547 Background: In ccRCC, carbonic anhydrase 9 (CA9) and prostate-specific membrane antigen (PSMA) have emerged as potential therapeutic targets. CA9 is overexpressed in ccRCC tumor cells, while PSMA, classically linked to prostate cancer, is expressed in ccRCC tumor-associated vasculature. An ongoing phase 1/2 trial (NCT06245915) is evaluating AB-2100, a programmable circuit T cell therapy using a PSMA-gated CA9 chimeric antigen receptor (CAR), for the treatment of ccRCC. We analyzed CA9 and PSMA expression in primary and metastatic ccRCC, assessing whether expression is retained in metastases and after prior treatment with immunotherapy (IO) and/or tyrosine kinase inhibitors (TKI). Methods: Single-plex immunohistochemistry (IHC) for CA9 and PSMA was performed on serial ccRCC samples. CA9 was scored by the percentage of tumor cells with membranous staining (0–100%), and PSMA by vascular staining positivity (0–3+). A total of 103 ccRCC samples (17 primary, 86 metastatic) from 78 patients (pts) were analyzed. Paired Wilcoxon test compared matched primary-metastatic pairs (n=16; most recent if multiple available). Independent metastatic samples (one per patient, most recent if multiple available, n=77) were analyzed using the Wilcoxon rank-sum test to compare CA9 and PSMA expression in treatment-naive versus treatment-exposed samples. Correlation between the two markers was also evaluated (Spearman’s ρ). Results: Across the 78 pts, 61 received a TKI-based therapy, 46 an IO-containing regimen, and 13 a HIF-2α inhibitor. Of the 103 samples, 54.4% (n=56) were treatment-naive and 45.6% (n=47) were collected after prior exposure to systemic therapy. CA9 expression was uniformly high (median=100%, IQR: 99-100%, range: 10-100%) across 103 evaluable samples. Among matched pairs (n=16), there was no significant loss of CA9 expression in metastatic samples compared to their corresponding primary tumors (paired Wilcoxon test, p = 0.78). PSMA was detected in the tumor vasculature in 102 evaluable cases (median = 2, IQR: 2-3), with comparable expression between 16 matched pairs (paired Wilcoxon test, p = 0.48). No correlation between CA9 and PSMA was found (Spearman's ρ = -0.17). Expression of both markers was not significantly affected by prior TKI- or IO-containing regimens (Table 1). Conclusions: CA9 and PSMA expression is consistently retained in metastatic ccRCC compared to primary tumors, even after systemic treatment. CA9 remains expressed in tumor cells, while PSMA is observed in the tumor vasculature, supporting the rationale for their therapeutic targeting in advanced ccRCC. Treatment-exposed vs naive samples. CA9 PSMA Treatment Group n Median p* n Median p* Naive (ref) 35 100.0 — 35 2.0 — TKI only 11 100.0 0.879 11 2.0 0.231 TKI-containing regimen 35 100.0 0.119 34 2.0 0.434 IO-containing regimen 26 100.0 0.152 26 3.0 0.293 *P-values from Wilcoxon rank-sum test vs naive (reference group).

Annotation of immunologic gene function in vivo typically requires the generation of knockout mice, which is time consuming and low throughput. We previously developed CHimeric IMmune Editing (CHIME), a CRISPR–Cas9 bone marrow delivery system for constitutive, ubiquitous deletion of single genes. Here we describe X-CHIME, four new CHIME-based systems for modular and rapid interrogation of gene function combinatorially (C-CHIME), inducibly (I-CHIME), lineage-specifically (L-CHIME) or sequentially (S-CHIME). We use C-CHIME and S-CHIME to assess the consequences of combined deletion of Ptpn1 and Ptpn2 , an embryonic lethal gene pair, in adult mice. We find that constitutive deletion of both PTPN1 and PTPN2 leads to bone marrow hypoplasia and lethality, while inducible deletion after immune development leads to enteritis and lethality. These findings demonstrate that X-CHIME can be used for rapid mechanistic evaluation of genes in distinct in vivo contexts and that PTPN1 and PTPN2 have some functional redundancy important for viability in adult mice.

PD-1 is a key negative regulator of CD8+ T cell activation and is highly expressed by exhausted T cells in cancer and chronic viral infection. Although PD-1 blockade can improve viral and tumor control, physiological PD-1 expression prevents immunopathology and improves memory formation. The mechanisms driving high PD-1 expression in exhaustion are not well understood and could be critical to disentangling its beneficial and detrimental effects. Here, we functionally interrogated the epigenetic regulation of PD-1 using a mouse model with deletion of an exhaustion-specific PD-1 enhancer. Enhancer deletion exclusively alters PD-1 expression in CD8+ T cells in chronic infection, creating a 'sweet spot' of intermediate expression where T cell function is optimized compared to wild-type and Pdcd1-knockout cells. This permits improved control of chronic infection without additional immunopathology. Together, these results demonstrate that tuning PD-1 via epigenetic editing can reduce CD8+ T cell dysfunction while avoiding excess immunopathology.

Baseline innate immune signatures can influence protective immunity following vaccination. Here, we used systems transcriptional analysis to assess the molecular mechanisms underlying differential immunogenicity and protective efficacy results of a clinical trial of the radiation-attenuated whole sporozoite PfSPZ Vaccine in African infants. Innate immune activation and myeloid signatures at pre-vaccination baseline correlated with protection from Plasmodium falciparum infection in placebo controls, while the same signatures predicted susceptibility to infection among infants who received the highest and most protective dose of the PfSPZ Vaccine. Machine learning identified monocytes and an antigen presentation signature as pre-vaccination features predictive of malaria infection after highest-dose PfSPZ vaccination. Consistent with these human data, innate stimulation in vivo conferred protection against malaria infection in mice while diminishing the CD8+ T cell response to radiation-attenuated sporozoites. These data establish a dichotomous role of innate stimulation for malaria protection and induction of protective immunity of whole-sporozoite malaria vaccines.

Abstract T cell exhaustion resulting from chronic antigen stimulation and an immunosuppressive tumor microenvironment limits the efficacy of T cell therapies in the solid tumor setting. The onset of T cell exhaustion is associated with distinct epigenetic and transcriptional changes. We hypothesized that genetic perturbations which shift T cells away from exhaustion associated states could increase the potency of immunotherapies. To this end, we utilized pooled, in vitro CRISPR/Cas9-based screening paired with deep sequencing readouts to characterize perturbation dependent T cell states in the context of chronic antigen stimulation. In order to achieve this, we developed a lentivirus-based workflow to perform CRISPRko, CRISPRi and CRISPRa pooled screens in human CAR T-cells that allowed for assessment of T cell phenotypes mediated by knockout, knockdown or overexpression of a large pool of target genes with single cell transcriptome readout. Subjecting these engineered CAR T-cells to an antigen-specific, cell-based repetitive stimulation assay (RSA) led to the progressive loss of T cell proliferation and effector function enabling in vitro modeling of T cell exhaustion. Moreover, characterization of the CAR T-cells by single cell sequencing recapitulated key hallmarks of the transcriptional and epigenetic landscape of T cell exhaustion. We also discovered T cell intrinsic gene perturbations that govern T cell states in the context of chronic antigen stimulation. These results demonstrate the power of pooled CRISPR screening with single cell readouts to identify novel target genes to enhance CAR T-cell therapies. Citation Format: Sahil Joshi, Glenn Wozniak, John Gagnon, Kristina Vucci, Allyson Merrell, Mandi Simon, Catherine Oh, Andrew Cardozo, David DeTomaso, Julie Chow, Grace Zheng, Angela Boroughs, Keith Joho, Pratiksha Thakore, Soyoung Oh, Jake Freimer, Ashley Cass, Vibhavari Sail, Carla Tocchini, Marian Sandoval, Andrea Liu, Eric Cui, Matt Drever, Brendan Galvin, Jeff Milush, Levi Gray-Rupp, Emily Wheeler, Bob Chen, Jacob Levine, Celine Eidenschenk, Jill Schartner, Katie Geiger-Schuller, Jan-Christian Huetter, Sascha Rutz, Orit Rozenblatt-Rosen, Ira Mellman, W. Nicholas Haining. Pooled CRISPR screening coupled with single-cell sequencing identifies modifiers of CAR T cell state in the context of chronic antigen stimulation [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 7034.

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 The therapeutic potential of T cell therapies for treating solid tumors can be limited by intrinsic T cell exhaustion mechanisms and the suppressive tumor microenvironment. To better understand these challenges and identify genetic perturbations to enhance T cell functions, we developed in vivo exhaustion models and performed pooled CRISPR/Cas9-based screens with detailed characterization of T cell states through single cell RNA-sequencing (scRNA-seq). In vivo Xenograft exhaustion models were developed in the format of T cell receptor (TCR) T cells, with tumor cell lines expressing TCR-specific antigen (NY-ESO-1) respectively. Individual perturbations were introduced into T cells via non-viral editing and pooled before transferred into mice with established tumors of various sizes that provided continuous antigen exposure to drive exhaustion, while providing sufficient number of T cells from tumor and spleen tissues for scRNA-seq. The pooled CRISPR in vivo screens in human TCR T cells enabled us to evaluate T cell phenotypes resulting from gain-of-function, loss-of-function, or combinations of these modifications across a wide array of target genes. We identified known and previously uncharacterized combinatorial perturbations that were enriched or depleted and were associated with distinct transcriptional phenotypes over the course of chronic stimulation. This approach allows us to bridge the gaps in our understanding of how T cells can be reprogrammed to overcome exhaustion and function more effectively in the challenging environment of solid tumors. Citation Format: Dina Polyak, Jessica Fuhriman, Josephine Susanto, Allyson Merrell, Mandi Simon, Catherine Oh, Andrew Cordazo, David DeTomasso, Carla Tocchini, Vibhavari Sail, Eric Cui, Jeff Milush, Levi Gray-Rupp, Brendan Galvin, Christopher Murriel, Grace Zheng, Angela C. Boroughs, Pratiksha Thakore, Soyoung Oh, Jake Freimer, Bob Chen, Celine Eidenschenk, Emily Wheeler, Jacob Levine, Jan-Christian Huetter, Jill Schartner, Katie Geiger-Schuller, Orit Rozenblatt-Rosen, Sascha Rutz, Ira Mellman, W. Nicholas Haining. Enhancing TCR T cell function in solid tumors through in vivo combinatorial screens and single-cell analysis [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 7042.

Abstract In solid tumors, CAR T cell efficacy is limited by on-target off-tumor toxicity, as well as functional suppression by the tumor microenvironment (TME). AB-1015 is an integrated circuit T cell (ICT cell) developed for use in ovarian cancer. The AB-1015 transgene cassette includes two functional modules: A) a sequential ”AND” logic gate designed to limit off-tumor toxicity through dual tumor antigen recognition, which consists of a priming receptor (PrimeR) against ALPG/P and an inducible Mesothelin(MSLN)-targeted chimeric antigen receptor (CAR) that is upregulated in response to PrimeR engagement; as well as B) a dual shRNA-miR targeting FAS and PTPN2 to resist TME suppression and improve ICT cell function. The AB-1015 is generated by CRISPR-based, targeted insertion of a single transgene into a safe harbor locus. AB-1015 is currently being studied in a phase I clinical trial (NCT05617755) for patients with platinum-resistant ovarian cancer. The heterogeneity in antigen expression among different tumor cells within solid tumors can hinder the efficacy of targeted CAR T cell therapy. To investigate this, we initially assessed the percentage of tumor cells expressing ALPG/P and MSLN in ovarian cancer samples using immunohistochemistry (IHC). Most tumor cores evaluated had detectable MSLN expression; however, the percentage of ALPG/P expressing tumor cells was heterogeneous. To model the heterogeneity of ALPG/P observed in the tumor samples, we developed a human ovarian carcinoma mixed-cell model system with an increasing proportion of ALPG/P expressing cells. The human ovarian carcinoma cell line, OVCAR3, expresses high levels of MSLN with ≤5% endogenous expression of ALPG/P. In the mixed-cell model system, these cells were then engineered to express ALPG/P (OVCAR3-ALPG) and mixed with the parental OVCAR3 cells at increasing ratios. In this system, we demonstrated that CAR induction is tightly linked to the percentage of ALPG/P-positive tumor cells in co-culture. We demonstrated that as few as 5-15% of ALPG-positive tumor cells were capable of inducing sufficient CAR expression to eliminate an otherwise MSLN-positive tumor culture. These preclinical data show that very few ALPG-positive tumor cells are needed to elicit anti-tumor activity from AB-1015, suggesting potential efficacy of AB-1015 in ovarian cancer with low percentage of ALPG/P expressing cells, similar to the antigen heterogeneity found in patients’ tumors. Citation Format: Dina Polyak, Erik Beckman, Preethi Bala Balakrishnan, Hongruo Yun, Xinyan Tang, Jun Feng, Nickolas Attansio, Ashley Cass, Stephen Santoro, Alba Gonzalez-Junca, W. Nicholas Haining, Christopher L. Murriel. Logic-gated CAR T cell product AB-1015 response to ovarian cancer models with heterogeneous levels of ALPG/P antigen [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B070.

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.

Resistance to immune checkpoint inhibitors represents a major therapeutic challenge, as only 40% of patients with melanoma (and less with other tumor types) have a long-term response to anti-PD-1 therapy. Resistance can arise because of somatic mutations in cancer cells that allow evasion of T cell-mediated killing. One commonly acquired resistance mutation in melanoma, loss of beta-2 microglobulin (B2m), prevents T cell killing by hiding cancer cells from CD8+ T cell recognition. To understand the failed immune response against resistant tumors, we used single-cell RNA-seq to characterize tumor-infiltrating immune cells in antigen presentation-deficient human melanoma biopsies and CRISPR-modified mouse melanoma tumors. Our data demonstrate an increase in immunosuppressive M2-like macrophages and absence of CD8+ T cells in B2m-null tumors. To overcome this resistance, we treated tumor-bearing mice with CD40 agonist antibody, which promotes differentiation of macrophages towards a pro-inflammatory phenotype and increases dendritic cell priming of CD8+ T cells. Treatment with CD40 agonist reduced tumor growth and improved tumor clearance in B2m-null melanoma and colorectal cancer models. To determine how CD40 agonist treatment works, we depleted different immune populations from the tumor microenvironment. We hypothesized that by depleting M2 macrophages, CD40 agonist treatment would remove an immunosuppressive brake to allow natural killer (NK) cells to kill tumor cells lacking MHC expression. To our surprise, NK cells were not required for the efficacy of CD40 agonist. Instead CD8+ T cells were required, even though the CD8+ T cells cannot directly recognize the tumor cells. scRNA-seq identified a transcriptionally unique state of CD8+ T cells that is recruited to the tumor microenvironment after CD40 agonist treatment. These CD8+ T cells produce IFNγ, which is required for the efficacy of CD40 agonist treatment. These data demonstrate that CD8+ T cells, a key mediator of anti-tumor immunity, can still be recruited to control tumors deficient in antigen presentation. More broadly, they suggest that strategies to activate CD8+ T cells may be effective even in the context of acquired resistance to checkpoint inhibitor therapy. Citation Format: Brian C. Miller, Yacine Choutri, Rose Al Abosy, Amy Huang, Emily K. Cox, Matthew P. Zimmerman, Wan Lin Chong, Katherine J. Vietor, Jenna Collier, Sarah A. Weiss, Debattama Sen, W. Nicholas Haining, Arlene H. Sharpe. Overcoming resistance to immunotherapy due to loss of antigen presentation [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 6362.

Figure S1. Characterization of cells and CDK4/6 inhibitors; Figure S2. CDK6 phosphorylates serine residues of the regulatory domain of NFAT4 (NFATc3); Figure S3. Analysis of lung tumor immune infiltrates after CDK4/6 inhibition from KrasG12D (Kras), KrasG12DLkb1 (KL) or KrasG12DTrp53fl/fl (KP) mice; Figure S4. T cell proliferation and cytokine/chemokine profiling of KrasG12DTrp53fl/fl GEMM mice; Figure S5. Tumor antigen experienced T cells are more sensitive to CDK4/6 inhibition; Figure S6. Short-term CDK4/6 inhibition alters the cell cycle status of tumor infiltrating T cells; Figure S7. CDK4/6 inhibition induces changes in the expression of activation and suppression marker genes in tumor-infiltrating T cells; Figure S8. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody elicits anti-tumor immunity; Figure S9. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody on established tumor; Figure S10. Effect of TCR stimulation and CDK4/6 inhibition on phosphorylation of NFkB; Supplementary Table S1; Supplmentary Table S2; Supplementary Table S3: Selected genes reported to be regulated by NFAT