Cancer Immunotherapy by Targeting 4-1BB with Novel Immunostimulatory Bispecific Antibodies

In recent decades immunotherapy has revolutionised cancer treatment and rejuvenated the field of cancer immunology. A key factor to this success has been the development of monoclonal antibodies (mAbs) that promote cytotoxic T cell responses against tumours by binding T cell inhibitory molecules known as immune checkpoints. Nonetheless, immune checkpoint inhibitor mAbs are only effective in a small fraction of tumour types with most cancer patients failing to respond. Thus, the need for additional therapeutic approaches is highlighted. Immunostimulatory mAbs targeting co-stimulatory receptors on T cells hold promise as combination agents targeting different yet complementary immune regulatory pathways. Notably, antibodies targeted towards the tumour necrosis factor receptor superfamily member 4-1BB show potential, with two anti-human 4-1BB (h4-1BB) mAbs known as Urelumab and Utomilumab already in the clinic. However, further clinical development of agonist 4-1BB antibodies is currently being hampered by immune-related toxicities associated with on-target off-tumour activity. Moreover, insight into what confers co-stimulatory potential and agonistic activity to anti-h4-1BB antibodies is currently lacking. In attempt to show anti-cancer efficacy can be decoupled from on-target off-tumour toxicity as well as improve understanding of what confers co-stimulatory potential and agonistic activity to anti-h4-1BB antibodies, we developed a panel of novel bispecific antibody (BsAb) constructs targeted towards h4-1BB and human B7-H3 (hB7-H3); B7-H3 is a tumour-associated antigen that is commonly over-expressed on both tumour cells and tumour stroma. All BsAb constructs were designed to target different h4-1BB epitopes across different cysteine-rich domains to allow us to investigate the effects of epitope, binding distance from the membrane and binding affinity on co-stimulatory potential and agonistic activity. Using in vitro assays to measure cellular binding to h4-1BB and hB7-H3 as well as nuclear factor kappa B pathway activation downstream from h4-1BB, we have so far shown that all our novel BsAb constructs have co-stimulatory potential and agonistic activity, facilitated by their ability to cross-link hB7-H3. Co-stimulatory potential and agonistic activity appear to be epitope-independent and unaffected by both binding distance from the membrane and binding affinity. Overall, this work highlights the potential for using novel anti-h4-1BB anti-hB7-H3 BsAb constructs of this format in providing 4-1BB co-stimulation and inducing T cell activation in a tumour microenvironment. Furthermore, it presents a point from which to continue BsAb development and characterisation.