Development of a potent high-affinity human therapeutic antibody via novel application of recombination signal sequence–based affinity maturation

Therapeutic antibody development requires discovery of an antibody molecule with desired specificities and drug-like properties. For toxicological studies, a therapeutic antibody must bind the ortholog antigen with a similar affinity to the human target to enable relevant dosing regimens, and antibodies falling short of this affinity design goal may not progress as therapeutic leads. Herein, we report the novel use of mammalian recombination signal sequence (RSS)–directed recombination for complementarity-determining region–targeted protein engineering combined with mammalian display to close the species affinity gap of human interleukin (IL)-13 antibody 731. This fully human antibody has not progressed as a therapeutic in part because of a 400-fold species affinity gap. Using this nonhypothesis-driven affinity maturation method, we generated multiple antibody variants with improved IL-13 affinity, including the highest affinity antibody reported to date (34 fM). Resolution of a cocrystal structure of the optimized antibody with the cynomolgus monkey (or nonhuman primate) IL-13 protein revealed that the RSS-derived mutations introduced multiple successive amino-acid substitutions resulting in a de novo formation of a π–π stacking–based protein–protein interaction between the affinity-matured antibody heavy chain and helix C on IL-13, as well as an introduction of an interface-distant residue, which enhanced the light chain–binding affinity to target. These mutations synergized binding of heavy and light chains to the target protein, resulting in a remarkably tight interaction, and providing a proof of concept for a new method of protein engineering, based on synergizing a mammalian display platform with novel RSS-mediated library generation. Therapeutic antibody development requires discovery of an antibody molecule with desired specificities and drug-like properties. For toxicological studies, a therapeutic antibody must bind the ortholog antigen with a similar affinity to the human target to enable relevant dosing regimens, and antibodies falling short of this affinity design goal may not progress as therapeutic leads. Herein, we report the novel use of mammalian recombination signal sequence (RSS)–directed recombination for complementarity-determining region–targeted protein engineering combined with mammalian display to close the species affinity gap of human interleukin (IL)-13 antibody 731. This fully human antibody has not progressed as a therapeutic in part because of a 400-fold species affinity gap. Using this nonhypothesis-driven affinity maturation method, we generated multiple antibody variants with improved IL-13 affinity, including the highest affinity antibody reported to date (34 fM). Resolution of a cocrystal structure of the optimized antibody with the cynomolgus monkey (or nonhuman primate) IL-13 protein revealed that the RSS-derived mutations introduced multiple successive amino-acid substitutions resulting in a de novo formation of a π–π stacking–based protein–protein interaction between the affinity-matured antibody heavy chain and helix C on IL-13, as well as an introduction of an interface-distant residue, which enhanced the light chain–binding affinity to target. These mutations synergized binding of heavy and light chains to the target protein, resulting in a remarkably tight interaction, and providing a proof of concept for a new method of protein engineering, based on synergizing a mammalian display platform with novel RSS-mediated library generation. Recent advances in experimental and computational methods (1Baker D. What has de novo protein design taught us about protein folding and biophysics?.Protein Sci. 2019; 28: 678-683Google Scholar, 2Silva D.A. Yu S. Ulge U.Y. Spangler J.B. Jude K.M. Labao-Almeida C. Ali L.R. Quijano-Rubio A. Ruterbusch M. Leung I. Biary T. Crowley S.J. Marcos E. Walkey C.D. Weitzner B.D. et al.De novo design of potent and selective mimics of IL-2 and IL-15.Nature. 2019; 565: 186-191Google Scholar, 3Koepnick B. Flatten J. Husain T. Ford A. Silva D.A. Bick M.J. Bauer A. Liu G. Ishida Y. Boykov A. Estep R.D. Kleinfelter S. Norgard-Solano T. Wei L. Players F. et al.De novo protein design by citizen scientists.Nature. 2019; 570: 390-394Google Scholar) are making it possible to complement well-established in vivo and in vitro methods of antibody discovery with novel in silico approaches to refine affinity and specificity of potential therapeutics (4Nimrod G. Fischman S. Austin M. Herman A. Keyes F. Leiderman O. Hargreaves D. Strajbl M. Breed J. Klompus S. Minton K. Spooner J. Buchanan A. Vaughan T.J. Ofran Y. Computational design of epitope-specific functional antibodies.Cell Rep. 2018; 25: 2121-2131.e5Google Scholar). The complexities of predicting protein structure and interactions of large molecular weight multichain molecules, however, remain a challenge for the field (5Vasquez M. Krauland E. Walker L. Wittrup D. Gerngross T. Connecting the sequence dots: Shedding light on the genesis of antibodies reported to be designed in silico.mAbs. 2019; 11: 803-808Google Scholar) as is design of improved affinity binding of an antibody to a therapeutically relevant target (6Kiyoshi M. Caaveiro J.M. Miura E. Nagatoishi S. Nakakido M. Soga S. Shirai H. Kawabata S. Tsumoto K. Affinity improvement of a therapeutic antibody by structure-based computational design: Generation of electrostatic interactions in the transition state stabilizes the antibody-antigen complex.PLoS One. 2014; 9e87099Google Scholar, 7Lippow S.M. Wittrup K.D. Tidor B. Computational design of antibody-affinity improvement beyond in vivo maturation.Nat. Biotechnol. 2007; 25: 1171-1176Google Scholar, 8Mahajan S.P. Meksiriporn B. Waraho-Zhmayev D. Weyant K.B. Kocer I. Butler D.C. Messer A. Escobedo F.A. DeLisa M.P. Computational affinity maturation of camelid single-domain intrabodies against the nonamyloid component of alpha-synuclein.Sci. Rep. 2018; 8: 17611Google Scholar). Many currently utilized in silico affinity engineering technologies rely on the availability of structures and application of rational design (9Kuroda D. Tsumoto K. Antibody affinity maturation by computational design.Methods Mol. Biol. 2018; 1827: 15-34Google Scholar, 10Fischman S. Ofran Y. Computational design of antibodies.Curr. Opin. Struct. Biol. 2018; 51: 156-162Google Scholar), but these approaches are limited for highly complex multipass membrane proteins, where a high-resolution crystal structure may remain elusive, underscoring the need for nonhypothesis-driven affinity maturation approaches. In humans, antibodies are generated from a heavy-chain (HC) gene and one of two light-chain (LC) genes (kappa or lambda). Sequences encoding antibody variable regions are formed from precursor gene segments in a process called V(D)J recombination. At the HC locus, different combinations of V, D, and J segments (or V and J segments at the LC) are joined together in a manner dependent on recombination signal sequences (RSSs) (11Sakano H. Huppi K. Heinrich G. Tonegawa S. Sequences at the somatic recombination sites of immunoglobulin light-chain genes.Nature. 1979; 280: 288-294Google Scholar, 12Sakano H. Kurosawa Y. Weigert M. Tonegawa S. Identification and nucleotide sequence of a diversity DNA segment (D) of immunoglobulin heavy-chain genes.Nature. 1981; 290: 562-565Google Scholar, 13Kurosawa Y. von Boehmer H. Haas W. Sakano H. Trauneker A. Tonegawa S. Identification of D segments of immunoglobulin heavy-chain genes and their rearrangement in T lymphocytes.Nature. 1981; 290: 565-570Google Scholar), the recombination-activating gene 1 (RAG1)/recombination-activating gene 2 (RAG2) complex (14Schatz D.G. Oettinger M.A. Baltimore D. The V(D)J recombination activating gene, RAG-1.Cell. 1989; 59: 1035-1048Google Scholar, 15Oettinger M.A. Schatz D.G. Gorka C. Baltimore D. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination.Science. 1990; 248: 1517-1523Google Scholar) and nonhomologous end joining. DNA segments are joined together imperfectly and deletions, insertions, and/or substitutions dependent on terminal deoxynucleotidyl transferase (16Baltimore D. Is terminal deoxynucleotidyl transferase a somatic mutagen in lymphocytes?.Nature. 1974; 248: 409-411Google Scholar, 17Desiderio S.V. Yancopoulos G.D. Paskind M. Thomas E. Boss M.A. Landau N. Alt F.W. Baltimore D. Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells.Nature. 1984; 311: 752-755Google Scholar) (TdT) and nonhomologous end joining greatly enhance diversity. These mechanisms, in addition to variations in HC–LC pairing, allow B cells to generate large antibody repertoires. Affinity maturation is a process for improving antibody affinity for a specific antigen. In vivo, the immune system enables affinity maturation of antibodies by somatic hypermutation of antibody genes and clonal selection of B cells (18Tonegawa S. Somatic generation of antibody diversity.Nature. 1983; 302: 575-581Google Scholar), and V(D)J recombination is restricted to complementarity-determining region 3 (CDR3) diversity generation. In vitro, this process can be mimicked by repeated cycles of gene mutation and selection of phage or yeast display libraries (19Gai S.A. Wittrup K.D. Yeast surface display for protein engineering and characterization.Curr. Opin. Struct. Biol. 2007; 17: 467-473Google Scholar). RSS is nature's most powerful diversification system that the body leverages for the development of B-cell and T-cell immunological diversity (11Sakano H. Huppi K. Heinrich G. Tonegawa S. Sequences at the somatic recombination sites of immunoglobulin light-chain genes.Nature. 1979; 280: 288-294Google Scholar). Its adaption to an in vitro mammalian protein engineering platform enables mutations resulting in insertions, deletions, and/or substitutions of one or multiple amino acids at any desired position within an antibody or a protein (Fig. S1A) and has been applied to in vitro T-cell receptor T-cell engineering in the past (20Oh J. Warshaviak D.T. Mkrtichyan M. Munguia M.L. Lin A. Chai F. Pigott C. Kang J. Gallo M. Kamb A. Single variable domains from the T cell receptor beta chain function as mono- and bifunctional CARs and TCRs.Sci. Rep. 2019; 9: 17291Google Scholar). Here, we apply this mutagenesis strategy to the affinity maturation of a fully human antibody. HuTARG technology is a novel RSS recombination–based protein engineering platform coupled to cell surface display in a mammalian cell culture system. Briefly, DNA encoding CDRs in the HCs and LCs of antibody 731 (Ab731) were engineered by standard molecular biology methods to contain RSS sites (Figs. 1A and S1A). The resulting plasmid pool contained individual CDRs, where an RSS integration was targeted; successful integration of RSS signals (21Lee A.I. Fugmann S.D. Cowell L.G. Ptaszek L.M. Kelsoe G. Schatz D.G. A functional analysis of the spacer of V(D)J recombination signal sequences.PLoS Biol. 2003; 1E1Google Scholar) into each CDR was confirmed by terminal restriction fragment length polymorphism (T-RFLP) (Figs. 1A and S1B). The resulting constructs were stably integrated as a pool into the HuTARG cell line using the Cre-Lox system (22Sauer B. Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1.Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5166-5170Google Scholar). HuTARG cells are recombination-competent mammalian cells, where the RAG-1-mediated recombinase activity (14Schatz D.G. Oettinger M.A. Baltimore D. The V(D)J recombination activating gene, RAG-1.Cell. 1989; 59: 1035-1048Google Scholar, 23Grawunder U. Leu T.M. Schatz D.G. Werner A. Rolink A.G. Melchers F. Winkler T.H. Down-regulation of RAG1 and RAG2 gene expression in preB cells after functional immunoglobulin heavy chain rearrangement.Immunity. 1995; 3: 601-608Google Scholar) is induced under tetracycline treatment (24Yao F. Svensjo T. Winkler T. Lu M. Eriksson C. Eriksson E. Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells.Hum. Gene Ther. 1998; 9: 1939-1950Google Scholar). The induction of recombination results in each cell undergoing a unique rearrangement that involves the removal of the RSS cassette and, in the presence of TdT, double-strand break repair, resulting in imperfect joining of recombined segments followed by sequence variation in human immunoglobulin G (IgG) antibody. This antibody is expressed on the surface of the cells (Fig. 1B). As HC and LC sequences were targeted separately, two individual pools of vectors were generated and used to create diversified HC and LC antibody cell surface display libraries. Since recombination occurs in the cell directly and there is no need for a transformation step or individual integration step, the limitation of library complexity is determined by cell number. In this case, we utilized a library complexity of 3.4 × 108 cells for the diversified HC and 3.2 × 106 cells for diversified LC. Improved affinity variants (as determined by higher than parental antibody binding) were isolated by three rounds of fluorescence-activated cell sorting (FACS) by cell-surface staining with 36 pM of recombinant soluble nonhistone protein (NHP) IL-13 (Fig. 1B, right panel). Variant antibody sequences selected to have improved affinity were found to include insertions and substitutions that were relatively evenly distributed across the enriched LCs consistent with the frequency of RSS integration (Fig. S2). Rescued antibody sequences were cloned and transiently expressed on the surface of human embryonic kidney 293T (HEK293T) cells to rank their binding to soluble human and NHP IL-13 by FACS analysis (Fig. 1C). Geometric mean of for binding to the target protein were in three based on cell-surface expression strategy is in the HC variants that improved NHP IL-13 two were confirmed by (Fig. to have an affinity higher than their and (Fig. and A similar analysis was for LC variants and in the of three LC sequences and with higher than parental antibody binding (Fig. and These LC variants were combined by with the HC variants in possible (Fig. This in the of three and which improved affinity to NHP and human IL-13 (Fig. and and higher binding than antibodies with individual mutations in the LC or was for of from human cells with human or NHP antibodies were found to be potent against with that were limited by the of IL-13 in the (Fig. In the parental no activity against NHP of highest affinity variants by HC and LC mutations with IL-13 affinity and not in a new not affinity that of pM to NHP IL-13 and to human IL-13, a improvement and a 1 and the molecular of the interaction of the antibody with NHP and human IL-13, we determined the crystal structure of NHP IL-13 in complex with fragment at a of (Fig. and for and to the The crystal structure of NHP IL-13 in complex with revealed that the of IL-13 is to the and is between the HCs and The surface of is than that for C. J. of Scholar). The of an higher of for the than at Mol. Biol. Scholar), an and for the two The crystal structure an to the affinity of NHP and human from the parental three in the and two successive on the 1 and The of the formation of a π–π the engineered from π–π interactions with of to similar the with and IL-13 and in (Fig. is that the presence of and in of parental and to the of the this was to because of the with the presence of two The of to the binding is by surface of which of the surface a for rather than a of affinity or two we are by interactions from the (Fig. As the and with to two we to in this region the in binding interactions that between NHP and human In the crystal from NHP IL-13 is between and the IL-13 from to The structure that the tight by these in a that with (Fig. In human IL-13, we a in of (Fig. and from the IL-13 is restricted and to a with the of resulting in binding with experimental In case, and to a the of is by the with the the of a and as to the parental which has a (Fig. and and a In the of the parental antibody in complex with human IL-13, the higher affinity of the interaction an of the presence of a This with NHP IL-13, where the affinity of the interaction the to and the an This affinity of the parental antibody NHP The binding of for human and NHP IL-13 is by protein–protein interactions not or standard molecular engineering using computational methods and as confirmed by molecular surface methods applied to the NHP IL-13 complex in the region of were found to be and to the between the we were to the of mutations of the LC to and HC to that an of be and are not in a This is on one to the and length of the interactions the and on the to the of the CDRs the affinity is to using in silico molecular methods or molecular The generated for and are consistent with the and mutation which affinity NHP IL-13 with parental a at position which we to the these were after the was and the In vitro affinity maturation is a used to affinity of therapeutic protein to their technologies in the field include targeted are and of fragment is by mutagenesis or mutagenesis M. Y. W. H. E. F. in therapeutic antibody affinity 2019; Scholar, I. Antibody affinity maturation by Mol. Biol. 248: Scholar, J. I. C. W. K. J. T. Affinity maturation of human antibodies by light chain yeast Scholar). As these methods rely on display technologies dependent on yeast or phage the library is limited to a of successful G. antibodies by phage display Scholar). In as the engineering occurs on a molecular and is expressed on yeast or or not be Using an in vitro mutagenesis strategy in a mammalian we were to a fully human therapeutic antibody with the highest affinity reported in the a cocrystal structure to allow a mutagenesis strategy based on rational The mutations introduced by RSS-mediated recombination CDRs of the in in with the the which is not as is the of for B cells the in the region of is for most antibody Scholar). and were the of mutations in the FACS cells (Fig. S2). the affinity of (as well as at to the human IL-13 protein is one of the highest in the reported an in antibody with affinity, by technology S. L. S. J. of an in vivo generated affinity fully human antibody to Scholar). of antibodies include affinity maturation by mutagenesis of a and of antibodies and by technology H. J. K. E. M. M. L. a antibody for the treatment of 3: Scholar). engineered antibody was reported to have a of to the by an method Wittrup K.D. of antibody with Natl. Acad. Sci. U. S. A. Scholar, S.M. Tidor B. Wittrup K.D. improvement with in a affinity Mol. Biol. Scholar). the structure of IL-13 is A. T.J. G. S.A. of an Scholar), the of the nonhypothesis-driven RSS affinity maturation is that was not The RSS-derived mutations are unique and introduced multiple successive amino-acid resulting in a de novo formation of a π–π stacking–based protein–protein interaction between the affinity-matured HC and the helix C on IL-13, as well as an interface-distant which the affinity to target. These mutations synergistically binding from the HCs and which have been to a using in silico we the NHP species affinity gap between the therapeutic and an molecule to the therapeutic Antibody sequence diversification was using RSS recombination in cell Using standard molecular biology an RSS cassette of a a spacer region of and a RSS was into a plasmid encoding the HC and LC sequence of the antibody to be affinity This was used to a of each with an RSS cassette at a unique within or of the LC. RSS cassette contained a restriction which was used to successful RSS into the plasmid by A similar was used to a of with RSS cassette insertions the HC recombination was used to stably of from each into the HuTARG mammalian cell line engineered to and complex under the of of recombination by tetracycline in of the RSS cassette and the imperfect joining of and DNA nonhomologous end of antibody sequences to variation in length and sequence at within the encoding a functional antibody gene antibody on the cell surface a joining in expression of the which selection cells antibodies this two mammalian display libraries were one with a LC and sequence diversity in the HC CDRs and the with a HC and sequence diversity in the LC HC and LC HuTARG recombined libraries were enriched for binding to soluble recombinant IL-13 by multiple rounds of FACS on FACS The recombined cells with cell line parental on the surface were with of NHP IL-13 followed by with IL-13 with sorting was for a of NHP IL-13 binding to cell surface antibody expression based on the parental cell line as a to that the higher three rounds of the enriched cells were and the antibody sequences were determined by antibodies were cloned as cell surface for or standard antibody and expressed in cells DNA encoding the antibodies was expressed transiently in The cells were at 1 × 106 in for 3 on a in a was using with 1 were after with expressed as for binding to NHP IL-13, cells were with pM of recombinant IL-13 with as for binding to human IL-13, cells were with pM of recombinant human IL-13 protein followed by analysis and mean using a of vectors encoding RSS within antibody CDR sequences or LC) were using a and an of the resulting with the restriction in at a unique within the RSS cassette that the of the determined by fragment analysis determined within the antibody the the the into the antibody sequence the RSS cassette was with an was used to the by standard method (1Baker D. What has de novo protein design taught us about protein folding and biophysics?.Protein Sci. 2019; 28: 678-683Google Scholar, S. L. S. J. of an in vivo generated affinity fully human antibody to Scholar). Briefly, for were by human IL-13 or NHP IL-13 S. L. S. J. of an in vivo generated affinity fully human antibody to Scholar) with a of and the to at for to The in the at was by the with The was with as S. L. S. J. of an in vivo generated affinity fully human antibody to Scholar). For each antibody a of two were using different to generate an and a For the affinity multiple were at 1 1 and The were with the in the analysis experimental are in the crystal structure that the of this have been in the The is generated for this are within this and or are This at Mol. Biol. Scholar, S. L. S. J. of an in vivo generated affinity fully human antibody to Scholar, K. K. analysis of in and Scholar, W. and D Biol. Scholar, K.D. A.G. A. S.J. et of the and D Biol. Scholar, 2007; Scholar, B. K. and development of D Biol. Scholar, G. N. D.C. et A system for structure D Biol. Scholar, E. K. of from Mol. Biol. 2007; Scholar, K. T. Warshaviak D. C. D. Antibody structure using a of and 2014; Scholar, W. D Biol. Scholar). A. I. S. T. A. and I. N. F. are and of at the of C. is an of The have a application based on results in the and for on the and for for this A. K. and I. N. F. C. I. S. T. and A. S. and M. A. I. D. and I. N. F. with and