Affinity CD16 Research Articles

Effect of placental circulating T cells expressing CD16 on multiple hematological and solid tumor cancers through combination with various monoclonal antibodies.

2554 Background: PT-CD16VS is an allogeneic cell therapy derived from human postpartum placental circulating T (PT) cells that are genetically modified to express CD16 and endogenous T cell receptor (TCR) knockout. PT-CD16VS cells can be combined with various monoclonal antibodies to engage in anti-tumor antibody-dependent cellular cytotoxicity (ADCC) against diverse cancers with a “universal receptor” approach. Here we report the characterization and preclinical evaluation of PT-CD16VS in combination with monoclonal antibodies against both hematological and solid tumor cancers. Methods: PT cells were activated, transduced with a lentiviral vector containing a construct expressing a high affinity CD16 variant, CD16VS, and transfected to knock out the TCR. In vitro,functional activity of PT-CD16VS cells in combination with monoclonal antibodies was assessed in cytotoxicity, cytokine release, and proliferation assays. Invivo, PT-CD16VS was combined with Rituximab in a Raji xenograft model in NSG mice, and with Trastuzumab in a subcutaneous NCI-N87 xenograft model in NSG mice in which tumor volume was measured and tumor samples were evaluated for PT-CD16VS infiltration. Results: In vitro, PT-CD16VS exhibited potent ADCC and cytokine release when combined with Rituximab against CD20+ Burkitt's lymphoma (Daudi and Raji), with Trastuzumab against HER2+ gastric carcinoma (NCI-N87), with Trastuzumab or Avelumab against HER2+/PDL-1+ non-small cell lung carcinoma (NCI-H1975) and Trastuzumab-resistant metastatic mammary adenocarcinoma (JIMT-1), and with Cetuximab against EGFR+ triple negative metastatic mammary adenocarcinoma (MDA-MB-231). Moreover, examples of PT-CD16VS antigen-specific proliferation were demonstrated against Raji and NCI-N87 when cells were combined with Rituximab and Trastuzumab, respectively. In vivo, in combination with Rituximab in the Raji mouse model, PT-CD16VS exhibited significant survival benefit compared to vehicle and Rituximab alone treated groups. In the subcutaneous NCI-N87 solid tumor model, PT-CD16VS with Trastuzumab demonstrated significant reduction in tumor volume compared to vehicle, Trastuzumab, and Enhertu groups. In addition, PT-CD16VS infiltration into the tumor was shown to be Trastuzumab dependent, with infiltrating cells expressing high levels of CD16 and Ki67. Conclusions: Our results show that PT-CD16VS have potent in vitro and in vivo ADCC activity, and a single drug product has the potential to be combined with various monoclonal antibodies to target multiple cancers across hematological and solid tumor indications. This “universal receptor” with antibody-dependent targeting approach, together with the benefits of an allogeneic cell platform, may democratize accessibility of such therapies for patients.

Abstract 1235: Impact of anti-CD3 and tumor-target binder affinities on in-vitro potency, in-vivo efficacy, and cytokine release

Abstract Background: First generation T cell bispecifics (TCBs) were developed by utilizing high CD3 and tumor target binder affinities to induce a potent response. Instrumentalizing this, TCBs have clinically proven to elicit efficacious target cell killing and anti-tumor efficacy, but they encountered hurdles related to cytokine release. Recent studies suggested that reducing CD3 binder affinity could lead to an efficient tumor killing with lower cytokine levels. However, the answer to whether cytokine release is mainly dependent on CD3 binder affinity or a multifactorial phenomenon remains ambiguous. Here, we aim to characterize the efficacy-safety relationship of different TCBs through investigating the interplay between CD3 binder affinity and the affinity of the tumor target-specific binder. For that purpose, a series of TCBs with varying CD3 affinities, and two target binders of high and low affinities were generated in the 2:1 TCB format, resulting in a bivalent binding to the tumor antigen and a monovalent one to CD3, which offers a unique opportunity to enhance the activity of TCBs. Methods: Firstly, 2:1 TCBs comprising either the high or low affinity target binder and varying CD3 binder affinities were co-cultured in-vitro with human peripheral blood mononuclear cells (PBMCs), and human tumor cell lines, in order to assess T cell mediated cytotoxicity and cytokine release at different time points. Additionally, the impact on in-vivo efficacy, cytokine release, and pharmacokinetics was assessed in humanized mice implanted with a patient-derived xenograft model. Results: Based on the dose-response in the cytotoxicity assay, the CD3 binders were classified into high, intermediate, and low levels of affinity. As expected, we observed that the in-vitro T cell cytotoxicity, in-vivo tumor growth inhibition (TGI), and cytokine release levels were maximal with the highest affine CD3 binder. Simultaneously, we have observed a decrease in cytotoxicity response with decreasing CD3 affinity compared to the high affinity binder. However, this was accompanied by an atypical decrease in cytokine release. Particularly, the TCB comprising the intermediate CD3 affinity binder illustrated a comparable in-vitro cytotoxicity and in-vivo TGI to the high affinity binder with a marked decrease in cytokine release [4x reduction in-vitro and 6x reduction in-vivo]. Upon testing the TCBs comprising the low affinity target binder with the same CD3 binders, we observed an additional decrease in cytokine release with the decreasing CD3 affinity. In conclusion, we designed and generated TCBs with varying affinities in both the CD3 and target binding arms. Reducing cytokine release while maintaining adequate efficacy is feasible through CD3 binder affinity attenuation; however, the target binder affinity should be taken into consideration when interpreting our findings and designing new molecules. Citation Format: Omar Abdelmotaleb, Anneliese Schneider, Thomas Hofer, Johannes Sam, Martin Lechmann, Inja Waldhauer, Ali Bransi, Miro Eigenmann, Anne Freimoser-Grundschober, Christian Gassner, Alex Odermatt, Peter Brünker, Sara Colombetti, Christian Klein. Impact of anti-CD3 and tumor-target binder affinities on in-vitro potency, in-vivo efficacy, and cytokine release [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 1235.

Abstract 5232: Placental circulating T cells expressing CD16 in combination with trastuzumab demonstrate robust anti-tumor antibody-dependent cellular cytotoxicity (ADCC) against gastric cancer

Abstract Introduction: Celularity is developing PT-CD16VS as a novel platform for use in combination with approved monoclonal antibodies for the potential treatment of multiple cancers. PT-CD16VS is an allogeneic cell therapy derived from human postpartum placental circulating T (P-T) cells that are genetically modified to express a proprietary CD16 variant and endogenous T cell receptor (TCR) knockout. Here we report the characterization and preclinical evaluation of PT-CD16VS against HER2+ gastric cancer cells in combination with Trastuzumab. In vitro safety studies evaluating the potential for on-target/off-tumor activity of PT-CD16VS against HER2 low expressing lung epithelial cells (LEC) and human dermal fibroblasts (HDF) are also highlighted. Methods: P-T cells were activated and transduced with a lentiviral vector containing a CD16 construct expressing a high affinity CD16 variant, CD16VS. Following transduction, P-T cells were transfected to knock out the TCR. In vitro, the functional activity of PT-CD16VS cells in combination with Trastuzumab was assessed against the HER2+ gastric cancer NCI-N87 cell line in cytotoxicity, cytokine release, and proliferation assays. For on-target/off-tumor studies, similar assays were utilized to assess activity against the HER2 low expressing LEC and HDF cell lines. In vivo, PT-CD16VS with Trastuzumab was evaluated in a subcutaneous NCI-N87 xenograft model in NSG mice. Tumor volume was measured and tumor samples were evaluated for PT-CD16VS infiltration. Results: High CD16 transduction efficiency (>60%) was achieved across donors. In vitro, PT-CD16VS plus Trastuzumab demonstrated potent cytotoxicity against NCI-N87 cells at low E:T ratios (0.625:1) and at 12 hours of co-culture with moderate cytokine secretion at 24-hour of co-culture. PT-CD16VS proliferated following 48-hour incubation with NCI-N87 cells and Trastuzumab, as compared to an IgG1 control. PT-CD16VS in combination with Trastuzumab did not elicit on-target/off-tumor cytotoxicity or cytokine release in response to low HER2 expression on normal LEC or HDF cells. In vivo, PT-CD16VS with Trastuzumab demonstrated significant reduction in tumor volume compared to vehicle, Trastuzumab alone, and the positive control Enhertu groups. In addition, PT-CD16VS infiltration into the tumor was shown to be Trastuzumab dependent, with infiltrating cells expressing high levels of CD16 and Ki67. Conclusions: Our results show that PT-CD16VS has robust in vitro and in vivo ADCC activity against gastric cancer tumor cells when combined with Trastuzumab. Furthermore, PT-CD16VS activity is specific to tumor HER2 antigen expression and does not target low HER2 expression on normal healthy cells. PT-CD16VS is being developed in combination with various monoclonal antibodies for the treatment of multiple cancers. Citation Format: Kathy Karasiewicz-Mendez, Joseph Gleason, Kristina Tess, Mansour Djedaini, Shengchen Lin, Chenfei Huang, Shuyang He, Robert Hariri, Adrian Kilcoyne. Placental circulating T cells expressing CD16 in combination with trastuzumab demonstrate robust anti-tumor antibody-dependent cellular cytotoxicity (ADCC) against gastric cancer [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 5232.

Abstract 2369: EVOLVE-105, a differentiated CD20-targeted CD2 co-stimulatory T cell engager engineered for the treatment of B cell malignancies

Abstract Despite the recent approvals of CD20-targeted CD3 bispecific molecules in follicular lymphoma (Lunsumio™, mosunetuzumab) and diffuse large B cell lymphoma (Columvi™, glofitamab; and Epkinly™, epcoritamab), significant opportunities to improve patient outcomes in these indications remain. Targeted induction of T cell costimulatory receptors (signal 2) as a complement to T cell activation mediated by T cell receptor signal 1 is an attractive strategy to sustain and optimize the redirected T cell effector response to these tumors. Drug combinations of CD3-bispecifics and tumor-targeted costimulatory biologics such as CD19-41BBL and CD19-CD28 costimulatory agonists are promising approaches that have shown preclinical efficacy and are being investigated in the clinic. EVOLVE-105 is a CD20-targeted trispecific T cell engager biotherapeutic with an integrated, affinity-tuned CD3 and a CD2 co-stimulatory agonist. The advantages of physical integration include an increased probability of simultaneous T cell receptor and CD2 receptor engagement on each T cell and a simplified patient management strategy, compared to drug combinations of molecules individually targeting each receptor. EVOLVE-105 displays improved human T cell activation, increased T cell expansion, and tumor cell killing activity without a substantial increase in cytokine release compared to CD20xCD3 bispecifics with matching CD3 affinity which does not supply CD2 costimulation. EVOLVE-105 also displays superior potency compared to the CD20xCD3 clinical benchmarks mosunetuzumab, epcoritamab, and odronextamab. Importantly, the tumor-killing potency of EVOLVE-105 was within two-fold of the activity of glofitamab, while inducing less cytokine release, suggesting its potential to establish an improved therapeutic index. In cynomolgus monkeys, EVOLVE-105 demonstrated favorable tolerability and pharmacokinetics consistent with an attractive clinical developability profile. Sustained, dose-dependent B cell depletion in peripheral blood, lymph nodes, and spleen was observed. Circulating cytokine excursions, including IL-6, IFNγ, and TNFα at efficacious doses were markedly reduced by EVOLVE-105 treatment, in contrast to the circulating levels of cytokines reported for epcoritamab and glofitamab at their efficacious doses. EVOLVE-105 demonstrates favorable biophysical properties conducive to its development as intravenous and subcutaneous formulations. The emerging profile of EVOLVE-105 suggests its potential as a differentiated CD20-targeted immunotherapy capable of providing superior, more durable tumor control with a preferred cytokine release profile, compared to current CD20-targeted T cell engager therapies. Citation Format: Xingyue An, Mohosin Sarkar, Stella Martomo, Maria Hackett, Guixian Jin, Tiffany Liang, Danielle Klaskin, Tracy Lichter, Nana Adjoa Pels, Abudukadier Abulizi, Evelyn Teran, Afsana Sabrin, Oksana A. Sergeeva, Amber Fearnley, Hayden Karp, Julio Rodriguez, Eric M. Tam, Louis Matis, Jay S. Fine, Jeremy S. Myers. EVOLVE-105, a differentiated CD20-targeted CD2 co-stimulatory T cell engager engineered for the treatment of B cell malignancies [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 2369.

Abstract 3192: A novel long acting pegylated T cell engager targeting CD3 and cd19 with effective tumor killing and no potential CRS risk in CD19+ hematologic malignancies

Abstract The development of bispecific T cell engagers represents a highly effective immunotherapy strategy for the treatment of hematologic malignancies. However, Blinatumomab, a bispecific T cell engager of CD3/CD19, has disadvantages in clinical setting due to the adverse pharmacokinetics (PK), which resulted in less than 2 hours of in vivo elimination half-life, neurotoxicity, and cytokine release syndrome. In this study, a bispecific antibody JY108 was developed to target CD19 and CD3e with much reduced affinities towards both targets (revealed by Biacore assay results). JY108 was prepared by a patented pegylation bispecific linker technology, which could address the limitations of current therapeutic approaches. In vitro cytotoxicity assay results demonstrated that the EC50s of the drug specific cytotoxicity of T cells mediated by JY108 are in the concentrations of ng/ml, while blinatumomab mediated cytotoxicity to the same CD19+ cells are in the concentrations of pg/ml. Despite the weaker in vitro cytotoxicity than Blinatumomab, JY108 presents comparable tumor inhibition effects with Blinatumomab in different in vivo studies, which include the PBMC reconstructed models with established MEC-1 or Farage tumors, the delayed drug administration on human PBMCs/tumor co-transplanted model of Raji. In addition, JY108 prolonged the survival of animals with Nalm6 brain metastasis more significantly than Blinatumomab at the same dosage. Furthermore, the PK study revealed that at the different dosages, the circulation T1/2s of JY108 in mice are above 20 hours, which is expected to result in more significant efficacious benefits in clinical setting. Meanwhile, the completed package of GLP-toxicity study did not observe any drug related tox to the model animals (including no cytokine release in vivo, no neurotoxicity, no respiratory toxicity, etc.), and JY108 did not induce significant cytokine release than the blank control to the whole blood or PBMCs from healthy donor in vitro. In the efficacious concentrations of exerting cytotoxicity to CD19+ tumor cells in the presence of PBMC, JY108 did not induce significant cytokine release than the blank control, while blinatumomab always induced significant cytokine release. We postulated that the lower CD3 affinity of JY108 most probably resulted in the decoupling of the signaling for activating the gene expression of cytokines from the secretion of the polarized lytic granules by T cells, which resulted in a much milder T cell activation by JY108. Currently, JY108 is a phase I trial candidate and awaits further evaluations. Citation Format: Shumin (Sam) Liu, Yu (Yvonne) Wen, Weidong Lyu, Shuqiang Yin, Yang Lei, Dechun Wu, Zibin Wu, Qiudong Zhuo. A novel long acting pegylated T cell engager targeting CD3 and cd19 with effective tumor killing and no potential CRS risk in CD19+ hematologic malignancies [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 3192.

Abstract 3601: Improving killing of renal cell carcinoma through the combined effects of TCR engineered CD4+ and CD8+ T cells

Abstract Patients with late-stage kidney cancer have poor long-term survival rates. Our lab isolated a low affinity CD8 dependent TCR named BZ-4 from CTLs of a patient who had immune-mediated regression of renal cell carcinoma (RCC) following an allogeneic stem cell transplant and found this TCR recognized a human endogenous retrovirus type E (HERV-E) derived 10-mer peptide, CT-RCC-1. The BZ-4 clone belongs to the Vβ7.1 family and binds to CT-RCC-1 in an HLA-A11 dependent manner. Adoptive infusion of CD8+ BZ-4 transduced T cells is currently being explored in an NIH phase I trial (NCT03354390). Because CD4+ T cells have been shown to augment the anti-tumor effects of TCR-modified CD8+ T cells, we hypothesized that CD4+ T cells genetically engineered to co-express BZ-4 and CD8 would recognize and kill RCC tumors expressing the CT-RCC-1 and would improve the in vivo proliferation and tumor cytotoxicity of CD8+ BZ-4 T cells. CD4+ T cells isolated from healthy donor PBMCs were transfected via Sleeping Beauty electroporation with the control MART-1 reactive TCR (DMF5), BZ-4, or BZ-4 alongside the CD8 co-receptor (BZ-4-8). CD8+ T cells were similarly transfected with DMF5 or BZ-4. Following magnetic bead enrichment, 66% to 84% (mean 77%) of transfected T cells stained positive for Vβ7.1. TCR engineered T cells were subsequently tested for their ability to lyse a) luciferase transduced HLA A-11 expressing RCC tumors and b) a T2 cell line modified to express both HLA A-11 and luciferase that was peptide-pulsed with decreasing concentrations (5 × 10−1, 5 × 10−2, 5 × 10−3 & 5 × 10−4 ug/mL) of CT-RCC-1. CD4+ T cells expressing BZ-4 or BZ-4-8 as well as CD8+ T cells expressing BZ-4 or DMF5 were used either alone or in combination in cytotoxicity assays. For combined T cell groups, the total effector/target ratio was maintained at the same ratio as individual T cell groups. For T-2 targets, CD4+ BZ-4 T cells exhibited no cytolytic activity in contrast to both CD8+ BZ-4 T cells and CD4+ BZ-4-8 T cells, which were highly cytolytic to target cells. For RCC tumor targets, CD4+ BZ-4 T cells exhibited no cytolytic activity at 48, 72 and 96 hours in contrast to both CD8+ BZ-4 T cells and CD4+ BZ-4-8 T cells, which exhibited high levels of tumor killing at these timepoints. Remarkably, CD4+ BZ-4-8 T cells alone or in combination with CD8+ BZ-4 T cells demonstrated tumor cytotoxicity that was comparable to that observed with CD8+ BZ-4 T cells alone. In conclusion, these data show that CD4+ BZ-4 T cells transfected with the CD8 co-receptor acquire the ability to lyse RCC tumors at levels comparable to that observed in CD8+ BZ-4 T cells. Additional experiments are planned to evaluate whether mixtures of CD8+ BZ-4 T cells with CD4+ BZ-4-8 T cells will augment the ability of TCR modified T cells to proliferate in vivo and kill human RCC tumors compared to CD8+ BZ-4 T cells alone in tumor bearing NSG mice. Citation Format: Muna Igboko, Long Chen, Stefan Barisic, Elena Cherkasova, David Allan, Mala Chakraborty, Joseph Clara, Angie Parrizzi, Kyra Carney, Rosa Nadal Rios, Robert Reger, Richard Childs. Improving killing of renal cell carcinoma through the combined effects of TCR engineered CD4+ and CD8+ T cells [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 3601.

Using protein geometry to optimize cytotoxicity and the cytokine window of a ROR1 specific T cell engager.

T cell engaging bispecific antibodies have shown clinical proof of concept for hematologic malignancies. Still, cytokine release syndrome, neurotoxicity, and on-target-off-tumor toxicity, especially in the solid tumor setting, represent major obstacles. Second generation TCEs have been described that decouple cytotoxicity from cytokine release by reducing the apparent binding affinity for CD3 and/or the TAA but the results of such engineering have generally led only to reduced maximum induction of cytokine release and often at the expense of maximum cytotoxicity. Using ROR1 as our model TAA and highly modular camelid nanobodies, we describe the engineering of a next generation decoupled TCE that incorporates a "cytokine window" defined as a dose range in which maximal killing is reached but cytokine release may be modulated from very low for safety to nearly that induced by first generation TCEs. This latter attribute supports pro-inflammatory anti-tumor activity including bystander killing and can potentially be used by clinicians to safely titrate patient dose to that which mediates maximum efficacy that is postulated as greater than that possible using standard second generation approaches. We used a combined method of optimizing TCE mediated synaptic distance and apparent affinity tuning of the TAA binding arms to generate a relatively long but persistent synapse that supports a wide cytokine window, potent killing and a reduced propensity towards immune exhaustion. Importantly, this next generation TCE induced significant tumor growth inhibition in vivo but unlike a first-generation non-decoupled benchmark TCE that induced lethal CRS, no signs of adverse events were observed.

Low-affinity CD8+ T cells provide interclonal help to high-affinity CD8+ T cells to augment alloimmunity

Following solid organ transplantation, small precursor populations of polyclonal CD8+ T cells specific for any graft-expressed antigen preferentially expand their high-affinity clones. This phenomenon, termed “avidity maturation,” results in a larger population of CD8+ T cells with increased sensitivity to alloantigen, posing a greater risk for graft rejection. Using a mouse model of minor-mismatched skin transplantation, coupled with the tracking of 2 skin graft-reactive CD8+ T cell receptor-transgenic tracer populations with high and low affinity for the same peptide-major histocompatibility complex, we explored the conventional paradigm that CD8+ T cell avidity maturation occurs through T cell receptor affinity-based competition for cognate antigen. Our data revealed “interclonal CD8-CD8 help,” whereby lower/intermediate affinity clones help drive the preferential expansion of their higher affinity counterparts in an interleukin-2/CD25-dependent manner. Consequently, the CD8-helped high-affinity clones exhibit greater expansion and develop augmented effector functions in the presence of their low-affinity counterparts, correlating with more severe graft damage. Finally, interclonal CD8-CD8 help was suppressed by costimulation blockade treatment. Thus, high-affinity CD8+ T cells can leverage help from low-affinity CD8+ T cells of identical specificity to promote graft rejection. Suppressing provision of interclonal CD8-CD8 help may be important to improve transplant outcomes.

Zfp36l1 establishes the high-affinity CD8 T-cell response by directly linking TCR affinity to cytokine sensing.

How individual T cells compete for and respond to IL-2 at the molecular level, and, as a consequence, how this shapes population dynamics and the selection of high-affinity clones is still poorly understood. Here we describe how the RNA binding protein ZFP36L1, acts as a sensor of TCR affinity to promote clonal expansion of high-affinity CD8 T cells. As part of an incoherent feed-forward loop, ZFP36L1 has a nonredundant role in suppressing multiple negative regulators of cytokine signaling and mediating a selection mechanism based on competition for IL-2. We suggest that ZFP36L1 acts as a sensor of antigen affinity and establishes the dominance of high-affinity T cells by installing a hierarchical response to IL-2.

Abstract IA004: Preclinical development of DuoBody®-CD3xB7H4, a novel CD3 bispecific antibody for the treatment of solid cancers

Abstract Several clinical trials are currently underway to investigate the effectiveness of CD3 bispecific antibodies (bsAb) in solid tumors. Given the number of approved CD3 bsAb therapies thus far, the development of these bsAbs appears to be more challenging in solid cancer compared to hematological malignancies. The choice of an appropriate tumor-specific target and CD3 affinity are important considerations for the development of effective and safe CD3 bsAbs. The immune checkpoint protein B7H4 is differentially expressed between normal and tumor tissue, showing high expression in various solid cancers. Therefore, B7H4 provides an attractive target for a CD3 bsAb. We explored the preclinical mechanism of action (MoA) and pharmacodynamic (PD) markers of two B7H4-targeting CD3 bsAbs with different CD3 affinities. By crosslinking CD3 on T cells with B7H4 on tumor cells, CD3xB7H4 bsAbs will induce T-cell mediated cytotoxicity in B7H4-expressing tumor cells, associated with T-cell activation and cytokine production. In comparative preclinical characterization studies in vitro, we showed that both bsAbs induced target-specific, dose-dependent, and complete tumor cell kill in a panel of tumor cell lines expressing varying levels of B7H4, albeit with different potency. Moreover, both molecules demonstrated an acceptable safety profile in cynomolgus monkeys. DuoBody-CD3xB7H4 (GEN1047), the variant with lower CD3 affinity, was selected as clinical candidate, to leverage its observed propensity for lower cytokine production. In a patient-derived xenograft model of ovarian cancer in NCG-HIS mice examined in vivo, DuoBody-CD3xB7H4 showed dose-dependent antitumor activity, which was associated with increased numbers of intratumoral T cells and with peripheral PD biomarkers of T-cell activation and cytokine production. Currently, DuoBody-CD3xB7H4 is being investigated in a first-in-human clinical trial for the treatment of solid tumors known to express B7H4 (NCT05180474), in which the MoA and PD, including biomarkers of response, will be clinically explored. Citation Format: Louise A Koopman, Farshid Alemdehy, Madelon Paauwe, Laura Smits-de Vries, Frosso Karaiskaki, Marcel Brandhorst, Patrick Franken, Theo S Plantinga, Mischa A Houtkamp, Stefanie A.H. de Poot, Esther C.W. Breij. Preclinical development of DuoBody®-CD3xB7H4, a novel CD3 bispecific antibody for the treatment of solid cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA004.

CAR-NK Specific Targeting of Clonal Tcrvβ Chain to Treat T-Cell Non-Hodgkin's Lymphoma

Background T-cell Non-Hodgkin Lymphoma (T-NHL) is a diverse group of aggressive malignancies which are minimally researched and have poorer patient outcomes than those with B-cell NHL (Stuver et. al., 2022, Foss et. al., 2011). Therapies are characterized by pan T cell cytotoxicity, poor response rates and the requirement for multiple levels of treatments resulting in toxicity and suppression of the immune system that can lead to life threatening infections (Bellei et. Al., 2018). Clinical trials are considered a first line option for advanced disease, speaking to the lack of viable treatment options. One possible target for T cells is the T-cell receptor (TCR). TCRs possess tremendous diversity owing to diverse variable β chain (Vβ) (n=48) and α chain (Vα) (n=46) gene segments, that recombine with D and J gene segments to form a functional TCR. T Multiplexed PCR of the TCR gene has been used to identify the presence of a distinct malignant clone and facilitate diagnosis of T-NHL (Savage et. al., 2008). Thus, the Vβ chains represent a therapeutic target that discriminates the clonally expanded malignant T cells from the vast majority of healthy T-cells. Lucero et. al. (2023), showed specific targeting of T cell receptor Vβ chains, for T-NHL patients with an expanded TCRVβ clone using a naked antibody approach. Similar modalities have used BiTE and CAR-T. One downside of the naked antibody and BiTE approach (Paul et. Al., 2021) is that they require patients to have functional effector NK and CD8 T cells, and these cells are often not present in advanced T-NHL diseases. The expanded Vβ chain could also be targeted by CAR-T cell therapy; however, this approach is hampered by concerns of toxicity and poor autologous T cell quality due to the patients' disease (Marofi et. al., 2021). Therefore, we propose CAR-NK cell therapy utilizing the TCRVβ chain (Fig 1A) for patients with advanced T-NHL against the clonally expanded TCRVβ which will allow for an off the shelf approach that bypasses the need for autologous effector cells. Methods To show proof of concept, TCRVβ13 CAR-NK cells were manufactured from an NK-92 cell line, HaNK, which expresses a high affinity CD16 V158 FcγRIIIa receptor (Jochems, et. al., 2016). CAR transduction was confirmed via flow cytometry. Along with the CAR, NK/T-cell lymphoma cell lines, EL4 (mouse) and Jurkat (human), were transduced through a lentiviral vector containing a plasmid to express our Vβ of interest, Vβ13 (EL4-V13 and Jurkat-V13, respectively) (Lucero, 2023). We performed In vitro cytotoxicity co-culture assays on NK/T-cell lymphoma cell lines, EL4-V13 and Jurkat-V13, to confirm functional activity in CAR-HaNK cells compared to wild type HaNK cells at three different effector to target ratios. From the in vitro cytotoxicity assay, assays on supernatants from each of the co-cultures for changes of the cytokines INF-gamma and TNF-alpha levels to assess functional NK activity are in progress. Results The in vitro co-culture cytotoxicity assays showed increased cytotoxicity of the TCRVβ CAR-HaNK cells in comparison to HaNK cells in the presence of the TCRVβ for both EL-4-V13 and Jurkat-V13 cell lines. Targeting of the EL-4 cell line without the transduced TCRVβ showed similar percent cytotoxicity in CAR-HaNK cells and HaNK cells, while EL-4-V13 cell line showed significant increase in cytotoxicity when co-cultured with CAR-NK cells in comparison to the HaNK cells. In the 1:1 co-culture of either CAR-HaNK or HaNK cells with EL-4-V13 there was a 27% increase in cytotoxicity in the CAR-NK in comparison to the HaNK cells (P<0.0001), the 5:1 showed 71% increase in CAR-NK (P<0.0001), and the 10:1 showed an 80.5% increase in CAR-HaNK (P<0.0001) (Fig 1B). Similarly, in the human cell line Jurkat-V13, co-cultures with CAR-HaNK cells showed an increase in cytotoxicity in the 1:1 of 30% (P=0.0169), the 5:1 of 37% (P=0.0074), and 10:1 of 57.7% (P<0.0001) when comparing to HaNK cells (Fig 1B). Conclusions The goal of these preclinical studies is to leverage advances in CAR development with the efficacy of targeting TCRVβ as a direct and safe therapeutic option for patients with T-NHL. If successful, this will propel both cell-directed therapies and a novel target to a disease characterized by few efficacious, durable and safe therapies.

Updated Safety and Efficacy Results of Abbv-383, a BCMA x CD3 Bispecific T-Cell Redirecting Antibody, in a First-in-Human Phase 1 Study in Patients with Relapsed/Refractory Multiple Myeloma

Updated Safety and Efficacy Results of Abbv-383, a BCMA x CD3 Bispecific T-Cell Redirecting Antibody, in a First-in-Human Phase 1 Study in Patients with Relapsed/Refractory Multiple Myeloma

GB261, an Fc-Function Enabled and CD3 Affinity De-Tuned CD20/CD3 Bispecific Antibody, Demonstrated a Highly Advantageous Safety/Efficacy Balance in an Ongoing First-in-Human Dose-Escalation Study in Patients with Relapsed/Refractory Non-Hodgkin Lymphoma

Introduction: GB261 is a novel and highly differentiated CD20/CD3 bispecific T cell engager antibody computationally designed to maintain Fc effector function, i.e., antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) to broaden the mechanisms of action (MOA) for tumor cell killing. Furthermore, the “imbalanced” design of GB261 integrates de-tuned CD3 binding to reduce CRS incidence and improve safety features of the Fc effector function. Extensive pre-clinical studies have shown that GB261 has a highly advantageous safety/efficacy balance.Here, we present the preliminary clinical safety and efficacy results of an ongoing phase I/II study for GB261 (NCT04923048). Methods: This is an open-label, multicenter (China and Australia), dose-escalation/expansion phase 1/2 study of GB261 to evaluate the safety, tolerability and efficacy in patients with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma(B-NHL) and Chronic Lymphocytic Leukemia (CLL). Adult patients were eligible when they have CD20+ r/r B-NHL or CLL with no available standard of care treatments, adequate organ function, and no CNS involvement or other CNS disease, infections or other active/serious medical issues that may affect compliance or interpretation of results. GB261 was administered in 21-day cycles, weekly (QW) for the first 6 doses followed by every 3 weeks (Q3W) until disease progression, or other situation defined in protocol. Response assessment was based on Lugano 2014 and LYRIC 2016 criteria. Results: As of June 17, 2023, 47 r/r B-NHL patients (DLBCL:76.6%; FL:23.4%) were enrolled at flat or step-up doses of GB261 ranging from 1mg to 300 mg. Median age was 60.0 years (range: 28, 81), 55.3% of patients were male. Median prior lines of therapy were 3 (range: 1, 10). 78.7% of patients were refractory to any anti-CD20 therapy, 70.2% refractory to their last systemic therapy. Median time since last prior therapy to first study treatment was 1.9 months. In efficacy evaluable patients (n=22) from 3mg to 100mg, with at least 75% dose exposure before the first radiographic assessment, the median duration of study follow-up was 4.5 months (95%CI: 4.0, 7.4). The overall response rate (ORR) was 73% (16/22), and complete response rate (CRR) was 45.5% (10/22). ORR and CRR were 100% and 100% in 3mg, 56% and 22% in 10mg, 67% and 33% in 30mg. At 100mg dose, there were 5 evaluable patients, with ORR 100% (5/5), CRR 80% (4/5) and PR (20%, 1/5; mosunetuzumab-refractory rrDLBCL patient). Median time to response (TTR) was 1.3 months (95%CI: 1.2, 1.5), the same as median time to CR. Median duration of response (DOR) was not reached. In safety evaluable patients (n=47), the median duration of study follow-up was 4.1 months (95%CI: 2.9, 5.3). Treatment-emergent adverse events (TEAEs) were 95.7%, treatment-related adverse events were 85.1%. The most common TEAEs were COVID-19 infection (40.4%; grade 1 or 2: 27.6%; grade >=3: 12.8%) and neutropenia (31.9%; grade 1or 2: 14.9%; grade >=3: 17.0%). AE related treatment discontinuation and death were reported in 2 patients, which were all due to COVID-19 pneumonia. CRS occurred in 12.8% (6/47) patients, was mild and transient. CRS in 100mg were 14.3% (2/14). All cases of CRS were grade 1 (8.5%, 4/47) or 2 (4.3%, 2/47), no grade 3 (Lee et al., ASTCT criteria), no interruptions of treatment, and no administration of Tocilizumab. The median duration of CRS was 7 hours. No ICANS were reported. The PK profile of GB261 appeared to be linear across dose ranges tested (1mg-100mg). Effective half-life appeared to be 2-3 weeks which supports every 3-4 weeks dosing. Conclusion: GB261, a novel and highly differentiated CD20/CD3 bispecific antibody, is the first clinical stage Fc+ CD20/CD3 T cell engager. In heavily pretreated B-NHL patients, GB261 showed a highly advantageous safety/efficacy balance, consistent with the MOA. The safety profile is excellent especially for the CRS which is very mild, transient and less frequent compare with other CD20/CD3 bispecific antibodies. The response after GB261 treatment was early, deep and durable. At the 100mg dose level, 80% of patients achieved CR and with favorable safety. Additionally, clinical benefit seen in other CD20/CD3 bispecific antibody failed patients provides clinical support to the unique and differentiated MOA of GB261.

Asymmetric anti-CLL-1×CD3 bispecific antibody, ABL602 2+1, with attenuated CD3 affinity endows potent antitumor activity but limited cytokine release

BackgroundAcute myeloid leukemia (AML) is a type of leukemia in adults with a high mortality rate and poor prognosis. Although targeted therapeutics, chemotherapy, and hematopoietic stem cell transplantation can improve...

DISCOVERY OF A CYNOMOLGUS MONKEY-CROSS-REACTIVE ANTI-HUMAN CD3 MAB FOR T CELL ENGAGERS

Abstract Background Anti-CD3 based T cell engager antibodies can redirect cytotoxic activity of T cells in a non-MHC restricted fashion to kill tumor cells effectively. Therefore, the discovery of an anti-CD3 antibody capable of activating T cells in the presence of tumor cells is highly desirable. Recently, many anti-CD3 bispecific antibodies (bsAbs) entered clinical trials. Despite the promising efficacy of anti-CD3 bsAbs, safety issues arose and establishing a proper therapeutic window between efficacy and safety became a challenge. One of the safety concerns for anti-CD3 bsAbs is the cytokine release syndrome due to T-cell activation. Recent studies have shown that this safety challenge can be mitigated by selecting an anti-CD3 antibody with the appropriate binding epitope, CD3 affinity and binding kinetics (on and off rate). Methods by using WuXi Biologics’ state-of-the-art hybridoma platform, an anti-CD3 Ab was discovered through a combination of immunization and screening strategies. Results the selected anti-CD3 Ab demonstrates moderate affinity and fast-on fast-off binding kinetics against both human and cynomolgus CD3 molecules. Once constructed into T cell engagers (TCEs) using this anti-CD3 Ab with TAA binding arms in WuXiBody® format, the obtained TCEs mediated efficient anti-tumor activity, but induced low levels of cytokine production by T cells. Conclusions WuXi Biologics has discovered an anti-CD3 Ab with desired binding properties to human CD3. As shown in two showcases, the TCEs constructed using this anti-CD3 Ab can elicit efficient T cell cytotoxicity against tumor cells but low levels of cytokine release. The cross-reactivity of the anti-CD3 Ab enables preclinical assessments of toxicity in NHP.

Abstract CT013: Safety and efficacy from the phase 1/2 first-in-human study of REGN5459, a BCMA×CD3 bispecific antibody with low CD3 affinity, in patients with relapsed/refractory multiple myeloma

Abstract Background: MM remains incurable and new tx options are needed, especially in heavily pre-treated R/R pts. REGN5459, a BCMAxCD3 bispecific Ab, binds to BCMA on MM cells and with low affinity to CD3 on T cells, triggering T-cell activation and plasma cell depletion with low cytokine release preclinically. This first-in-human study aimed to assess the safety, tolerability, and preliminary anti-tumor activity of REGN5459 monotherapy in pts with R/R MM. Methods: Eligible pts had received ≥3 prior lines of tx including an anti-CD38 Ab, proteasome inhibitor, and immunomodulatory drug, and had exhausted all available tx options. Pts could receive REGN5459 until progression/intolerable toxicity. Primary objectives were to assess safety, tolerability, dose limiting toxicities (DLTs), and determine the recommended Ph 2 dose (RP2D) of REGN5459 (Ph 1) and assess efficacy of REGN5459 (Ph 2) per ORR. Results: As of June 9, 2022, 43 pts were enrolled (Ph 1, 33; Ph 2, 10): median age 67 yrs (range, 26-85), 51% female, 16% high cytogenetic risk, 14% extramedullary plasmacytoma, 37% R-ISS Stage III disease, 61% triple-class refractory, and median of 5 (range, 2-9) prior lines of tx. In Ph 1, one DLT was reported in a pt receiving the highest dose (900 mg; Gr 3 hypoxia, pt later found to have primary lung cancer). RP2D was identified as 480 mg. All pts enrolled had ≥1 TEAE, 74% had Gr ≥3 TEAEs. TEAEs all-Gr in ≥30% of pts were CRS (54%), fatigue (44%), neutropenia (37%), anemia (35%), cough (30%), and diarrhea (30%). Gr ≥3 TEAEs in ≥15% of pts were neutropenia (37%), anemia (26%), lymphopenia (23%), thrombocytopenia (19%), and hypertension (16%). CRS Gr 1, 2, and 3 were reported in 47%, 2%, and 5%, respectively; there was no Gr 4 or 5 CRS. No Gr 3 CRS with RP2D. Tocilizumab was used in 19% and steroids in 9% of pts. One pt developed ICANS (2%, Gr 2). Incidence of serious TEAEs and TEAEs leading to tx discontinuation was 63% and 16%. Infections occurred in 61% (37% Gr ≥3). After data-cutoff, two deaths due to COVID pneumonia and COVID infection have been reported. ORR was 67% (58% ≥VGPR) for the entire cohort and 100% (85% ≥VGPR; 15% sCR; 39% CR) among pts receiving the RP2D (n=13). Median follow-up was 7 mos (range, 1-26) with longest response ongoing for 22+ mos. Median time to response was 0.8 mos. Median DOR was NR (95% CI, 12-NE); 12-mo DOR for RP2D was 66.7% (95% CI, 5.4-94.5). Of pts in ≥CR with available MRD results (n=8), 50% were MRD negative at the 10−5 threshold. Conclusion: These initial data show that REGN5459 has acceptable safety/tolerability in R/R MM with most CRS of low grade and low incidence of ICANS. Modulation of CD3 affinity on bispecific Abs to maximize tumor killing, and mitigate CRS and T-cell exhaustion, warrants further research. Efficacy in this heavily pre-treated cohort was encouraging, with 100% ORR with the RP2D. Updated data will be presented at the meeting. Citation Format: Attaya Suvannasankha, Prashant Kapoor, Matthew J. Pianko, Joshua Richter, Anita D'Souza, Larry D. Anderson, Andrew Magyar, Oluwaseun Aina, Anita Boyapati, Damien Cronier, Nikhil Singh, Karen Rodriguez-Lorenc, Glenn S. Kroog, Hans C. Lee. Safety and efficacy from the phase 1/2 first-in-human study of REGN5459, a BCMA×CD3 bispecific antibody with low CD3 affinity, in patients with relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT013.

BCMAxCD3 Bispecific Yields Robust Responses in Myeloma.

In emerging data from a phase I/II trial, patients with relapsed/refractory multiple myeloma responded well to REGN5459, an investigational BCMA-targeting bispecific T-cell engager. However, despite the drug's low affinity for CD3 on T cells, this design did not mitigate the risk of cytokine release syndrome, a common toxicity.

Abstract 3183: Combination of anti-EGFR antibody cetuximab with NKX101, an allogeneic NKG2D-L targeting NK cell therapy, enhances potency and in vitro cytotoxicity against solid tumors

Abstract Background: Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism of cell-mediated immune defense against opsonized target cells wherein the Fc-fragment of an antibody binds to the CD16 Fc receptor (FcγRIIIa) on NK cells, triggering NK cell activation and targeted killing. Cetuximab is a human IgG1 antibody targeting epidermal growth factor receptor (EGFR) that is approved for the treatment of colorectal and head and neck cancers. Binding of cetuximab to EGRF+ cell lines has been shown to trigger ADCC mediated by NK cells. NKX101 is an investigational allogeneic NK cell therapy in development for the treatment of cancer. NKX101 is engineered to express an NKG2D-based chimeric antigen receptor (CAR) and membrane-bound IL-15. To investigate the combination of NKX101 and cetuximab, we developed in vitro assays to (i) demonstrate enhanced activity against cancer cells via CD16-mediated ADCC activity, (ii) compare ADCC activity of NKX101 cells generated from high-affinity CD16 and low-affinity CD16 donors, and (iii) evaluate the synergistic interaction between NKX101 and cetuximab. Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. NucRed-labeled EGFR+ NKG2D-L+ human cancer cell lines were pre-incubated with cetuximab or isotype control and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte instrument. For synergy assessments, a dose response matrix of NKX101 cells and cetuximab was used to generate cytotoxicity values. Synergy scores for combinations were determined using the Synergy Finder Plus web application. To determine donor CD16 V/F 158 genotypes, genomic DNA was isolated from each donor and assayed using a TaqMan CD16 genotyping kit. Results: We demonstrate that cetuximab increases the anti-tumor effect of NKX101 in a dose-dependent manner. Assessment of the interaction between NKX101 and cetuximab in vitro revealed that the two agents can combine to kill cancer cells in a synergistic manner. Blocking CD16 on NKX101 cells with a neutralizing antibody significantly decreased potency of the combination, suggesting that the improvement in potency observed is a direct result of CD16-mediated ADCC activity. To determine if common CD16 polymorphisms influence NKX101 ADCC activity, we compared NKX101 cells generated from CD16 158 V/V, V/F, and F/F donors. No significant differences in potency were observed with NKX101 cells expressing the high affinity CD16 158 V/V genotype compared with those derived from CD16 lower affinity 158 V/F or 158 F/F donors. In summary, we show that the combination of an engineered allogeneic NK cell therapy, NKX101, with cetuximab leads to an increase in the specific killing of cancer cells in vitro, supporting further investigation into this combination for the treatment of solid tumors. Citation Format: Cynthia Cho, Kyle Hansen, Max Zhang, Carmel Chan, James Trager. Combination of anti-EGFR antibody cetuximab with NKX101, an allogeneic NKG2D-L targeting NK cell therapy, enhances potency and in vitro cytotoxicity against 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 3183.

Abstract 1882: SCR-9171, a MUC17-targeted bispecific T cell engager molecule for gastrointestinal cancer

Abstract Background: Although advances treatments have significantly improved the survival of patients with gastrointestinal cancer in recent decades, there is still an unmet medical need for more effective treatments. A promising strategy is the use of bispecific antibodies that recruit T cells to kill cancer cells by simultaneously binding to CD3 on T cells and tumor associated antigens. CD3-based T cell engagers are highly potent therapeutic molecules with T cell cytotoxicity activities. But high CRS risk and T cell exhaustion has been observed for high affinity CD3-based T cell engagers in clinicals. Effective and safe use of these therapeutics depends on selective targeting of cancer cells and optimal CD3 affinity. MUC17, a member of the mucin family, is a transmembrane glycoprotein only expressed on the apical membrane of normal gastrointestinal mucosal epithelial cells. MUC17 is overexpressed in 23%-52% of patients with gastric cancer. Compared to the adjacent normal tissues, the expression of MUC17 protein is consistently higher in gastric cancer tissues, suggesting that MUC17 is an attractive therapeutic target for gastric cancer. Method & Results: We created MUC17/CD3 T cell engaging bispecific antibodies by starting with a nanobody humanized from camel and a low affinity CD3 antibody. Here we report the design and the promising preclinical activity of SCR-9171 molecule in vitro and in vivo. SCR-9171 induced T cell activation and had potent cytotoxicity of MUC17 positive tumor cells in vitro. However, the cytokine release was much lower than the positive control, which established with a high affinity CD3 antibody. Importantly, in mouse models with reconstituted human immune cells, SCR-9171 showed strong antitumor efficacy against MUC17-positive tumors through the activation of T cells. Tolerability and pharmacokinetics of MUC17/CD3 bispecific antibody are being assessed in cynomolgus monkey. The results demonstrated that SCR-9171 was well tolerated in a one-month repeat-dose toxicology study at 3mg/kg with no associated clinical signs or changes in body weight. Conclusion: These findings suggest that MUC17/CD3 bispecific antibody is a potential therapeutic molecule for MUC17-positive solid tumor. Citation Format: Guangcun Cheng, Yayuan Fu, Hua Zheng, ng Li, Xiaolei He, Xi Jiao, Shuhui Luan, Shuai Wang, Lei Jiang, Wenjing Li, Yun Zhang, Lei Liu, Renhong Tang. SCR-9171, a MUC17-targeted bispecific T cell engager molecule for gastrointestinal cancer [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 1882.

Abstract 2942: ZW171, a T cell-engaging, bispecific antibody for the treatment of mesothelin-expressing solid tumors

Abstract 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.