Zymeworks (Canada)

Germinal centers (GCs) are specialized lymphoid structures in which activated B cells undergo clonal selection and B cell receptor (BCR) somatic hypermutation to generate high-affinity antibodies. Previous work has shown that T cells expressing choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine (ACh), are linked to the production of high-affinity antibodies in the GC response. However, whether B cells in the GC also express ChAT, and the details of the interplay of cholinergic circuits within the GC, remain unclear. Here we show that Chat expressed by GC B cells contributes to the early accumulation of high-affinity GC B cells following antigen encounter. We identify key transcriptional regulators of Chat expression in GC B cells and demonstrate that ACh receptor (AChR) expression is dynamically coordinated during B cell activation. In vitro, we show that ACh binding to muscarinic AChRs limits plasma cell differentiation and dampens BCR signal transduction to fine-tune the threshold for affinity-based positive selection. Together, these findings reveal a previously unrecognized regulatory axis that operates early during GC selection and uses cholinergic signals to shape B cell fate decisions and humoral immunity.

IntroductionIn the phase 3 EVOKE-01 trial (NCT05089734), sacituzumab govitecan (SG) exhibited a numerical improvement in overall survival and tolerability compared with docetaxel in patients with metastatic NSCLC previously treated with platinum-based chemotherapy and programmed cell death protein (ligand) 1 inhibitors, although results were not statistically significant. This analysis evaluated health-related quality of life (HRQoL) data from EVOKE-01.MethodsPatients (N = 603) were randomized 1:1 to SG (n = 299) or docetaxel (n = 304) in 21-day cycles. HRQoL was assessed by the NSCLC Symptom Assessment Questionnaire (NSCLC-SAQ), European Organization for Research and Treatment of Cancer Quality of Life questionnaire–Core 30 (QLQ-C30), and EuroQol 5 Dimension 3-level questionnaire. Least square mean changes from baseline at week 25, time to first meaningful deterioration or death, and time to confirmed deterioration were analyzed.ResultsSG exhibited significantly and meaningfully better effects than docetaxel on NSCLC-SAQ shortness of breath (SoB), fatigue, total score, and on QLQ-C30 role functioning, fatigue, and dyspnea. Time to first meaningful deterioration or death favored SG over docetaxel for NSCLC-SAQ SoB (hazard ratio [95% confidence interval ]: 0.75 [0.61–0.91]), fatigue (0.70 [0.57–0.86]), and total score (0.80 [0.66–0.97]); QLQ-C30 fatigue (0.80 [0.66–0.96]) and dyspnea (0.74 [0.60–0.90]); and EuroQol visual analog scale (0.79 [0.65–0.96]). The time to confirmed deterioration favored SG over docetaxel for NSCLC-SAQ SoB (0.59 [0.44–0.77]) and fatigue (0.70 [0.52–0.95]), and QLQ-C30 fatigue (0.75 [0.59–0.95]).ConclusionsThese exploratory results suggest that SG may benefit HRQoL over docetaxel, supporting SG as an active therapeutic agent for metastatic NSCLC post platinum-based and programmed cell death protein (ligand) 1 inhibitor therapy.

TPS3160 Background: Mesothelin (MSLN) is a membrane glycoprotein overexpressed in several solid tumors, making it a promising target for cancer treatments, including T cell engagers (TCEs). ZW171 is a humanized trivalent bispecific TCE antibody that targets a threshold level of MSLN expression with 2 binding sites and CD3e receptor on T cells with 1 binding site. Preclinical studies of ZW171 demonstrated favorable pharmacology, pharmacokinetics (PK), and toxicology, showing it preferentially kills MSLN-overexpressing cells, activates T cells without significant toxicity, inhibits tumor growth, and is well tolerated in cynomolgus monkeys, suggesting its potential for treating MSLN-expressing tumors 1 while sparing healthy tissues with low levels of expression. This first-in-human, phase 1, ongoing study (ZWI-ZW171-101) evaluates safety, tolerability, PK, and anti-tumor activity of ZW171 in participants with advanced solid tumors. Methods: This 2-part study enrolls eligible adult participants with unresectable MSLN-expressing ovarian cancer (OC), non-small cell lung cancer (NSCLC), or other MSLN-expressing cancers, with measurable disease per RECIST v1.1, ECOG PS score of 0 to 1, adequate organ function, and a minimum life expectancy of 12 weeks. Participants with additional progressing malignancies, recent transplants, clinically significant ongoing toxicity, uncontrolled renal, pancreatic or liver disease, or active autoimmune diseases requiring high-dose corticosteroids or immunosuppressive drugs are excluded. Part 1 evaluates the safety and tolerability of ZW171 and Part 2 evaluates the anti-tumor activity while continuing to evaluate safety and tolerability. Part 1 is dose escalation to identify maximum tolerated dose (using modified toxicity probability interval [mTPI-2] design, n=40) among participants with OC or NSCLC receiving subcutaneous ZW171 monotherapy on days 1, 8, and 15 of 3-week (21-day) cycles. Approximately 6 dose levels will be explored based on safety and tolerability. Step-up dosing will be used for cycle 1. Dose level 1, determined by QSP-based MABEL approach 2 , is administered at 4.2 µg (day 1), 12.6 µg (day 8), and 38.0 µg (day 15). Part 2 is dose expansion in participants with OC, NSCLC, and other MSLN-expressing cancers (MSLN expression evaluated retrospectively). Primary objectives are to evaluate safety and tolerability of ZW171 and determine the maximum tolerated dose. Key secondary objectives are to assess PK, anti-drug antibodies, and anti-tumor activity. This is a global study with sites in North America, Europe, and Asia; and actively enrolling participants into Part 1.

Abstract Background: Biparatopic antibodies and antibody-drug conjugates (ADCs) can provide many functional benefits over their traditional monoparatopic counterparts including increased binding, greater cell surface decoration, superior receptor blockade, and enhanced internalization and payload delivery; however, not all antigens are amenable to biparatopic targeting. We sought to leverage our prior knowledge of biparatopic antibody development and our AzymetricTM technology to develop a novel biparatopic ADC against protein tyrosine kinase 7 (PTK7) that could provide a functional enhancement over monoparatopic ADCs. PTK7 is an attractive ADC target due to preliminary clinical activity demonstrated with cofetuzumab peledotin in non-small cell lung cancer, ovarian cancer, and metastatic breast cancer and evidence that PTK7 is overexpressed in other solid tumor indications including esophageal cancer, colorectal cancer, head and neck cancer, and cervical cancer. Materials and Methods: Twenty-nine PTK7-targeting paratopes were isolated from a mouse antibody discovery campaign and paratope pairs were combined into a panel of biparatopic antibodies using AzymetricTM Het_Fc mutations. A high-throughput screen was used to assess the functional properties of these antibodies and to identify the top paratope combinations. ADCs were produced by conjugating the top biparatopic and monoparatopic antibodies to ZD06519, a topoisomerase 1 inhibitor payload used in ZW191, an ADC targeting FRα under evaluation in a phase 1 clinical trial (NCT06555744). ADC activity was investigated in a series of in vitro and in vivo experiments using ovarian, lung, and breast cancer models. Results: Across multiple cancer cell lines with varying PTK7 expression we compared a lead monoparatopic antibody and cofetuzumab with the top biparatopic antibody which demonstrated greater cell surface decoration and better internalization. At clinically relevant doses, the biparatopic ZD06519 ADC was more efficacious than cofetuzumab pelidotin in triple negative breast cancer and lung cancer cell line derived xenograft models. Additional studies of the PTK7 biparatopic ADC in patient-derived xenograft models and a non-human primate tolerability study are planned. Citation Format: Luying Yang, Vincent Fung, Alex Wu, Dunja Urosev, Saki Konomura, Tik Nga Tong, Katina Mak, Elizabeth M. Porter, Diego A. Alonzo, Catrina M. Kim, Janice P. Tsui, Linglan Fu, Lemlem Degefie, Kaylee Wu, Matthew Bonderud, Jamie R. Rich, Stuart D. Barnscher. Design and development of biparatopic antibody-drug conjugates against protein tyrosine kinase 7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1565.

Abstract Bispecific T cell engagers (TCEs) have exhibited clinical successes in the treatment of hematological cancers, while treatment of solid tumors remains a challenge. Treatment of solid tumors with conventional CD3-engaging TCEs can result in limited T cell proliferation and recruitment to the tumor site, and treatment related T cell anergy, thus restricting the ability of bispecific TCEs to inhibit growth of these poorly infiltrated tumors and rapidly growing tumors. Next generation tumor-targeting, trispecific T cell engagers with integrated costimulation (TriTCE Co-Stim) have the potential to provide more durable responses and re-invigorate T cell responses by mediating integrated signaling through CD3 (signal 1) and co-stimulation through CD28 (signal 2). Superagonist anti-CD28 antibodies activate T cells but resulted in clinical toxicities with severe cytokine release syndrome (CRS). Therefore, achieving a balance between signal 1 and 2 is critical for optimal T cell activation and proliferation. Using our AzymetricTM and EFECTTM platforms, we generated heterodimeric TriTCE Co-Stim antibody formats with various geometries and affinities to optimize T cell response and widen the therapeutic window for the treatment of solid tumors. We have previously described screening and interrogation of different TriTCE Co-stim formats and paratope affinities, and the selection of a lead format with optimized CD3 and CD28 T cell activation. Here, we have further expanded our validation of the TriTCE Co-stim platform with our lead CLDN18.2 TriTCE co-stim format, to include a tolerability assessment in nonhuman primates.Our lead TriTCE Co-Stim format exhibited a favorable safety profile in vitro and mediated target-dependent induction of T cell activity, with enhanced proliferation, survival, and T cell-mediated cytotoxic potency compared to bispecific TCEs. In a predictive in vivo model of CRS, our lead TriTCE Co-Stim did not exhibit systemic toxicity or peripheral cytokine release. TriTCE Co-Stim mediated enhanced tumor growth regression in vivo with an increase of T cells within the tumor and no increase of T cells within the periphery. Repeat doses of the lead TriTCE Co-Stim molecule at 3 mg/kg were well-tolerated in nonhuman primates with no abnormal clinical signs. Minor decreases in body weight and food consumption were observed. Clinical pathology changes were consistent with a mild systemic inflammatory response. In summary, our lead TriTCE co-stim molecule has enhanced target-dependent antitumor activity vs. bispecific benchmarks and is tolerated a murine CRS model and repeat dose non-human primate study. These data suggest TriTCE Co-stim may provide tolerable and more durable antitumor responses and contribute to improved clinical outcomes. Citation Format: Lisa Newhook, Purva Bhojane, Kurt Stahl, Nichole E. Escalante, Peter Repenning, Diego Perez Escanda, Polly Shao, Maya C. Poffenberger, Alec Robinson, Kesha Patel, Alexandra Livernois, Chayne L. Piscitelli, Nicole Afacan, Paul A. Moore, Nina E. Weisser, Thomas Spreter von Kreudenstein. TriTCE Co-Stim: A next generation trispecific T cell engager platform with integrated CD28 costimulation, engineered to improve responses in the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6719.

Abstract Heteromorphic sex chromosomes (XY or ZW) present problems of gene dosage imbalance between the sexes, and with the autosomes. Mammalian X chromosome inactivation was long thought to imply a critical need for dosage compensation in vertebrates. However, the universal importance of sex chromosome dosage compensation was questioned by mRNA abundance measurements that demonstrated sex chromosome transcripts are neither balanced between the sexes or with autosomes in monotreme mammals or birds. Here, we demonstrate unbalanced mRNA levels of X genes in platypus males and females that correlate with differential loading of histone modifications, and confirm that transcripts of Z genes are unbalanced between males and females also in chicken. However, we found that in both species, median male to female protein abundance ratios were 1:1, implying an additional level of post-transcriptional control. We conclude that parity of sex chromosome output is achieved in birds, as well as all mammal groups, by a combination of transcriptional and post-transcriptional control, consistent with an essential role for sex chromosome dosage compensation in vertebrates.

Cannabinoids are a promising therapeutic avenue for chronic pain. However, clinical trials often fail to report analgesic efficacy of cannabinoids. Inhibition of voltage gate calcium (Cav ) channels is one mechanism through which cannabinoids may produce analgesia. We hypothesized that cannabinoids and cannabinoid receptor agonists target different types of Cav channels through distinct mechanisms. Electrophysiological recordings from tsA-201 cells expressing either Cav 3.2 or Cav 2.2 were used to assess inhibition by HU-210 or cannabidiol (CBD) in the absence and presence of the CB1 receptor. Homology modelling assessed potential interaction sites for CBD in both Cav 2.2 and Cav 3.2. Analgesic effects of CBD were assessed in mouse models of inflammatory and neuropathic pain. HU-210 (1μM) inhibited Cav 2.2 function in the presence of CB1 receptor but had no effect on Cav 3.2 regardless of co-expression of CB1 receptor. By contrast, CBD (3μM) produced no inhibition of Cav 2.2 and instead inhibited Cav 3.2 independently of CB1 receptors. Homology modelling supported these findings, indicating that CBD binds to and occludes the pore of Cav 3.2, but not Cav 2.2. Intrathecal CBD alleviated thermal and mechanical hypersensitivity in both male and female mice, and this effect was absent in Cav 3.2 null mice. Our findings reveal differential modulation of Cav 2.2 and Cav 3.2 channels by CB1 receptors and CBD. This advances our understanding of how different cannabinoids produce analgesia through action at different voltage-gated calcium channels and could influence the development of novel cannabinoid-based therapeutics for treatment of chronic pain.

This manuscript describes the development and characterization of a second-generation "helping hand" solubilizing tag. This semipermanent tag aids solubility of peptides during purification and handling steps and is compatible with common native chemical ligation conditions. The utility of this tag is demonstrated by the synthesis of the Shiga toxin B subunit.