Human Antibody Bispecifics through Phage Display Selection

Communication Cite This: Biochemistry XXXX, XXX, XXX−XXX

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Human Antibody Bispecifics through Phage Display Selection Ansha Luthra,†,‡ David B. Langley,† Peter Schofield,†,‡ Jennifer Jackson,† Mahmoud Abdelatti,† Romain Rouet,† Damien Nevoltris,† Ohan Mazigi,†,‡ Ben Crossett,§ Mary Christie,∥,∇ and Daniel Christ*,†,‡ †

Garvan Institute of Medical Research, 384 Victoria Road, Darlinghurst, Sydney, New South Wales 2010, Australia Faculty of Medicine, St Vincent’s Clinical School, The University of New South Wales, Darlinghurst, Sydney, New South Wales 2010, Australia § Sydney Mass Spectrometry, Charles Perkins Centre, The University of Sydney, Camperdown, Sydney, New South Wales 2006, Australia ∥ Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, New South Wales 2010, Australia

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S Supporting Information *

antibody VH variable domains on phage (Figure 1A). We first studied the variable domain of clone 4D5 derived from the human antibody therapeutic Trastuzumab (Herceptin).12 Using error-prone PCR13 to introduce random mutations into the 4D5 domain, we generated a repertoire of 2 × 108 VH variants. This phage repertoire was then incubated with 10 μM soluble 4D5 VL domain (cognate), which had been labeled with a PEO4 biotin affinity tag. Alternatively, the repertoire was incubated with 50 μM unlabeled VL competitor domain in addition to the labeled cognate domain. For this purpose, we utilized the DPK9 κ germline domain as a competitor. This domain closely resembles the 4D5 VL domain, from which it differs at hypervariable CDR positions;12,14 with the majority of human antibody therapeutics falling into the Vκ subclass,15 it also represents an excellent general choice of competitor VL domain. After three rounds of selection using 1011 input phage particles, 4D5 VH sequences were determined: this revealed a strong enrichment of mutations at a positional hotspot at position 100b (numbering according to Kabat)16 (Figure 1B, lower panel). Hotspot identification was strongly dependent on the presence of the DPK9 competitor domain during the selection process, with no detectable enrichment observed in the absence of a competitor (Figure 1B, upper panel). Position 100b is located at the C-terminal base of the complementarity determining region 3 (VH CDR3). To investigate the structural implications of the observed hotspot mutations, we determined the structure of WT 4D5 in a Fab format. (Protein Data Bank accession code: 6MH2. No crystals were observed for mutant A100bV Fab.) Crystals were obtained in space group P1, diffracting to 2.8 Å (Table S1). In the 4D5 Fab structure, the WT alanine residue at position 100b projects toward, but only partially fills, an intrinsic pocket lined by the side chains of residues A34, Y36, L46, Y49, and H91 of the 4D5 VL domain (Figure 1C). Structural analysis suggests that the selected mutations in the 4D5 VH (A100bV, A100bT, and A100bP) optimize interactions with this pocket

ABSTRACT: We developed a repertoire approach to generate human antibody bispecifics. Using phage display selection of antibody heavy chains in the presence of a competitor light chain and providing a cognate light chain with an affinity handle, we identified mutations that prevent heavy/light chain mispairing. The strategy allows for the selection of human antibody chains that autonomously assemble into bispecifics.

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ntibodies with a specificity for two different targets are preferred for many therapeutic applications.1 However, the production of bispecifics is complicated by the fact that antibodies of the commonly used IgG isotype are complex multidomain proteins, formed by heavy and light chains. Efforts to introduce dual specificities into a single antibody commonly result in chain misassembly.1 A strategy maintaining the native antibody structure was developed over 20 years ago, focusing on the introduction of complementary mutations in the CH3 domain of each heavy chain.2 This well-validated “knobs-into-holes” strategy promotes the heterodimerization of heavy chains, thereby enabling dual heavy chain specificities within a single antibody molecule.3,4 However, this approach does not eliminate the problem of potentially incorrect heavy chain−light chain pairings. Several elegant approaches to the heavy chain−light chain mispairing problem have been proposed, including constant domain fusions,5 chemical ligation,6 domain swaps,7 computationally reshaped Fab interfaces,8 and the use of common light9 or a common heavy10 chain. However, all of these rational approaches require either non-natural linkers, the introduction of a large number of changes in conserved antibody regions (which might increase immunogenicity) or the restriction of chain diversity (which might reduce affinity and epitope coverage).11 Here we develop a different strategy based on repertoire selection and outline a phage display approach to overcome the heavy chain−light chain misassembly problem. To address this issue, we developed a high-throughput selection system that is based on the display of human © XXXX American Chemical Society

Received: January 15, 2019 Revised: March 1, 2019

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DOI: 10.1021/acs.biochem.9b00037 Biochemistry XXXX, XXX, XXX−XXX

Communication

Biochemistry

Figure 1. (A) Selection for improved antibody variable domain heavy−light pairing (VH-VL) by phage display. Display of antibody VH repertoire on the surface of phage pIII coat protein, followed by incubation with soluble biotinylated cognate antibody VL in the presence of excess VL competitor, followed by affinity capture and DNA sequencing. (B) Mutational hotspot at position 100b (in red) as revealed by selection in the presence of VL competitor. Alanine to valine substitution A100bV was the most frequently observed substitution. (C) Structure determination of VH WT in a Fab antibody fragment format and structural model of optimized VH-VL interface of the V100b mutant.

by introducing slightly larger side chains as an alanine replacement, resulting in an improved heavy−light surface complementarity (Figure 1C, Figure S1). As a result, the affinity of the cognate 4D5 heavy−light pairing was increased from 7 μM to 980 nM for the frequently observed A100bV variant (Figure 2A). In marked contrast, this pocket was not observed in the DPK9 competitor domain, and the affinity of the noncognate A100bV/DPK9 pair was considerably decreased (from 3 to 11 μM). Importantly, antigen binding was fully retained for the analyzed mutant (Figure S2). Thus, by using a combination of both negative and positive selection on phage, we increased the selectivity of the desired VH-VL assembly by more than 20-fold in a single domain format for A100bV. In comparison, the less frequently observed A100bT and A100bP mutations decreased the affinity for competitor VL, but did not detectably increase the affinity for cognate VL (Figure 2A, Figure S3). We next hypothesized that, due to the large observed increase in specificity, the single A100bV mutation might be sufficient to steer the assembly of heavy−light pairs, not only in the context of single antibody domains but also in the context of full length monoclonals (Figure 3A). For this experiment, we focused on hIgG1, by far the most commonly used isotype in human antibody therapeutics. As anticipated, we observed no detectable monospecific Trastuzumab when coexpressing Trastuzumab 4D5 heavy and light chains in the presence of the DPK9 competitor light chain, with IgG product containing

Figure 2. Characterization of optimized VH domains in a single domain format as determined by biolayer interferometry. (A) Kinetic and thermodynamic binding constants of 4D5 VH variants (magenta) to cognate VL 4D5 domain (light magenta) as well as VL DPK9 competitor (yellow). The A100bV hotspot mutation increases the affinity of the cognate interaction, while simultaneously decreasing the affinity for the noncognate competitor VL.

either the DPK9 light chain only or a mixture of DPK9 and the cognate 4D5 light chain (Figure 3B,C, top panels). However, this was not observed when expressing the optimized A100bV heavy chain; here, the ratio of incorrectly to correctly B

DOI: 10.1021/acs.biochem.9b00037 Biochemistry XXXX, XXX, XXX−XXX

Communication

Biochemistry

Figure 3. Characterization of optimized A100bV VH domain in a human IgG1 format. (A) Production of human IgG1 through coexpression of two different light chains in Expi293 cells. 4D5 heavy chain (magenta), 4D5 cognate light chain (light magenta), DPK9 competitor light chain (yellow), and anti-PD1 heavy and light chains (purple) shown. (B) Deconvoluted mass spectra of the assembled IgG products. (C) Variant yield as determined by mass spectrometry.

assembled chains was fully reversed, with no competitor-only IgG product observed (Figure 3B,C, center panel). These results illustrate that by introducing a single mutation in the heavy chain hypervariable region 3, the proportion of cognate heavy−light chain pairs can be increased from zero to more than 70 percent. In a further step, we coexpressed the optimized Trastuzumab 4D5 A100bV heavy chain in combination with the heavy chain of the anti-PD1 monoclonal Pembrolizumab (Keytruda),17 as well as the two respective light chains (designated L2 and L1, respectively). To induce heavy chain heterodimerization, we inserted previously described mutations into the CH3 domains of each pair. The corresponding two heavy chains and two light chains were cotransfected into Expi293 cells, purified by protein G chromatography and analyzed by mass spectrometry. (Figure 3B,C, lower panel). This revealed that the vast majority of the antibody preparation (around 85%) consisted of the correctly assembled bispecific antibody. We then investigated the binding of this PD1xHER2 bispecific and parental PD1 (Pembrolizumab) and HER2 (Trastuzumab) antibodies to a surface containing the immobilized PD1 antigen, followed by addition of the HER2 antigen. As outlined in Figure 4, binding to both antigens was observed for the PD1xHER2 bispecific antibody, but not the parental monospecific antibodies. In summary, while the selection of antibodies with optimized antigen interactions has been a mainstay of biotechnology for over 30 years,18−21 combinatorial strategies for the optimization of the VH-VL interaction had so far remained elusive. The lack of specific VH-VL assembly results in heavy−light mispairing and has greatly hindered the generation of more complex antibody formats, such as human bispecifics. Here we outline a repertoire strategy on the phage that allows for the selection of optimized antibody VH-VL pairs. By using both positive and negative selection pressures, this strategy allows for the identification of

Figure 4. Dual antigen binding of the optimized A100bV VH domain in human IgG1 format (top), as well as anti-PD1 (middle) and antiHER2 (lower) mAbs as determined by biolayer interferometry.

optimized antibody domains with superior chain specificity. When coexpressed in Expi293 cells, these engineered domains autonomously assemble into human bispecifics. Unlike previous approaches, the strategy outlined here does not rely on non-natural chain connectivity or extensive mutation of otherwise conserved antibody framework regions. Indeed, in the case of the 4D5 Trastuzumab VH domain analyzed here, a single point mutation at the base of CDR3 was sufficient to increase the specificity of the VH-VL interaction by more than 20-fold, highlighting the power of selection approaches. Further improvements in specificity could conceivably be achieved through a combination with rational strategies and/or by applying the selection strategy to additional domains (such as CH1-CL). Indeed, the unbiased and selection-based nature of the approach may be particularly suitable for such combination strategies. In addition to IgG formats, improved VH-VL interactions are also likely to be highly beneficial for antibody fragments including BiTEs, Fv, and scFv fragments, which often suffer from aggregation and undesired oligomerization behaviors. We conclude that the C

DOI: 10.1021/acs.biochem.9b00037 Biochemistry XXXX, XXX, XXX−XXX

Communication

Biochemistry

Engineering Bifunctional Antibodies with Constant Region Fusion Architectures. J. Am. Chem. Soc. 139, 18607−18615. (6) Kim, C. H., Axup, J. Y., Dubrovska, A., Kazane, S. A., Hutchins, B. A., Wold, E. D., Smider, V. V., and Schultz, P. G. (2012) Synthesis of bispecific antibodies using genetically encoded unnatural amino acids. J. Am. Chem. Soc. 134, 9918−9921. (7) Schaefer, W., Regula, J. T., Bahner, M., Schanzer, J., Croasdale, R., Durr, H., Gassner, C., Georges, G., Kettenberger, H., Imhof-Jung, S., Schwaiger, M., Stubenrauch, K. G., Sustmann, C., Thomas, M., Scheuer, W., and Klein, C. (2011) Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies. Proc. Natl. Acad. Sci. U. S. A. 108, 11187−11192. (8) Lewis, S. M., Wu, X., Pustilnik, A., Sereno, A., Huang, F., Rick, H. L., Guntas, G., Leaver-Fay, A., Smith, E. M., Ho, C., HansenEstruch, C., Chamberlain, A. K., Truhlar, S. M., Conner, E. M., Atwell, S., Kuhlman, B., and Demarest, S. J. (2014) Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. 32, 191−198. (9) Carter, P. (2001) Bispecific human IgG by design. J. Immunol. Methods 248, 7−15. (10) Fischer, N., Elson, G., Magistrelli, G., Dheilly, E., Fouque, N., Laurendon, A., Gueneau, F., Ravn, U., Depoisier, J. F., Moine, V., Raimondi, S., Malinge, P., Di Grazia, L., Rousseau, F., Poitevin, Y., Calloud, S., Cayatte, P. A., Alcoz, M., Pontini, G., Fagete, S., Broyer, L., Corbier, M., Schrag, D., Didelot, G., Bosson, N., Costes, N., Cons, L., Buatois, V., Johnson, Z., Ferlin, W., Masternak, K., and KoscoVilbois, M. (2015) Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG. Nat. Commun. 6, 6113. (11) Rouet, R., and Christ, D. (2014) Bispecific antibodies with native chain structure. Nat. Biotechnol. 32, 136−137. (12) Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B., Henner, D., Wong, W. L., Rowland, A. M., Kotts, C., Carver, M. E., and Shepard, H. M. (1992) Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc. Natl. Acad. Sci. U. S. A. 89, 4285− 4289. (13) Cadwell, R. C., and Joyce, G. F. (1992) Randomization of genes by PCR mutagenesis. Genome Res. 2, 28−33. (14) Cox, J. P., Tomlinson, I. M., and Winter, G. (1994) A directory of human germ-line V kappa segments reveals a strong bias in their usage. Eur. J. Immunol. 24, 827−836. (15) Jain, T., Sun, T., Durand, S., Hall, A., Houston, N. R., Nett, J. H., Sharkey, B., Bobrowicz, B., Caffry, I., Yu, Y., Cao, Y., Lynaugh, H., Brown, M., Baruah, H., Gray, L. T., Krauland, E. M., Xu, Y., Vasquez, M., and Wittrup, K. D. (2017) Biophysical properties of the clinicalstage antibody landscape. Proc. Natl. Acad. Sci. U. S. A. 114, 944−949. (16) Kabat, E., Wu, T. T., Perry, H. M., Kay, S., and Gottesman, C. F. (1992) Sequences of Proteins of Immunological Interest, 5 ed., DIANE Publishing. (17) Kwok, G., Yau, T. C., Chiu, J. W., Tse, E., and Kwong, Y. L. (2016) Pembrolizumab (Keytruda). Hum. Vaccines Immunother. 12, 2777−2789. (18) Marks, J. D., Hoogenboom, H. R., Bonnert, T. P., McCafferty, J., Griffiths, A. D., and Winter, G. (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581−597. (19) McCafferty, J., Griffiths, A. D., Winter, G., and Chiswell, D. J. (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552−554. (20) Huse, W. D., Sastry, L., Iverson, S. A., Kang, A. S., Alting-Mees, M., Burton, D. R., Benkovic, S. J., and Lerner, R. A. (1989) Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246, 1275−1281. (21) Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341, 544−546.

selection strategy outlined here greatly improves the generation of bispecifics and other complex human antibody formats.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.9b00037. Additional experimental methods, X-ray data collection, processing, and model statistics, VL surface differences, equilibrium binding affinities and kinetic association and dissociation constants, and expression of Herceptin WT IgG and Herceptin A100bV IgG variant (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Romain Rouet: 0000-0003-4210-9613 Daniel Christ: 0000-0002-7313-3977 Present Address ∇

School of Life and Environmental Sciences, The University of Sydney, New South Wales, 2006, Australia Funding

This work was supported by the Program Grant 1113904, the Project Grant 1148051, the Development Grants 1113790 and 1076356, and the Fellowship 105146 from the National Health and Medical Research Council (NHMRC) and the Grants 160104915, 160100608, and 140103465 from the Australian Research Council (ARC). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.L. performed experiments. M.A. constructed phage repertoires. J.J., R.R., D.N., and O.M. assisted with protein expression and biophysical analyses. P.S. supervised affinity measurements. D.L. and M.C. supervised structure analyses, designed experiments and analyzed data. A.L. and B.C. optimized, performed, and analyzed mass spectrometry experiments. D.C. conceived the phage method. A.L. and D.C. designed experiments and wrote the paper. All authors reviewed the manuscript.

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REFERENCES

(1) Kontermann, R. (2012) Dual targeting strategies with bispecific antibodies. MAbs 4, 182. (2) Ridgway, J. B., Presta, L. G., and Carter, P. (1996) Knobs-intoholes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng., Des. Sel. 9, 617−621. (3) Gunasekaran, K., Pentony, M., Shen, M., Garrett, L., Forte, C., Woodward, A., Ng, S. B., Born, T., Retter, M., Manchulenko, K., Sweet, H., Foltz, I. N., Wittekind, M., and Yan, W. (2010) Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. J. Biol. Chem. 285, 19637−19646. (4) Merchant, A. M., Zhu, Z., Yuan, J. Q., Goddard, A., Adams, C. W., Presta, L. G., and Carter, P. (1998) An efficient route to human bispecific IgG. Nat. Biotechnol. 16, 677−681. (5) Du, J., Cao, Y., Liu, Y., Wang, Y., Zhang, Y., Fu, G., Zhang, Y., Lu, L., Luo, X., Kim, C. H., Schultz, P. G., and Wang, F. (2017) D

DOI: 10.1021/acs.biochem.9b00037 Biochemistry XXXX, XXX, XXX−XXX