This paper focuses on the use of novel technologies and innovative trial designs to accelerate evidence generation and increase pharmaceutical Research and Development (R&D) productivity, at Bristol Myers Squibb. We summarize learnings with case examples, on how we prepared and continuously evolved to address the increasing cost, complexities, and external pressures in drug development, to bring innovative medicines to patients much faster. These learnings were based on review of internal efforts toward accelerating R&D focusing on four key areas: adopting innovative trial designs, optimizing trial designs, leveraging external control data, and implementing novel methods using artificial intelligence and machine learning.

PURPOSE The advent of new therapeutic modalities highlighted deficiencies in the traditional maximum tolerated dose approach for oncology drug dose selection and prompted the Food and Drug Administration (FDA)'s Project Optimus initiative, which suggests that sponsors take a holistic approach, including efficacy, safety, and pharmacokinetic (PK) and pharmacodynamic data, in conjunction with integrated exposure-response (ER) analyses. However, this method comes with an inherent challenge of the collation of the multisource data. To address this issue, an ER-based clinical utility score (CUS) framework, combining benefit and risk into a single measurement, was developed. METHODS Model-predicted outcomes for each clinically relevant end point, informed by ER modeling, are converted to a CUS using a user-defined utility function. Thereafter, individual CUS is integrated into a single score with user-defined weighting for each end point. The user-defined weighting feature allows the user to incorporate expert knowledge/understanding into weighing the product's benefit versus risk profile. RESULTS To validate the framework, data were leveraged from over 50 oncology programs from 2019 to 2023 on the basis of FDA new drug application/biologics license application review packages and/or related literature studies. Five representative cases were selected for in-depth evaluation. Results showed that the optimal benefit-risk ratio (highest CUS) was consistently observed at PK exposures synonymous with recommended doses. A recurring theme across cases was a greater emphasis on safety over efficacy in oncology drug dose determination. CONCLUSION The ER-based CUS framework offers a strategic tool to navigate the complexities of dose selection in oncology programs. It serves as a pillar to the importance of integrative data analysis, aligning with the vision of Project Optimus , and demonstrates its potential in guiding dose optimization by balancing therapeutic benefits against risk.

472 Background: Relapsed/refractory clear cell renal cell carcinoma (ccRCC) progressing after treatment with CPI and VEGF inhibitor remains an area of unmet need. We have developed AB-2100, an autologous, integrated circuit T (ICT) cell engineered to include three new features: a sequential “AND” logic gate that requires the IO presence of two antigens in the tumor microenvironment (TME) to trigger T cell killing; a shRNA-miR module to enhance resistance to suppressive TME via constitutive knockdown of FAS and TGFBR2; and a constitutive synthetic pathway activator (SPA) that increases STAT3 signaling for enhanced T cell cytotoxicity and expansion. Methods: A previous clinical study of CA9-specific CAR-T cell therapy was limited by on-target, off-tumor toxicity. To overcome this, AB-2100 includes a sequential “AND” logic gate that consists of a priming receptor (PrimeR) targeting PSMA, and a CA9-targeted CAR that is upregulated upon PrimeR engagement with PSMA expressed on the tumor neovasculature. A series of assays were performed to assess the specificity and potency of AB-2100: dual-antigen specificity of the logic gate was assessed in vitro and in vivo against CA9+ and PSMA+CA9+ tumors; vascular priming was modeled by co-culturing AB-2100 cells with PSMA-expressing endothelial cells and CA9+ tumor cells; a FAS cross-linking assay was conducted to assess the impact of FAS knockdown; the enhanced anti-tumor activity conferred by TGFBR2 shRNA and SPA modules were assessed in a 786-O xenograft model; and AB-2100 potency was measured in a subcutaneous renal A498 xenograft. Results: AB-2100 selectively kills tumors that express both CA9 and PSMA, and not tumors that express CA9 alone, as assessed by in vitro cytotoxicity against single or dual antigen expressing tumor cell lines and by a dual flank xenograft model. Furthermore, we confirmed that co-culture with PSMA-expressing endothelial cells was sufficient to upregulate CA9 CAR expression and enable tumor cell killing. Finally, AB-2100 containing both shRNA-miR and SPA modules demonstrated enhanced anti-tumor activity in xenograft RCC models. Conclusions: These data demonstrate that AB-2100 selectively targets tumors co-expressing PSMA and CA9, and can overcome multiple suppressive mechanisms in the TME. These results support the evaluation of AB-2100 in the clinic for the treatment of advanced or metastatic ccRCC.

Abstract Common challenges of CAR-T cell therapies in solid tumors, such as clear cell renal cell carcinoma (ccRCC), include insufficient therapeutic potency and tumor specificity. AB-2100 is an autologous integrated circuit T cell product generated via CRISPR-mediated knock-in of a single transgene into a safe-harbor locus. AB-2100 encodes a transcriptionally regulated sequential AND gate that comprises a priming receptor (PrimeR) specific for PSMA and an inducible CAR targeting CA9 antigen, which is widely expressed on ccRCC tumor cells. AB-2100’s sequential AND logic-gate design aims to confer tumor-specific activity by priming off of PSMA-expressing tumor vasculature to induce CA9 CAR expression. The logic gate is intended to increase the safety profile of AB-2100 given that PSMA and CA9 are predicted to have limited co-expression in normal tissues. Additional enhancements in T cell functionality include short-hairpin RNAs (shRNA) against Fas and TGFBR2 designed to prevent tumor microenvironment (TME)-mediated suppression, and a synthetic pathway activator (SPA) designed to drive constitutive STAT3 signaling and to enhance T cell expansion and antitumor activityMechanism of action studies demonstrate that AB-2100 can prime off of PSMA-expressing endothelial cells and induce tumor-specific killing of CA9 tumor cells, leading to the eradication of ccRCC targets in vitro. AB-2100 also exhibited selective killing of dual antigen expressing tumors in vivo using a dual-flank subcutaneous xenograft model. Evaluation of AB-2100 in the subcutaneous A498 xenograft model demonstrated that inclusion of the SPA resulted in significant increase in antitumor efficacy. Furthermore, a rechallenge xenograft model demonstrated that presence of SPA increased the long term functional persistence of AB-2100. In summary, preclinical data demonstrate that AB-2100 selectively targets tumors co-expressing PSMA and CA9, and can overcome multiple suppressive mechanisms in the tumor microenvironment. These results support the ongoing evaluation of AB-2100 for the treatment of advanced or metastatic ccRCC. Citation Format: Suchismita Mohanty, Jeremy Chen, Alma Gomez, Angela Boroughs, Irene Scarfo, Laura Lim, Kevin Dang, Marvin Chew, Rakesh Sudhakar, Michelle Nguyen, Thomas Gardner, Beatriz Millare, James Zhang, Darrian Moskowitz, Stanley Zhou, Neroli H. Xie, Nickolas Attanasio, Amanda Fearon, Ivan Chan, Vibhavari Sail, Vince Thomas, Jennesa Smith, Jennifer McDevitt, Levi Gray-Rupp, Alba Gonzalez, Christopher Murriel, W. Nicholas Haining. AB-2100, a PSMA-inducible CA9-specific CAR T cell product intended for the treatment of ccRCC provides long-term tumor responses in preclinical mouse model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B037.

Abstract Common challenges of CAR-T cell therapies in solid tumors, such as clear cell renal cell carcinoma (ccRCC), include insufficient therapeutic potency and lack of tumor specificity. We have developed AB-2100, an autologous integrated circuit T (ICT) cell product, generated via CRISPR-mediated knock-in of a single transgene into a safe-harbor locus. AB-2100 encodes a transcriptionally regulated sequential AND gate that comprises a priming receptor (PrimeR) specific for PSMA and an inducible CAR targeting CA9 antigen, which is widely expressed on local and metastatic lesions. AB-2100’s sequential AND logic-gate confers tumor-specific activity by priming off of PSMA-expressing tumor vasculature to induce CA9 CAR expression. This unique feature of the logic gate is intended to increase the safety profile of AB-2100 given that PSMA and CA9 are predicted to have limited co-expression in normal tissues. Additional functionality includes short-hairpin RNAs (shRNA) against Fas and TGFBR designed to prevent tumor-mediated resistance, and a synthetic pathway activator (SPA) that drives constitutive STAT3 signaling and enhanced T cell cytotoxicity and expansion. Mechanism of action studies demonstrate that AB-2100 can prime off of PSMA-expressing endothelial cells and induce tumor-specific killing of CA9 tumor cells, leading to the eradication of ccRCC targets in vitro. AB-2100 also exhibited selective killing of dual antigen expressing tumors in vivo using a dual-flank subcutaneous xenograft model. Preclinical xenograft studies also demonstrated that TGFBR shRNA and SPA modules enhanced antitumor activity of ICTs. When AB-2100 potency was evaluated in the subcutaneous A498 xenograft model, treatment with AB-2100 resulted in complete and durable anti-tumor responses. In summary, preclinical data demonstrate that AB-2100 selectively targets tumors co-expressing PSMA and CA9, and can overcome multiple suppressive mechanisms in the tumor microenvironment. These results support the evaluation of AB-2100 in the clinic for the treatment of advanced or metastatic ccRCC. Citation Format: Suchismita Mohanty, Jeremy Chen, Alma Gomez, Angela Boroughs, Irene Scarfo, Laura Lim, Kevin Dang, Marvin Chew, Rakesh Sudhakah, Michelle Nguyen, Thomas J. Gardner, Beatriz Millare, James Zhang, Darrian Moskowitz, Stanley Zhou, Neroli H. Xie, Nickolas Attanasio, Amanda Fearon, Ivan Chan, Vibhavari Sail, Vince Thomas, Jennesa Smith, Jennifer McDevitt, Levi Gray-Rupp, Alba Gonzalez, Christopher Murriel, W. Nicholas Haining. AB-2100, a PSMA-inducible CA9-specific CAR T cell product for the treatment of ccRCC provides long-term tumor responses in preclinical mouse model [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 38.

Background: CD38 is a transmembrane protein that is often overexpressed in multiple myeloma (MM) and other hematologic cancers. The success of anti-CD38 monoclonal antibodies, like daratumumab and isatuximab, in the treatment of MM supports the potential of CD38-targeting therapy candidates in development, including chimeric antigen receptor (CAR) engineered natural killer (NK) cells. Because CD38 is expressed on normal hematopoietic cells and other tissues, it is important to assess the affinity and epitope-specificity of the CD38-targeting moiety to limit potential on-target/off-tumor toxicities. To determine how different biophysical characteristics impact CAR activity, we identified a diverse panel of CD38-specific antibodies, which were shown to display a wide range of affinities and epitopes towards CD38 and converted these clones into CARs to evaluate their expression and target-specific and non-specific activation in a high-throughput (HTP) CD38-knockout (CD38 KO) Jurkat screen. Methods: Antibodies against CD38 were identified through hybridoma screening. Humanized mice were immunized with the extracellular domain of CD38, and lymphocytes from lymph nodes were fused with SP2/0 cells to generate hybridoma cells. Supernatants from single cell sorted hybridoma clones were screened against CD38+ and CD38 KO Jurkat cells and were assessed for affinity and epitope via surface plasmon resonance detection with the Carterra LSA. 96 unique clones were converted into CARs and transduced into CD38 KO Jurkat cells. CAR-expressing Jurkat cells were assayed for CAR expression and activation (via CD69 expression) when cultured overnight in the presence or absence of MM.1R target cells. Seven CARs were selected for additional characterization in primary NK cells. NK cells from healthy donors were expanded on our proprietary NKSTIM cell line, knocked out for CD38 using a CRISPR-Cas system, and transduced with CD38 CAR constructs. CD38 CAR NK-mediated cytotoxicity against MM.1S target cells was assessed by IncuCyte® S3 live cell analysis system. Results: Hybridoma screening identified many unique CD38-specific antibodies, displaying a wide range of affinities and epitopes towards CD38. Despite these biophysical differences, many of these antibodies, when converted to CARs, confer target-specific activity. Multiple epitopes on CD38 may be targeted and even low affinity antibodies were sufficient for target-specific CAR activation. Furthermore, the seven selected CAR candidates that were expressed in CD38 KO donor NK cells displayed potent in vitro target cell killing against MM.1S target cells. Conclusions: In summary, we have identified and characterized a range of CD38-specific antibodies. When converted into CARs, these targeting moieties mediate target-specific activation, despite differences in CD38 epitope and in affinity, demonstrating the utility of HTP CAR screening. Moreover, expression of the top seven CAR candidates in CD38 KO NK cells resulted in potent target cell killing, supporting further pre-clinical evaluation in relevant models. Citation Format: Emily N Kang, Jacinda T Chen, Bing Li, Kate Jamboretz, Nitin Patel, Daniel Bedinger, Kyle T Pratt, Jessica Hsieh, Luxuan TE Buren, Chao Guo, Ivan Chan, James B Trager, Sasha Lazetic. Screening and characterization of CD38 chimeric antigen receptors for the development of natural killer cell-based therapies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A044.

Background Clinically effective CAR T cell therapy for solid tumors may require substantial T cell engineering to increase specificity and potency. We have developed AB-2100, an autologous, integrated circuit T (ICT) cell that encodes multiple synthetic 'modules' to overcome challenges in the treatment of clear cell renal cell carcinoma (ccRCC). AB-2100 includes a sequential 'AND' logic gate designed to limit off-tumor toxicity through dual tumor antigen recognition, a short hairpin ribonucleic acid-microRNA (shRNA-miR) module for the constitutive expression of shRNA-miRs for knockdown of FAS and TGFBR2, and a constitutive synthetic pathway activator (SPA) that drives constitutive STAT3 signaling for enhanced T cell cytotoxicity and expansion. Methods On-target, off-tumor toxicity was previously observed with constitutive CA9 CAR T cell therapy. To overcome this, the AB-2100 sequential 'AND' logic gate consists of a priming receptor (PrimeR) specific for PSMA and an inducible CA9-targeted CAR that is expressed upon PrimeR engagement with PSMA on the tumor neovasculature of ccRCC. This unique feature of the logic gate increases the safety profile of AB-2100 given that PSMA and CA9 are not expressed in the same normal tissues. Dual-antigen specificity of the logic gate was assessed in vitro and in vivo via CA9+ and PSMA+CA9+-786-O tumors established on contralateral flanks. To model vascular priming, AB-2100 cells were co-cultured with PSMA-expressing endothelial HUVEC cells and K562-CA9 cells. An in vitro FAS cross-linking assay was conducted to assess the impact of FAS knockdown on FAS-mediated apoptosis. The enhanced anti-tumor activity conferred by TGFBR2 shRNA and SPA modules were assessed in a subcutaneous 786-O xenograft model. Lastly, AB-2100 potency was measured in a subcutaneous renal A498 xenograft tumor model. Results In vitro cytotoxicity against single or dual antigen expressing tumor cell lines, as well as a dual flank xenograft model demonstrate that AB-2100 selectively kills tumors that express both CA9 and PSMA, and not tumors that express CA9 alone. Furthermore, we confirmed that AB-2100 was able to prime off of PSMA-expressing endothelial HUVEC cells and kill K562-CA9 tumor cells. Finally, AB-2100 containing shRNA-miR and SPA modules demonstrated enhanced anti-tumor activity in xenograft RCC models (786-O and A498). Conclusions Preclinical data demonstrate that AB-2100 can selectively target antigens that cannot be safely targeted by conventional CARs, and overcome multiple suppressive mechanisms in the tumor microenvironment. These results support the evaluation of AB-2100 in the clinic for the treatment of advanced or metastatic ccRCC.

Abstract Multiple myeloma (MM) is a progressive hematological cancer with a 5-year survival rate of 53% (1). Novel therapeutic strategies are being developed to target specific MM surface antigens. Yet, changes in antigen expression through MM progression are poorly understood in the clinic and have not been well characterized in preclinical models. Here, we were interested in understanding how BCMA, CD38, CD138 and HLA-DR surface expression patterns may be affected as MM progresses in preclinical models. To do so, ARH-77 and RPMI-8226 cells were inoculated subcutaneously while MM.1S, MM.1R, U266 and NCI-H929 cells were dosed intravenously into NSG mice. Tumor xenograft or bone marrow, whole blood and spleen were processed for flow cytometry analysis. Antigen expression was measured at different stages of tumor progression as well as in vitro. Soluble BCMA levels were assessed in the mouse serum by ELISA. ARH-77 and RPMI-8226 subcutaneous xenografts show preferential growth when inoculated in PBS and 50% growth factor-reduced Matrigel, respectively. BCMA expression was reduced in both ARH-77 and RPMI-8226 xenografts compared to in vitro culture. CD38 and HLA-DR expression increased as ARH-77/PBS and RPMI-8226/Matrigel tumor volume progressed. All 4 antigens were detected in the bone marrow from MM.1S, MM.1R and U266-bearing mice, as early as 5-6 weeks post intravenous injection. In contrast, antigens were not detected in any tissue from mice injected with NCI-H929, likely indicating failed engraftment. At 5 weeks, antigens were detected in the whole blood and spleen in MM.1S-bearing mice only, suggesting organ invasion and a more advanced disease-stage compared to MM.1R or U266. Experiments are being carried out to test antigen expression in tissue at late-stage tumor progression as well as serum BCMA levels in the intravenous MM models. These data highlight antigen expression differences in MM cells when analyzed in mouse tissue compared to in vitro culture. Like the widely variable expression observed between patients (2, 3), BCMA and CD138 were differentially expressed in the mouse bone marrow between models. Our observations suggest that commonly targeted antigens in MM vary kinetically in vivo and can be measured by flow cytometry. The present findings also support the use of RPMI-8226 and MM.1S cell lines when screening for antigen-specific immunotherapies and combinatorial studies for MM treatment. References: 1.Howlader et al. https://seer.cancer.gov/csr/1975_2017 2.Brudno et al. J Clin Oncol. 2018 3.Kawano et al. Int J Oncol. 2012 Citation Format: Nadege Morisot, Julian Tam, Nicole Dailey, Tina Davis, Jacinda Chen, Janeen Islar, Luxuan Buren, Nitin Patel, Sasha Lazetic, Ivan Chan, James B. Trager, Joanne B. Tan. Surveying surface antigen expression in multiple myeloma preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6004.

The dramatic success of CAR T cell therapies in the treatment of certain B cell malignancies has led to a wave of novel cell therapies for cancer. All approved engineered cell therapies are derived from autologous T cells. This has posed barriers for patient access to qualified treatments that include a lengthy production time, inconsistent product characteristics, uncertain manufacturability, and a high cost of manufacturing. To address these obstacles, allogeneic “universal” cell therapies, derived from healthy donors or differentiated pluripotent cells are engineered and expanded in large quantities for off-the-shelf treatment. In allogeneic CAR T cell therapy, T cell receptor (TCR) knockout is required to prevent graft-versus-host disease of alloreactive T cells targeting against host tissue. Allogeneic cells may have a very limited half-life following infusion, however, as they are rapidly targeted by the patient’s own immune system causing host-versus-graft disease. Gene editing is used to increase the persistence of allogeneic T cells by enabling them to evade host T cell or natural killer (NK) cell surveillance. A conventional method of preventing host T cell rejection is to knockout (KO) the β-2 microglobulin (β2M) to diminish the expression of MHC class I protein. To further minimize rejection, overexpression of nonclassical MHC class I protein, HLA-E, is engineered into allogeneic T cells to evade host NK cell rejection. Strategies to improve allogenic CAR T cell persistence through immune evasion have implications for developing a therapeutic product consisting of both CAR T and CAR NK cells. Here we show the use of different methods to investigate the effectiveness of the immune evasion strategies. We found that the effectiveness of HLA-E expression in the suppression of NK cell rejection is highly correlated with the expression of CD94/NKG2A on the host NK cells. Host NK cells with low CD94/NKG2A expression will rapidly recognize and kill allogeneic CAR-T cells even when HLA-E is overexpressed. As it is not possible to control CD94/NKG2A expression in patient NK cells, we sought to identify alternative factors to allow allogeneic cells to evade rapid immune recognition without requiring long term suppression of immune response. To mitigate the donor-to-donor variation on alloreactivity, we developed a strategy to identify NK ligands that can effectively diminish host NK activation in the presence of allogeneic CAR T cells with TCR and B2M gene KOs. A platform using K562 cell line was developed to screen a wide variety of NK inhibitory peptides and synthetic ligands to identify their inhibitory activities. To date, we have identified several synthetic proteins that demonstrate enhanced NK inhibitory function. These synthetic proteins can be used to complement, or to replace HLA-E and enable more extended persistence of an allogeneic cell product in a broader patient population. Citation Format: Meriam Vejiga, Don Wang, Guangnan Li, Kyle Pratt, Phung Gip, James Trager, Ivan Chan. Immune masking strategies to extend the pharmacokinetics of allogeneic cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5511.

Background Healthy adult peripheral blood natural killer (PBNK) cells are mature cytotoxic innate lymphocytes possessing an inherent capacity for tumor cell killing, making them attractive candidates for adoptive cell therapy. These NK cells are also amenable to chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for off-the-shelf therapy since they are not known to cause graft-versus-host disease, unlike T cells. Approved CAR cell therapies are custom-made from each patient’s own T cells, a process that can limit patient eligibility and contribute to product variability. In this study, we compare PBNK cells to umbilical cord blood NK (CBNK) cells to evaluate both as candidate starting materials for clinical and commercial supply of CAR NK cells. Methods PBNK and CBNK cells were expanded using either a 14-day protocol and a single stimulation with Nkarta’s NKSTIM cell line plus IL-2, or with 5 stimulations over 70 days.1 IL-12 and IL-18 were added at the beginning and end of the 70-day expansion to drive memory-like NK cell differentiation. We transduced NK cells to express CD19-targeted CAR and membrane-bound IL-15 following the first NKSTIM pulse. We measured cytotoxicity against 3 tumor cell lines by IncuCyte, and phenotyped cells for NK markers including differentiation markers CD57 and NKG2C, and NKG2A and KIR. Results Purified NK cells from 1 PBNK donor and 4 CBNK donors were successfully expanded and engineered to express high levels of CAR. The 70-day final product (FP) CBNK cells were CD57-KIRlo/- and NKG2A+, consistent with an immature phenotype, whereas the FP PBNK cells were educated, at more than 80% NKG2A-KIR+. CBNK cells expanded to approximately 11-million-fold, whereas PBNK cells surpassed 250-billion-fold expansion, without appearing to have reached a terminal expansion limit. At the end of the study, Nkarta’s standard 14-day process (SP) cells [1], and FP PBNK cells were as potent or trended towards greater potency than CBNK cells against 3 different tumor targets in a 72-hour IncuCyte assay. Furthermore, FP PBNK cells were as or more potent than SP PBNK cells, depending on the tumor target. Conclusions We demonstrate healthy donor-derived PBNK cells can expand over 250 billion-fold while maintaining potency. These results show robust expansion capability of educated, potent NK cells and provide a rationale for the development of off-the-shelf CAR NK cell therapies using NK cells from donors selected to provide optimal product characteristics. Reference Whang M, Xie M, Jamboretz K, Lamar H, Guo C, Rahman N, Chan I, Whiteley E, Brandenberger R, Lazetic S, Trager J. 151 Potentiating the large-scale expansion and engineering of peripheral blood-derived CAR NK Cells for off-the-shelf application. J Immunother Cancer. 2021; 9: 151.

With rising costs and prolonged timelines for drug development, more innovative trial designs are critical to improve efficiency. Use of available historical control data in a new trial can reduce the number of control patients and accordingly reduce costs and timelines. A major limitation of historical data borrowing is potential prior-data conflict. This difference can increase false decision rates and confound the outcome interpretation. The potential inflation in both type I error rate and type II error rate should be clearly characterized and controlled during the trial design stage. In this paper, we develop a simple approach to incorporating historical control data in clinical trial design and analysis. First we provide a simple statistical approach to evaluating design properties when using a Bayesian approach to incorporating historical data. We then propose a six-step process for trial design including selection and summarization of historical control data, sample size determination with and without borrowing, design property evaluation, and how much historical data should be borrowed to control false positive/negative rate inflation based on trial variability. A detailed procedure to select historical control data is also provided. Finally, we use an example to illustrate our approach. The simplicity of methodology (no simulation required), the streamlined processes for data selection, and explicit evaluation of the impact of prior-data conflict on type I error rate and power make the proposed approach statistically rigorous and easy to understand and implement.

Background Peripheral blood natural killer (NK) cells are attractive candidates for adoptive cell therapy. NK cells possess innate ability for tumor cell killing and are also amenable to genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for allogeneic, off-the-shelf therapy since, unlike T cells, they are neither HLA-restricted nor known to cause graft-versus-host disease. Cytokine inducible SH2-containing protein (CISH) is a negative regulator of interleukin 15 (IL-15) signaling in natural killer (NK) cells. Here we show the potential application of CISH gene-knockout CAR NK cells targeting CD70 and expressing a membrane-bound form of IL-15. CD70 is an antigen that is aberrantly expressed in a variety of malignant settings, including renal cell carcinoma (RCC), while its expression in normal tissues is restricted to a subset of lymphoid cell types. Methods To target CD70 on RCC cells, we generated CD70-CAR NK cells with CISH deletion. Using the CRISPR/Cas9 system, we knocked out CISH expression in isolated peripheral blood NK cells from healthy donors. Since CD70 expression is present on activated NK cells, we also targeted CD70 for CRISPR knockout to avoid fratricide. We then expanded these edited NK cells by using IL-2 and stimulation using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added during expansion to drive memory-like NK cell differentiation. We transduced the expanded NK cells to express engineered CD70-targeted CAR and mbIL-15. We assessed CAR expression, NK cell persistence, and NK cell activity against RCC target cells using end-point cytotoxicity assays and IncuCyte. Results CISH gene-knockout CD70-CAR NK cells could be produced efficiently and exhibited extended persistence in culture. After engineering and expansion, CD70-CAR transduction efficiency was 60–80%. CD70-CAR NK cells displayed potent cytotoxicity against CD70-expressing renal cancer derived cell lines. Interestingly, cytotoxicity assays demonstrated that CISH gene-knockout CD70-CAR NK cells were partially resistant to TGFß and adenosine inhibition of cytotoxicity. Furthermore, CISH gene-knockout CD70-CAR NK cells maintained their activity during prolonged culture. Conclusions In summary, we show CISH gene-knockout CD70-CAR NK cells demonstrate potent anti-tumor activity against relevant solid tumor cell lines and partially provide resistance to tumor microenvironment inhibition. These data support the further exploration of CISH gene-knockout CD70 CAR NK cells for clinical application.

Background Peripheral blood natural killer (NK) cells are mature cytotoxic innate lymphocytes possessing an inherent capacity for tumor cell killing, thus making them attractive candidates for adoptive cell therapy. These NK cells are also amenable to CRISPR and chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for off-the-shelf therapy since they are not known to cause graft-versus-host disease, unlike T cells. Presently, approved CAR cell therapy is custom-made from each patient‘s own T cells, a process that can limit patient pool, narrow therapeutic window, and contribute to product variability. In this study, we investigate whether peripheral blood NK cells from a selected donor can be edited, engineered, and expanded sufficiently for off-the-shelf use in a wide patient population. Methods Using the CRISPR/Cas9 system, we knocked out CISH expression in isolated peripheral blood NK cells from 3 healthy donors. Subsequently, we expanded edited NK cells by using IL-2 and sequential stimulations using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added twice during expansion to drive memory-like NK cell differentiation. We transduced the expanded NK cells to express engineered CD19-targeted CAR and mbIL-15 during an interval between the first and second NKSTIM pulses. We assessed NK cell cytotoxicity against Nalm6 target cells by IncuCyte. Results Isolated peripheral blood NK cells from 3 healthy donors were successfully edited using CRISPR/Cas9, engineered to express high levels of CAR, extensively expanded using a series of NKSTIM pulses in the presence of IL-2, and differentiated into memory-like NK cells using IL-12 and IL-18. Interestingly, NK cells from the 3 donors exhibited distinct outcomes. NK cells from one donor reached a peak expansion limit of approximately 7-million-fold before undergoing contraction whereas NK cells from two donors continued to expand over the length of the study surpassing 100-million-fold expansion, without appearing to have reached a terminal expansion limit. At the end of the study, NK cells from one donor exceeded 1-billion-fold expansion and maintained 88% cytolytic activity compared to Nkarta’s standard process control in a 72-hour IncuCyte assay. Conclusions In this study, we demonstrate that healthy donor-derived peripheral blood NK cells are capable of expanding over billion-fold while maintaining potency. These results provide a rationale for the development of off-the-shelf CAR NK cell therapies using NK cells from donors selected to provide optimal product characteristics. Ethics Approval Human samples were collected with written informed consent by an approved vendor.

BackgroundNK cells expanded on membrane-bound (mb) IL-15 and 41BBL expressing K562 stimulatory cells (NKSTIM) for clinical use can be genetically modified to express activating chimeric receptors.1 2 3 NK cells activated in the presence of IL-12, IL-15 and IL-18 develop cytokine induced memory-like (CIML) phenotype and function; these cells have shown clinical promise.4 Additionally, HSCT AML transplants using NK KIR Haplotype Group B donors with better and best Group B profiles (≥2 activating genes) show better survival.5 6 Here we investigate whether KIR profiles impact healthy allogeneic donor NK cell function and phenotype when these cells are expanded on NKSTIM in the presence of IL-12 and IL-18 (12–18).MethodsHealthy donor PBMC NK were genotyped for HLA and KIR and expanded on K562-mbIL15-41BBL stimulatory cells with IL-2 alone or with IL-2 plus IL-12 and IL-18 (12–18). Expanded NK were transduced with CAR constructs including CD19, and then evaluated for NK cell expansion, cytokine secretion, RNA profiles, cytotoxicity against tumor lines, and cell surface phenotypes. Expanded CD19 NK donors with varying numbers of activating KIR vs inhibitory KIR were tested for effector function, and these donors were then tested for in vivo efficacy and pharmacokinetics. A KIR ranking score was developed by considering both the number of activating and inhibitory KIR genes expressed by each donor. This score was correlated with functional properties of CAR NK cells.ResultsAddition of 12–18 to the K562-mbIL15-41BBL stimulatory cells improves CD19-CAR NK potency 2-fold relative to the stimulatory cell line alone (P=.02) while NK cell expansion is unchanged. 12–18 also drove an increase in effector cytokine accumulation on exposure of CAR-NK to CD19 tumor. CIML CAR NK cells from donors with higher KIR scoring also had higher cytotoxicity (Pearson’s R=0.74, P=0.006); this correlation was not observed following expansion in the absence of 12–18. 12–18 also drove more potent in vivo activity against tumor with an increased presence of circulating NK cells over 4 weeks in the mice.ConclusionsCIML CAR NK cells derived from donors with favorable KIR scoring have greater cytotoxic activity, effector cytokine production, and in vivo pharmacokinetics and efficacy. These findings may provide an important criterion for donor selection in the development of more robust and potent engineered NK cells for clinical use.ReferencesLapteva N, Durett AG, Sun J, Rollins LA, Huye LL, Fang J, Dandekar V, Mei Z, Jackson K, Vera J, Ando J, Ngo MC, Coustan-Smith E, Campana D, Szmania S, Garg T, Moreno-Bost A, Vanrhee F, Gee AP, Rooney CM. Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications. Cytotherapy 2012;14(9):1131–1143.Chihaya I, Iwamoto S, Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood 2005;106:376–383.Yang Y, Connolly J, Shimasaki N, Mimura K, Kono K, Campana D. A Chimeric Receptor with NKG2D Specificity Enhances Natural Killer Cell Activation and Killing of Tumor Cells. Cancer Res 2013;73(6):1777–1786.Romee R, Rosario M, Berrien-Elliott MM, Wagner JA, Jewell BA, Schappe T, Leong JW, Abdel-Latif S, Schneider SE, Willey S, Neal CC, Yu L, Oh ST, Lee YS, Mulder A, Claas F, Cooper MA, Fehniger TA. Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia. Sci Trans Med 2016;8(357): 357ra123.Cooley S, Weisdorf DJ, Guethlein LA, Klein JP, Wang T, Le CT, Marsh SGE, Geraghty D, Spellman S, Haagenson MD, Ladner M, Trachtenberg E, Parham P, and Miller JS. Donor selection for natural killer cell receptor genes leads to superior survival after unrelated transplantation for acute myelogenous leukemia. Blood 2010;116(14):2414–2419.Cooley S, Weisdorf DJ, Guethlein LA, Klein JP, Wang T, Marsh SGE, Spellman S, Haagenson MD, Saeturn K, Ladner M, Trachtenberg E, Parham P, and Miller JS. Donor Killer Cell Ig-like Receptor B Haplotypes, Recipient HLA-C1, and HLA-C Mismatch Enhance the Clinical Benefit of Unrelated Transplantation for Acute Myelogenous Leukemia. JI, 2014;192(10):4592–600.Ethics ApprovalAnimal studies were conducted with IACUC approval.

Background Natural killer (NK) cells are highly effective and fast-acting cytolytic cells capable of eradicating target cells with limited adverse effects such as cytokine release syndrome (CRS) or graft-versus-host disease. Chimeric antigen receptors (CARs)-engineered NK cells have been recently used against leukemia with encouraging clinical outcomes.1 The surface antigen CD19, expressed by B-lymphoblasts, represents an ideal CAR target against B cell acute lymphoblastic leukemia (B-ALL). We developed a highly potent CD19 -directed CAR NK cell therapy, NKX019, with an extended in vivo half-life aimed at killing CD19-expressing target. Methods NK cells isolated from healthy PBMCs were expanded in the presence of NKSTIM cells, IL-2, IL-12, IL-18 and transduced with both a CD19-targeted CAR construct and a membrane-bound form of IL-15 (mbIL-15). Control (non-engineered) NK cells were produced in parallel. Cytotoxic activity of NKX019 against CD19+ B-ALL cell line (REH), pre-B ALL cell line (Nalm-6), allogeneic PBMCs was assessed using Incucyte® or flow cytometry. NSG mice bearing either Nalm-6.fluc (Nalm6) or REH.fluc (REH) tumor received different concentrations of NKX019 or control NK cells. In-life analysis of tumor-bearing and naïve NSG mice include: 1) bioluminescence imaging, 2) clinical observations, 3) serum cytokines and 4) CAR+ NK cell persistency. Results NKX019 showed enhanced cytolytic activity against REH and Nalm-6 tumor cells compared to control NK cells and CAR19+ T cells. The superiority of NKX019 over CAR19+ T cells was more pronounced at the earlier time point (24 hours) with near identical calculated EC50 observed at 72 hours for both cell types. Increased cytolytic activity of NKX019 was limited to CD19+ cells in bulk PBMCs. Consistent with our in vitro observations, NKX019 controlled Nalm-6 and REH tumor growth in doses as low as 2 × 106 cells/kg for up to 30 days with no apparent increase in cytokines commonly associated with CRS. Increased Nalm-6 tumor growth coincided with an apparent decrease in measurable NKX019 in the periphery. In tumor-naïve NSG mice, NKX019 was detectable in the blood for up to 9 weeks post-infusion consistent with its extended half-life. Conclusions NKX019 expresses mbIL-15 and is produced in the presence of IL-12 and IL-18, resulting in enhanced in vitro expansion and longer in vivo half-life than non-engineered NK cells. NKX019 also exhibited advantages compared to CAR19+ T cells including faster cytotoxic kinetics and limited production of cytokines associated with CRS. A first-in-human trial of NKX019 in B cell malignancies is planned for 2021. Ethics Approval The animal procedures described in this abstract were conducted in accordance with Explora BioLabs Animal Care and Use Protocol approved by Explora BioLabs Institutional Animal Care and Use Committee. Reference Liu, et al. 2020 NEJM

IL-10 is regarded as an anti-inflammatory cytokine but it is also essential for the cytotoxicity and proliferation of antigen-activated CD8 T cells. Activation of the T cell receptor induces the expression of IL-10 receptors and PD-1 on CD8 T cells. This provides the mechanistic rationale for combining AM0010 and anti-PD1 for the treatment of cancer pts. Tolerability and anti-tumor activity of AM0010 alone and in combination with chemotherapies or immune checkpoint inhibitors was explored in a multi-basket phase 1 clinical trial. Pts with advanced renal cell cancer (RCC) were treated with AM0010 alone (daily SC) or in combination with pembrolizumab (q3wk IV). Tumor responses were monitored following irRC. Immune responses were measured by analysis of serum cytokines, activation of blood derived T cells, peripheral T cell clonality. Nineteen pts. with RCC (15 evaluable), were treated with AMO010 alone (20 mg/kg) and eight were treated in combination with pembrolizumab (2mg/kg). Both regimens were tolerated well (observation period 15 months). All TrAEs were transient and TrAEs leading to study discontinuation were not observed. There was no colitis, pneumonitis, or endocrine disruptions. G3/4 TrAEs in monotherapy included anemia (9), hypertriglyceridemia (3), thrombocytopenia (2), ALT/AST increase (2) and fatigue (2). AM0010 combination with Pembrolizumab did not increase TrAEs. Objective responses (PR/CR) were observed in 4 of 15 evaluable RCC pts. in monotherapy (27%) and in 4 of 8 patients in AM0010 /pembrolizumab (50%). Progression free survival (PFS) was 3 and 9.4 months, respectively. AM0010 alone and in combination with anti-PD1 increased Th1 cytokines (IL-18, IFNg, IL-7) as well as the number and proliferation of PD1+ activated CD8 T cells while decreasing the proliferation of FoxP3+ Tregs and TGFb in the blood. AM0010 / anti-PD1 induced de-novo oligoclonal expansion of T cell clones in the blood without affecting total lymphocyte counts. AM0010 alone or in combination with anti-PD1 is well-tolerated. The clinical activity and the observed CD8 T cell activation encourages the continued exploration of AM0010 in combination with anti-PD1.

### P189 Rational combinations of intratumoral T cell and myeloid agonists mobilize abscopal responses in prostate cancer #### Casey Ager1, Matthew Reilley2, Courtney Nicholas1, Todd Bartkowiak1, Ashvin Jaiswal1, Michael Curran1 ##### 1Department of Immunology, University of Texas MD Anderson