The term bispecific antibody (bsAb) is used to describe a large family of molecules designed to recognize two different epitopes or antigens. BsAbs come in many formats, ranging from relatively small proteins, merely consisting of two linked antigen-binding fragments, to large immunoglobulin G (IgG)-like molecules with additional domains attached. An attractive bsAb feature is their potential for novel functionalities — that is, activities that do not exist in mixtures of the parental or reference antibodies. In these so-called obligate bsAbs, the physical linkage of the two binding specificities creates a dependency that can be temporal, with binding events occurring sequentially, or spatial, with binding events occurring simultaneously, such as in linking an effector to a target cell. To date, more than 20 different commercialized technology platforms are available for bsAb creation and development, 2 bsAbs are marketed and over 85 are in clinical development. Here, we review the current bsAb landscape from a mechanistic perspective, including a comprehensive overview of the pipeline.
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Nisonoff, A., Wissler, F. C. & Lipman, L. N. Properties of the major component of a peptic digest of rabbit antibody. Science 132, 1770–1771 (1960).
Riethmuller, G. Symmetry breaking: bispecific antibodies, the beginnings, and 50 years on. Cancer Immun. 12, 12 (2012).
Fudenberg, H. H., Drews, G. & Nisonoff, A. Serologic demonstration of dual specificity of rabbit bivalent hybrid antibody. J. Exp. Med. 119, 151–166 (1964).
Brinkmann, U. & Kontermann, R. E. The making of bispecific antibodies. mAbs 9, 182–212 (2017).
Ha, J. H., Kim, J. E. & Kim, Y. S. Immunoglobulin Fc heterodimer platform technology: from design to applications in therapeutic antibodies and proteins. Front. Immunol. 7, 394 (2016).
Spiess, C., Zhai, Q. & Carter, P. J. Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol. Immunol. 67, 95–106 (2015).
Godar, M., de Haard, H., Blanchetot, C. & Rasser, J. Therapeutic bispecific antibody formats: a patent applications review (1994–2017). Expert Opin. Ther. Pat. 28, 251–276 (2018).
Staerz, U. D., Kanagawa, O. & Bevan, M. J. Hybrid antibodies can target sites for attack by T cells. Nature 314, 628–631 (1985).
Perez, P., Hoffman, R. W., Shaw, S., Bluestone, J. A. & Segal, D. M. Specific targeting of cytotoxic T cells by anti-T3 linked to anti-target cell antibody. Nature 316, 354–356 (1985).
Heiss, M. M. et al. The trifunctional antibody catumaxomab for the treatment of malignant ascites due to epithelial cancer: results of a prospective randomized phase II/III trial. Int. J. Cancer 127, 2209–2221 (2010).
Borlak, J., Langer, F., Spanel, R., Schondorfer, G. & Dittrich, C. Immune-mediated liver injury of the cancer therapeutic antibody catumaxomab targeting EpCAM, CD3 and Fcgamma receptors. Oncotarget 7, 28059–28074 (2016).
Gokbuget, N. et al. Blinatumomab for minimal residual disease in adults with B-precursor acute lymphoblastic leukemia. Blood 131, 1522–1531 (2018).
Kantarjian, H. et al. Blinatumomab versus chemotherapy for advanced acute lymphoblastic leukemia. N. Engl. J. Med. 376, 836–847 (2017).
de Bruin, R. C. G. et al. A bispecific nanobody approach to leverage the potent and widely applicable tumor cytolytic capacity of Vgamma9Vdelta2-T cells. Oncoimmunology 7, e1375641 (2017).
Oldenburg, J. et al. Emicizumab prophylaxis in hemophilia A with inhibitors. N. Engl. J. Med. 377, 809–818 (2017).
Labrijn, A. F. & Parren, P. W. Hitting Ebola, to the power of two. Science 354, 284–285 (2016).
Mullard, A. Bispecific antibody pipeline moves beyond oncology. Nat. Rev. Drug Discov. 16, 666–668 (2017).
Milstein, C. & Cuello, A. C. Hybrid hybridomas and their use in immunohistochemistry. Nature 305, 537–540 (1983).
Birch, J. R. & Racher, A. J. Antibody production. Adv. Drug Deliv. Rev. 58, 671–685 (2006).
Demarest, S. J. & Glaser, S. M. Antibody therapeutics, antibody engineering, and the merits of protein stability. Curr. Opin. Drug Discov. Devel. 11, 675–687 (2008).
Lowe, D. et al. Aggregation, stability, and formulation of human antibody therapeutics. Adv. Protein Chem. Struct. Biol. 84, 41–61 (2011).
Harwood, S. L. et al. ATTACK, a novel bispecific T cell-recruiting antibody with trivalent EGFR binding and monovalent CD3 binding for cancer immunotherapy. Oncoimmunology 7, e1377874 (2017).
Blanco-Toribio, A. et al. Generation and characterization of monospecific and bispecific hexavalent trimerbodies. mAbs 5, 70–79 (2013).
Compte, M. et al. A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity. Nat. Commun. 9, 4809 (2018).
Chames, P. & Baty, D. Bispecific antibodies for cancer therapy. Curr. Opin. Drug Discov. Devel. 12, 276–283 (2009).
Chan, A. C. & Carter, P. J. Therapeutic antibodies for autoimmunity and inflammation. Nat. Rev. Immunol. 10, 301–316 (2010).
Wu, C. et al. Molecular construction and optimization of anti-human IL-1alpha/beta dual variable domain immunoglobulin (DVD-Ig) molecules. mAbs 1, 339–347 (2009).
Bonisch, M. et al. Novel CH1:CL interfaces that enhance correct light chain pairing in heterodimeric bispecific antibodies. Protein Eng. Des. Sel. 30, 685–696 (2017).
Lewis, S. M. et al. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. 32, 191–198 (2014).
Lindhofer, H., Mocikat, R., Steipe, B. & Thierfelder, S. Preferential species-restricted heavy/light chain pairing in rat/mouse quadromas. Implications for a single-step purification of bispecific antibodies. J. Immunol. 155, 219–225 (1995).
Mazor, Y. et al. Improving target cell specificity using a novel monovalent bispecific IgG design. mAbs 7, 377–389 (2015).
Schaefer, W. et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies. Proc. Natl Acad. Sci. USA 108, 11187–11192 (2011).
Wu, X. et al. Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies. mAbs 7, 364–376 (2015).
Cooke, H. et al. EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies. mAbs 10, 1248–1259 (2018).
Choi, H. J., Kim, Y. J., Lee, S. & Kim, Y. S. A heterodimeric Fc-based bispecific antibody simultaneously targeting VEGFR-2 and Met exhibits potent antitumor activity. Mol. Cancer Ther. 12, 2748–2759 (2013).
Davis, J. H. et al. 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 (2010).
De Nardis, C. et al. A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1. J. Biol. Chem. 292, 14706–14717 (2017).
Gunasekaran, K. et al. Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. J. Biol. Chem. 285, 19637–19646 (2010).
Leaver-Fay, A. et al. Computationally designed bispecific antibodies using negative state repertoires. Structure 24, 641–651 (2016).
Merchant, A. M. et al. An efficient route to human bispecific IgG. Nat. Biotechnol. 16, 677–681 (1998).
Moore, G. L. et al. A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. mAbs 3, 546–557 (2011).
Skegro, D. et al. Immunoglobulin domain interface exchange as a platform technology for the generation of Fc heterodimers and bispecific antibodies. J. Biol. Chem. 292, 9745–9759 (2017).
Von Kreudenstein, T. S. et al. Improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design. mAbs 5, 646–654 (2013).
Wei, H. et al. Structural basis of a novel heterodimeric Fc for bispecific antibody production. Oncotarget 8, 51037–51049 (2017).
Fischer, N. et al. Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG. Nat. Commun. 6, 6113 (2015).
Smith, E. J. et al. A novel, native-format bispecific antibody triggering T cell killing of B cells is robustly active in mouse tumor models and cynomolgus monkeys. Sci. Rep. 5, 17943 (2015).
Jackman, J. et al. Development of a two-part strategy to identify a therapeutic human bispecific antibody that inhibits IgE receptor signaling. J. Biol. Chem. 285, 20850–20859 (2010).
Shatz, W. et al. An efficient route to bispecific antibody production using single-reactor mammalian co-culture. mAbs 8, 1487–1497 (2016).
Spiess, C. et al. Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies. Nat. Biotechnol. 31, 753–758 (2013).
Huang, S. et al. Structural and functional characterization of MBS301, an afucosylated bispecific anti-HER2 antibody. mAbs 10, 864–875 (2018).
Labrijn, A. F. et al. Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. Proc. Natl Acad. Sci. USA 110, 5145–5150 (2013).
Strop, P. et al. Generating bispecific human IgG1 and IgG2 antibodies from any antibody pair. J. Mol. Biol. 420, 204–219 (2012).
Wec, A. Z. et al. A “Trojan horse” bispecific antibody strategy for broad protection against ebolaviruses. Science 354, 350–354 (2016).
De Gast, G. C. et al. Clinical experience with CD3 x CD19 bispecific antibodies in patients with B cell malignancies. J. Hematother 4, 433–437 (1995).
Tibben, J. G. et al. Pharmacokinetics, biodistribution and biological effects of intravenously administered bispecific monoclonal antibody OC/TR F(ab’)2 in ovarian carcinoma patients. Int. J. Cancer 66, 477–483 (1996).
Bargou, R. et al. Tumor regression in cancer patients by very low doses of a T cell-engaging antibody. Science 321, 974–977 (2008).
Loffler, A. et al. A recombinant bispecific single-chain antibody, CD19 x CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes. Blood 95, 2098–2103 (2000).
Klinger, M. et al. Immunopharmacologic response of patients with B-lineage acute lymphoblastic leukemia to continuous infusion of T cell-engaging CD19/CD3-bispecific BiTE antibody blinatumomab. Blood 119, 6226–6233 (2012).
Topp, M. S. et al. Safety and activity of blinatumomab for adult patients with relapsed or refractory B-precursor acute lymphoblastic leukaemia: a multicentre, single-arm, phase 2 study. Lancet Oncol. 16, 57–66 (2015).
Chatenoud, L. et al. In vivo cell activation following OKT3 administration. Systemic cytokine release and modulation by corticosteroids. Transplantation 49, 697–702 (1990).
Xu, D. et al. In vitro characterization of five humanized OKT3 effector function variant antibodies. Cell. Immunol. 200, 16–26 (2000).
Woodle, E. S. et al. Phase I trial of a humanized, Fc receptor nonbinding OKT3 antibody, huOKT3gamma1(Ala-Ala) in the treatment of acute renal allograft rejection. Transplantation 68, 608–616 (1999).
Labrijn, A. F. et al. Efficient generation of bispecific murine antibodies for pre-clinical investigations in syngeneic rodent models. Sci. Rep. 7, 2476 (2017).
Vafa, O. et al. An engineered Fc variant of an IgG eliminates all immune effector functions via structural perturbations. Methods 65, 114–126 (2014).
Schlothauer, T. et al. Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein Eng. Des. Sel. 29, 457–466 (2016).
Moore, P. A. et al. Application of dual affinity retargeting molecules to achieve optimal redirected T cell killing of B cell lymphoma. Blood 117, 4542–4551 (2011).
Tita-Nwa, F. et al. Cytokine-induced killer cells targeted by the novel bispecific antibody CD19xCD5 (HD37xT5.16) efficiently lyse B-lymphoma cells. Cancer Immunol. Immunother. 56, 1911–1920 (2007).
Pessano, S., Oettgen, H., Bhan, A. K. & Terhorst, C. The T3/T cell receptor complex: antigenic distinction between the two 20-kd T3 (T3-delta and T3-epsilon) subunits. EMBO J. 4, 337–344 (1985).
Leong, S. R. et al. An anti-CD3/anti-CLL-1 bispecific antibody for the treatment of acute myeloid leukemia. Blood 129, 609–618 (2017).
Bortoletto, N., Scotet, E., Myamoto, Y., D’Oro, U. & Lanzavecchia, A. Optimizing anti-CD3 affinity for effective T cell targeting against tumor cells. Eur. J. Immunol. 32, 3102–3107 (2002).
List, T. & Neri, D. Biodistribution studies with tumor-targeting bispecific antibodies reveal selective accumulation at the tumor site. mAbs 4, 775–783 (2012).
Mandikian, D. et al. Relative target affinities of T-cell-dependent bispecific antibodies determine biodistribution in a solid tumor mouse model. Mol. Cancer Ther. 17, 776–785 (2018).
Chatenoud, L. CD3-specific antibody-induced active tolerance: from bench to bedside. Nat. Rev. Immunol. 3, 123–132 (2003).
Reusch, U. et al. Characterization of CD33/CD3 tetravalent bispecific tandem diabodies (TandAbs) for the treatment of acute myeloid leukemia. Clin. Cancer Res. 22, 5829–5838 (2016).
Comeau, M. R. et al. Abstract 1786: APVO436, a bispecific anti-CD123 x anti-CD3 ADAPTIR™ molecule for redirected T cell cytotoxicity, induces potent T cell activation, proliferation and cytotoxicity with limited cytokine release. Cancer Res. 78, 1786 (2018).
Offner, S., Hofmeister, R., Romaniuk, A., Kufer, P. & Baeuerle, P. A. Induction of regular cytolytic T cell synapses by bispecific single-chain antibody constructs on MHC class I-negative tumor cells. Mol. Immunol. 43, 763–771 (2006).
Baeuerle, P. A. Development of T-cell-engaging bispecific antibody blinatumomab (Blincyto®) for treatment of B-cell malignancies. Successful Drug Discov. https://doi.org/10.1002/9783527808694.ch5 (2018).
Purbhoo, M. A., Irvine, D. J., Huppa, J. B. & Davis, M. M. T cell killing does not require the formation of a stable mature immunological synapse. Nat. Immunol. 5, 524–530 (2004).
Liddy, N. et al. Monoclonal TCR-redirected tumor cell killing. Nat. Med. 18, 980–987 (2012).
Oberst, M. D. et al. CEA/CD3 bispecific antibody MEDI-565/AMG 211 activation of T cells and subsequent killing of human tumors is independent of mutations commonly found in colorectal adenocarcinomas. mAbs 6, 1571–1584 (2014).
Laszlo, G. S. et al. Cellular determinants for preclinical activity of a novel CD33/CD3 bispecific T cell engager (BiTE) antibody, AMG 330, against human AML. Blood 123, 554–561 (2014).
Lopez-Albaitero, A. et al. Overcoming resistance to HER2-targeted therapy with a novel HER2/CD3 bispecific antibody. Oncoimmunology 6, e1267891 (2017).
Hammond, S. A. et al. Selective targeting and potent control of tumor growth using an EphA2/CD3-Bispecific single-chain antibody construct. Cancer Res. 67, 3927–3935 (2007).
Friedrich, M. et al. Regression of human prostate cancer xenografts in mice by AMG 212/BAY2010112, a novel PSMA/CD3-Bispecific BiTE antibody cross-reactive with non-human primate antigens. Mol. Cancer Ther. 11, 2664–2673 (2012).
Li, J. et al. Membrane-proximal epitope facilitates efficient T cell synapse formation by anti-FcRH5/CD3 and is a requirement for myeloma cell killing. Cancer Cell 31, 383–395 (2017).
Pfosser, A., Brandl, M., Salih, H., Grosse-Hovest, L. & Jung, G. Role of target antigen in bispecific-antibody-mediated killing of human glioblastoma cells: a pre-clinical study. Int. J. Cancer 80, 612–616 (1999).
Bluemel, C. et al. Epitope distance to the target cell membrane and antigen size determine the potency of T cell-mediated lysis by BiTE antibodies specific for a large melanoma surface antigen. Cancer Immunol. Immunother. 59, 1197–1209 (2010).
Ahmed, M., Cheng, M., Cheung, I. Y. & Cheung, N. K. Human derived dimerization tag enhances tumor killing potency of a T cell engaging bispecific antibody. Oncoimmunology 4, e989776 (2015).
Slaga, D. et al. Avidity-based binding to HER2 results in selective killing of HER2-overexpressing cells by anti-HER2/CD3. Sci. Transl Med. 10, eaat5775 (2018).
Bacac, M. et al. CD20 Tcb (RG6026), a novel “2:1” T cell bispecific antibody for the treatment of B cell malignancies. Blood 128, 1836 (2016).
Hiemstra, I. H. et al. Duobody-CD3xCD20 shows unique and potent preclinical anti-tumor activity in vitro and in vivo, and is being evaluated clinically in patients with B-cell malignancies. Blood 132, 1664–1664 (2018).
Bacac, M. et al. A novel carcinoembryonic antigen T-cell bispecific antibody (CEA TCB) for the treatment of solid tumors. Clin. Cancer Res. 22, 3286–3297 (2016).
Fisher, T. S. et al. A CD3-bispecific molecule targeting P-cadherin demonstrates T cell-mediated regression of established solid tumors in mice. Cancer Immunol. Immunother. 67, 247–259 (2018).
Ishiguro, T. et al. An anti-glypican 3/CD3 bispecific T cell-redirecting antibody for treatment of solid tumors. Sci. Transl Med. 9, eaal4291 (2017).
Benonisson, H. et al. CD3-bispecific antibody therapy turns solid tumors into inflammatory sites but does not install protective memory. Mol. Cancer Ther. 18, 312–322 (2019).
Braig, F. et al. Resistance to anti-CD19/CD3 BiTE in acute lymphoblastic leukemia may be mediated by disrupted CD19 membrane trafficking. Blood 129, 100–104 (2017).
Junttila, T. T. et al. Antitumor efficacy of a bispecific antibody that targets HER2 and activates T cells. Cancer Res. 74, 5561–5571 (2014).
Feucht, J. et al. T cell responses against CD19+pediatric acute lymphoblastic leukemia mediated by bispecific T cell engager (BiTE) are regulated contrarily by PD-L1 and CD80/CD86 on leukemic blasts. Oncotarget 7, 76902–76919 (2016).
Osada, T. et al. CEA/CD3-bispecific T cell-engaging (BiTE) antibody-mediated T lymphocyte cytotoxicity maximized by inhibition of both PD1 and PD-L1. Cancer Immunol. Immunother. 64, 677–688 (2015).
Krupka, C. et al. Blockade of the PD-1/PD-L1 axis augments lysis of AML cells by the CD33/CD3 BiTE antibody construct AMG 330: reversing a T cell-induced immune escape mechanism. Leukemia 30, 484–491 (2016).
Argiles, G. et al. Novel carcinoembryonic antigen T cell bispecific (CEA-TCB) antibody: preliminary clinical data as a single agent and in combination with atezolizumab in patients with metastatic colorectal cancer (mCRC) [abstract LBA-004]. Ann. Oncol. 28, mdx302.003 (2017).
Webster, J. et al. Blinatumomab in combination with immune checkpoint inhibitors of PD-1 and CTLA-4 in adult patients with relapsed/refractory (R/R) CD19 positive B-cell acute lymphoblastic leukemia (ALL): preliminary results of a phase I study. Blood 132, 557 (2018).
Topp, M. S. et al. Safety and preliminary antitumor activity of the anti-PD-1 monoclonal antibody REGN2810 alone or in combination with REGN1979, an anti-CD20 x anti-CD3 bispecific antibody, in patients with B-lymphoid malignancies. Blood 130, 1495 (2017).
Wolchok, J. D. et al. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. N. Engl. J. Med. 377, 1345–1356 (2017).
Dovedi, S. J. et al. MEDI5752: a novel bispecific antibody that preferentially targets CTLA-4 on PD-1 expressing T cells [abstract 2776]. Cancer Res. 78, 2776 (2018).
Hedvat, M. et al. Simultaneous checkpoint — checkpoint or checkpoint — costimulatory receptor targeting with bispecific antibodies promotes enhanced human T cell activation [abstract P664]. Presented at the 2018 Society for Immunotherapy of Cancer (SITC) (2018).
Lutterbuese, R. et al. T cell-engaging BiTE antibodies specific for EGFR potently eliminate KRAS- and BRAF-mutated colorectal cancer cells. Proc. Natl Acad. Sci. USA 107, 12605–12610 (2010).
Ross, S. L. et al. Bispecific T cell engager (BiTE(R)) antibody constructs can mediate bystander tumor cell killing. PLOS ONE 12, e0183390 (2017).
Kebenko, M. et al. A multicenter phase 1 study of solitomab (MT110, AMG 110), a bispecific EpCAM/CD3 T cell engager (BiTE(R)) antibody construct, in patients with refractory solid tumors. Oncoimmunology 7, e1450710 (2018).
Duell, J. et al. Frequency of regulatory T cells determines the outcome of the T cell-engaging antibody blinatumomab in patients with B-precursor ALL. Leukemia 31, 2181–2190 (2017).
Kabelitz, D., Lettau, M. & Janssen, O. Immunosurveillance by human gammadelta T lymphocytes: the emerging role of butyrophilins. F1000Res 6, 782 (2017).
Tosolini, M. et al. Assessment of tumor-infiltrating TCRVgamma9Vdelta2 gammadelta lymphocyte abundance by deconvolution of human cancers microarrays. Oncoimmunology 6, e1284723 (2017).
Gentles, A. J. et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat. Med. 21, 938–945 (2015).
Don Yun, H. et al. Trispecific killer engager CD16xIL15xCD33 potently induces NK cell activation and cytotoxicity against neoplastic mast cells. Blood Adv. 2, 1580–1584 (2018).
Schmohl, J. U., Felices, M., Taras, E., Miller, J. S. & Vallera, D. A. Enhanced ADCC and NK cell activation of an anticarcinoma bispecific antibody by genetic insertion of a modified IL-15 cross-linker. Mol. Ther. 24, 1312–1322 (2016).
Oberg, H. H. et al. Tribody [(HER2)2xCD16] is more effective than trastuzumab in enhancing gammadelta T cell and natural killer cell cytotoxicity against HER2-expressing cancer cells. Front. Immunol. 9, 814 (2018).
Reusch, U. et al. A novel tetravalent bispecific TandAb (CD30/CD16A) efficiently recruits NK cells for the lysis of CD30+tumor cells. mAbs 6, 728–739 (2014).
Pahl, J. H. W. et al. CD16A activation of NK cells promotes NK cell proliferation and memory-like cytotoxicity against cancer cells. Cancer Immunol. Res. 6, 517–527 (2018).
Dheilly, E. et al. Selective blockade of the ubiquitous checkpoint receptor CD47 is enabled by dual-targeting bispecific antibodies. Mol. Ther. 25, 523–533 (2017).
Kruse, R. L. et al. In situ liver expression of HBsAg/CD3-bispecific antibodies for HBV immunotherapy. Mol. Ther. Methods Clin. Dev. 7, 32–41 (2017).
Meng, W. et al. Targeting human-cytomegalovirus-infected cells by redirecting T cells using an anti-CD3/anti-glycoprotein B bispecific antibody. Antimicrob. Agents Chemother. 62, e01719–17 (2018).
Fabozzi, G., Pegu, A., Koup, R. A. & Petrovas, C. Bispecific antibodies: potential immunotherapies for HIV treatment. Methods 154, 118–124 (2018).
Pegu, A. et al. Activation and lysis of human CD4 cells latently infected with HIV-1. Nat. Commun. 6, 8447 (2015).
Brozy, J. et al. Antiviral activity of HIV gp120-targeting bispecific T cell engager antibody constructs. J. Virol. 92, e00491–18 (2018).
Sloan, D. D. et al. Targeting HIV reservoir in infected CD4 T cells by dual-affinity re-targeting molecules (DARTs) that bind HIV envelope and recruit cytotoxic T cells. PLOS Pathog. 11, (e1005233 (2015).
Sung, J. A. et al. Dual-affinity re-targeting proteins direct T cell-mediated cytolysis of latently HIV-infected cells. J. Clin. Invest. 125, 4077–4090 (2015).
Wu, G. et al. HDAC inhibition induces HIV-1 protein and enables immune-based clearance following latency reversal. JCI Insight 2, e92901 (2017).
Orlic, D. et al. Bone marrow cells regenerate infarcted myocardium. Nature 410, 701–705 (2001).
Chong, J. J. et al. Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature 510, 273–277 (2014).
Barbash, I. M. et al. Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution. Circulation 108, 863–868 (2003).
Li, Z. et al. Pretargeting and bioorthogonal click chemistry-mediated endogenous stem cell homing for heart repair. ACS Nano 12, 12193–12200 (2018).
Ziegler, M. et al. Platelet-targeted delivery of peripheral blood mononuclear cells to the ischemic heart restores cardiac function after ischemia-reperfusion injury. Theranostics 7, 3192–3206 (2017).
Engelman, J. A. et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316, 1039–1043 (2007).
Moores, S. L. et al. A novel bispecific antibody targeting EGFR and cMet is effective against EGFR inhibitor-resistant lung tumors. Cancer Res. 76, 3942–3953 (2016).
Grugan, K. D. et al. Fc-mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced killing of lung cancer cells. mAbs 9, 114–126 (2017).
Cho, B. C. et al. JNJ-61186372 (JNJ-372), an EGFR-cMET bispecific antibody, in advanced non-small cell lung cancer (NSCLC): an update on phase I results [abstract 1497P]. Ann. Oncol. 29, mdy292.118 (2018).
Geuijen, C. A. W. et al. Unbiased combinatorial screening identifies a bispecific IgG1 that potently inhibits HER3 signaling via HER2-guided ligand blockade. Cancer Cell 33, 922–936 (2018).
Li, Y. et al. ABT-165, a dual variable domain immunoglobulin (DVD-Ig) targeting DLL4 and VEGF, demonstrates superior efficacy and favorable safety profiles in preclinical models. Mol. Cancer Ther. 17, 1039–1050 (2018).
Regula, J. T. et al. Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases. EMBO Mol. Med. 8, 1265–1288 (2016).
Weisser, N., Wickman, G., Davies, R. & Rowse, G. Preclinical development of a novel biparatopic HER2 antibody with activity in low to high HER2 expressing cancers [abstract 31]. Cancer Res. 77, 31 (2017).
American Association for Cancer Research. ZW25 effective in HER2-positive cancers. Cancer Discov. 9, 8 (2018).
Li, J. Y. et al. A biparatopic HER2-targeting antibody-drug conjugate induces tumor regression in primary models refractory to or ineligible for HER2-targeted therapy. Cancer Cell 29, 117–129 (2016).
Coskun, T. et al. Fibroblast growth factor 21 corrects obesity in mice. Endocrinology 149, 6018–6027 (2008).
Xu, J. et al. Acute glucose-lowering and insulin-sensitizing action of FGF21 in insulin-resistant mouse models—association with liver and adipose tissue effects. Am. J. Physiol. Endocrinol. Metab. 297, E1105–E1114 (2009).
Wu, A. L. et al. Amelioration of type 2 diabetes by antibody-mediated activation of fibroblast growth factor receptor 1. Sci. Transl Med. 3, 113ra126 (2011).
Kolumam, G. et al. Sustained brown fat stimulation and insulin sensitization by a humanized bispecific antibody agonist for fibroblast growth factor receptor 1/betaklotho complex. EBioMedicine 2, 730–743 (2015).
Fon Tacer, K. et al. Research resource: comprehensive expression atlas of the fibroblast growth factor system in adult mouse. Mol. Endocrinol. 24, 2050–2064 (2010).
Arora, P. S. et al. A bispecific agonistic antibody to FGF-R1/KlothoB improves the cardiometabolic profile in otherwise healthy obese subjects—preliminary results from the first-in-human single ascending dose study [abstract #1096]. Presented at the American Diabetes Association’s 77th Scientific Sessions (2017).
Sampei, Z. et al. Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity. PLOS ONE 8, e57479 (2013).
Kitazawa, T. et al. Factor VIIIa-mimetic cofactor activity of a bispecific antibody to factors IX/IXa and X/Xa, emicizumab, depends on its ability to bridge the antigens. Thromb. Haemost. 117, 1348–1357 (2017).
Shima, M. et al. Factor VIII-mimetic function of humanized bispecific antibody in hemophilia A. N. Engl. J. Med. 374, 2044–2053 (2016).
Mahlangu, J. et al. Emicizumab prophylaxis in patients who have hemophilia A without inhibitors. N. Engl. J. Med. 379, 811–822 (2018).
Raso, V. & Griffin, T. Hybrid antibodies with dual specificity for the delivery of ricin to immunoglobulin-bearing target cells. Cancer Res. 41, 2073–2078 (1981).
Raso, V., Brown, M. & McGrath, J. Intracellular targeting with low pH-triggered bispecific antibodies. J. Biol. Chem. 272, 27623–27628 (1997).
Yu, Y. J. et al. Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target. Sci. Transl Med. 3, 84ra44 (2011).
Yu, Y. J. et al. Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates. Sci. Transl Med. 6, 261ra154 (2014).
Niewoehner, J. et al. Increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle. Neuron 81, 49–60 (2014).
Bezabeh, B. et al. Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties. mAbs 9, 240–256 (2017).
DiGiandomenico, A. et al. A multifunctional bispecific antibody protects against Pseudomonas aeruginosa. Sci. Transl Med. 6, 262ra155 (2014).
Thanabalasuriar, A. et al. Bispecific antibody targets multiple Pseudomonas aeruginosa evasion mechanisms in the lung vasculature. J. Clin. Invest. 127, 2249–2261 (2017).
Tabor, D. E. et al. Pseudomonas aeruginosa PcrV and Psl, the molecular targets of bispecific antibody MEDI3902, are conserved among diverse global clinical isolates. J. Infect. Dis. 218, 1983–1994 (2018).
de Goeij, B. E. et al. Efficient payload delivery by a bispecific antibody-drug conjugate targeting HER2 and CD63. Mol. Cancer Ther. 15, 2688–2697 (2016).
Schmidt, E. G. W. et al. Direct demonstration of a neonatal Fc receptor (FcRn)-driven endosomal sorting pathway for cellular recycling of albumin. J. Biol. Chem. 292, 13312–13322 (2017).
Van Roy, M. et al. The preclinical pharmacology of the high affinity anti-IL-6R Nanobody(R) ALX-0061 supports its clinical development in rheumatoid arthritis. Arthritis Res. Ther. 17, 135 (2015).
Harris, K. E. et al. Sequence-based discovery demonstrates that fixed light chain human transgenic rats produce a diverse repertoire of antigen-specific antibodies. Front. Immunol. 9, 889 (2018).
Logtenberg, T. O. N., Pinto Rui, D. & Houtzager, E. Antibody producing non-human mammals. US Patent 9951124B2 (2018).
McWhirter, J. et al. Common light chain mouse. US Patent 20120021409A1 (2012).
Van Blarcom, T. et al. Productive common light chain libraries yield diverse panels of high affinity bispecific antibodies. mAbs 10, 256–268 (2018).
Nixon, A. E., Sexton, D. J. & Ladner, R. C. Drugs derived from phage display: from candidate identification to clinical practice. mAbs 6, 73–85 (2014).
Xiao, X. et al. A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG. mAbs 9, 996–1006 (2017).
Harms, B. D., Kearns, J. D., Iadevaia, S. & Lugovskoy, A. A. Understanding the role of cross-arm binding efficiency in the activity of monoclonal and multispecific therapeutic antibodies. Methods 65, 95–104 (2014).
Zheng, S. et al. Cross-arm binding efficiency of an EGFR x c-Met bispecific antibody. mAbs 8, 551–561 (2016).
Steinmetz, A. et al. CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications. mAbs 8, 867–878 (2016).
Kitazawa, T. et al. A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model. Nat. Med. 18, 1570–1574 (2012).
Jimeno, A. et al. A first-in-human phase 1a study of the bispecific anti-DLL4/anti-VEGF antibody navicixizumab (OMP-305B83) in patients with previously treated solid tumors. Invest. New Drugs https://doi.org/10.1007/s10637-018-0665-y (2018).
Panowski, S. H. et al. Preclinical evaluation of a potent anti-BCMA CD3 bispecific molecule for the treatment of multiple myeloma. Blood 128, 383–383 (2016).
Gaudet, F. et al. Development of a CD123xCD3 bispecific antibody (JNJ-63709178) for the treatment of acute myeloid leukemia (AML). Blood 128, 2824–2824 (2016).
de Vries Schultink, A. H. M. et al. Translational PK-PD modeling analysis of MCLA-128, a HER2/HER3 bispecific monoclonal antibody, to predict clinical efficacious exposure and dose. Invest. New Drugs 36, 1006–1015 (2018).
Shiraiwa, H. et al. Engineering a bispecific antibody with a common light chain: Identification and optimization of an anti-CD3 epsilon and anti-GPC3 bispecific antibody, ERY974. Methods 154, 10–20 (2018).
Moore, G. L. et al. A robust heterodimeric Fc platform engineered for efficient development of bispecific antibodies of multiple formats. Methods 154, 38–50 (2018).
Grosso, J. F. & Jure-Kunkel, M. N. CTLA-4 blockade in tumor models: an overview of preclinical and translational research. Cancer Immun. 13, 5 (2013).
Loisel, S. et al. Relevance, advantages and limitations of animal models used in the development of monoclonal antibodies for cancer treatment. Crit. Rev. Oncol. Hematol. 62, 34–42 (2007).
Marques, A. & Muller, S. Mouse models of autoimmune diseases. Curr. Drug Discov. Technol. 6, 262–269 (2009).
Li, B. et al. CD89-mediated recruitment of macrophages via a bispecific antibody enhances anti-tumor efficacy. Oncoimmunology 7, e1380142 (2017).
Amann, M. et al. Therapeutic window of MuS110, a single-chain antibody construct bispecific for murine EpCAM and murine CD3. Cancer Res. 68, 143–151 (2008).
Lutterbuese, R. et al. Potent control of tumor growth by CEA/CD3-bispecific single-chain antibody constructs that are not competitively inhibited by soluble CEA. J. Immunother. 32, 341–352 (2009).
Ruf, P. et al. Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model. J. Transl Med. 10, 219 (2012).
Schlereth, B. et al. Potent inhibition of local and disseminated tumor growth in immunocompetent mouse models by a bispecific antibody construct specific for murine CD3. Cancer Immunol. Immunother. 55, 785–796 (2006).
Wu, C. et al. Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat. Biotechnol. 25, 1290–1297 (2007).
Lo, M. et al. Effector-attenuating substitutions that maintain antibody stability and reduce toxicity in mice. J. Biol. Chem. 292, 3900–3908 (2017).
Li, J. et al. IFNgamma-induced chemokines are required for CXCR3-mediated T-cell recruitment and antitumor efficacy of anti-HER2/CD3 bispecific antibody. Clin. Cancer Res. 24, 6447–6458 (2018).
Dimasi, N. et al. Development of a trispecific antibody designed to simultaneously and efficiently target three different antigens on tumor cells. Mol. Pharm. 12, 3490–3501 (2015).
Gantke, T. et al. Trispecific antibodies for CD16A-directed NK cell engagement and dual-targeting of tumor cells. Protein Eng. Des. Sel. 30, 673–684 (2017).
Schoonjans, R. et al. Fab chains as an efficient heterodimerization scaffold for the production of recombinant bispecific and trispecific antibody derivatives. J. Immunol. 165, 7050–7057 (2000).
Nyakatura, E. K. et al. Design and evaluation of bi- and trispecific antibodies targeting multiple filovirus glycoproteins. J. Biol. Chem. 293, 6201–6211 (2018).
Khan, S. N. et al. Targeting the HIV-1 spike and coreceptor with bi- and trispecific antibodies for single-component broad inhibition of entry. J. Virol. 92, e00384–18 (2018).
Kugler, M. et al. A recombinant trispecific single-chain Fv derivative directed against CD123 and CD33 mediates effective elimination of acute myeloid leukaemia cells by dual targeting. Br. J. Haematol. 150, 574–586 (2010).
Wang, X. B. et al. A new recombinant single chain trispecific antibody recruits T lymphocytes to kill CEA (carcinoma embryonic antigen) positive tumor cells in vitro efficiently. J. Biochem. 135, 555–565 (2004).
Castoldi, R. et al. TetraMabs: simultaneous targeting of four oncogenic receptor tyrosine kinases for tumor growth inhibition in heterogeneous tumor cell populations. Protein Eng. Des. Sel. 29, 467–475 (2016).
Steinhardt, J. J. et al. Rational design of a trispecific antibody targeting the HIV-1 Env with elevated anti-viral activity. Nat. Commun. 9, 877 (2018).
Xu, L. et al. Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques. Science 358, 85–90 (2017).
Klein, C., Schaefer, W. & Regula, J. T. The use of CrossMAb technology for the generation of bi- and multispecific antibodies. mAbs 8, 1010–1020 (2016).
Hu, S. et al. Four-in-one antibodies have superior cancer inhibitory activity against EGFR, HER2, HER3, and VEGF through disruption of HER/MET crosstalk. Cancer Res. 75, 159–170 (2015).
Wu, X., Yuan, R., Bacica, M. & Demarest, S. J. Generation of orthogonal Fab-based trispecific antibody formats. Protein Eng. Des. Sel. 31, 249–256 (2018).
Keyt, B., Presta Leonard, G., Zhang, F. E. N. & Baliga, R. Modified J-chain. US Patent 20170283510A1 (2017).
Kaveri, S. V., Silverman, G. J. & Bayry, J. Natural IgM in immune equilibrium and harnessing their therapeutic potential. J. Immunol. 188, 939–945 (2012).
Patel, A. et al. An engineered bispecific DNA-encoded IgG antibody protects against Pseudomonas aeruginosa in a pneumonia challenge model. Nat. Commun. 8, 637 (2017).
Stadler, C. R. et al. Elimination of large tumors in mice by mRNA-encoded bispecific antibodies. Nat. Med. 23, 815–817 (2017).
Bakker, J. M., Bleeker, W. K. & Parren, P. W. Therapeutic antibody gene transfer: an active approach to passive immunity. Mol. Ther. 10, 411–416 (2004).
Wing, A. et al. Improving CART-cell therapy of solid tumors with oncolytic virus-driven production of a bispecific T cell engager. Cancer Immunol. Res. 6, 605–616 (2018).
Kohler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 (1975).
Huston, J. S. et al. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl Acad. Sci. USA 85, 5879–5883 (1988).
Holliger, P., Prospero, T. & Winter, G. “Diabodies”: small bivalent and bispecific antibody fragments. Proc. Natl Acad. Sci. USA 90, 6444–6448 (1993).
Mallender, W. D. & Voss, E. W. Jr. Construction, expression, and activity of a bivalent bispecific single-chain antibody. J. Biol. Chem. 269, 199–206 (1994).
Gruber, M., Schodin, B. A., Wilson, E. R. & Kranz, D. M. Efficient tumor cell lysis mediated by a bispecific single chain antibody expressed in Escherichia coli. J. Immunol. 152, 5368–5374 (1994).
Ridgway, J. B., Presta, L. G. & Carter, P. ‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 9, 617–621 (1996).
Coloma, M. J. & Morrison, S. L. Design and production of novel tetravalent bispecific antibodies. Nat. Biotechnol. 15, 159–163 (1997).
Schuurman, J. et al. Normal human immunoglobulin G4 is bispecific: it has two different antigen-combining sites. Immunology 97, 693–698 (1999).
van der Neut Kolfschoten, M. et al. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange. Science 317, 1554–1557 (2007).
Oberg, H. H. et al. Novel bispecific antibodies increase T-cell cytotoxicity against pancreatic cancer cells. Cancer Res. 74, 1349–1360 (2014).
The authors thank R. Roovers and H. van der Vliet for helpful comments on the manuscript and A. Cook and V. P. Rath for access to the Beacon Targeted Therapies database.
A.F.L. and M.L.J. are employees of Genmab, a biotechnology company that develops therapeutic antibodies including bispecific antibodies and bispecific antibody technology. They own warrants and/or stock. P.W.H.I.P. is an employee of Lava Therapeutics, a start-up biotechnology company that develops therapeutic antibodies including bispecific antibodies and bispecific antibody technology. He obtains stock options as part of his employment.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- Combinatorial bsAbs
Bispecific antibodies (bsAbs) that display an activity or functionality that can also be obtained by combining separate antibodies with the same specificities (for example, a parental or reference antibody mixture).
- Obligate bsAbs
Bispecific antibodies (bsAbs) that display an activity or functionality that is dependent on the physical linkage of the two specificities (and cannot be obtained by combining separate antibodies with the same specificities). The dual-targeting concepts mediated by these bsAbs are considered obligate concepts.
- Chain-association issue
The co-expression of two different heavy (H) and two different light (L) chains results in a complex mixture of sixteen possible H2L2 recombinations, representing ten different antibodies. Only one of these antibodies (represented by two possible H2L2 recombinations) corresponds with the desired bispecific antibody (maximal yield 12.5% in the mixture). This issue is addressed by strategies forcing cognate HL-pairing and/or promoting heterodimerization of the two different H chains.
The number of antigen-binding sites in an antibody molecule. The design of a bispecific antibody (bsAb) format influences the number of binding sites per target. A bivalent bsAb with one binding site for each target is denoted as 1 + 1. Incorporating additional binding sites can lead to trivalent (2 + 1) and tetravalent (2 + 2 or 1 + 3) designs.
- Antibody fragments
The antibody molecule consists of different domains that can be expressed separately and used as modular building blocks. The domains involved in antigen recombination are often used as binding moieties in the design of antibody-based therapeutics. Examples include domain antibodies (heavy chain-only variable domain (VHH)) and single-chain Fv fragments (scFvs), antigen-binding fragments (Fabs), single-chain Fab fragments (scFabs) and, more recently, single-chain Fc fragments (scFcs).
- Cross-arm binding efficiency
An increase in apparent affinity when a bispecific antibody binds to the second target or receptor following its binding to the first target or receptor on the same cell.
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Labrijn, A.F., Janmaat, M.L., Reichert, J.M. et al. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov 18, 585–608 (2019). https://doi.org/10.1038/s41573-019-0028-1
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