| Biolata EpCAM is overexpressed in a wide range of tumors, making it an ideal target for pan-cancer therapies. However, it is also widely expressed in healthy epithelial tissues. EpCAM has been clinically validated using locally administered catumaxomab (EpCAM × CD3 bispecific antibody),17 which has shown good efficacy. However, systemic administration of catumaxomab led to severe CRS and hepatotoxicity. Similarly, clinical trials with solitomab were terminated before reaching efficacious concentrations due to toxicity.12 TCEs are very potent molecules that redirect the cytotoxic activity of T cells in the absence of MHC restriction and are an emerging new class of cancer therapies. TCEs are designed to recruit T cells via a common signaling molecule, such as CD3, against tumor cells bearing a frequently expressed tumorassociated antigen (TAA). To date, twelve TCEs have been approved; nine for hematological cancers targeting BCMA, CD19, CD20, or GPCR5D, and only three for the treatment of solid tumors targeting EpCAM, gp100 and DLL3 (The Antibody Society. Therapeutic monoclonal antibodies approved or in regulatory review. (02/19/2024); www.antibo dysociety.org/antibody-therapeutics-product-data). TCEs targeting solid tumors require much higher peripheral levels to achieve efficacious concentrations inside solid tumor tissue18,19 which can lead to severe on-target offtumor toxicities. Since TCEs can recognize very low levels of TAAs, it is not uncommon to observe on-target, off-tumor toxicities for TAAs expressed in normal tissues.8, 13, 20–22 This has hampered the development of bispecific antibodies using CD3-directed TCEs against targets, such as EpCAM, which are widely expressed in normal tissues. The only approved TCE targeting EpCAM, Catumaxomab, has limited application due to severe toxicity10 and it has been withdrawn for the market for commercial reasons. We used our CAB technology14 to develop conditionally active EpCAM-specific TCEs that bind to their respective targets only under TME conditions. CAB antibodies and CAB bispecifics have optimized binding domains that have no or greatly reduced binding in normal tissue and preserve high-affinity binding in the TME without covalent modification of the antibody requiring enzymatic activity for activation. Since CAB antibody binding is controlled by extracellular pH and the respective conditional binding of the PaCS molecules, it becomes inactive when leaving the TME for the normal alkaline microenvironment and active once it reenters the acidic TME again.14 A wide range of bispecific antibody formats have been described in the literature.23 These formats vary in size from 25 kDa (nanobodies) to 300 kDa (chemically linked IgGs) and can generally be grouped into hetero-dimeric and homodimeric molecules. We elected to use a homo-dimeric format based on an IgG1 backbone (targeting the TAA) and an scFv domain fused to the C-terminus of the light chain for binding to CD3. The presence of an Fc allows purification of bispecific antibodies using protein A as the initial step, similar to a standard IgG molecule. The addition of the CAB-CD3 binding domain to the C-terminus of the light chain allows for the easy conversion of any antibody into a MonoCAB (CAB function at CD3 binding domain) TCE without additional engineering, thus creating a platform for the rapid generation of MonoCAB TCE against any tumor target. In this report, we describe the development of a dual CAB EpCAM × CD3 bispecific antibody, BF-588-DualCAB, and compare its properties with those of a MonoCAB version (BF-588-MonoCAB) that has conditionally active binding only on the CD3-binding domains, and the parental nonCAB molecule, BF-588-WT. Clones BF-588-DualCAB and BF-588-MonoCAB showed tumor-selective binding to human and cynomolgus EpCAM and CD3 in vitro, with a pH inflection point at pH6.6 ( = 50% binding signal compared to pH6.0) and very little binding at normal physiological alkaline pH (=pH7.4) in an affinity ELISA assay. The binding of BF-588-WT was similar under all the pH conditions tested. Binding kinetics analyzed by SPR showed a decrease in both affinity from pH6.0 to pH7.4, and a concomitant decrease in the maximum signal, similar to other CABs described previously.14 BF-588-DualCAB and BF-588-MonoCAB demonstrated the ability to induce T cell activation and killing of cancer cells in vitro in a pH-dependent manner, while the non-CAB parental clone showed activity independent of the pH conditions. All three clones were tested in a cell line-derived xenograft (CDX) humanized mouse model using HCT116 human colon cancer cells in NOG mice engrafted with human PBMCs. Treatment with BF-588-WT, BF-588-MonoCAB, or BF-588- DualCAB at 1 mpk (twice weekly for four weeks) induced 100% tumor regression in this model. BF-588-DualCAB also inhibited the tumor growth of a BT474 human breast cancer cell line in NCG mice humanized with human PBMCs at 0.5 mpk (twice weekly for four weeks). The immunotoxicity and tolerability of these molecules were tested in a single-dose toxicity study in cynomolgus monkeys. As expected, the non-CAB clone BF-588-WT proved to be very toxic and was not tolerated at 0.05 mpk (only dose tested). Cytokine levels increased dramatically within six hours of administration, and the animals were euthanized. BF-588- MonoCAB showed an improved toxicity profile and was welltolerated up to 0.25 mpk. At this dose cytokine levels increased to similar levels as observed with the non-CAB molecule at 0.05 mpk, but the animals showed only minimal elevation of liver enzymes and had no clinical symptoms. Clone BF-588- DualCAB was tested up to 2.5 mpk and later, in a repeat dose study up to five mpk. Despite its robust ability to activate T cells and eliminate EpCAM-expressing target cells at a similar minimal efficacious dose as the non-CAB and MonoCAB clones, BF-588-DualCAB had an extremely low immunotoxicity profile. Cytokine levels were slightly elevated at 2.5 mpk (IL-6) or barely above the baseline (IL-2 and MCP1). In the repeat dose study animals were dosed with BF-588- DualCAB up to 5 mg/kg/week for 4 weeks and no mortality or morbidity, no macroscopic or microscopic changes were observed, and no cytokine-related AEs were observed, indicating that the maximum tolerated dose for BF-588-DualCAB was greater than five mpk. Our data show that the tolerability of EpCAM-specific TCE is improved by more than five-fold by using a conditionally active CD3-binding domain in combination with a non-CAB EpCAM-binding domain. The full potential improvement in safety and tolerability, however, is reached in the DualCAB format, which can be dosed at > 20-fold higher concentrations compared to MonoCAB and > 100-fold higher concentrations compared to the non-CAB parent. The current results clearly demonstrate that BF-588- DualCAB represents a novel, potentially highly effective targeted agent for the treatment of cancer patients with EpCAMoverexpressing tumors. Recently, our team initiated a Phase 1 clinical trial using this synergistic, DualCAB TCE (named BA3182) to evaluate the potential of this therapeutic candidate as a pan-cancer therapy. |
