Mesothelin (MSLN) is a tumor associated antigen overexpressed in many cancer indications and is an attractive target for immunotherapies including bispecific T cell engagers (TCE) and chimeric antigen receptor T (CART) cells. While MSLN-targeting immunotherapies have shown signs of clinical activity, their success has been hindered by dose-limiting toxicities associated with on-target off-tumor effects and cytokine release syndrome (CRS). To overcome these issues, we engineered ZW171, a MSLN-targeting TCE, with enhanced safety and antitumor activity. ZW171 is a 2+1 IgG1-like antibody, built with AzymetricTM and EFECTTM technologies, consisting of two MSLN binding domains and one low affinity CD3ε binding domain. We previously showed that the unique geometry and 2+1 design of ZW171 facilitates tumor selective binding and potent preferential killing of MSLN-mid and -high target cells, while sparing MSLN-low expressing target cells, and enhanced antitumor activity compared to other 2+1 TCE formats and the clinical benchmark HPN536 in MSLN-expressing PBMC-engrafted CDX models. To address the evolving clinical landscape and assess antitumor activity in additional indications and more translationally relevant models, we assessed ZW171 activity in advanced patient-derived organoid and xenograft models and benchmarked to other MSLN-targeting TCE including AMG 305, JNJ-79032421 and CT95. Additionally, activity in expanded MSLN-positive indications including pancreatic, endometrial, and gastric cancer, and the in the presence of soluble MSLN (sMSLN), which is observed in the serum of patients and can impede MSLN-targeted antibody-based therapies, was assessed in vitro. Ex vivo, ZW171 mediated potent tumor cell killing and T cell activation in patient-derived ovarian cancer organoid models. ZW171 induced complete tumor regressions in established patient-derived in vivo MSLN-positive non-small cell lung cancer and pancreatic cancer models. Comparison of ZW171 to AMG 305, JNJ-79032421 and CT95 showed reduced binding to T cells and equivalent or greater antitumor activity against MSLN-overexpressing cells. In vitro, ZW171 demonstrated potent MSLN-dependent killing in MSLN-positive indications including pancreatic, endometrial, and gastric cancer, and maintained activity in the presence of clinically relevant sMSLN concentrations observed in patient serum. Overall, ZW171 demonstrates differentiated and potent antitumor activity in a range of MSLN-expressing cancers. ZW171 is being evaluated in a Phase 1 clinical trial in MSLN-expressing solid tumors (NCT06523803). Nicole J. Afacan, Patricia Zwierzchowski, Wingkie Wong, Maya Poffenberger, Chayne Piscitelli, Thomas Spreter von Kreudenstein, Nina E. Weisser. ZW171, a differentiated 2+1 T cell-engaging bispecific antibody with antitumor activity in a range of mesothelin-expressing cancers [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 3503.

Small cell lung cancer (SCLC) is a highly aggressive and difficult-to-treat malignancy with limited treatment options. Delta-like ligand 3 (DLL3), a cell surface protein overexpressed in SCLC, has emerged as a promising therapeutic target. Bispecific T cell engagers (TCE) targeting DLL3, including tarlatamab which has received accelerated approval, have demonstrated anti-tumor activity in the clinic with an improved duration of response compared to current standard of care, including chemotherapy and immunotherapy. Despite these advances, we believe there is opportunity to improve the rate and depth of response as the clinical activity of bispecific T cell engagers may be limited by low T cell infiltration and poor T cell function characteristic of SCLC tumors, and by the emergence of treatment-related T cell anergy due to T cell stimulation via signal 1 (CD3) only. The incorporation of signal 2 co-stimulation, via CD28 signaling, has the potential to improve response rates by stimulating increased T cell activation, proliferation and survival. To address treatment challenges and enhance the durability and sustainability of T cell activation, we engineered ZW209, a trispecific co-stimulatory T cell engager (TriTCE Co-stim) that optimally engages CD3 and CD28 and redirects and enhances cytotoxic T cell responses to DLL3-expressing tumor cells while maintaining a desired safety profile. Our development candidate, ZW209, is designed to optimally engage CD3 and CD28 in an obligate cis manner, supported by a lack of T cell cross-linking and fratricide. The addition of CD28 co-stimulation in ZW209 demonstrated enhanced DLL3-dependent cytokine induction and T cell proliferation with improved antitumor activity relative to clinical bispecific TCEs benchmarks. In a serial, repeated challenge in vitro cytotoxicity assay, ZW209 displayed superior T cell fitness and anti-tumor activity to a bispecific control and clinical benchmark. In vivo, ZW209 exhibited potent anti-tumor activity in multiple humanized SCLC xenograft models. In aqueous and solid phase cytokine release assays, ZW209 exhibited minimal DLL3-independent cytokine production. Importantly, a ZW209 cynomolgus cross-reactive surrogate molecule displayed a favorable safety profile as it was well tolerated in non-human primates with repeat dosing at 10 mg/kg with no abnormal clinical signs. In summary, these preclinical data support that ZW209 promotes improved anti-tumor activity against DLL3-positive cancer cells relative to competitor bispecific TCEs. By integrating CD28 co-stimulation into our trispecific T cell engager, ZW209 has the potential to improve the rate and duration of response for better clinical outcomes. Desmond Lau, Peter Repenning, Diana Canals Hernaez, Alec Robinson, Diego Perez Escanda, John Zhang, Hamed Shirvani, Catherine Wu, Kurt Stahl, Aditi Deshmukh, Nichole Escalante, Mariana Rocha, Begonia Silva Moreno, Lisa Newhook, Purva Bhojane, Paul A. Moore, Nina E. Weisser, Thomas Spreter von Kreudenstein. ZW209, a DLL3 targeted trispecific T cell engager with integrated CD28 co-stimulation, demonstrates safety and potent preclinical efficacy in models of small cell lung cancer [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 7318.

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 Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer with a poor prognosis and high unmet medical need. Current standards of care, including chemotherapy, targeted therapy and immunotherapy, are associated with limited duration of response. Furthermore, SCLC is characterized by an immunosuppressive microenvironment and poor T cell infiltration which present additional treatment challenges. Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand that is aberrantly overexpressed in SCLC and other neuroendocrine tumors, but minimally expressed in normal tissues, and is a new therapeutic target. Bispecific T cell engagers (TCE) targeting DLL3 have entered the clinic and demonstrated promising anti-tumor activity in SCLC patients; however, we believe there is room to improve on the rate and depth of response. To address treatment challenges and enhance the durability and sustainability of T cell activation, we have engineered a trispecific co-stimulatory T cell engager (TriTCE Co-stim) molecule that optimally engages CD3 and CD28 and redirects and enhances cytotoxic T cell responses to DLL3-expressing tumor cells while maintaining a desired safety profile. A panel of DLL3xCD3xCD28 trispecific antibodies (Abs) was engineered with a conventional anti-CD28 agonist paratope in a variety of Ab formats, geometries, paratope affinities and specificities. Geometry and design features of the tri-specific Abs were informed by prior TriTCE Co-stim molecule optimization1. AzymetricTM and EFECTTM platforms were used to facilitate heterodimeric multi-chain antibody assembly with silenced Fc gamma function. Lead TriTCE Co-stim Abs were selected for enhanced DLL3-dependent antitumor cytotoxic activity and increased T cell function including T cell activation, proliferation and cytokine production, compared to competitor bispecific TCEs. DLL3-independent activity and cytokine release were assessed with human PBMCs in solid and solution phase assays to evaluate the potential for cytokine release induction. In summary, we have identified multiple TriTCE Co-Stim Ab formats with improved DLL3-dependent cytotoxicity and T cell activation over competitor bispecific TCEs across multiple DLL3-expressing tumors. By screening various Ab formats, geometries and paratope affinities, we have selected lead DLL3 TriTCE Co-stim Abs displaying target-dependent activation of T cells with potent anti-tumor activity which may translate to improved and more durable antitumor responses in the treatment DLL3-expressing cancers. Citation Format: Peter Repenning, Desmond Lau, Diana C. Hernaez, Alec Robinson, Diego P. Escanda, Mariana Rocha, Begonia S. Moreno, John Zhang, Polly Shao, Nichole Escalante, Lisa Newhook, Purva Bhojane, Chayne L. Piscitelli, Paul A. Moore, Thomas Spreter Von Kreudenstein, Nina E. Weisser. DLL3 TriTCE Co-Stim: A next generation trispecific T cell engager with integrated CD28 costimulation for the treatment of DLL3-expressing cancers [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 6716.

CD3-bispecific T cell engager (TCE) therapies have exhibited clinical utility against hematological malignancies, but successes in solid tumor indications have been limited. Compared to heme malignancies, treatment of solid tumors is hindered by immunosuppressed microenvironments that can be refractory to traditional CD3-bispecific TCEs. Immunosuppression in the tumor microenvironment limits treatment responses in part due to the expression of inhibitory immune checkpoints, such as PD-1 on exhausted T cells and PD-L1 on tumor cells. To improve T cell responses and anti-tumor activity in immunosuppressed solid tumors, we generated trispecific TCE antibodies (Abs) that target a tumor associated antigen (TAA), CD3 and PD-L1 (via a PD1 moiety) to stimulate tumor-directed T cell killing and checkpoint blockade at the tumor site. In this engineering approach we harnessed the flexibility of our AzymetricTM technology to screen multiple antibody formats, geometries, paratopes, and PD-1 domain affinities in parallel. We screened the TriTCE CPI antibodies, targeting different TAAs, for tumor-directed T cell cytotoxicity and CPI activity on TAA+PD-L1+ and TAA-PD-L1+ tumor cells. We identified multiple TriTCE CPI Abs that induced potent TAA-dependent T cell killing of TAA+PD-L1+, but not TAA-PD-L1+, tumor cells. Evaluation of CPI using a PD-1/PD-L1 checkpoint reporter gene assay identified antibody formats that stimulated simultaneous TAA dependent T cell engagement and enhanced checkpoint inhibition superior to bispecific Ab plus anti-PD-L1 Ab combination treatments. Additionally, in a human PBMC-engrafted xenograft model, TriTCE CPI Abs showed increased anti-tumor activity compared to a bispecific Ab control +/- anti-PD-L1 Ab treatment. Furthermore, the benefits of increased anti-tumor activity and CPI was observed across multiple TriTCE CPI Abs targeting different TAAs. We generated multiple TriTCE CPI Abs that combine tumor-dependent T cell cytotoxicity with checkpoint blockade, which may translate to improved T cell responses in immunosuppressed solid tumors. The evaluation of multiple Ab formats, geometries and paratope affinities allowed for optimization of TAA-dependent cytotoxicity and CPI to identify Abs with enhanced anti-tumor activity and superior site-specific CPI, key factors that may contribute to a wide therapeutic index and improved clinical outcomes. Citation Format: Maya C. Poffenberger, Meghan M. Verstraete, Anna Von Rossum, Patricia Zwierzchowski, Matteo Zago, Veronica Luu, Sifa Arrafi, Siran Cao, Harsh Pratap, Chayne L. Piscitelli, Nina E. Weisser, Thomas Spreter von Kreudenstein. TriTCE CPI, next generation trispecific T cell engagers with integrated checkpoint inhibition (CPI) for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2982.

Abstract Bispecific T cell engager (TCE) therapies have exhibited clinical utility against hematological cancers, but limited success in solid tumors. Treatment of solid tumors has additional challenges - including immunosuppressive environments and low T cell infiltration - limiting the antitumor activity of CD3-bispecific TCEs. Conventional T cell activation and sustained proliferation requires signaling via CD3 (signal 1) and costimulatory molecules (signal 2), such as CD28. The balance between signals 1 and 2 is critical for optimal T cell activation – signal 1 in the absence of signal 2 results in T cell anergy, while overactivation via signals 1 and 2 can lead to T cell dysfunction and cytokine release, as observed with toxicities associated with αCD28 superagonist antibodies (Abs). Optimal signal 2 costimulation via CD28 results in improved T cell fitness, activation and proliferation. To improve T cell responses in solid tumors, we developed costimulatory trispecific TCEs (TriTCE Co-stim) that engage CD3, CD28 and a tumor-associated antigen (TAA). Our novel TriTCE Co-stim Abs were generated using the AzymetricTM and EFECTTM platforms to facilitate heterodimeric TriTCE Co-stim assembly and to knockout Fc gamma receptor interactions, respectively. To limit potential CD28-mediated toxicities, we evaluated a conventional αCD28 agonist paratope and generated a paratope library with varying affinities for CD28. TriTCE Co-stim Abs were engineered with various formats, geometries, paratope affinities, and TAA specificities. To understand the impact of TriTCE Co-stim Abs on T cell activation, we assessed in vitro cytotoxicity, cytokine production and proliferation of primary human CD3 T cells in co-culture with TAA-expressing cancer cell lines. A human PBMC-engrafted xenograft model was used to assess in vivo antitumor activity in a TAAhigh tumor model. TriTCE Co-stim Abs exhibited a range of cytotoxic potency, with several formats exhibiting greater potency than bispecific TCEs, and induced greater cytotoxicity of tumor cells in long term co-cultures at low effector to target ratios. TriTCE Co-stim Abs exhibited TAA-dependent cytokine release and T cell proliferation, with enhanced IL-2 production and proliferation compared to that induced by bispecific TCEs. Tumor growth regression was observed in vivo following treatment with different TriTCE Co-stim Ab formats. In summary, we identified TriTCE Co-stim Ab formats that exhibit improved proliferation and antitumor activity against multiple TAA targets compared to bispecific TCE, which may translate to improved and more durable antitumor responses in solid tumors with low T cell infiltration. The evaluation of multiple formats, geometries and paratope affinities allowed optimization of activity and selectivity to promote maximal therapeutic index and efficacy, key factors that may contribute to improved clinical outcomes. Citation Format: Lisa Newhook, Purva P. Bhojane, Peter W. Repenning, Diego Perez Escanda, Nichole K. Escalante, Patricia Zwierzchowski, Alec Robinson, Lauren Clifford, Harsh Pratap, David N. Douda, Chayne L. Piscitelli, Nicole J. Afacan, Thomas Spreter von Kreudenstein, Nina E. Weisser. TriTCE Co-stim, next generation costimulatory trispecific T cell engagers for the treatment of solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5121.

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.

Mesothelin (MSLN) is a GPI-linked membrane glycoprotein that is overexpressed in many cancer indications for which there is a high unmet medical need including pancreatic, mesothelioma, and ovarian. While MSLN-targeting agents have shown early signs of clinical activity, therapies with improved safety and efficacy are still needed. T cell engager (TCE) therapies have exhibited clinical utility against hematological malignancies, but limited success against solid tumors due to dose-limiting toxicities associated with cytokine release syndrome (CRS) and on-target off-tumor effects. To improve the therapeutic intervention of MSLN-expressing tumors, we engineered a bispecific TCE, designed to have an improved therapeutic window by enhancing both safety and anti-tumor activity profiles. To avoid dose-limiting toxicities related to CRS observed with high-affinity anti-CD3 paratopes (e.g. based on OKT3 or SP34), we engineered an anti-CD3 paratope (ZW_CD3_1) with low affinity CD3 binding and, in the context of a bispecific TCE, potent redirected T cell cytotoxicity. Compared to a bispecific antibody containing the high CD3 affinity SP34 paratope, the ZW_CD3_1 anti-CD3 paratope showed reduced and less potent T cell binding, stimulated less cytokine release yet elicited equivalent T cell mediated killing of MSLN-expressing tumor cells. Using the bispecific AzymetricTM platform, the ZW_CD3_1 paratope was incorporated into a panel of bispecific antibodies with different geometries, formats, and anti-MSLN paratope affinities then screened for T cell mediated lysis of target cells and production of cytokines. Based on reiterative screening and engineering, we determined the MSLN paratope affinity and the 2+1 format, consisting of two anti-MSLN single chain variable fragments (scFvs) and one anti-CD3 fragment antigen binding (Fab) domain, as the lead candidate format. In vivo, the lead format also showed significantly greater anti-tumor activity than other 2+1 bispecific formats. Following additional engineering and screening a lead MSLN x CD3 2 +1 bispecific antibody, ZW171, was selected for development. We further characterized the mechanistic and anti-tumor activity of ZW171 and showed that incubation of human T cells and tumor cells with ZW171 led to potent preferential killing of MSLN-mid and -high target cells while sparing MSLN-low expressing target cells. ZW171 only stimulated T cells to proliferate and produce cytokines in the presence of MSLN-expressing tumor cells, mitigating the risk of peripheral T cell activation and CRS. Importantly, ZW171 exhibited potent tumor growth inhibition in multiple established tumor models. Collectively, these data suggest that ZW171 has the potential to be an efficacious and safe therapeutic for the treatment of MSLN-expressing cancers. Citation Format: Nicole J. Afacan, Chayne Piscitelli, Patricia Zwierzchowski, Siran Cao, Janessa Li, Wingkie Wong, Kara White-Moyes, Thomas Spreter von Kreudenstein, Nina E. Weisser. ZW171, a T cell-engaging, bispecific antibody for the treatment of mesothelin-expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2942.

Abstract HER2-directed therapies have improved clinical outcomes for many patients with HER2-positive breast and gastric cancer. Despite these successes, there remains a need to develop improved HER2-targeted therapies for these and other HER2-expressing tumors, particularly in the setting of recurrent or metastatic disease. Zanidatamab (ZW25) is a humanized, bispecific, immunoglobulin (Ig) G1-like antibody directed against the juxtamembrane extracellular domain (ECD4) and the dimerization domain (ECD2) of human epidermal growth factor receptor 2 (HER2), the same domains targeted by trastuzumab (T) and pertuzumab (P), respectively. Data from the ongoing phase 1 study (NCT02892123) demonstrate that zanidatamab is well tolerated and has single agent activity in patients with advanced HER2-expressing cancers that have progressed after standard of care (SOC) therapies, including HER2-targeted agents such as T, P, and trastuzumab emtansine.1,2 We have previously shown that the unique design and bispecific binding of zanidatamab results in multiple mechanisms of action including increased antibody binding density, potent effector function, improved receptor internalization and HER2 downregulation relative to T.3 To better understand the mechanism by which zanidatamab differentiates itself from T, P and T+P, we recently expanded our mechanistic evaluations including cell surface HER2 aggregation, complement-dependant cytotoxicity (CDC) and inhibition of both tumor cell growth and intracellular signaling. Single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM) was used to map HER2 receptor distribution and quantitate the size, density, and frequency of receptor clusters induced by antibody binding. In vitro assessments were performed in a panel of HER2-expressing cell lines using standard assays including CDC with human complement serum and inhibition of both tumor cell growth and intracellular signaling. Using dSTORM, we observed that zanidatamab binding resulted in enhanced HER2 aggregation and distinct HER2 capping on the tumor cell surface compared to T, P or T+P. Evaluation of CDC activity in HER2-overexpressing tumor cells demonstrated that zanidatamab, but not T, P or T+P, elicited CDC suggesting that the enhanced HER2 aggregation and capping on the tumor cell surface provides high avidity docking sites to which C1 binds and is activated. Zanidatamab showed further differentiation in the inhibition of both tumor growth and intracellular signaling of HER2-overexpressing cells compared to T, P and T+P. Zanidatamab has novel cell surface binding and additional mechanisms of action compared to T, P and T+P. Zanidatamab is actively being evaluated in clinical trials in multiple HER2-expressing solid tumors, including a registration-enabling clinical trial in HER2 gene amplified biliary tract cancer (NCT04466891). Citation Format: Nina E. Weisser, Grant Wickman, Libin Abraham, Jason O'Toole, Bryant Harbourne, Joy Guedia, Chi Wing Cheng, Peter Chan, Duncan Browman, Michael R. Gold, Neil Josephson, Surjit Dixit, Gerry Rowse. The bispecific antibody zanidatamab's (ZW25's) unique mechanisms of action and durable anti-tumor activity in HER2-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1005.

A self-consistent model of β-mannan oligosaccharides bound to a monoclonal antibody, C3.1, that protects mice against Candida albicans has been developed through chemical mapping, NMR spectroscopic, and computational studies. This antibody optimally binds di- and trisaccharide epitopes, whereas larger oligomers bind with affinities that markedly decrease with increasing chain length. The (1→2)-β-linked di-, tri-, and tetramannosides bind in helical conformations similar to the solution global minimum. Antibody recognition of the di- and trisaccharide is primarily dependent on the mannose unit at the reducing end, with the hydrophobic face of this sugar being tightly bound. Recognition of a tetrasaccharide involves a frameshift in the ligand interaction, shown by strong binding of the sugar adjacent to the reducing end. We show that frameshifting may also be deliberately induced by chemical modifications. Molecular recognition patterns similar to that of mAb C3.1, determined by saturation transfer difference-NMR, were also observed in polyclonal sera from rabbits immunized with a trisaccharide glycoconjugate. The latter observation points to the importance of internal residues as immunodominant epitopes in (1→2)-β-mannans and to the viability of a glycoconjugate vaccine composed of a minimal length oligosaccharide hapten.

The use of recombinant antibody fragments (rAbF) as therapeutic agents is compromised by shorter serum persistences than IgG therapeutics and their inability to mediate Fc-dependent effector functions. Here, we show that the strategy of complex formation between epitope-tagged rAbFs and anti-epitope IgG monoclonal antibodies (mAb) can improve the therapeutic potential of rAbFs by both enhancing their serum persistence and conferring on them the ability to recruit Fc-mediated effector functions. These two mechanistic aspects of this strategy were demonstrated using c-myc- and 6xHis-tagged Fab and scFv rAbFs, both directed against Pseudomonas aeruginosa O6ad, in combination with two different murine anti-epitope tag IgGs, anti-5xHis IgG (Penta-His) and anti-c-myc IgG (9E10). Further enhancement of this strategy for the employment of rAbFs as therapeutics is discussed.

Antibodies (Abs) are some of the most powerful tools in therapy and diagnostics and are currently one of the fastest growing classes of therapeutic molecules. Recombinant antibody (rAb) fragments are becoming popular therapeutic alternatives to full length monoclonal Abs since they are smaller, possess different properties that are advantageous in certain medical applications, can be produced more economically and are easily amendable to genetic manipulation. Single-chain variable fragment (scFv) Abs are one of the most popular rAb format as they have been engineered into larger, multivalent, bi-specific and conjugated forms for many clinical applications. This review will show the tremendous versatility and importance of scFv fragments as they provide the basic antigen binding unit for a multitude of engineered Abs for use as human therapeutics and diagnostics.

Peptide mimotopes have been investigated as surrogate antigens of carbohydrate (CHO) targets on pathogen and tumor cells in vaccine and therapeutic discovery. One of the main bottlenecks in peptide mimotope discovery is the inability of initial screening regimes to differentiate between true mimotopes and non-mimotopes. As a result, subsequent in vivo analysis of putative peptide mimotopes is often inefficient requiring the use of experimental animals during a lengthy in vivo immunization process. Here, we demonstrate a rapid preliminary screening method to identify putative mimotopes using a recombinant antibody (rAb) library, which may increase the probability of identifying peptides that will elicit a CHO-cross-reactive response in vivo. A human naïve rAb library was screened against both an established peptide mimotope and a non-mimotope of the Group B Streptococcus (GBS) type III polysaccharide to determine if selected antibodies cross-reacted with the original GBS polysaccharide. We were able to differentiate between these two peptides because peptide-binding Abs that cross-reacted to GBS was isolated only with the peptide mimotope. We discuss the feasibility of using this method to significantly increase the breadth of screening and reduce the discovery time for peptide mimotopes.