From Bispecific to Trispecific Antibodies in Cancer Immunotherapy

Bispecific and trispecific antibodies are essentially antibodies bioengineered to contain two or three distinct antigen-binding domains, which allow these recombinant molecules to bind specifically to more than one target. Harnessing the versatility of multi-specific antibodies, an immunotherapy strategy relies on using bispecific and, more recently, trispecific antibodies to close the gap between cancer and cytotoxic T cells, promoting cancer cell lysis.

Work supporting this approach dates back to the mid-1980s. For example, Staerz and Bevan proposed that bispecific antibodies could "focus effector T cell-activity" to target virally-infected or cancer cells for elimination. Currently, Blinatumomab is the only bispecific antibody approved since 2014 in the US for cancer treatment, indicated for B-cell acute lymphoblastic leukemia (B-ALL).

Adapted from "Bispecific Antibody Design" by (2021). Retrieved from

"T cell engagers" are bispecific antibodies for cancer immunotherapy- Blinatumomab is a first-generation bispecific approved by the FDA for the treatment of B-ALL. Mosunetuzumab is a second-generation bispecific being tested as an investigational drug for the treatment of various lymphoma indications. Different from the tandem-scFv format of Blinatumomab, Mosunetuzumab is a full-length bispecific with improved pharmacokinetic properties. Additionally, the Fc fragment in Mosunetuzumab has been bioengineered to silence its effector functions, preventing off-target toxicities. The binding of bispecific antibodies to the T cell antigen and tumor antigen supports forming a "cytolytic synapse" where the release of molecules such as perforin and granzymes leads to tumor cell killing (Pytlik et al. 2020).

Blinatumomab is a bispecific tandem single-chain variable fragment (scFv) that binds to CD3 and CD19 on T cells and cancer cells, respectively (Liu et al. 2021). Blinatumomab's successful clinical outcomes have prompted the development of similar "T cell engaging antibodies" targeting other antigen pairs, such as CD3/CD20 and CD3/BCMA, which are being tested clinically as therapies for lymphoma and myeloma (Labrijn et al. 2019, Liu et al. 2021).

Developing a Trispecific Antibody Targeting Solid Tumors

While T cell-engaging bispecific antibodies effectively treat hematological malignancies, similar strategies have not been as successful with solid tumors. Several factors are thought to contribute to T cell engagers' lower efficacy in solid tumors, including the reduced availability of ideal tumor-specific targets, immunosuppressive nature of the tumor microenvironment, and tumor physical barriers reducing antibody access (Austin et al. 2021). To address these challenges, Austin and colleagues at Harpoon Therapeutics have bioengineered a new T cell-engager consisting of a trispecific rather than a bispecific antibody.

The newly developed "Trispecific T-cell Activating Construct" or TriTAC binds specifically to CD3e, human serum albumin (HSA), and prostate-specific membrane antigen (PSMA). Structurally, the trispecific antibody was designed to have one scFv fragment (CD3e) and two single-domain antibodies (sdAbs) to HSA and PSMA, having a compact molecular size of ~53 KDa.

Austin and colleagues performed extensive testing to elucidate the impact of the trispecific configuration on antigen-binding and tumor cell killing. Additionally, by leveraging a "single substitution library method," they evaluated how anti-CD3e affinity affected cytotoxic activity and pharmacokinetics of various TriTAC candidates.

Overall, their findings allowed them to develop a trispecific antibody candidate, HPN424, to treat metastatic castration-resistant prostate cancer. Austin et al. demonstrated both in vitro and in vivo that their lead candidate, HPN424, effectively engaged T cells to induce prostate cancer cell killing. Lastly, in a non-human primate model, Austin et al. demonstrated that HPN424 had a prolonged half-life of over three days and remained stable and biologically active while circulating for one week.

HPN424 is currently being tested in Phase 1 clinical studies for safety and efficacy. Investigators are hopeful that the prolonged half-life of HPN424 may support weekly dosing, representing a significant improvement over bispecific antibodies (e.g., Pasotuxizumab) currently under clinical investigation that require daily administration.


Austin, R. J. et al. Tritacs, a novel class of t-cell–engaging protein constructs designed for the treatment of solid tumors. Mol. Cancer Ther. (2021) doi:10.1158/1535-7163.MCT-20-0061.

Labrijn, A. F., Janmaat, M. L., Reichert, J. M. & Parren, P. W. H. I. Bispecific antibodies: a mechanistic review of the pipeline. Nature Reviews Drug Discovery (2019) doi:10.1038/s41573-019-0028-1.

Liu, J. et al. Therapeutic Advances in Oncology. Int. J. Mol. Sci. 2021, 22, 2008.

Pytlik R. et al. Current Immunotherapy Approaches in Non-Hodgkin Lymphomas. Vaccines (Basel). 2020 Nov 27;8(4):708. doi: 10.3390/vaccines8040708.

Staerz, U. D. & Bevan, M. J. Hybrid hybridoma producing a bispecific monoclonal antibody that can focus effector T-cell activity. Proc. Natl. Acad. Sci. U. S. A. (1986) doi:10.1073/pnas.83.5.1453.


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