The Evolution of Bispecific Antibody Engineering

Face the unknown Key bispecific antibody discoveries Revolution of hybridoma technology Quadroma technology enabled BsAb innovation FDA approved first mAb therapy ScFv transformed antibody engineering Antibody diversity shapes the therapeutic landscape BsAb pairing puzzle solved Antibody architecture unraveled 1959 1975 1983 1986 1988 1998 2024 Learn more Revolution of hybridoma technology Quadroma technology enabled BsAb innovation FDA approved first mAb therapy ScFv transformed antibody engineering Antibody diversity shapes the therapeutic landscape BsAb pairing puzzle solved Antibody architecture unraveled 1975 1983 1986 1988 1998 2024 Learn more Face the unknown Key bispecific antibody discoveries 1959 Antibody architecture unraveled Until the late 1950s, our understanding of the structure and function of antibodies was extremely vague. This changed in 1959 when American biologist Gerald Edelman and British biochemist Rodney Porter independently described the chemical structure of antibodies for the first time. This groundbreaking discovery led to the two scientists being jointly awarded the Nobel Prize in Physiology and Medicine in 1972. Reference Antigen binding domain Constant region Fc region IgG (M, 150 000) Revolution of hybridoma technology Quadroma technology enabled BsAb innovation FDA approved first mAb therapy ScFv transformed antibody engineering Antibody diversity shapes the therapeutic landscape BsAb pairing puzzle solved Antibody architecture unraveled 1959 1983 1986 1988 1998 2024 Learn more Face the unknown Key bispecific antibody discoveries 1975 Revolution of hybridoma technology Even with the newfound understanding of antibody structure, scientists were still restricted by an inability to isolate and purify specific antibodies. In 1975, Georges Köhler and César Milstein published a report in Nature describing the development of hybridoma technology. This method, which supported the large-scale production of monoclonal antibodies (mAbs) directed against specific antigens for the first time, and paved the way for mAb commercialization. Hybridoma technology had a profound impact on research and diagnostics, heralding a new era of mAb therapies. Spleen cells Hybridomas Culture in HAT medium selective for positive cells Harvest monoclonal antibodies Myeloma cells Reference Revolution of hybridoma technology Quadroma technology enabled BsAb innovation FDA approved first mAb therapy ScFv transformed antibody engineering Antibody diversity shapes the therapeutic landscape BsAb pairing puzzle solved Antibody architecture unraveled 1959 1975 1986 1988 1998 2024 Learn more Face the unknown Key bispecific antibody discoveries 1983 Quadroma technology enabled BsAb innovation This revolutionary work was continued by Milstein, who pioneered the development of hybrid-hybridoma (quadroma) technology with his colleague Cuello. They published their findings in 1983. The development of quadroma technology represented an early attempt to produce BsAbs. BsAbs are designed to bind two specific antigens or epitopes simultaneously to redirect immune cells, such as T cells, neutrophils, and macrophages, to tumor cells, resulting in tumor cell lysis. Reference DVD-lg “Knobs into Holes” approach Revolution of hybridoma technology Quadroma technology enabled BsAb innovation FDA approved first mAb therapy ScFv transformed antibody engineering Antibody diversity shapes the therapeutic landscape BsAb pairing puzzle solved Antibody architecture unraveled 1959 1975 1983 1988 1998 2024 Learn more Face the unknown Key bispecific antibody discoveries 1986 FDA approved first mAb therapy In 1986, the focus shifted back to mAbs as the FDA approved the first-ever mAb-based therapy, muromonab-CD3 (Orthoclone OKT3). OKT3 is a murine mAb therapy given to reduce acute rejection in patients undergoing organ transplants. OKT3 had great potential, but induced immune responses in patients, leading to major adverse effects. This setback spurred further research into techniques aimed at transforming rodent antibodies into structures more similar to human antibodies. The next antibody therapy would not be approved until eight years later, in 1994. Reference Mouse monoclonal Chimeric monoclonal Humanized/ CDR-grafted monoclonal Phage display synthetic monoclonal Fully human/ transgenic human monoclonal e.g. Murmonab (“Muromomab”) (Orthoclone OKT3) 1975 Köhler & Milstein Rituximab (Rituxan) 1984 Morrison et al. Bevacizumab (Avastin) 1986 Jones et al. Adalimumab (Humira) 1990 McCafferty et al. Panitumumab (Vectibix) 1994 Lonberg et al. & Green et al. Decreasing likelyhood of immune reaction towards the antibody Revolution of hybridoma technology Quadroma technology enabled BsAb innovation FDA approved first mAb therapy ScFv transformed antibody engineering Antibody diversity shapes the therapeutic landscape BsAb pairing puzzle solved Antibody architecture unraveled 1959 1975 1983 1986 1998 2024 Learn more Face the unknown Key bispecific antibody discoveries 1988 Reference ScFv transformed antibody engineering Alongside ongoing efforts to develop effective antibody-based therapies, research efforts continued to work towards development of novel antibody variants aimed at addressing the limitations of existing approaches. In 1988, Huston and colleagues described the invention of the single-chain variable fragment (scFv). An scFv is a fusion protein of the variable regions of heavy and light immunoglobulin chains connected by a flexible linker. The scFv retains the specificity of the original immunoglobulin while minimizing the challenges caused by the refolding of twochain species, such as incorrect domain pairing and aggregation. C N N VH C VL VL VH Revolution of hybridoma technology Quadroma technology enabled BsAb innovation FDA approved first mAb therapy Antibody diversity shapes the therapeutic landscape BsAb pairing puzzle solved Antibody architecture unraveled 1975 1983 1986 1988 2024 Learn more Face the unknown Key bispecific antibody discoveries 1998 BsAb pairing puzzle solved Building on earlier work, the 1990s provided further development of BsAb technology to address the difficulties scientists frequently faced surrounding expressing two different antibodies with the correct pairing. In 1998, Margaret Merchant and team described the development of an efficient production pipeline for BsAbs, combining engineered disulfide bonds with previously identified “knobs-into-holes” mutations. Overcoming hurdles in BsAb production represented a huge win, given the potential for these antibodies as therapeutic agents for cancer treatment. trifunctional antibody chemically linked F(ab')2 BiTE (bi-specific T-cell engager) Fab (fragment antigen binding) F(ab')2 Fab' scFv (single-chain variable fragment) di-scFv sdAb (single domain antibody) Reference Antibody diversity shapes the therapeutic landscape Learn more 2024 12 10 8 6 FDA antibody approvals Canonical antibody Antibody—drug conjugate Bispecific Fragment Other 4 2 0 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Reference Antibody diversity shapes the therapeutic landscape Together, these significant milestones and transformative discoveries in antibody research and development led us to where we are today. Today, most approved antibodies on the market are full-length antibodies of the IgG1, IgG2, or IgG4 subclass. Numerous alternative formats are available to address different clinical situations, including nine globally approved bispecific antibodies, with 180 more in preclinical trials and 50 in clinical trials. cytiva.com Cytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corporation or an affiliate doing business as Cytiva. Any other third-party trademarks are the property of their respective owners. © 2025 Cytiva For local office contact information, visit cytiva.com/contact CY42851-12Mar25-IG