Zymeworks Inc. announced five presentations including new data from its preclinical development-stage programs at the 2024 American Association for Cancer Research Annual Meeting being held in San Diego, California April 5-10, 2024. ZW191 is a FR-targeting antibody-drug conjugate differentiated by its novel antibody and novel topoisomerase I inhibitor payload. The compelling preclinical activity profile supports ZW191 development across multiple tumor types, including FR-high/mid/low ovarian cancers and other FR-expressing indications, including non-small cell lung cancer, endometrial cancer, and triple-negative breast cancer.

An investigational new drug submission or foreign equivalent is planned for 2024. Key Results: Superior internalization, payload delivery, and spheroid penetration to other FR-targeted multi-specific antibodies. Bystander active payload drives activity in settings with heterogeneous FR expression.

Well-tolerated, with a highest non-severely toxic dose of 60 mg/kg in non-human primates. Inter-patient and intra-tumoral target expression heterogeneity is an obstacle in the design of ADCs that target a single tumor associated antigen. While bystander active payloads mitigate intra-tumoral target heterogeneity, bispecific ADCs that target two different TAAs independently provide an additional approach to overcome limitations associated with the expression profile of any single target antigen.

Critical to this bispecific ADC approach however is identification of the optimal bispecific antibody format suited for payload delivery to two independent tumor antigens. To address this challenge a novel design and screening approach of bispecific ADCs was employed, using FR and NaPi2b as an exemplary target pair, independently expressed across various cancer types. Key Results: Leveraging AzymetricTM, bioconjugation, analytical mass spectrometry, and high throughput functional screening, a workflow for the rapid generation and characterization of bispecific ADCs was established to identify optimal format, paratope, and valency.

A library of 48 bispecific ADC molecules co-targeting FR and NaPi2b was rapidly generated covering multiple paratopes, antibody formats, and valency bins (1+1, 2+1, 2+2). Functional screening of the library across multiple cancer cell lines expressing FR and/or NaPi2b revealed ranges in binding, internalization, and cytotoxicity that were dependent on epitope, valency, and format geometry. Antibody-drug conjugates are an effective class of cancer therapeutics comprised of a linker-payload conjugated to a monoclonal antibody targeting a TAA, to enable the delivery of the cytotoxic payload to cancer cells.

Current standard in vitro monolayer models do not sufficiently reflect in vivo tumor tissue complexity, particularly in consideration of the interaction between protein-based therapeutics such as antibodies in a three-dimensional (3D) environment. To address this, methodology to yield in vitro 3D spheroid models from cancer cell lines in a rapid, robust, and uniform manner was developed. Subsequently methods were integrated to evaluate the tissue penetration capability and cytotoxic activity of structurally distinct antibodies or ADCs bearing various payload classes targeting multiple TAAs.

Key Results: A readily implementable method for the rapid generation of cancer cell line spheroids was established and applied to over 50 distinct immortalized cancer cell lines derived from more than 10 tissue types, demonstrating varying morphological features with a success rate of >95%. In vitro assays were developed to evaluate the spheroid penetration capability and 3D cytotoxic activity of multispecific antibodies and ADCs, enabling the interrogation of various antibody formats and payload classes. These 3D models and assays serve as valuable functional tools that provide improved translation between in vitro and in vivo activity, supporting the characterization of therapeutic ADC candidates and their pipeline advancement.

Low T cell infiltration and T cell anergy are challenges associated with the treatment of solid tumors with conventional CD3- 1 engaging bispecific T cell engagers. By optimizing "Signal 1" (CD3) and "Signal 2" within the context of a single molecule, co-stimulatory trispecific TCEs have the potential to increase therapeutic responses beyond that achievable by conventional CD3-based TCEs by stimulating T cell proliferation in patients with poorly infiltrated tumors and providing more durable anti-tumor control by enhancing T cell activation. Key Results: Relative to comparator bispecific TCEs, the lead CLDN18.2 TriTCE Co-Stim molecule mediates enhanced T cell- mediated killing of tumor cells at low E:T ratios, exhibits sustained T cell mediated activity in serial challenge assays and supports superior antitumor activity in humanized models of gastric cancer.

TriTCE Co-stim design facilitates obligate cis T cell binding and activation of CD28 that requires co-engagement of CD3 with no detectable cytokine levels observed upon incubation with human peripheral blood mononuclear cells in the absence of tumor target engagement. CLDN18.2 TriTCE Co-stim was well tolerated in non-human primates upon repeat dosing, with minimal peripheral cytokine elevations and no on-target histopathological changes observed. 2 Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer with a poor prognosis and high unmet medical need .

DLL3 is a therapeutic target that is selectively expressed in SCLC and other neuroendocrine tumors. Bispecific TCEs targeting DLL3 have entered the clinic and demonstrated encouraging anti-tumor activity in SCLC patients3,4; however, SCLC is frequently characterized by an immunosuppressive microenvironment and poor T cell infiltration, which may limit clinical 5 activity of CD3 engagers . DLL3 TriTCE Co-Stim is a TriTCE designed to optimally engage CD3 and CD28 to redirect and enhance cytotoxic T cell responses to DLL3-expressing tumor cells while maintaining a desired safety profile.

This approach has the potential to improve outcomes for patients, especially those with poorly infiltrated tumors, by increasing the depth and durability of response. Using Zymeworks' TriTCE Co-stim platform in combination with its AzymetricTM and EFECTTM technologies, the company generated a panel of DLL3 TriTCE Co-Stim antibody formats and evaluated multiple formats, geometries, and paratope affinities, which allowed for optimization of selectivity and activity to promote a widened therapeutic index with enhanced anti-tumor activity. Key Results: Induces greater in vitro cytotoxicity and improves T cell proliferation and survival compared to bispecific TCEs.

Displays no cross-linking of T cells and exhibits obligate cis T cell binding of CD28, requiring co-engagement of CD3. Mediates improved in vivo tumor regression in an established SCLC humanized xenograft model relative to a clinical benchmark TCE.