumab (right box) [65]. PDB codesfor VEGF in complex with VEGFR1, Fab of G6, B20-4,bevacizumab, YADS1 and YADS2 are 1FLT, 2FJG, 2FJH, 1BJI, 1TZHand 1TZI, respectively. The figure is courtesy of C Wiesmann.VEGF: Vascular endothelial growth factor; VEGFR: VEGF receptor.

VEGFR1-d2 Bevacizumab-Fab

G6-Fab

B20-4-Fab

YADS1-Fab

YADS2-Fab

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possibility of getting the opposite effect. A TNF receptorfamily molecule, such as BR3, is activated when oligomerizedon the trimeric ligand, B cell activating factor (BAFF) [83]. Inorder to obtain antagonist to BR3, both synthetic antibodyphage library and hybridoma approaches were utilized in arecent report [64]. Antibodies that block the interaction ofBAFF/BR3 were found from both methods. To assess theiractivities on the B cell, two kinds of functional assays wereperformed. One is an antagonistic activity assay whereantibody was added in the presence of activating level ofBAFF. The second is an agonist assay where no BAFF wasadded to test whether the antibody itself would turn on theBR3 signaling events. Phage-derived antibodies (CB and itsaffinity-improved variants, CB2 and CB3s) are shown to beantagonists, but a variant of a hybridoma-derived antibody(2.1 IgG) was agonistic. From mapping and structural studiesof binding epitopes, the antibody from phage libraries wasshown to mimic the BAFF ligand binding to BR3 by centringthe main interaction on the tip of a conserved β-hairpin loop(Figure 4A). It blocks BAFF binding by occupying an epitopeon BR3 that encompass all the BAFF-binding sites on BR3.On the other hand, the hybridoma antibodies centre theirepitopes on residues that are not conserved between humanand mouse BR3 and appear to have minimal or none overlapswith BAFF epitope (Figure 4A). In the absence of structures,the mechanism of how these antibodies block BAFF bindingis not clear. The discovery that one hybridoma antibody (2.1)is agonistic demonstrates the importance of epitopes inmaking an effective antagonist.

In some cases, it may be necessary to use monovalentantibody fragment such as Fab to block the targetedinteraction as pure antagonist; however, one drawback of Fabis that it will not be able to harness the Fc-mediated effectorfunction of the full-length antibody. There are many differentways to extend the serum half-life of Fab for therapeuticpurpose, for example, conjugation to polyethylene glycol(PEGylation) or fusion to albumin binding peptide [84-87].

6. Epitopes of agonistic antibodies

Binding epitope is also important for developing agonisticantibodies. Dimerization triggers signaling of many differentcell receptors [88]. However, bivalent crosslinking by antibodymay not be sufficient to activate the receptor. Human growthhormone receptor (GHR) is the hallmark example where theligand-mediated dimerization is elucidated at the molecularlevel [89]. There are many anti-GHR (ECD) antibodies, someof which block growth hormone and some of which do not.However, most can not activate signaling of the cells expressingfull-length GHR (G Fuh, unpublished data) [90]. There is onlyone antibody reported that can directly activate GHR. Thisantibody binds to a very different region from where humangrowth hormone binds [91]. There seem to be some particularrequirements for agonist epitopes and it is not straightforwardin predicting what would make an agonistic epitope.

Generating antibodies with many varieties of epitopes will helpin finding desired agonistic antibody.

7. Epitopes and Fc-mediated effector function

Antibody can activate effector responses by Fc interactionwith complement and with Fcγ receptors (FcγRs) [92-94].Fc-mediated effector function plays an important role inantibody therapeutic development [3,95]. Many are working onthe variation in Fc by mutation or glycosylation pattern (e.g.,reduction of fucosylation) to improve the Fc effectorfunction [96-100]. The most compelling evidence of the impactof effector function is the clinical association of rituximabefficacy in B cell mediated diseases with the FcγRIIIaallotypes [101-103]. The patients of the FcγRIIIa allotype withhigher affinity for Fc responded better to the antibodytreatment. It is strongly suggested that enhancing theFc–Fc receptor interaction improves the therapeutic effect.

Whether the binding epitope can play a role in theefficiency of Fc-mediated function is a new interesting area ofresearch. One recent study showed a set of anti-CD20antibodies generated by immunizing the human Ig transgenicxenomice (although rituximab was derived from immunizingregular mice) [104]. The authors claimed that the antibodieshave modified complement-dependent cytotoxicity comparedwith rituximab due to different interactions with CD20 [105].The effector function can also be important for an antibodythat targets soluble antigen. Where and how antibodies binddetermine the shape and size of the immune complex(especially if the antigen is multimeric). This could determinethe efficiency of the interaction with complement system andits function, such as the clearance of the immunecomplex [106,107]. The antibody-mediated clearance of theantigen target is particularly important for an antigen that isan infectious agent or toxin [108].

8. Epitopes of cross-species antibodies from synthetic antibody libraries

The cross human/mouse antibody can validate the targetingin mouse preclinical models before determining its costlydevelopment track. For example, it is important whendeveloping therapeutics for human cancer and the targetmolecule is a stroma-derived factor. The same is true if thetarget molecule is a receptor on host endothelial cellsresponding to angiogenesis modulator, for example,VEGFR2; the antibody to validate the targeting in mousemodels (implanted with human xenograft) would have to bedirected toward the mouse antigen. Ideally, it would becross-reactive to human antigen.

The in vitro process of antibody generation does not havethe tolerance mechanism as the in vivo immunization, so ithas the advantage over mice hybridoma in findingcross-species binding antibody, especially cross human/mouseantigen. The hybridoma process, using either regular rodents

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or xenomice, is restricted in their epitope finding by thetolerance mechanism. Mice with the orthologue gene deletionmay be needed in order to obtain the following: the antibodyto the murine antigen; the antibody to human antigen that ishighly homologous to its murine orthologue; or thehuman/murine cross-species antibodies. However, theresources and time needed to develop the knockout mice is

great and the knockout has to be viable. If immunization isthe only alternative, hamster or rabbit could be goodalternatives. There is a report showing that a certain strain ofrabbit is capable of generating a human/murine VEGFR2cross-binding antibody [109]; however, the immune tolerancemechanism in these animals can still limit the epitopes thatcan be targeted.

Figure 4. Cross-species BR3 binding of the synthetic antibody, CB [64]. (A) Epitopes are determined using alanine scanningmutagenesis of hBR3. Residues that are important for binding the phage library-derived CB2 (pink) or hybridoma-derived antibodies,11G9 (blue), 2.1 (orange) and 9.1 (green) and ligand BAFF (circled) [120], are highlighted on a tube representation of BR3 (1P0T) [83]. Thehybridoma antibodies bind mainly to non-conserved residues (underlined) of hBR3. CB2 and BAFF bind to the conserved residues andshare common binding sites. Based on structure of BR3 complexed with CB3s or BAFF, CB3s approaches and binds the conservedβ-hairpin loop in a similar manner as BAFF (blue arrow) (CB2 is the first affinity-improved variant and CB3s is the second affinity-improvedvariant of the original clone CB from phage libraries). (B) Paratopes on the CB3s for binding hBR3 or mBR3 are mapped by shotgunalanine mutatgenesis. The structural paratope for hBR3 are encirled with dotted line. Residues not-conserved between hBR3 and mBR3are shown with side chains (in green) and human/murine residues in the mBR3 paratope graph. Residues important for binding arecoloured in red (very important, F > 30), orange (important, 10 < F < 30) or yellow (modestly important, 3 < F < 10) on CB3s (as asurface) based the crystal structure of the complex of CB3s and hBR3. The F values represent the preference for a wild-type residue (overalanine mutant) based on the shotgun scanning method [121]. Residues coloured in purple are also very important, but the data werefrom a separate mutagenesis experiment. CB3s accommodates some region of hBR3 and mBR3 with identical binding energetics(encircle with an oval on hBR3 binding surface) and some region with different energetics. PDB code for BR3 in complex with BAFF orCB3s is 1P0T or 2HFG, respectively. The figure is courtesy of SG Hymowitz and MA Starovasnik.BAFF: B cell activating factor; hBR3: Human BR3; mBR3: Murine BR3.

HC LC

H31N

G36E

F > 3010 < F < 303 < F < 10

HC LC

A34S

B.

L28

V29

L38

R39

G36

P21

A22

11G911G9

9.19.12.12.1

CB2CB2

A.

BAFFBAFF

L38F

L27P

hBR3-binding functional paratope mBR3-binding functional paratope

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Bevacizumab is hybridoma-derived and provides anexample that shows the exquisite mechanism of in vivotolerance at the atomic level. Human and murine VEGF are> 85% identical. The structural and functional mapping ofhuman VEGF in complex with Fab of bevacizumab [76,110]

shows that the binding activity centres on the only residuewithin the epitope not conserved between the two species:Gly88. Murine VEGF has serine instead of glycine at thisposition. The tight packing of the bevacizumab around Gly88explains why serine, which has two additional non-hydrogenatoms (Cβ and O), would not fit in. In fact, mutating Gly88to alanine in human VEGF, which would add only onenon-hydrogen atom (Cβ) in between the two moleculestotally disrupted the interaction [76]. In contrast, G6 andB20-4, which are the phage library-derived antibodies toVEGF (described above), are crossreactive to human andmurine VEGF. They bind to two distinct but overlappingepitopes, and both epitopes are highly conserved and similarto epitopes for VEGF receptor (Figure 3).

Although VEGF is a proven target clinically for certain cancerand age-related macular degeneration, the cross-species bindingG6 and B20-4 are important to improve the understanding ofthe precise role of VEGF in angiogenesis related biologicalprocesses. These include the developmental processes andpathogenesis [111,112]. Some human tumour xenografts (grownin mice) were resistant to bevacizumab due to the involvementof host stroma-derived VEGF (murine VEGF), whichbevacizumab does not inhibit [113]. With the cross-speciesVEGF antibodies, more tumour types, including genetic murinetumour models, can now be studied to identify the tumourtypes that can be targeted, and the tumour models that are trulyresistant to the treatment of VEGF antibodies. Identification ofresistant tumour models is important because the factors thatcontribute to the resistance can then be investigated. Further,having these antibodies that bind distinct epitopes on VEGFalso allow investigation into the potential effect of epitopes onthe efficacy of anti-VEGF in tumour inhibition.

The crossreactivity depends on the molecules in question,and it is difficult to attain when there is low degree ofhomology. For example, the human and murine BR3 shareonly 57% identity. Only one of the clones (CB) originallyisolated from the synthetic antibody libraries by bindingmurine BR3 showed some marginal crossreactivity, withbarely detectable binding to human BR3 [64]. CDR residueswere subsequently mutated to improve affinity for murineBR3 (the binding for human BR3 was too weak to yield anyenrichment in phage selection). Among the affinity-improvedclones, it was observed that one type of mutation improvedaffinity for only mBR3, whereas the other type of mutationimproved binding to both human and murine BR3 tonanomolar affinity. The structural and functional studies ofthe interaction were performed to understand thecrossreactivity (Figure 4). It was concluded that the property ofcross-species binding by CB3s was achieved, first, by a hotspot binding interaction with the conserved residues in the

BR3 β-hairpin, and, second, by accommodating the rest ofthe molecule of both species through the changes made to theoriginal antibody. If the hotspot of the original antibody clonewere focused on murine BR3 residues that are not conservedwith human, it would have been difficult to evolve the clonesto become crossreactive. The cross-species anti-BR3 played avital role to expedite the target validation of BR3.

Targeting highly conserved and biologically importantepitopes have other potential benefits. Prediction is that theseepitopes are less likely to differ between individuals from genepolymorphism or to mutate in the disease states. For example,in tumour progression, mutation can occur to enhance thetumorigenesis and drug resistance or alter the target moleculeof interest by changing its binding toward the cognate ligand.However, if the biological function of the target molecule isimportant for the tumour to progress, the latter is less likely tooccur. Antibodies that target conserved epitopes, which areimportant for the target’s biological function, might haveincreased potential as successful therapeutics.

9. Expert opinion and conclusion

From the success of these synthetic antibody libraries, itappears that many different approaches with relatively defineddiversity can generate highly functional synthetic orsemisynthetic libraries. The common theme is a focus on fourfeatures: high diversity in amino acid composition and/orlength in CDR-H3; diversity in selected positions in otherCDRs; utilization of frameworks that are frequently employedin nature; and sufficiently large sized libraries (109 – 1010).The success and performances of the antibody libraries havebeen based on the affinities of acquired antibody as thecriteria to judge whether there is sufficient chemical diversityin the libraries. As affinity improvement process has becomequite robust and much less of a bottleneck, it is more useful iflibraries can find clones that bind to many different epitopes.This is important as it offers the opportunities to find the‘right’ clone to move forward in developing therapeutics. Acollection of synthetic antibody libraries with different andwell-defined diversity designs would be needed to be able toobtain a good mix of antibody leads that bind differently.

To develop a therapeutic antibody with synthetic antibodylibraries, it is a concern that if the sequence of antibody isunusual or ‘not-human like’, it will be immunogenic in patients.Immunogenicity of antibodies, however, is a complex issue. Theimmune system utilizes somatic mutations within the variabledomain, especially in CDRs, in addition to non-templatedinsertion and deletion within CDR-H3 in its repertoire; thismakes the definition of what is ‘human’ in the variable domainnot clearcut. The rate of immunogenic response to existingtherapeutic antibodies of different types, such as chimaeric,humanized or human, does not necessarily indicate what kind ofantibody is most ‘human’ [114]. For example, adalimumab isderived from natural repertoire libraries and is considered fullyhuman, but it raised immune reaction in a good portion

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(5 – 12%) of patients [115]. However, combined treatment ofadalimumab with methotrexate, an immunosuppressor,decreases the rate of antiadalimumab response to ∼ 1% [116].There are other examples where fully human proteins areimmunogenic in patients [117]; some are due to the poor proteinbehaviour such as aggregation. Among the factors that influencewhether antiantibody response will be an issue are: proteinbehaviour in large-scale production; the types of disease thetherapeutics are treating; dosing frequency; duration oftreatment; route of administration; and the presence of othertherapeutics in the treatment. In considering theimmunogenicity potential of synthetic antibodies where thehuman framework scaffolds are utilized, synthetically designingCDRs to mimic natural sequences is an attractive approach.Indeed, it is more preferable than using unnatural diversity.However, the ‘unnatural’ diversity can offer some opportunities,due to the unique chemical diversity that the libraries contain,and antibodies with different and perhaps desirable epitopes canbe preferably isolated. Choosing stable and easy-to-express

human framework scaffolds that are commonly used in natureto be the library template(s) contributes much to generateantibodies that behave well in large-scale production, and thiscould in turn reduce the potential of immunogenicity.

Synthetic antibody libraries have come of age. Definedtemplates and defined diversity increase the speed of discoveryand optimization of new antibodies. Cross-species antibodiesare becoming the tools of choice to validate therapeutictargeting. Different epitopes can be selectively targeted byusing libraries of different diversity designs. Increased varietyof epitopes will illuminate the relationship between epitopesand the function of antibody to modulate biological processesin vitro and in vivo. Thus, more choices of antibodies offeropportunities to find ones with the most beneficial features astherapeutics. Furthermore, the ability to produce Fab or IgGin large quantities allows researchers to dissect the moleculardetails of the binding interactions of these antibodies. Thisunderstanding will increase the potential of future librariesand future antibodies.

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• Good example of target validation with cross human/mouse antibody derived from synthetic antibody phage libraries.

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• Description of synthetic antibody libraries with highly restricted diversity.

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• Description of the correlation of CDR-L1 length and antigen size.

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• See [92].

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• Comprehensive review of the understanding of Fc receptor biology.

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• Review of the potential of dialing Fc-mediated effector function for therapeutic antibodies.

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•• The comprehensive mapping of Fc binding sites for Fc receptors and its implications.

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105. TEELING JL, MACKUS WJ, WIEGMAN LJ et al.: The biological activity of human CD20 monoclonal antibodies is linked to unique epitopes on CD20. J. Immunol. (2006) 177(1):362-371.

• Description of the potential effect of antibody binding sites on the efficiency of Fc-mediated function.

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• Description of how cross-species VEGF antibodies assisted the targeting of angiogenesis in diseases such as cancer.

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•• An excellent review of the immunogenicity issue for the different types of antibody therapeutics.

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AffiliationGermaine FuhGenentech, Inc., Department of Protein Engineering, 1 DNA Way, South San Francisco, CA 94080, USAE-mail: [email protected]

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Synthetic antibodies as therapeutics