The use of monoclonal antibodies as therapeutics requires optimizing several of their key attributes. These include binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, and compatibility with the attachment of additional antibody domains (bispecific antibodies) and cytotoxic drugs (antibody–drug conjugates). Addressing these and other challenges requires the use of systematic design methods that complement powerful immunization and in vitro screening methods. We review advances in designing the binding loops, scaffolds, domain interfaces, constant regions, post-translational and chemical modifications, and bispecific architectures of antibodies and fragments thereof to improve their bioactivity. We also highlight unmet challenges in antibody design that must be overcome to generate potent antibody therapeutics.


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Literature Cited

  1. Gitlin D. 1.  1966. Current aspects of the structure, function, and genetics of the immunoglobulins. Annu. Rev. Med. 17:1–22 [Google Scholar]
  2. Bradbury AR, Sidhu S, Dubel S, McCafferty J. 2.  2011. Beyond natural antibodies: the power of in vitro display technologies. Nat. Biotechnol. 29:245–54 [Google Scholar]
  3. Reichert JM. 3.  2013. Which are the antibodies to watch in 2013?. mAbs 5:1–4 [Google Scholar]
  4. Lowe D, Dudgeon K, Rouet R, Schofield P, Jermutus L, Christ D. 4.  2011. Aggregation, stability, and formulation of human antibody therapeutics. Adv. Protein Chem. Struct. Biol. 84:41–61 [Google Scholar]
  5. Perchiacca JM, Tessier PM. 5.  2012. Engineering aggregation-resistant antibodies. Annu. Rev. Chem. Biomol. Eng. 3:263–86 [Google Scholar]
  6. Ratanji KD, Derrick JP, Dearman RJ, Kimber I. 6.  2014. Immunogenicity of therapeutic proteins: influence of aggregation. J. Immunotoxicol. 11:99–109 [Google Scholar]
  7. Connolly BD, Petry C, Yadav S, Demeule B, Ciaccio N. 7.  et al. 2012. Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter. Biophys. J. 103:69–78 [Google Scholar]
  8. Pantazes RJ, Maranas CD. 8.  2010. OptCDR: a general computational method for the design of antibody complementarity determining regions for targeted epitope binding. Protein Eng. Des. Sel. 23:849–58 [Google Scholar]
  9. Li T, Pantazes RJ, Maranas CD. 9.  2014. OptMAVEn—a new framework for the de novo design of antibody variable region models targeting specific antigen epitopes. PLOS ONE 9:e105954 [Google Scholar]
  10. Moroncini G, Kanu N, Solforosi L, Abalos G, Telling GC. 10.  et al. 2004. Motif-grafted antibodies containing the replicative interface of cellular PrP are specific for PrPSc. PNAS 101:10404–9 [Google Scholar]
  11. Heppner FL, Musahl C, Arrighi I, Klein MA, Rulicke T. 11.  et al. 2001. Prevention of scrapie pathogenesis by transgenic expression of anti-prion protein antibodies. Science 294:178–82 [Google Scholar]
  12. Peretz D, Williamson RA, Kaneko K, Vergara J, Leclerc E. 12.  et al. 2001. Antibodies inhibit prion propagation and clear cell cultures of prion infectivity. Nature 412:739–43 [Google Scholar]
  13. Solforosi L, Bellon A, Schaller M, Cruite JT, Abalos GC, Williamson RA. 13.  2007. Toward molecular dissection of PrPC–PrPSc interactions. J. Biol. Chem. 282:7465–71 [Google Scholar]
  14. Perchiacca JM, Ladiwala AR, Bhattacharya M, Tessier PM. 14.  2012. Structure-based design of conformation- and sequence-specific antibodies against amyloid β. PNAS 109:84–89 [Google Scholar]
  15. Petkova AT, Leapman RD, Guo Z, Yau WM, Mattson MP, Tycko R. 15.  2005. Self-propagating, molecular-level polymorphism in Alzheimer's β-amyloid fibrils. Science 307:262–65 [Google Scholar]
  16. Ladiwala AR, Bhattacharya M, Perchiacca JM, Cao P, Raleigh DP. 16.  et al. 2012. Rational design of potent domain antibody inhibitors of amyloid fibril assembly. PNAS 109:19965–70 [Google Scholar]
  17. Barbas CF 3rd, Languino LR, Smith JW. 17.  1993. High-affinity self-reactive human antibodies by design and selection: targeting the integrin ligand binding site. PNAS 90:10003–7 [Google Scholar]
  18. Smith JW, Hu D, Satterthwait A, Pinz-Sweeney S, Barbas CF 3rd. 18.  1994. Building synthetic antibodies as adhesive ligands for integrins. J. Biol. Chem. 269:32788–95 [Google Scholar]
  19. Kirkham PM, Neri D, Winter G. 19.  1999. Towards the design of an antibody that recognises a given protein epitope. J. Mol. Biol. 285:909–15 [Google Scholar]
  20. Koerber JT, Thomsen ND, Hannigan BT, Degrado WF, Wells JA. 20.  2013. Nature-inspired design of motif-specific antibody scaffolds. Nat. Biotechnol. 31:916–21 [Google Scholar]
  21. Stoevesandt O, Taussig MJ. 21.  2013. Phospho-specific antibodies by design. Nat. Biotechnol. 31:889–91 [Google Scholar]
  22. Lippow SM, Wittrup KD, Tidor B. 22.  2007. Computational design of antibody-affinity improvement beyond in vivo maturation. Nat. Biotechnol. 25:1171–76 [Google Scholar]
  23. Marvin JS, Lowman HB. 23.  2003. Redesigning an antibody fragment for faster association with its antigen. Biochemistry 42:7077–83 [Google Scholar]
  24. Clark LA, Boriack-Sjodin PA, Eldredge J, Fitch C, Friedman B. 24.  et al. 2006. Affinity enhancement of an in vivo matured therapeutic antibody using structure-based computational design. Protein Sci. 15:949–60 [Google Scholar]
  25. Tharakaraman K, Robinson LN, Hatas A, Chen YL, Siyue L. 25.  et al. 2013. Redesign of a cross-reactive antibody to dengue virus with broad-spectrum activity and increased in vivo potency. PNAS 110:E1555–64 [Google Scholar]
  26. Kiyoshi M, Caaveiro JM, Miura E, Nagatoishi S, Nakakido M. 26.  et al. 2014. 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 9:e87099 [Google Scholar]
  27. Kuroda D, Shirai H, Jacobson MP, Nakamura H. 27.  2012. Computer-aided antibody design. Protein Eng. Des. Sel. 25:507–21 [Google Scholar]
  28. Selzer T, Albeck S, Schreiber G. 28.  2000. Rational design of faster associating and tighter binding protein complexes. Nat. Struct. Biol. 7:537–41 [Google Scholar]
  29. Schreiber G, Fersht AR. 29.  1996. Rapid, electrostatically assisted association of proteins. Nat. Struct. Biol. 3:427–31 [Google Scholar]
  30. Worn A, Pluckthun A. 30.  2001. Stability engineering of antibody single-chain Fv fragments. J. Mol. Biol. 305:989–1010 [Google Scholar]
  31. Honegger A. 31.  2008. Engineering antibodies for stability and efficient folding. Handbook of Experimental Pharmacology 181 Y Chernajovsky, A Nissim 47–68 Dordrecht, Neth: Springer [Google Scholar]
  32. Helms LR, Wetzel R. 32.  1995. Destabilizing loop swaps in the CDRs of an immunoglobulin VL domain. Protein Sci. 4:2073–81 [Google Scholar]
  33. Yasui H, Ito W, Kurosawa Y. 33.  1994. Effects of substitutions of amino acids on the thermal stability of the Fv fragments of antibodies. FEBS Lett. 353:143–46 [Google Scholar]
  34. Zabetakis D, Anderson GP, Bayya N, Goldman ER. 34.  2013. Contributions of the complementarity determining regions to the thermal stability of a single-domain antibody. PLOS ONE 8:e77678 [Google Scholar]
  35. Perchiacca JM, Bhattacharya M, Tessier PM. 35.  2011. Mutational analysis of domain antibodies reveals aggregation hotspots within and near the complementarity determining regions. Proteins 79:2637–47 [Google Scholar]
  36. Ewert S, Honegger A, Pluckthun A. 36.  2004. Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering. Methods 34:184–99 [Google Scholar]
  37. Miller BR, Demarest SJ, Lugovskoy A, Huang F, Wu X. 37.  et al. 2010. Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng. Des. Sel. 23:549–57 [Google Scholar]
  38. Barthelemy PA, Raab H, Appleton BA, Bond CJ, Wu P. 38.  et al. 2008. Comprehensive analysis of the factors contributing to the stability and solubility of autonomous human VH domains. J. Biol. Chem. 283:3639–54 [Google Scholar]
  39. Wirtz P, Steipe B. 39.  1999. Intrabody construction and expression III: engineering hyperstable VH domains. Protein Sci. 8:2245–50 [Google Scholar]
  40. Tan PH, Sandmaier BM, Stayton PS. 40.  1998. Contributions of a highly conserved VH/VL hydrogen bonding interaction to scFv folding stability and refolding efficiency. Biophys. J. 75:1473–82 [Google Scholar]
  41. Monsellier E, Bedouelle H. 41.  2006. Improving the stability of an antibody variable fragment by a combination of knowledge-based approaches: validation and mechanisms. J. Mol. Biol. 362:580–93 [Google Scholar]
  42. Haidar JN, Yuan QA, Zeng L, Snavely M, Luna X. 42.  et al. 2012. A universal combinatorial design of antibody framework to graft distinct CDR sequences: a bioinformatics approach. Proteins 80:896–912 [Google Scholar]
  43. Wang N, Smith WF, Miller BR, Aivazian D, Lugovskoy AA. 43.  et al. 2009. Conserved amino acid networks involved in antibody variable domain interactions. Proteins 76:99–114 [Google Scholar]
  44. Jordan JL, Arndt JW, Hanf K, Li G, Hall J. 44.  et al. 2009. Structural understanding of stabilization patterns in engineered bispecific Ig-like antibody molecules. Proteins 77:832–41 [Google Scholar]
  45. Das R, Baker D. 45.  2008. Macromolecular modeling with Rosetta. Annu. Rev. Biochem. 77:363–82 [Google Scholar]
  46. Chennamsetty N, Voynov V, Kayser V, Helk B, Trout BL. 46.  2009. Design of therapeutic proteins with enhanced stability. PNAS 106:11937–42 [Google Scholar]
  47. Rothlisberger D, Honegger A, Pluckthun A. 47.  2005. Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability. J. Mol. Biol. 347:773–89 [Google Scholar]
  48. Wang T, Duan Y. 48.  2011. Probing the stability-limiting regions of an antibody single-chain variable fragment: a molecular dynamics simulation study. Protein Eng. Des. Sel. 24:649–57 [Google Scholar]
  49. Kim DY, Kandalaft H, Ding W, Ryan S, van Faassen H. 49.  et al. 2012. Disulfide linkage engineering for improving biophysical properties of human VH domains. Protein Eng. Des. Sel. 25:581–89 [Google Scholar]
  50. Saerens D, Conrath K, Govaert J, Muyldermans S. 50.  2008. Disulfide bond introduction for general stabilization of immunoglobulin heavy-chain variable domains. J. Mol. Biol. 377:478–88 [Google Scholar]
  51. Reiter Y, Brinkmann U, Webber KO, Jung SH, Lee B, Pastan I. 51.  1994. Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv. Protein Eng. 7:697–704 [Google Scholar]
  52. Young NM, MacKenzie CR, Narang SA, Oomen RP, Baenziger JE. 52.  1995. Thermal stabilization of a single-chain Fv antibody fragment by introduction of a disulphide bond. FEBS Lett. 377:135–39 [Google Scholar]
  53. Wang X, Kumar S, Buck PM, Singh SK. 53.  2013. Impact of deglycosylation and thermal stress on conformational stability of a full length murine IgG2a monoclonal antibody: observations from molecular dynamics simulations. Proteins 81:443–60 [Google Scholar]
  54. Ewert S, Honegger A, Pluckthun A. 54.  2003. Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach. Biochemistry 42:1517–28 [Google Scholar]
  55. Lee CC, Perchiacca JM, Tessier PM. 55.  2013. Toward aggregation-resistant antibodies by design. Trends Biotechnol. 31:612–20 [Google Scholar]
  56. Perchiacca JM, Lee CC, Tessier PM. 56.  2014. Optimal charged mutations in the complementarity-determining regions that prevent domain antibody aggregation are dependent on the antibody scaffold. Protein Eng. Des. Sel. 27:29–39 [Google Scholar]
  57. Perchiacca JM, Ladiwala AR, Bhattacharya M, Tessier PM. 57.  2012. Aggregation-resistant domain antibodies engineered with charged mutations near the edges of the complementarity-determining regions. Protein Eng. Des. Sel. 25:591–601 [Google Scholar]
  58. Wu SJ, Luo J, O'Neil KT, Kang J, Lacy ER. 58.  et al. 2010. Structure-based engineering of a monoclonal antibody for improved solubility. Protein Eng. Des. Sel. 23:643–51 [Google Scholar]
  59. Dudgeon K, Rouet R, Kokmeijer I, Schofield P, Stolp J. 59.  et al. 2012. General strategy for the generation of human antibody variable domains with increased aggregation resistance. PNAS 109:10879–84 [Google Scholar]
  60. Jespers L, Schon O, Famm K, Winter G. 60.  2004. Aggregation-resistant domain antibodies selected on phage by heat denaturation. Nat. Biotechnol. 22:1161–65 [Google Scholar]
  61. Pepinsky RB, Silvian L, Berkowitz SA, Farrington G, Lugovskoy A. 61.  et al. 2010. Improving the solubility of anti-LINGO-1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis. Protein Sci. 19:954–66 [Google Scholar]
  62. Miklos AE, Kluwe C, Der BS, Pai S, Sircar A. 62.  et al. 2012. Structure-based design of supercharged, highly thermoresistant antibodies. Chem. Biol. 19:449–55 [Google Scholar]
  63. Kayser V, Chennamsetty N, Voynov V, Forrer K, Helk B, Trout BL. 63.  2011. Glycosylation influences on the aggregation propensity of therapeutic monoclonal antibodies. Biotechnol. J. 6:38–44 [Google Scholar]
  64. Schaefer JV, Pluckthun A. 64.  2012. Engineering aggregation resistance in IgG by two independent mechanisms: lessons from comparison of Pichia pastoris and mammalian cell expression. J. Mol. Biol. 417:309–35 [Google Scholar]
  65. Buck PM, Kumar S, Singh SK. 65.  2013. Insights into the potential aggregation liabilities of the b12 Fab fragment via elevated temperature molecular dynamics. Protein Eng. Des. Sel. 26:195–205 [Google Scholar]
  66. Lauer TM, Agrawal NJ, Chennamsetty N, Egodage K, Helk B, Trout BL. 66.  2012. Developability index: a rapid in silico tool for the screening of antibody aggregation propensity. J. Pharm. Sci. 101:102–15 [Google Scholar]
  67. Buck PM, Kumar S, Wang X, Agrawal NJ, Trout BL, Singh SK. 67.  2012. Computational methods to predict therapeutic protein aggregation. Methods Mol. Biol. 899:425–51 [Google Scholar]
  68. Tan PH, Chu V, Stray JE, Hamlin DK, Pettit D. 68.  et al. 1998. Engineering the isoelectric point of a renal cell carcinoma targeting antibody greatly enhances scFv solubility. Immunotechnology 4:107–14 [Google Scholar]
  69. Jespers L, Schon O, James LC, Veprintsev D, Winter G. 69.  2004. Crystal structure of HEL4, a soluble, refoldable human VH single domain with a germ-line scaffold. J. Mol. Biol. 337:893–903 [Google Scholar]
  70. Arbabi-Ghahroudi M, To R, Gaudette N, Hirama T, Ding W. 70.  et al. 2009. Aggregation-resistant VHs selected by in vitro evolution tend to have disulfide-bonded loops and acidic isoelectric points. Protein Eng. Des. Sel. 22:59–66 [Google Scholar]
  71. Ewert S, Cambillau C, Conrath K, Pluckthun A. 71.  2002. Biophysical properties of camelid VHH domains compared to those of human VH3 domains. Biochemistry 41:3628–36 [Google Scholar]
  72. Desjarlais JR, Lazar GA. 72.  2011. Modulation of antibody effector function. Exp. Cell Res. 317:1278–85 [Google Scholar]
  73. Strohl WR. 73.  2009. Optimization of Fc-mediated effector functions of monoclonal antibodies. Curr. Opin. Biotechnol. 20:685–91 [Google Scholar]
  74. Shields RL, Namenuk AK, Hong K, Meng YG, Rae J. 74.  et al. 2001. High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR. J. Biol. Chem. 276:6591–604 [Google Scholar]
  75. Sondermann P, Huber R, Oosthuizen V, Jacob U. 75.  2000. The 3.2-A crystal structure of the human IgG1 Fc fragment–FcγRIII complex. Nature 406:267–73 [Google Scholar]
  76. Idusogie EE, Presta LG, Gazzano-Santoro H, Totpal K, Wong PY. 76.  et al. 2000. Mapping of the C1q binding site on Rituxan, a chimeric antibody with a human IgG1 Fc. J. Immunol. 164:4178–84 [Google Scholar]
  77. Dall'Acqua WF, Cook KE, Damschroder MM, Woods RM, Wu H. 77.  2006. Modulation of the effector functions of a human IgG1 through engineering of its hinge region. J. Immunol. 177:1129–38 [Google Scholar]
  78. Tao MH, Canfield SM, Morrison SL. 78.  1991. The differential ability of human IgG1 and IgG4 to activate complement is determined by the COOH-terminal sequence of the CH2 domain. J. Exp. Med. 173:1025–28 [Google Scholar]
  79. Norderhaug L, Brekke OH, Bremnes B, Sandin R, Aase A. 79.  et al. 1991. Chimeric mouse human IgG3 antibodies with an IgG4-like hinge region induce complement-mediated lysis more efficiently than IgG3 with normal hinge. Eur. J. Immunol. 21:2379–84 [Google Scholar]
  80. Canfield SM, Morrison SL. 80.  1991. The binding affinity of human IgG for its high affinity Fc receptor is determined by multiple amino acids in the CH2 domain and is modulated by the hinge region. J. Exp. Med. 173:1483–91 [Google Scholar]
  81. Mimoto F, Igawa T, Kuramochi T, Katada H, Kadono S. 81.  et al. 2013. Novel asymmetrically engineered antibody Fc variant with superior FcγR binding affinity and specificity compared with afucosylated Fc variant. mAbs 5:229–36 [Google Scholar]
  82. Lazar GA, Dang W, Karki S, Vafa O, Peng JS. 82.  et al. 2006. Engineered antibody Fc variants with enhanced effector function. PNAS 103:4005–10 [Google Scholar]
  83. Mossner E, Brunker P, Moser S, Puntener U, Schmidt C. 83.  et al. 2010. Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity. Blood 115:4393–402 [Google Scholar]
  84. Liu Z, Gunasekaran K, Wang W, Razinkov V, Sekirov L. 84.  et al. 2014. Asymmetrical Fc engineering greatly enhances antibody-dependent cellular cytotoxicity (ADCC) effector function and stability of the modified antibodies. J. Biol. Chem. 289:3571–90 [Google Scholar]
  85. Moore GL, Chen H, Karki S, Lazar GA. 85.  2010. Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions. mAbs 2:181–89 [Google Scholar]
  86. Idusogie EE, Wong PY, Presta LG, Gazzano-Santoro H, Totpal K. 86.  et al. 2001. Engineered antibodies with increased activity to recruit complement. J. Immunol. 166:2571–75 [Google Scholar]
  87. Natsume A, In M, Takamura H, Nakagawa T, Shimizu Y. 87.  et al. 2008. Engineered antibodies of IgG1/IgG3 mixed isotype with enhanced cytotoxic activities. Cancer Res. 68:3863–72 [Google Scholar]
  88. Raju TS. 88.  2008. Terminal sugars of Fc glycans influence antibody effector functions of IgGs. Curr. Opin. Immunol. 20:471–78 [Google Scholar]
  89. Jefferis R. 89.  2009. Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action. Trends Pharmacol. Sci. 30:356–62 [Google Scholar]
  90. Simmons LC, Reilly D, Klimowski L, Raju TS, Meng G. 90.  et al. 2002. Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies. J. Immunol. Methods 263:133–47 [Google Scholar]
  91. Jung ST, Reddy ST, Kang TH, Borrok MJ, Sandlie I. 91.  et al. 2010. Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte–dendritic cells. PNAS 107:604–9 [Google Scholar]
  92. Sazinsky SL, Ott RG, Silver NW, Tidor B, Ravetch JV, Wittrup KD. 92.  2008. Aglycosylated immunoglobulin G1 variants productively engage activating Fc receptors. PNAS 105:20167–72 [Google Scholar]
  93. Hodoniczky J, Zheng YZ, James DC. 93.  2005. Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol. Prog. 21:1644–52 [Google Scholar]
  94. Matsumiya S, Yamaguchi Y, Saito J, Nagano M, Sasakawa H. 94.  et al. 2007. Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1. J. Mol. Biol. 368:767–79 [Google Scholar]
  95. Shields RL, Lai J, Keck R, O'Connell LY, Hong K. 95.  et al. 2002. Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular toxicity. J. Biol. Chem. 277:26733–40 [Google Scholar]
  96. Sondermann P, Pincetic A, Maamary J, Lammens K, Ravetch JV. 96.  2013. General mechanism for modulating immunoglobulin effector function. PNAS 110:9868–72 [Google Scholar]
  97. Natsume A, Shimizu-Yokoyama Y, Satoh M, Shitara K, Niwa R. 97.  2009. Engineered anti-CD20 antibodies with enhanced complement-activating capacity mediate potent anti-lymphoma activity. Cancer Sci. 100:2411–18 [Google Scholar]
  98. Gerdes CA, Nicolini VG, Herter S, van Puijenbroek E, Lang S. 98.  et al. 2013. GA201 (RG7160): a novel, humanized, glycoengineered anti-EGFR antibody with enhanced ADCC and superior in vivo efficacy compared with cetuximab. Clin. Cancer Res. 19:1126–38 [Google Scholar]
  99. Panowksi S, Bhakta S, Raab H, Polakis P, Junutula JR. 99.  2014. Site-specific antibody drug conjugates for cancer therapy. mAbs 6:34–45 [Google Scholar]
  100. Chari RV, Miller ML, Widdison WC. 100.  2014. Antibody–drug conjugates: an emerging concept in cancer therapy. Angew. Chem. Int. Ed. 53:3796–827 [Google Scholar]
  101. Kubota T, Niwa R, Satoh M, Akinaga S, Shitara K, Hanai N. 101.  2009. Engineered therapeutic antibodies with improved effector functions. Cancer Sci. 100:1566–72 [Google Scholar]
  102. Sievers EL, Senter PD. 102.  2013. Antibody–drug conjugates in cancer therapy. Annu. Rev. Med. 64:15–29 [Google Scholar]
  103. Wang L, Amphlett G, Blattler WA, Lambert JM, Zhang W. 103.  2005. Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry. Protein Sci. 14:2436–46 [Google Scholar]
  104. Doronina SO, Toki BE, Torgov MY, Mendelsohn BA, Cerveny CG. 104.  et al. 2003. Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat. Biotechnol. 21:778–84 [Google Scholar]
  105. Sun MM, Beam KS, Cerveny CG, Hamblett KJ, Blackmore RS. 105.  et al. 2005. Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides. Bioconjug. Chem. 16:1282–90 [Google Scholar]
  106. McDonagh CF, Turcott E, Westendorf L, Webster JB, Alley SC. 106.  et al. 2006. Engineered antibody–drug conjugates with defined sites and stoichiometries of drug attachment. Protein Eng. Des. Sel. 19:299–307 [Google Scholar]
  107. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD. 107.  et al. 2008. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 26:925–32 [Google Scholar]
  108. Junutula JR, Bhakta S, Raab H, Ervin KE, Eigenbrot C. 108.  et al. 2008. Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs. J. Immunol. Methods 332:41–52 [Google Scholar]
  109. Shen BQ, Xu K, Liu L, Raab H, Bhakta S. 109.  et al. 2012. Conjugation site modulates the in vivo stability and therapeutic activity of antibody–drug conjugates. Nat. Biotechnol. 30:184–89 [Google Scholar]
  110. Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH. 110.  et al. 2012. Synthesis of site-specific antibody–drug conjugates using unnatural amino acids. PNAS 109:16101–6 [Google Scholar]
  111. Hofer T, Skeffington LR, Chapman CM, Rader C. 111.  2009. Molecularly defined antibody conjugation through a selenocysteine interface. Biochemistry 48:12047–57 [Google Scholar]
  112. Boeggeman E, Ramakrishnan B, Pasek M, Manzoni M, Puri A. 112.  et al. 2009. Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection. Bioconjug. Chem. 20:1228–36 [Google Scholar]
  113. Strop P, Liu SH, Dorywalska M, Delaria K, Dushin RG. 113.  et al. 2013. Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem. Biol. 20:161–67 [Google Scholar]
  114. Jeger S, Zimmermann K, Blanc A, Grunberg J, Honer M. 114.  et al. 2010. Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase. Angew. Chem. Int. Ed. 49:9995–97 [Google Scholar]
  115. Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J. 115.  et al. 2004. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin. Cancer Res. 10:7063–70 [Google Scholar]
  116. Polson AG, Calemine-Fenaux J, Chan P, Chang W, Christensen E. 116.  et al. 2009. Antibody–drug conjugates for the treatment of non-Hodgkin's lymphoma: target and linker-drug selection. Cancer Res. 69:2358–64 [Google Scholar]
  117. Nolting B. 117.  2013. Linker technologies for antibody–drug conjugates. Methods Mol. Biol. 1045:71–100 [Google Scholar]
  118. Senter PD. 118.  2009. Potent antibody drug conjugates for cancer therapy. Curr. Opin. Chem. Biol. 13:235–44 [Google Scholar]
  119. Behrens CR, Liu B. 119.  2014. Methods for site-specific drug conjugation to antibodies. mAbs 6:46–53 [Google Scholar]
  120. Klein C, Sustmann C, Thomas M, Stubenrauch K, Croasdale R. 120.  et al. 2012. Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies. mAbs 4:653–63 [Google Scholar]
  121. Carter P. 121.  2001. Bispecific human IgG by design. J. Immunol. Methods 248:7–15 [Google Scholar]
  122. Holliger P, Winter G. 122.  1993. Engineering bispecific antibodies. Curr. Opin. Biotechnol. 4:446–49 [Google Scholar]
  123. Marvin JS, Zhu Z. 123.  2005. Recombinant approaches to IgG-like bispecific antibodies. Acta Pharmacol. Sin. 26:649–58 [Google Scholar]
  124. Gunasekaran K, Pentony M, Shen M, Garrett L, Forte C. 124.  et al. 2010. Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. J. Biol. Chem. 285:19637–46 [Google Scholar]
  125. Davis JH, Aperlo C, Li Y, Kurosawa E, Lan Y. 125.  et al. 2010. SEEDbodies: fusion proteins based on strand-exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies. Protein Eng. Des. Sel. 23:195–202 [Google Scholar]
  126. Merchant AM, Zhu Z, Yuan JQ, Goddard A, Adams CW. 126.  et al. 1998. An efficient route to human bispecific IgG. Nat. Biotechnol. 16:677–81 [Google Scholar]
  127. Ridgway JB, Presta LG, Carter P. 127.  1996. ‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 9:617–21 [Google Scholar]
  128. Lindhofer H, Mocikat R, Steipe B, Thierfelder S. 128.  1995. Preferential species-restricted heavy/light chain pairing in rat/mouse quadromas. Implications for a single-step purification of bispecific antibodies. J. Immunol. 155:219–25 [Google Scholar]
  129. Strop P, Ho WH, Boustany LM, Abdiche YN, Lindquist KC. 129.  et al. 2012. Generating bispecific human IgG1 and IgG2 antibodies from any antibody pair. J. Mol. Biol. 420:204–19 [Google Scholar]
  130. Dong J, Sereno A, Aivazian D, Langley E, Miller BR. 130.  et al. 2011. A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity. mAbs 3:273–88 [Google Scholar]
  131. Xu L, Kohli N, Rennard R, Jiao Y, Razlog M. 131.  et al. 2013. Rapid optimization and prototyping for therapeutic antibody-like molecules. mAbs 5:237–54 [Google Scholar]
  132. Orcutt KD, Ackerman ME, Cieslewicz M, Quiroz E, Slusarczyk AL. 132.  et al. 2010. A modular IgG-scFv bispecific antibody topology. Protein Eng. Des. Sel. 23:221–28 [Google Scholar]
  133. Coloma MJ, Morrison SL. 133.  1997. Design and production of novel tetravalent bispecific antibodies. Nat. Biotechnol. 15:159–63 [Google Scholar]
  134. Shen J, Vil MD, Jimenez X, Iacolina M, Zhang H, Zhu Z. 134.  2006. Single variable domain–IgG fusion. a novel recombinant approach to Fc domain-containing bispecific antibodies. J. Biol. Chem. 281:10706–14 [Google Scholar]
  135. Holliger P, Prospero T, Winter G. 135.  1993. “Diabodies”: small bivalent and bispecific antibody fragments. PNAS 90:6444–48 [Google Scholar]
  136. Zhu Z, Presta LG, Zapata G, Carter P. 136.  1997. Remodeling domain interfaces to enhance heterodimer formation. Protein Sci. 6:781–88 [Google Scholar]
  137. Spiess C, Merchant M, Huang A, Zheng Z, Yang NY. 137.  et al. 2013. Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies. Nat. Biotechnol. 31:753–58 [Google Scholar]
  138. Kostelny SA, Cole MS, Tso JY. 138.  1992. Formation of a bispecific antibody by the use of leucine zippers. J. Immunol. 148:1547–53 [Google Scholar]
  139. Atwell S, Ridgway JB, Wells JA, Carter P. 139.  1997. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J. Mol. Biol. 270:26–35 [Google Scholar]
  140. Schaefer W, Regula JT, Bahner M, Schanzer J, Croasdale R. 140.  et al. 2011. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies. PNAS 108:11187–92 [Google Scholar]
  141. Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F. 141.  et al. 2014. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. 32:191–98 [Google Scholar]
  142. Wu C, Ying H, Grinnell C, Bryant S, Miller R. 142.  et al. 2007. Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat. Biotechnol. 25:1290–97 [Google Scholar]
  143. Wu C, Ying H, Bose S, Miller R, Medina L. 143.  et al. 2009. Molecular construction and optimization of anti-human IL-1α/β dual variable domain immunoglobulin (DVD-Ig) molecules. mAbs 1:339–47 [Google Scholar]
  144. Zuo Z, Jimenez X, Witte L, Zhu Z. 144.  2000. An efficient route to the production of an IgG-like bispecific antibody. Protein Eng. 13:361–67 [Google Scholar]
  145. Weitzner BD, Kuroda D, Marze N, Xu J, Gray JJ. 145.  2014. Blind prediction performance of RosettaAntibody 3.0: grafting, relaxation, kinematic loop modeling, and full CDR optimization. Proteins 82:1611–23 [Google Scholar]
  146. Shirai H, Ikeda K, Yamashita K, Tsuchiya Y, Sarmiento J. 146.  et al. 2014. High-resolution modeling of antibody structures by a combination of bioinformatics, expert knowledge, and molecular simulations. Proteins 82:1624–35 [Google Scholar]
  147. Zhu K, Day T. 147.  2013. Ab initio structure prediction of the antibody hypervariable H3 loop. Proteins 81:1081–89 [Google Scholar]
  148. Fleishman SJ, Whitehead TA, Ekiert DC, Dreyfus C, Corn JE. 148.  et al. 2011. Computational design of proteins targeting the conserved stem region of influenza hemagglutinin. Science 332:816–21 [Google Scholar]
  149. Stranges PB, Machius M, Miley MJ, Tripathy A, Kuhlman B. 149.  2011. Computational design of a symmetric homodimer using β-strand assembly. PNAS 108:20562–67 [Google Scholar]
  150. Xia Z, Huynh T, Kang SG, Zhou R. 150.  2012. Free-energy simulations reveal that both hydrophobic and polar interactions are important for influenza hemagglutinin antibody binding. Biophys. J. 102:1453–61 [Google Scholar]

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