Antibody engineering

Antibody engineering Leonard G. Presta G e n e n t e c h Inc, South San Francisco, California, USA Current research into antibody engineering stresses the design of constructs that have both scientific and medical significance. Highlights of the past year include several successful humanizations of non-human antibodies, in which a human antibody is created that possesses the same binding specificity as the non-human one, and phage display of antibodies. This review is also published in Current Opinion in Biotechnology 1992, 3:395-399. Current Opinion in Structural Biology 1992, 2:593-596

Introduction The antibody molecule possesses a variety of properties which make it exquisitely amenable to protein engineering. The natural molecule consists of four peptide chains, two light chains and two heavy chains, which form a symmetrical 'Y-shape'. The basic functions of the antibody reside in separate domains: antigen binding in the Fab fragments (the 'arms' of the Y) and etfector functions in the hinge and Fc portions (the 'base' of the Y). Not only are these functions separate, they are separable. For example, one can enzymatically (or using molecular biology techniques) detach an Fab domain from the rest of the antibody. Taking this one step further, the antigen-binding moiety, which consists of the variable light and heavy domains, can be engineered as a single-chain molecule [1]. One can even create bispecific antibodies in which two Fab fragments are covalently linked, each having a unique antigen specificity [2]. The brunt of antibody engineering research has dealt with the antigen-binding domains. The astounding diversity of the antibody repertoire lends itself to generating molecules with desired antigen specificity, which can then be exploited for applications in medicine, diagnostics'or the development of antibody catalysts. It is even more astounding that this immense diversity is contained in only six spatially adjacent loops, referred to as hypervariable regions or complementarity-determining regions (CDRs), which bind antigen. The CDRs can vary in sequence, size and conformation, all of which are accommodated by a framework relatively constant in both sequence and conformation. From an engineering standpoint, nature has provided a rather stable canvas upon which we can paint a variety of pictures.

Humanized antibodies Although human monoclonal antibodies hold promise as therapeutic and diagnostic tools, their generation has so

far remained elusive. Possible solutions include the introduction of a human immune system into mice [3], the generation of human immunoglobulin libraries in bacteria, and the humanization of non-human monoclonal antibodies. The use of non-human antibodies as therapeutic agents in humans is attended primarily by three problems. When challenged with the presence of a non-human antibody, the human immune system mounts a response which can reduce the therapeutic value of the non-human antibody. Further, therapeutic efllcacy may also be reduced because the non-human antibody is cleared from serum relatively more rapidly than human ones. Finally, nonhuman antibodies generally show only weak recruitment of effector functions (e.g. antibody-dependent cytotoxicity) which may be necessary for bioactivity of the antibody. There are essentially two categories of engineered antibodies which can replace non-human therapeutic antibodies and overcome these problems. A chimeric antibody entails substitution of an entire intact variable domain of a human antibody with that from a nonhuman one. In contrast, a humanized (reshaped) antibody includes only substitution of the six CDRs of a human antibody with those from a non-human antibody. Inclusion of some non-CDR residues (referred to as framework residues) may also be required in order to maintain proper CDR conformation [4]. Although technically a humanized antibody is a chimera, i.e. a hybrid of non-human and human sequences, the use of the terms humanized (reshaped) and chimeric can immediately differentiate the two constructs. In some recent publications, this delineation has become obscured by the interchangeable use of the two terms [5]. The technique of antibody humanization was pioneered by Winter and colleagues [6,7]. One method involves searching the database of human antibody sequences to find one which .is close to that of the non-human

Abbreviation CDR--complementarity-determining region.

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594 Engineeringand design antibody of interest. Transplantation of the non-human CDRs onto the human framework will then (hopefully) minimize structural differences that might affect CDR conformation and, consequently, binding to the target antigen. Co et al. [8] used this regimen to generate two humanized antibodies. After choosing human frameworks closest to the murine ones, however, they changed a number of residues in these human frameworks to other residues found more commonly in human sequences. In addition, a number of human framework residues were then substituted with residues from the murine antibodies, these being chosen using computer model-. ing of the antibodies. The affinity and bioactivity of the humanized antibodies were comparable to those of the original murine forms. Whereas Co et al. evaluated only a single version of each of their two humanized antibodies, Kettleborough and coworkers [9"'] tested several versions of a humanized antibody. Out of the six human framework residues that they substituted with murine residues, two had no effect, three had moderate effect and one a significant effect on antigen binding affinity. It is noteworthy that this group originally designed a humanized version without the aid of computer modeling and it failed to bind to the receptor. Subsequently, the use of a computer model to redesign their first version resulted in variants which did exhibit binding. Unfortunately, their best version exhibited an avidity for receptor which was only ~ 60% of that of the parent murine antibody. The necessity of considering framework residues was underscored in the work of Tempest et al. [10.]. Employing a human framework closest to the murine one, an initial version, in which Only CDRs were transferred, exhibited no binding. Subsequently, the replacement of four consecutive residues in the variable heavy domain (residues 91-94) of the initial construct generated a version with reduced affinity but comparable in vitro and in vivo bioactivities. The studies described above used a single human framework per humanized antibody. Gorman et al. [ 11. ] compared the effects of two different human heavy-chain frameworks, with the same six CDRs, in binding human CD4. One framework (derived from KOL, with 72 % sequence identity between murine and human forms) provided a threefold reduction in binding to antigen, whereas the second (derived from NEW, with 47 % sequence identity) showed poor binding even at high concentrations. Though one might interpret this result as suggesting that the 'closest human sequence' approach is the one to take, a single incorrect framework residue can have a significant effect on the binding ability of the humanized version [9"]. As effects of framework residues were not considered in this study, drawing such a conclusion is inadvisable. In addition, searching for the closest human sequence does not always produce a sequence which contains all the correct framework residues [9.o]. Shalaby et al. [12..] reported the development of a biologically active, fully humanized bispecific F(ab') 2 fragment. This molecule consists of two unique Fab fragments covalently linked at their carboxyl termini. In contrast to the regimen followed by all other groups

reviewed herein, i.e. where the human sequence closest to the murine is used, this group used a single framework derived from human consensus sequences to design both humanized Fabs. In an interesting theoretical study, Padlan [13"] suggested a novel approach to humanization. All the studies described herein involved transfer of CDRs and ap: propriate framework residues from murine to human frameworks. Padlan proposed replacement of the exposed (surface) residues in the murine framework with human residues, suggesting that this method would retain the internal packing necessary for maintenance of CDR conformation. Although this approach is enticing, these types of constructs would have a greater proportion of murine residues, even though these would be buried. The immunogenicity of exposed residues is accepted and is, of course, the rationale behind humanization. The possible immunogenicity of buried residues, however, for example after degradation and processing, remains uncertain. Because humanized antibodies are a relatively new idea, few clinical studies using them or, more importantly, comparing their performance against that of the parent murine antibodies have been reported. Recent studies on humanized anti-Tac antibody have begun to correct this shortfall [14,15..]. In monkeys, the humanized antiTac exhibited lower immunogenicity and a longer mean serum half-life than the murine antibody. Another study reported in vivo targeting of a tumor using a humanized antibody conjugated with a stable radiolabel [16]. Although none of the seven patients developed responses against the humanized antibody, three showed an immune response against the conjugate. Such results are strong encouragement for continued efforts to humanize therapeutically relevant antibodies and antibody fragments.

Phage display of antibodies The recently developed technique of displaying proteins on bacteriophage has added an exciting new dimension to antibody engineering. These systems allow linkage of a protein to the DNA encoding the protein and, hence, provide a method for rapid screening for desired properties and subsequent retrieval of the sequence of the protein. The study of Clackson et aL [17] used a library of murine heavy and kappa light chains, derived from mice immunized with a small-molecule hapten, to screen for strong binders. In contrast, Marks et aL [18..] used a library derived from human peripheral blood lymphocytes from unimmunized donors. After screening their library against turkey eggwhite lysozyme, bovine serum albumin and a small-molecule hapten, they isolated antibody fragments which exhibited both good affinity and specificity to the lysozyme and hapten. Garrard et aL [19"] diluted a humanized antibody with control (non-binding) antibodies, all on phage, and showed that the humanized antibody could be excised from a million-fold excess of non-binding antibodies in only two rounds of sorting. Further, they showed that the strong-binding humanized antibody could also be preferentially enriched from mixtures of variants with lower affinities for the antigen.

Antibody engineeringPresta 595 Taken together, these reports suggest new methods not only for obtaining human antibodies but of antibody engineering. For example, if one had a human (or murine) antibody which exhibited antigen specificity but only tolerable binding affinity, phage display could be used to alter some residues in the CDRs and then rapidly screen for variants with improved binding.

Catalytic antibodies The vast repertoire of the immune system can be used to recruit an antibody with specificity for a small molecule hapten and this antibody can then be used to catalyze a chemical reaction for which the hapten represents the transition-state [20..]. During the past year, antibodies have been reported which catalyze imine formation [21], cia--trans isomerization [22], acyl transfer [23], transesterification [24], stereoselective formation of organofluorine compounds [25] and enantiofacial protonation during hydrolysis of enol esters [26]. Crystal structures of some of the numerous catalytic antibodies are anticipated, permitting further appreciation of the relationship between the antibody structure and the catalytic event. Until these structures become available, other techniques are being used. For example, enzymatic analysis has been used to suggest a catalytic role for a tyrosine residue during hydrolysis of phenyl acetate by a catalytic antibody [27.]. Further, introduction of a catalytic antibody with chorismate mutase activity into a strain of yeast deficient in this enzyme [28,29.] can be used in screening site-directed mutants of the catalytic antibody for improved efficacy, as well as dissecting the residues involved in catalysis.

Miscellaneous studies A small group of unique and interesting reports emerged over the past year. Biltetta et al. [30 o] introduced a 12residue peptide as the CDR3 of a murine heavy chain. Rabbits and mice immunized with this engineered antibody produced anti-antibodies specific not only for the engineered loop but also for a corresponding synthetic peptide and the antigen from which the tetrapeptide repeat was taken. It is notable that using the engineered antibody a response could be induced in animals which did not respond to the corresponding synthetic peptide. Such constructs could prove efficaceous in eliciting antibody-based vaccines. One of the purposes for humanizing a non-human antibody is to increase its half-life in the human system. An alternative approach was reported in which a murine monoclonal antibody was chemically modified with methoxypolyethylene glycol, which diminished its clearance rate and immunogenicity [31]. Saragovi et al. [32"] reported the design and synthesis of a mimetic derived from an antibody CDR. The CDR loop was used as the basis for design of cyclic peptide analogs. These analogs were tested for activity and this information used, in turn, to design a cyclic organic molecule which showed modest inhibition in a biologi-

cal assay. Though not exactly antibody engineering, this study underscores the possibility of taking information from antibody engineering, e.g. concerning the importance of specific residues or CDR conformation, and applying it to the design of a therapeutic small molecule to replace a therapeutically relevant antibody.

Conclusion Within the past year, antibody engineering has continued to expand in its scientific and utilitarian aspects. Although humanization of therapeutically important antibodies will inevitably be replaced by novel methods of generating human antibody libraries, insight into antibody structure-function relationships is being enhanced by this technique. Likewise, continued interest in catalytic antibodies and dissection of the factors influencing the reactions will augment more traditional chemical methods. One relatively untapped area is that of immunoglobulin effector functions. As information regarding the portions of immunoglobulins involved in these functions and differences among the immunoglobulin classes and subclasses emerges, antibody engineering can move towards creating novel constructs with designed binding specificity as well as designed effector functions,

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •. of outstanding interest 1.

BIRD RE, WALKER BW: Single Chain Antibody Variable Regions. Trends Biotechnol 1991, 9:132-137.

2.

FANGERMW, SEGAL DM, ROMET-LEMONNEJ: Bispecific Antibodies and Targeted Cellular Cytotoxicity. I m m u n o l Today 1991, 12:51-54.

3.

DUCHOSALMA, EMING SA, FISCHER P, LETURCQ D, BARBAS CF II1, MCCONAHEY PJ, CAOTHIEN RH, THORNTON GB, DIXON FJ, BURTON DR: Immunization of hu-PBL-SCID Mice and the Rescue of H u m a n Monoclonal Fab Fragments T h r o u g h Combinatorial Libraries. Nature 1992, 355:258-262.

4.

CHOTHIAC, LESK AM, TRAMONTANOA, LEVITF M, SMITH-GILL SJ, AtR G, SHEmFV S, PADtAN F.A, DAVIES D, TUUe WR, ~r A/= Conformations of lmmunoglobulin Hypervariable Regions. Nature 1989, 342:877-883.

5.

BUI£NS F, VANDAMME A, BERNAR H, NELLES L, LIJNEN RH, COLLEN D: Construction and Characterization of a Functional Chimeric M u r i n e - H u m a n Antibody Directed Against H u m a n Fibrin Fragment-D Dimer. Eur J Biochem 1991, 195:235 242.

6.

JONES PT, DEAR PH, FOOTE J, NEUBERGER MS, WINTER G: Replacing the Complementarity-determining Regions in a H u m a n Antibody with Those from a Mouse. Nature 1986, 321:522-525.

7.

REICHMANNL, CLARK M, WALDMANNH, WINTER G: Reshaping H u m a n Antibodies for Therapy. Nature 1988, 332:323-329.

8.

Co MS, DESCHAMPSM, WHITLEY RJ, QUEEN C: Humanized Antibodies for Antiviral Therapy. Proc N a t l A c a d Sci USA 1991, 88:2869-2873.

9.

KETIZEBOROUGHCA, SALDANHAJ, HEATH VJ, MORRISON CJ, BENDIGMM: Humanization of a Mouse Monoclonal Antibody

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Engineering and design by CDR-grafting: t h e Importance of Framework Residues on Loop Conformation. Protein Eng 1991, 4:773-783. This study is noteworthy because several humanized versions were generated to test the importance of framework residues on antigen binding and because computer modeling aided in redesigning a non-binding version. 10. *

TEMPESTPR, BREMNERP, LAMBERTM, TAYLORG, FURZEJM, CARR FJ, HARMS WJ: Reshaping a H u m a n Monoclonal Antibody to Inhibit H u m a n Respiratory Syncytial Virus Infection in vivo. Biotechnology 1991, 9:266-271. Of interest is the failure to bind antigen of a version in which onty CDR residues were transferred from a murine antibody to a human framework. This exemplifies the need to consider framework residues when humanizing antibodies. 11.

GORMAN SD, CLARK MR, ROUTLEDGE EG,

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WALDMANNH: Reshal6ing a Therapeutic CD4 Antibody. Proc

20. •,

LERNERRA, BENKOWC SJ, SCHULTZ PG: At the Crossroads of Chemistry and Immunology: Catalytic Antibodies. Science 1991, 252:659~,-6T. An excellent review of the field. 21.

COCHRANAG, PHAM T, SUGASAWARAR, SCHULTZPG: Antibodycatalyzed BImolecular Imine Formation. J Am Chem Soc 1991, 113:6670--6672.

22.

JACKSONDY, SCHULTZ PG: An Antibody-catalyzed cis-trans Isomerization Reaction. J A m Chem Soc 1991, 113:2319-2321.

23.

JANDAKD, WE1NHOUSE MI, DANON T, PACEU2 KA, SCHLOEDER DM: Antibody Bait and Switch Catalysis: a Survey of Antigens Capable of Inducing Abzymes w i t h Acyl-transfer Properties. J A m Chem Soc 1991, 113:5427 5434.

24.

WIRSCHINGP, ASHLEYJA, BENKOVlC SJ, JANDA KD, LERNER RA: An Unexpectedly Efficient Catalytic Antibody Operating by Ping-Pong and Induced Fit Mechanisms. Science 199l, 252:680-685.

25.

K1TAZUMET, LIN JW, TAKEDA M, YAMAZAKIT: Stereoselective Synthesis of Fluorinated Materials Catalyzed by an Antibody. J Am Chem Soc 1991, 113:2123-2126.

26.

FujII I, LERNERRA, JANDA KD: Enantiofacial Protonation by Catalytic Antibodies. J A m Chem Soc 1991, 113:8528-8529.

COBBOLD SP,

Natl Acad Sci USA 1991, 88:4181-4185. The use of two different h u m a n heavy-chain frameworks gave quite dif ferent results. The binding of one framework was only slightly reduced compared with the murine antibody, whereas the other showed very poor binding.

12. ••

SHAIABYMR, SHEPARDHM, PRESTA L, RODRIGUES ML, BEVERLEY PCL, FELDMANN M, CARTER P: Development of Humanized Bispecific Antibodies Reactive with Cytotoxic Lymphocytes and T u m o r Cells Overexpressing t h e HER2 Protooncogene. J Exp Med 1992, 175:217-225. The first report of a fully humanized bispecific antibody fragment. 13. ••

PADLANEA: A Possible Procedure for Reducing the Iramunogenicity of Antibody Variable Domains While Preserving Their Ligand-binding Properties. Mol Immunol 1991, 28:489-498. An intriguing alternative method of humanization, which requires further experimentation to evaluate its potential. 14.

Striking evidence that phage display can be used to screen, retrieve and enrich antibodies for desired antigen-binding properties.

27. •

MARTINMT, NAPPER aD, SCHULTZ PG, REES AD: Mechanistic Studies of a Tyrosine-dependent Catalytic Antibody. Bic~ chemistry 1991, 30:9757-9761. Enzymatic analysis of a catalytic antibody and a suggestion that a tyrosine residue of the antibody may be essential for catalysis. 28.

BROWNPS JR, PARENTEAUGL, DIRBAS FM, GARSiARJ, GOLDMAN CK, BUKOWSKIMA, JUNGHANSRP, QUEEN C, HAKIMIJ, BENJAMIN WR, ET AL: Anti-Tac-H, a Humanized Antibody to the Interleukin 2 Receptor, Prolongs Primate Cardiac AUograft Survival. Proc Natl Acad Sci USA 1991, 88:2663 2667.

29. •

HAKIMiJ, CHIZZONtTE R, LUKE DR, FAMILLETI1 PC, BAILON P, KONDASJA, PILSON KS, [,IN P, WEBER DV, SPENCE C, ET AL.: Reduced l m m u n o g e n i c i t y and Improved Pharmacokinetics of Humanized Anti-Tac in Cynomolgus Monkeys. J lmmunol 1991, 147:1352-1359. The most thorough published clinical data yet available for a humanized antibody. A good example of the usefulness of humanized antibodies as possible therapeutic agents.

30. •

15. ••

16.

HIRD V, VERHOEYENM, BADLE~YRA, PRICE D, SNOOK D, KOSMAS C, GOODEN C, BAMIASA, MEARESC, LAVENDERJP, ETAL.: T u m o r Localisation with a Radioactivity Labelled Reshaped H u m a n Monoclonal Antibody. B r J Cancer 1991, 64:911-914.

17.

CLACKSONT, HOOGENBOOMHR, GRIFFITHSAD, WINTERG: Making Antibody Fragments Using Phage Display Libraries. Nature 1991, 352:624-628.

18. ,,•

MARKSJD, HOOGENBOOM HR, BONNERT TP, MCCAFFERTYJ, GRIFFITHSAD, WINTER G: By-passing Immunization. H u m a n Antibodies from V-Gene Libraries Displayed on Phage. J Mol Biol 1991, 222:581-597. This technique may replace current technology in deriving h u m a n or humanized monoclonal antibodies. 19. •

GARRARDLJ, YANG M, O'CONNELL MP, KELLEYRF, HENNER DJ: Fab Assembly and Enrichment in a Monovalent Phage Display System. Biotechnology 1991, 9:1373-1377.

BOWDISHK, TANG Y, HICKS JB, HILVERT D: Yeast Expression of a Catalytic Antibody with Chorismate Mutase Activity. J Biol Chem 1991, 266:11901-11908.

TANGY, HICKSJB, HILVERTD: In vivo Catalysis of a MetaboUcally Essential Reaction by an Antibody. Proc Natl Acad Sci USA 1991, 88:8784-43786. Description of the use of a catalytic antibody with chorismate mutase activity in a yeast strain deficient in the enzyme. Such systems may be useful in screening site-directed mutants. B1LLET1"AR, HOLLINGDALEMR, 7~,NETr~ M: Immunogenicity of an Engineered Internal Image Antibody. Proc Natl Acad Sci USA 1991, 88:4713-4717. A novel approach which involves incorporating a 'foreign' peptide epi tope as part of an antibody CDR, and using this to elicit antibodies against the antigen from which the foreign sequence was taken. 31.

KITAMURAK, TAKAH&SHIT, YAMAGUCHIT, NOGUCHIA, NOGUCHI A, TAKASHINA K, TSURUM1 H, INAGAKE M, TOYOKUNI T, HAKOMORI S: Chemical Engineering of the Monoclonal Antibody A7 by Polyethylene Glycol for Targeting Cancer Chemotherapy. Cancer Res 1991, 51:4310-4315.

32.

SARAGOWHU, FITZPATRICKD, RAKTABUTRA, NAKANISH1H, KAHN

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M, GREENE MI: Design and Synthesis of a Mimetic from

an Antibody Complementarity-determining Region. Science 1991, 253:792-795. The use of antibody structure information to design small molecules of possible therapeutic utility is illustrated.

LG Presta, Department of Protein Engineering, MS 27, Genentech Inc, 460 Point San Bruno Blvd, South San Francisco, California 94080, USA.