Abstract: Memory CD8+ T cells populate non-lymphoid tissues (NLTs) following pathogen infection, but little is known about the establishment of endogenous tumor-specific tissue-resident memory T cells (T RM ) during cancer immunotherapy. Using a transplantable mouse model of prostate carcinoma, here we report that tumor challenge leads to expansion of naïve neoantigen-specific CD8+ T cells and formation of a small population of non-recirculating T RM in several NLTs. Primary tumor destruction by irreversible electroporation (IRE), followed by anti-CTLA-4 immune checkpoint inhibitor (ICI), promotes robust expansion of tumor-specific CD8+ T cells in blood, tumor, and NLTs. Parabiosis studies confirm that T RM establishment following dual therapy is associated with tumor remission in a subset of cases and protection from subsequent tumor challenge. Addition of anti-PD-1 following dual IRE + anti-CTLA-4 treatment blocks tumor growth in non-responsive cases. This work indicates that focal tumor destruction using IRE combined with ICI is a potent in situ tumor vaccination strategy that generates protective tumor-specific T RM .

Abstract: T cell receptor (TCR) transgenic mice represent an invaluable tool to study antigen-specific immune responses. In the pre-existing models, a monoclonal TCR is driven by a non-physiologic promoter and randomly integrated into the genome. Here, we create a highly efficient methodology to develop T cell receptor exchange (TRex) mice, in which TCRs, specific to the self/tumor antigen mesothelin ( Msln ), are integrated into the Trac locus, with concomitant Msln disruption to circumvent T cell tolerance. We show that high affinity TRex thymocytes undergo all sequential stages of maturation, express the exogenous TCR at DN4, require MHC class I for positive selection and undergo negative selection only when both Msln alleles are present. By comparison of TCRs with the same specificity but varying affinity, we show that Trac targeting improves functional sensitivity of a lower affinity TCR and confers resistance to T cell functional loss. By generating P14 TRex mice with the same specificity as the widely used LCMV-P14 TCR transgenic mouse, we demonstrate increased avidity of Trac -targeted TCRs over transgenic TCRs, while preserving physiologic T cell development. Together, our results support that the TRex methodology is an advanced tool to study physiological antigen-specific T cell behavior.

Abstract: Achieving robust responses with adoptive cell therapy for the treatment of the highly lethal pancreatic ductal adenocarcinoma (PDA) has been elusive. We previously showed that T cells engineered to express a mesothelin-specific T cell receptor (TCR Msln ) accumulate in autochthonous PDA, mediate therapeutic antitumor activity, but fail to eradicate tumors in part due to acquisition of a dysfunctional exhausted T cell state. Methods Here, we investigated the role of immune checkpoints in mediating TCR engineered T cell dysfunction in a genetically engineered PDA mouse model. The fate of engineered T cells that were either deficient in PD-1, or transferred concurrent with antibodies blocking PD-L1 and/or additional immune checkpoints, were tracked to evaluate persistence, functionality, and antitumor activity at day 8 and day 28 post infusion. We performed RNAseq on engineered T cells isolated from tumors and compared differentially expressed genes to prototypical endogenous exhausted T cells. Results PD-L1 pathway blockade and/or simultaneous blockade of multiple coinhibitory receptors during adoptive cell therapy was insufficient to prevent engineered T cell dysfunction in autochthonous PDA yet resulted in subclinical activity in the lung, without enhancing anti-tumor immunity. Gene expression analysis revealed that ex vivo TCR engineered T cells markedly differed from in vivo primed endogenous effector T cells which can respond to immune checkpoint inhibitors. Early after transfer, intratumoral TCR engineered T cells acquired a similar molecular program to prototypical exhausted T cells that arise during chronic viral infection, but the molecular programs later diverged. Intratumoral engineered T cells exhibited decreased effector and cell cycle genes and were refractory to TCR signaling. Conclusions Abrogation of PD-1 signaling is not sufficient to overcome TCR engineered T cell dysfunction in PDA. Our study suggests that contributions by both the differentiation pathways induced during the ex vivo T cell engineering process and intratumoral suppressive mechanisms render engineered T cells dysfunctional and resistant to rescue by blockade of immune checkpoints.

Abstract: Memory CD8+ T cells populate non-lymphoid tissues (NLT) and can protect against malignancies, but little is known about the establishment of tumor-specific tissue resident memory CD8+ T cells (TRM) by therapeutically initiated immune responses. We used a transplantable mouse model of prostate carcinoma and MHCI tetramers to track and quantify tumor-specific CD8+ T cells. Tumor challenge led to systemic expansion of endogenous, naïve tumor antigen-specific CD8+ T cells and established a small population of TRM (CD44hi, CD62Llo, CD69+, CD103+) in several NLTs. Previously, we demonstrated that debulking primary tumors using irreversible electroporation (IRE) induced expansion of tumor-specific T cells. We hypothesized that IRE would cooperate with checkpoint blockade to increase antigen-specific TRM and promote tumor clearance in vivo. Combination treatment with IRE followed by α-CTLA4 promoted a robust 50-fold expansion of proliferating (Ki67+) tumor-specific T cells in blood, a 10-fold increase of TRM in NLT, and was associated with complete remission of residual tumor. Conversely, monotherapy allowed tumor outgrowth and failed to boost T cell numbers, resulting in small populations of PD-1hi tumor-specific CD8+ T cells. Similarly, anti-PD-1 alone or following IRE was also ineffective, suggesting that combined IRE/α-CTLA4 therapy targets newly primed T cells. IRE also enhanced antigen presentation by XCR1+ dendritic cells, promoting T cell priming in regional lymph nodes. These results indicate that combining α-CTLA4 checkpoint immunotherapy with focal tumor ablation, such as IRE, capable of releasing high quantities and qualities of endogenous tumor antigens may serve as a potent in situ tumor vaccination strategy.

Abstract: Pancreatic ductal adenocarcinoma (PDA) is a lethal and metastatic malignancy resistant to therapy. Elucidating how pancreatic tumor-specific T cells differentiate and are maintained in vivo could inform novel therapeutic avenues to promote T cell antitumor activity. Here, we show that the spleen is a critical site harboring tumor-specific CD8 T cells that functionally segregate based on differential Cxcr3 and Klrg1 expression. Cxcr3+ Klrg1- T cells express the memory stem cell marker Tcf1, whereas Cxcr3-Klrg1 + T cells express GzmB consistent with terminal differentiation. We identify a Cxcr3+ Klrg1+ intermediate T cell subpopulation in the spleen that is highly enriched for tumor specificity. However, tumor-specific T cells infiltrating primary tumors progressively downregulate both Cxcr3 and Klrg1 while upregulating exhaustion markers PD-1 and Lag-3. We show that antigen-specific T cell infiltration into PDA is Cxcr3 independent. Further, Cxcr3-deficiency results in enhanced antigen-specific T cell IFNγ production in primary tumors, suggesting that Cxcr3 promotes loss of effector function. Ultimately, however, Cxcr3 was critical for mitigating cancer cell dissemination following immunotherapy with CD40 agonist + anti-PD-L1 or T cell receptor engineered T cell therapy targeting mesothelin. In the absence of Cxcr3, splenic Klrg1 + GzmB + antitumor T cells wain while pancreatic cancer disseminates suggesting a role for these cells in eliminating circulating metastatic tumor cells. Intratumoral myeloid cells are poised to produce Cxcl10, whereas splenic DC subsets produce Cxcl9 following immunotherapy supporting differential roles for these chemokines on T cell differentiation. Together, our study supports that Cxcr3 mitigates tumor cell dissemination by impacting peripheral T cell fate rather than intratumoral T cell trafficking.

Abstract: Genocea’s ATLAS platform is an empirical bioassay that uses patient autologous immune cells to identify both true neoantigens and Inhibigens࣪ for inclusion in or exclusion from neoantigen-targeted vaccines and cell therapies, respectively. In ATLAS, patient-derived antigen-presenting cells (APCs) are pulsed with E. coli expressing individual mutations identified from the patient mutanome ± listeriolysin O, enabling interrogation of both CD8+ and CD4+ T cell recognition. True neoantigens induce T cell activation and cytokine release, while Inhibigens lead to a downregulation of T cell responses and thus can promote tumor growth. Previous ATLAS screening of CD8+ T cells from mice carrying B16F10 mouse melanoma tumors identified both neoantigens and Inhibigens. Upon therapeutic vaccination, adjuvanted neoantigens generated immunogenicity and anti-tumor efficacy1. In contrast, therapeutic vaccination with multiple ATLAS-identified Inhibigens, alone or in combination with an otherwise-protective vaccine, led to accelerated tumor growth, impaired T cell responses, and abrogated tumor immune infiltration. Our current study further explores the mechanism of Inhibigen-specific responses through adoptive transfer of vaccine-experienced T cells into tumor-bearing recipient mice, as well as through analysis of T cell gene expression. Additionally, in order to determine whether Inhibigen identification and treatment translates into pro-tumor effects universally across tumor models, we performed ATLAS screening on CD4+ and CD8+ T cells isolated from mice bearing orthotopic KPC pancreatic cancer. Out of 73 total non-synonymous mutations, we successfully identified 14 CD4+ and 15 CD8+ true neoantigens, and 16 CD4+ and 18 CD8+ Inhibigens. This is the first known comprehensive characterization of endogenous antigens in this model. Therapeutic administration of neoantigens as adjuvanted peptide vaccines in KPC tumor-bearing mice led to smaller tumor sizes and reduced ascites volumes, whereas Inhibigen vaccination accelerated tumor growth. Mouse studies are ongoing and additional data will be presented. Taken together, our data from human cancer patients and two mouse cancer models support the importance of appropriate neoantigen selection and Inhibigen identification and exclusion from cancer therapies. Genocea’s GEN-011 neoantigen-targeted peripheral T cell (NPT) therapy candidate, designed using ATLAS-identified neoantigens and omitting Inhibigens, is being evaluated in an ongoing clinical trial (NCT04596033). Continued exploration of mechanisms of action of Inhibigen-specific responses may reveal new paradigms of cancer immune evasion. 1H Lam et al, Cancer Discov 2021;11:1-18 Citation Format: Hanna S. Starobinets, Victoria L. DeVault, Zoe C. Schmiechen, Ebony A. Miller, Eduardo Cruz, Meagan R. Rollins, Adam L. Burrack, Stephanie J. Rinaldi, Julie Arnold, Emily Tjon, Kyle Gonzalez, Dimitry Lineker, Hubert Lam, Ingunn M. Stromnes, Jessica B. Flechtner. ATLAS-identified Inhibigen-specific responses accelerate tumor growth in mouse melanoma and pancreatic cancer [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 2088.

Abstract: Background: Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy that is resistant to conventional therapies including monotherapy using PD-1 or PD-L1 inhibition. Combination agonistic anti-CD40 and PD-1/PD-L1 blockade have clinical promise in advanced cancer patients including PDA. The underlying mechanism(s) driving the therapeutic effects of this combination are ill-defined. Here, we create a syngeneic PDA animal model and utilize various genetic tools to assess how CD40 agonist, PD-L1 blockade or the combination impact tumor antigen-specific T cells using fluorescently-labeled peptide:MHC tetramers and cells in the tumor microenvironment. Molecular analyses of tumor cell escape variants is also performed. Methods: We recently developed a high-throughput orthotopic syngeneic KPC pancreatic cancer mouse model that expresses a novel model neoantigen in B6 mice described in Burrack et al., Cell Reports, 2019. We create fluorescently labeled peptide:H-2Db tetramers to track the fate of endogenous pancreatic tumor-antigen specific CD8+ T cells over time. Here, we use this model alone or mixed at a 1:1 ratio of KPC tumor cells that do not express the neoantigen to examine how agnostic anti-CD40 (a single dose, clone FGK145), anti-PDL1 (3 doses, clone 10F.932), or the combination impact tumor growth in the pancreas over time using bioluminescent imaging and high-resolution ultrasound. We use multiparameter flow cytometry to investigate how anti-CD40 +/- PD-L1 blockade impacts the phenotype, longevity and functionality of tetramer-binding T cells over time. We assess how other immune cell lineages are altered systemically and in the tumor microenvironment by quantifying myeloid subpopulations, B cells, NK cells and regulatory T cells following therapy. We use Batf3-/- mice and XCR1VenusDTR mice to assess the role of conventional type I dendritic cells (cDC1s) on therapeutic efficacy. We employ both cytokine and chemokine reporter strains to identify how anti-CD40 +/- PD-L1 blockade impacts inflammatory gene expression in immune cells enriched the tumor microenvironment. We examine the persistence and location of tetramer-binding T cells in the pancreas, lung and liver of mice following tumor eradication. Additionally, we re-derive resistant tumor cells from mice and evaluate the integrity of MHC class I antigen processing and presentation pathways. Finally, single cell sequencing is performed to assess the traits of subpopulations of tumor-antigen specific T cells that correlate with enhanced antitumor activity following therapy. Results: We show that anti-CD40 or anti-PD-L1 monotherapy have significant yet transient antitumor effects in mice with neoantigen+ PDA with distinct effects on tumor specific T cells. Objective responses occur in 100% of the monotherapy treated mice and survival is significantly prolonged. However, tumors recur in 100% of these animals. Tumor escape variants defective in MHC class I protein and Tap1 gene expression following IFN-gamma treatment ultimately emerge. In contrast, combination agonistic anti-CD40 + PD-L1 blockade synergize therapeutically resulting in cures in 60% of the animals and formation of pancreas resident memory T cells that specifically bind tetramer and express CD49a and CD103 following tumor eradication. Mechanistically, the combination selectively expands conventional type 1 dendritic cells (cDC1s) in the spleens and tumors of tumor-bearing animals. cDC1s in PDA are CD11c+MHCII+ and express CD8, CD103 and Xcr1. Using Batf3-/- mice or an Xcr1venusDTR transient cDC1 depletion model, we demonstrate a striking dependency on cDC1s for therapeutic benefit with anti-CD40 or PD-L1 blockade. Unexpectedly, we find that the expansion of cDC1s in pancreatic tumor-bearing animals is partially dependent on Xcr1 expression by DCs. Anti-CD40+PD-L1 blockade significantly expand the number of tetramer-binding T cells that express KLRG1 in PDA. The tetramer-binding T cells remain PD-1+ yet have lower expression of Lag3 and have heightened polyfunctionality as measured by cytokine production. Further studies using chemokine and cytokine reporter models, we uncover key differences in how anti-CD40 and anti-PD-L1 impact inflammatory gene expression by antigen presenting cells in PDA. Finally, we demonstrate the requirement for tumor neoantigen expression for efficacy because in mice that have tumors containing a 50:50 mixture of neoantigen+ pancreatic tumor cells with neoantigen- pancreatic tumor cells, combination anti-CD40 + PD-L1 blockade results in elimination of predominantly those tumor cells that express the neoantigen. Further single cell sequencing data on how this combination impacts tumor-antigen specific T cell subpopulations as well as epitope spreading will be discussed. Conclusions: These findings reveal for the first time to our knowledge that anti-CD40 + PD-L1 blockade synergize via the expansion of cDC1s in pancreatic tumor-bearing animals. Instead of anti-CD40 promoting priming of neoantigen-specific T cells, we find that this combination promotes the systemic expansion and intratumoral accumulation of KLRG1+ tumor-specific T cells that eradicate PDA and form pancreas resident CD49a+CD103+ memory T cells. Citation Format: Adam L. Burrack, Meagan R. Rollins, Ellen J. Spartz, Jackson F. Raynor, Iris Wang, Jason Mitchell, Tsuneyasu Kaisho, Brian Fife, Ross Kedl, Stephen Shen, Ingunn M. Stromnes. Mechanisms governing efficacy of combination CD40 agonist and anti-PD-L1 in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr NG12.

Abstract: T lymphocytes are capable of specific recognition and elimination of target cells. Physiological antigen recognition is mediated by the T cell receptor (TCR), which is an alpha beta heterodimer comprising the products of randomly rearranged V, D, and J genes. The exquisite specificity and functionality of T cells can be leveraged for cancer therapy: specifically, the adoptive transfer of T cells that express tumor‐reactive TCRs can induce regression of solid tumors in patients with advanced cancer. However, the isolation and expression of a tumor antigen‐specific TCRs is a highly involved process that requires identifying an immunogenic epitope, ensuring human cells are of the correct haplotype, performing a laborious T cell expansion process, and carrying out downstream TCR sequencing and cloning. Recent advances in single‐cell sequencing have begun to streamline this process. This protocol synthesizes and expands upon methodologies to generate, isolate, and engineer human T cells with tumor‐reactive TCRs for adoptive cell therapy. Though this process is perhaps more arduous than the alternative strategy of using chimeric antigen receptors (CARs) for engineering, the ability to target intracellular proteins using TCRs substantially increases the types of antigens that can be safely targeted. © 2020 Wiley Periodicals LLC. Basic Protocol 1 : Generation of human autologous dendritic cells from monocytes Basic Protocol 2 : In vitro priming and expansion of human antigen‐specific T cells Basic Protocol 3 : Cloning of antigen‐specific T cell receptors based on single‐cell VDJ sequencing data Basic Protocol 4 : Validation of T cell receptor expression and functionality in vitro Basic Protocol 5 : Rapid expansion of T cell receptor–transduced T cells and human T cell clones