Search

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.

Figure S2: Engineered murine T cells exhibit effector function in response to cognate peptide.

Supplementary Figures 1-4

Figure Legends for Supplemental Figures

Adoptive T-cell therapy using high-affinity T-cell receptors (TCR) to target tumor antigens has potential for improving outcomes in high-grade serous ovarian cancer (HGSOC) patients. Ovarian tumors develop a hostile, multicomponent tumor microenvironment containing suppressive cells, inhibitory ligands, and soluble factors that facilitate evasion of antitumor immune responses. Developing and validating an immunocompetent mouse model of metastatic ovarian cancer that shares antigenic and immunosuppressive qualities of human disease would facilitate establishing effective T-cell therapies. We used deep transcriptome profiling and IHC analysis of human HGSOC tumors and disseminated mouse ID8VEGF tumors to compare immunologic features. We then evaluated the ability of CD8 T cells engineered to express a high-affinity TCR specific for mesothelin, an ovarian cancer antigen, to infiltrate advanced ID8VEGF murine ovarian tumors and control tumor growth. Human CD8 T cells engineered to target mesothelin were also evaluated for ability to kill HLA-A2+ HGSOC lines. IHC and gene-expression profiling revealed striking similarities between tumors of both species, including processing/presentation of a leading candidate target antigen, suppressive immune cell infiltration, and expression of molecules that inhibit T-cell function. Engineered T cells targeting mesothelin infiltrated mouse tumors but became progressively dysfunctional and failed to persist. Treatment with repeated doses of T cells maintained functional activity, significantly prolonging survival of mice harboring late-stage disease at treatment onset. Human CD8 T cells engineered to target mesothelin were tumoricidal for three HGSOC lines. Treatment with engineered T cells may have clinical applicability in patients with advanced-stage HGSOC.

Supplementary Tables

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to therapies, including immune-checkpoint blockade. We investigated two distinct strategies to modulate tumor-associated macrophages (TAM) to enhance cellular therapy targeting mesothelin in an autochthonous PDA mouse model. Administration of an antibody to colony-stimulating factor (anti-Csf1R) depleted Ly6Clow protumorigenic TAMs and significantly enhanced endogenous T-cell intratumoral accumulation. Despite increasing the number of endogenous T cells at the tumor site, as previously reported, TAM depletion had only minimal impact on intratumoral accumulation and persistence of T cells engineered to express a murine mesothelin-specific T-cell receptor (TCR). TAM depletion interfered with the antitumor activity of the infused T cells in PDA, evidenced by reduced tumor cell apoptosis. In contrast, TAM programming with agonistic anti-CD40 increased both Ly6Chigh TAMs and the intratumoral accumulation and longevity of TCR-engineered T cells. Anti-CD40 significantly increased the frequency and number of proliferating and granzyme B+ engineered T cells, and increased tumor cell apoptosis. However, anti-CD40 failed to rescue intratumoral engineered T-cell IFNγ production. Thus, although functional modulation, rather than TAM depletion, enhanced the longevity of engineered T cells and increased tumor cell apoptosis, ultimately, anti-CD40 modulation was insufficient to rescue key effector defects in tumor-reactive T cells. This study highlights critical distinctions between how endogenous T cells that evolve in vivo, and engineered T cells with previously acquired effector activity, respond to modifications of the tumor microenvironment.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to most therapies, including immune checkpoint blockade. To elucidate mechanisms of immunotherapy resistance, we assessed immune parameters in resected human PDA. We demonstrate significant interpatient variability in T-cell number, localization, and phenotype. CD8+ T cells, Foxp3+ regulatory T cells, and PD-1+ and PD-L1+ cells were preferentially enriched in tertiary lymphoid structures that were found in most tumors compared with stroma and tumor cell nests. Tumors containing more CD8+ T cells also had increased granulocytes, CD163+ (M2 immunosuppressive phenotype) macrophages, and FOXP3+ regulatory T cells. PD-L1 was rare on tumor cells, but was expressed by CD163+ macrophages and an additional stromal cell subset commonly found clustered together adjacent to tumor epithelium. The majority of tumoral CD8+ T cells did not express molecules suggestive of recent T-cell receptor (TCR) signaling. However, 41BB+PD-1+ T cells were still significantly enriched in tumors compared with circulation. Tumoral CD8+PD-1+ T cells commonly expressed additional inhibitory receptors, yet were mostly T-BEThi and EOMESlo, consistent with a less terminally exhausted state. Analysis of gene expression and rearranged TCR genes by deep sequencing suggested most patients have a limited tumor-reactive T-cell response. Multiplex immunohistochemistry revealed variable T-cell infiltration based on abundance and location, which may result in different mechanisms of immunotherapy resistance. Overall, the data support the need for therapies that either induce endogenous, or provide engineered, tumor-specific T-cell responses, and concurrently relieve suppressive mechanisms operative at the tumor site. Cancer Immunol Res; 5(11); 978–91. ©2017 AACR.

S1. TLS composition and immunological features of resected PDA. S2. Gating strategy for analyzing CD244 (2B4) and Tim3 expression on CD8 T cells in blood of normal individuals (Nl), in blood of PDA patients (PDA blood) and in tumors. S3. Representative human PDA section stained with the multiplex immunohistochemical panel. S4. Representative images of human PDA from the multiplex without (top row, A and B) and with (bottom, C and D), the stromal vs. tumor regions demarcated and cell numbers quantified. S5. PD-L1+ stromal cells in PDA include CD163+ macrophages (Figure 6A) as well as an additional CD163- cell subset shown here. Supplementary Table 1. Antibodies used for tissue multiplex in Figure 6. Supplementary Table 2. Patient characteristics. Supplementary Table 3. TCRβ CDR3 sequence data in blood and tumors from PDA patients.

SummaryPancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to immunotherapy. The pancreatic tumor microenvironment is shaped and maintained by myeloid cells that outnumber tumor cells. Here, using monocyte fate-mapping PDA mouse models and human tumor tissues, we identify monocytes give rise to most heterogeneous macrophage subpopulations in PDA. We show that monocyte differentiation is governed by the local presence of CD4, but not CD8, T cells. We demonstrate that tumor specific CD4 T cells induce monocyte differentiation into antitumor MHCIIhiproinflammatory macrophages dependent on non-redundant IFNγ and CD40 signaling pathways that suppress tumor growth. Pancreatic tissue-resident macrophages exhibit an immunosuppressive pro-tumor state that is refractory to the modulatory effects of antitumor CD4 T cells. Intratumoral monocytes adopt a pro-tumor fate indistinguishable from tissue-resident macrophages following CD4 T cell depletion. Thus, tumor-specific CD4 T cell governance of monocyte fate promotes immune-mediated control of solid tumors.Highlights▪Circulating monocytes are progenitors to most heterogeneous macrophage subsets in PDA▪Monocyte-derived macrophage acquisition of an MHCIIhiphenotype is dependent on tumor-specific CD4 T cells▪In the absence of CD4 T cells, monocyte-derived macrophages acquire tissue resident macrophage traits and tumors rapidly progress▪IFNγ and CD40 signaling are nonredundant and critical determinants of intratumoral monocyte fate

Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly(1,2). To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1, which encodes a crucial DNA repair protein(3,4), in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence(5–7) in the immune system only. We show that Vav-iCre(+/−);Ercc1(−/fl) mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice(8–10). Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre(+/−);Ercc1(−/fl) or aged wild-type mice into young mice induced senescence in trans, whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre(+/−);Ercc1(−/fl) mice with rapamycin reduced markers of senescence in immune cells and improved immune function(11,12). These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing.

Chronic inflammation and immune evasion are hallmarks of cancer. Cancer promotes T-cell differentiation toward an exhausted, or dysfunctional state, which contributes to immune evasion. In this issue, Lutz and colleagues show that the proinflammatory cytokine IL18 correlates with poor patient prognosis and promotes CD8+ T-cell exhaustion in pancreatic cancer by enhancing IL2R signaling. This link between proinflammatory cytokines and T-cell exhaustion elucidates consequences of modulating cytokine signaling during cancer immunotherapy. See related article by Lutz et al. p. 421 (1) .

BackgroundAchieving 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 (TCRMsln) 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.MethodsHere, 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.ResultsPD-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.ConclusionsAbrogation 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.

Adoptive T-cell therapy using high-affinity T-cell receptors (TCR) to target tumor antigens has potential for improving outcomes in high-grade serous ovarian cancer (HGSOC) patients. Ovarian tumors develop a hostile, multicomponent tumor microenvironment containing suppressive cells, inhibitory ligands, and soluble factors that facilitate evasion of antitumor immune responses. Developing and validating an immunocompetent mouse model of metastatic ovarian cancer that shares antigenic and immunosuppressive qualities of human disease would facilitate establishing effective T-cell therapies. We used deep transcriptome profiling and IHC analysis of human HGSOC tumors and disseminated mouse ID8VEGF tumors to compare immunologic features. We then evaluated the ability of CD8 T cells engineered to express a high-affinity TCR specific for mesothelin, an ovarian cancer antigen, to infiltrate advanced ID8VEGF murine ovarian tumors and control tumor growth. Human CD8 T cells engineered to target mesothelin were also evaluated for ability to kill HLA-A2+ HGSOC lines. IHC and gene-expression profiling revealed striking similarities between tumors of both species, including processing/presentation of a leading candidate target antigen, suppressive immune cell infiltration, and expression of molecules that inhibit T-cell function. Engineered T cells targeting mesothelin infiltrated mouse tumors but became progressively dysfunctional and failed to persist. Treatment with repeated doses of T cells maintained functional activity, significantly prolonging survival of mice harboring late-stage disease at treatment onset. Human CD8 T cells engineered to target mesothelin were tumoricidal for three HGSOC lines. Treatment with engineered T cells may have clinical applicability in patients with advanced-stage HGSOC.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to therapies, including immune-checkpoint blockade. We investigated two distinct strategies to modulate tumor-associated macrophages (TAM) to enhance cellular therapy targeting mesothelin in an autochthonous PDA mouse model. Administration of an antibody to colony-stimulating factor (anti-Csf1R) depleted Ly6Clow protumorigenic TAMs and significantly enhanced endogenous T-cell intratumoral accumulation. Despite increasing the number of endogenous T cells at the tumor site, as previously reported, TAM depletion had only minimal impact on intratumoral accumulation and persistence of T cells engineered to express a murine mesothelin-specific T-cell receptor (TCR). TAM depletion interfered with the antitumor activity of the infused T cells in PDA, evidenced by reduced tumor cell apoptosis. In contrast, TAM programming with agonistic anti-CD40 increased both Ly6Chigh TAMs and the intratumoral accumulation and longevity of TCR-engineered T cells. Anti-CD40 significantly increased the frequency and number of proliferating and granzyme B+ engineered T cells, and increased tumor cell apoptosis. However, anti-CD40 failed to rescue intratumoral engineered T-cell IFNγ production. Thus, although functional modulation, rather than TAM depletion, enhanced the longevity of engineered T cells and increased tumor cell apoptosis, ultimately, anti-CD40 modulation was insufficient to rescue key effector defects in tumor-reactive T cells. This study highlights critical distinctions between how endogenous T cells that evolve in vivo, and engineered T cells with previously acquired effector activity, respond to modifications of the tumor microenvironment.

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.

Pancreatic cancer is a particularly lethal malignancy that resists immunotherapy. In this study, using a preclinical pancreatic cancer murine model, we demonstrate a progressive decrease in IFN-γ and granzyme B and a concomitant increase in Tox and IL-10 in intratumoral tumor-specific T cells. Intratumoral myeloid cells produced elevated IL-27, a cytokine that correlates with poor patient outcome. Abrogating IL-27 signaling significantly decreased intratumoral Tox+ T cells and delayed tumor growth yet was not curative. Agonistic αCD40 decreased intratumoral IL-27–producing myeloid cells, decreased IL-10–producing intratumoral T cells, and promoted intratumoral Klrg1+Gzmb+ short-lived effector T cells. Combination agonistic αCD40+αPD-L1 cured 63% of tumor-bearing animals, promoted rejection following tumor rechallenge, and correlated with a 2-log increase in pancreas-residing tumor-specific T cells. Interfering with Ifngr1 expression in nontumor/host cells abrogated agonistic αCD40+αPD-L1 efficacy. In contrast, interfering with nontumor/host cell Tnfrsf1a led to cure in 100% of animals following agonistic αCD40+αPD-L1 and promoted the formation of circulating central memory T cells rather than long-lived effector T cells. In summary, we identify a mechanistic basis for T cell exhaustion in pancreatic cancer and a feasible clinical strategy to overcome it.

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.

BackgroundPancreatic ductal adenocarcinoma (PDA) is a lethal malignancy characterized by a highly suppressive tumor microenvironment. Despite this, engineered T cell therapy has promise for effectively targeting PDA. To identify the underlying mechanisms of antigen-specific engineered T cell immunosuppression in PDA, we create novel TCR knock-in mouse models for a robust and standardized source of naïve mesothelin (Msln)-specific T cells.MethodsSpecifically, we integrate two murine mesothelin-specific TCRs into the physiologic Trac locus in primary murine T cells and zygotes using CRISPR/Cas9 and rAAV expressing the TCR DNA. Simultaneously using CRISPR/Cas9, Msln was disrupted to circumvent T cell tolerance.ResultsThis strategy resulted in the rapid generation of homozygous TCR Trac knock-in mice and with homozygous null mutations in Msln. In these TCR-exchanged (TRex) mice, most T cells expressed the 1045 (high affinity) or 7431 (low affinity) as determined by tetramer staining. TRex T cells exhibit a naïve phenotype and rapidly differentiate into effector T cells upon antigenic stimulation. While the high affinity 1045 TCR elicits function in CD4 T cells, the lower affinity 7431 T cells exhibit a higher functional avidity and less TCR downregulation when antigen is limiting. Historical TCR transgenic T cells, in which the TCR is randomly integrated into the genome, exhibit increased PD1, CD25, and CD69, decreased functionality, and a bias to CD25-Foxp3+ Treg as compared to T cells from TRex mice. Further, TCR Trac integration in primary T cells retain superior function following repetitive antigenic stimulations retrovirally transduced T cells. Adoptive transfer of 1045 TRex T cells significantly prolongs survival of mice bearing autochthonous PDA. When combined with a vaccine, 1045 TRex T cells cause involution of the fibroinflammatory tumor stroma.ConclusionsIn sum, we rapidly generate mice that physiologically express the desired TCR, circumventing the shortcomings of standard T cell engineering strategies and TCR transgenic models.Ethics ApprovalUniversity of Minnesota Institutional Animal Care and Use Committee approved all animal studies to Dr. Ingunn Stromnes (2005-38115A.) Generation of TCR knockin (KI) animals was performed in the Mouse Genetic Laboratory at the University of Minnesota.

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.

Patients with resectable tumor, either in the body or the tail of the pancreas, and cancer patients with a primary tumor adjacent to the splenic vasculature frequently undergo a splenectomy as standard of care during resection. The spleen provides an unutilized source of lymphocytes with potential utility for adoptive cellular therapy (ACT). In this report, spleen and peripheral blood (PB) cells from cancer patients were compared to one another and normal PB by flow cytometry with a focus on CD8+ T-cells, memory phenotype, and their relative expression of checkpoint proteins including program death ligand-1 (PD1). PD1 is both an activation marker for T-cells including antigen (Ag) specific responses, as well as a marker of T-cell exhaustion associated with co-expression of other checkpoint molecules such as lymphocyte activating gene-3 (LAG-3) and T-cell immunoglobulin and mucin domain containing-3 (TIM-3). In summary, the spleen is a rich source of CD8+PD1+ T-cells, with an 8-fold higher frequency compared to the PB. These CD8+ T-cells are predominantly central and transitional memory T-cells with associated effector phenotypes and low expression of TIM-3 and LAG-3 with potential utility for ACT".

Adoptive cellular therapy (ACT) has progressed; however, it remains ineffective for most solid tumors. Further, neoadjuvant patients frequently lack tumor tissue sufficient to isolate and expand tumor infiltrating lymphocytes (TIL’s) that are the primary source of T-cells for ACT. However, patients with tumors near the splenic vasculature often undergo a splenectomy, providing a cellular resource for ACT. We document herein that spleens provide a high frequency and number of CD8+ T-cells, with low expression of exhaustion markers (program death ligand-1 (PD-1), T-cell immunoglobulin and mucin domain containing-3 (TIM-3), and lymphocyte activation gene-3 (LAG-3)) relative to the peripheral blood (PB). The memory phenotypes of these CD8+ T-cells (predominantly central and transitional) support their potential for expansion and their high frequency of PD-1 expression indicates tumor specificity as PD-1, is a surrogate for tumor specificity. Further, spleens have a low frequency of myeloid derived suppressor cells (MDSCs) subsets vs. patient PB. Our preliminary studies with spleens from 19 abdominal tumor patients revealed that the spleens, relative to the PB from the same patients, have a significantly higher frequency of CD8+ and PD-1+CD8+ T-cells, including central and transitional memory T-cells. Further, the CD8+PD-1+ T-cells in the spleen also have a low expression of TIM-3 and LAG-3 suggesting minimal exhaustion as supported by ELISPOT analysis of tumor specific CD8+ T-cells responses. Based on our studies we suggest that resected spleens from cancer patients including pancreatic ductal adenocarcinoma (PDAC) patients can provide a valuable source for ACT due to their high frequency of CD8+PD-1+ T-cells.

We have been exploring in preclinical models and clinical trials methods to reproducibly provide therapeutic T-cell responses by transfer of genetically engineered T cells. Our largest clinical experience has been in treating human acute myelogenous leukemia (AML), in which we have utilized a high-affinity TCR specific for WT1, a protein associated with promoting leukemic transformation that is overexpressed in human leukemic stem cells, to genetically engineer CD8 T cells. We recently reported a study (Chapuis et al., Nat Med 2019) in which we treated leukemia patients at high risk of relapse (after hematopoietic cell transplant) that demonstrated all treated patients remain alive and relapse free at a median of 48 months, compared to a relapse rate of ~35% in the concurrent matched cohort (p Citation Format: Philip D. Greenberg, Kristin G. Anderson, Dan Egan, Sunil R. Hingorani, Luigi Nezi, Teresa Manzo, Shannon K. Oda, Kelly G. Paulson, Rachel Perret, Leah Schmidt, Tom M. Schmitt, Ingunn M. Stromnes, Aude G. Chapuis. Targeting pancreatic cancer with TCR-engineered T cells [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I11.

Metastasis and therapy resistance are cardinal features of pancreatic ductal adenocarcinoma, a commonly lethal malignancy. In this issue of Cancer Cell, Steele et al. show that CXCR2 expression and neutrophils are required for metastasis. In mice treated with advanced disease, inhibiting both CXCR2 and PD1 cooperatively but not individually prolongs survival.

The efficacy of rituximab treatment in multiple sclerosis has renewed interest in the role of B cells in CNS autoimmunity. In this study, we show that B cells are the predominant MHC class II+ subset in the naive CNS in mice, and they constitutively express proinflammatory cytokines. Incidence of experimental autoimmune encephalomyelitis induced by adoptive transfer was significantly reduced in C3HeB/Fej μMT (B cell–deficient) mice, suggesting an important role for CNS B cells in initiating inflammatory responses. Initial T cell infiltration of the CNS occurred normally in μMT mice; however, lack of production of T cell cytokines and other immune mediators indicated impaired T cell reactivation. Subsequent recruitment of immune cells from the periphery driven by this initial T cell reactivation did not occur in μMT mice. B cells required exogenous IL-1β to reactivate Th17 but not Th1 cells in vitro. Similarly, reactivation of Th1 cells infiltrating the CNS was selectively impaired compared with Th17 cells in μMT mice, causing an increased Th17/Th1 ratio in the CNS at experimental autoimmune encephalomyelitis onset and enhanced brain inflammation. These studies reveal an important role for B cells within the CNS in reactivating T cells and influencing the clinical manifestation of disease.

We have been exploring in preclinical models and clinical trials methods to reproducibly provide therapeutic T cell responses by transfer of genetically engineered T cells. Our largest clinical experience is in treating human Acute Myelogenous Leukemia (AML). After identifying that WT1, a gene associated with promoting leukemic transformation, is over-expressed in human leukemic stem cells, and demonstrating in a clinical trial that in vitro expanded WT1-specific CD8 T cell clones can be safely transferred, exhibit anti-leukemic activity, and provide therapeutic benefit to AML patients, we extensively screened normal human repertoires and isolated a high affinity TCR specific for WT1 for genetically engineering CD8 T cells to reproducibly create cells with high avidity for leukemic cells. We have initially pursued this strategy in a 2 Arm trial for leukemia patients either at high risk of relapse (Arm 1) after hematopoietic cell transplant (HCT) or who have already relapsed after HCT (Arm 2). The prophylactic arm is now completed, with very encouraging results- all patients treated with engineered T cells remain alive and relapse free at a median of 38 months, compared to a relapse rate of ~50% in a concurrent matched cohort. Results in relapsed patients (Arm 2) have been less effective, and we have used high-dimensional analyses including single cell RNAseq both to elucidate the reasons for failure to eradicate the leukemia and to design strategies to overcome these obstacles. Our results have identified several mechanisms by which the leukemia escapes, and we have been testing approaches that employ further genetic modification of the T cells to enhance efficacy. The predominant reason for leukemia progression despite targeted therapy with T cells is the inability of the T cells to persist and maintain function in the context of encountering a rapidly proliferating myeloid leukemia. This reflects both engagement of pathways inhibitory to T cells by the leukemic cells, and apoptosis of the T cells from repetitive stimulation. We are addressing this issue by creating immuno-modulatory fusion proteins (IFPs) that have the ectodomain of an inhibitory or death receptor fused to a survival costimulatory domain. Results with two such IFPs will be discussed, a CD200R/CD28 fusion that binds the inhibitory ligand CD200 commonly expressed on leukemic cells but provides a CD28 costimulatory signal and a Fas/4-1BB fusion that binds FasL but rather than induce death promotes proliferation and survival. A more uncommon reason explaining progression is loss of expression of the WT1 epitope being targeted. This has occurred in 2 patients, but for distinct reasons. In one patient this reflected loss of a component of the immunoproteasome, and we have now isolated a TCR that recognizes an epitope not dependent on the immunoproteasome. In a second patient the level of WT1 expression declined- interestingly, this patient was treated with Vidaza, which can increase expression of WT1, and post-Vidaza the transferred T cells persisting in the patient’s bone marrow recognized and responded in vivo to the relapsing leukemia. The approaches and technologies we are developing and testing in leukemia are also applicable to solid tumors, and preclinical studies in pancreatic and ovarian cancers will be discussed. Citation Format: Kristin G. Anderson, Dan Egan, Sunil R. Hingorani, Shannon K. Oda, Kelly Paulson, Rachel Perret, Leah Schmidt, Thomas Schmitt, Ingunn Stromnes, Aude Chapuis, Philip D. Greenberg. Utilizing synthetic biology and high-dimensional probing to address therapeutic obstacles and empower engineered T cells with the capacity to eradicate tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA17.

Effective cellular therapy for human malignancies requires first identifying and validating an appropriate antigenic target, and then establishing in each patient a tumor-reactive T cell response of high avidity and high magnitude that is safe and can infiltrate and retain function in the tumor microenvironment. We have been exploring in preclinical models and clinical trials methods to reproducibly provide such responses by transfer of genetically engineered T cells that acquire target specificity by virtue of an introduced high affinity TCR. To identify candidate antigens in leukema, we examined purified human leukemic stem cells for over-expression of genes based on comparisons to purified human hematopoietic stem cells as well as normal somatic tissues. Our analysis revealed that WT1, a gene known to be associated with promoting leukemic transformation, is expressed in comparative abundance in human leukemic stem cells. Preclinical studies were then performed in a mouse model, and revealed that CD8 T cells specific for this oncogene with even higher avidity than can be detected in normal repertoires could be safely administered, with no evidence of toxicity to the normal tissues known to express low but detectable levels of WT1. For our initial clinical trial, poor prognosis leukemia patients who relapsed after hematopoietic cell transplant (HCT) were treated with transfer of WT1-specific CD8 T cells clones isolated and expanded in vitro from the HCT donor. This study demonstrated that such T cells were safe, mediated in vivo anti-leukemic activity, and were associated with maintenance of long-term remissions in some patients, but generating sufficient numbers of WT1-specific CD8 T cells with high avidity for the target in each patient represented a substantive problem. Therefore, to create a more predictably effective standardized reagent for treatment of patients with a tumor that expresses the target antigen and shares the associated MHC restricting allele, we pursued methods to genetically engineer patient T cells to acquire high avidity for the tumor target. This requires identifying a high affinity TCR and producing a vector that can achieve high-level expression of the genes encoding the Vα and Vβ genes of a TCR demonstrated to have high affinity for the target epitope. Therefore, we screened a large number of normal repertoires for the presence of high avidity WT1-specific CD8 T cells, and selected the T cell clone expressing the highest affinity TCR. We then incorporated changes in the TCR genes such as codon optimization to enhance expression, and introduced a point mutation in each chain to create a disulfide bond that minimizes the potential problem of mispairing of the introduced TCR chains with the endogenous TCR chains. We have now have now initiated a trial in which this high affinity, WT1-specific, HLA-A2-restricted TCR is being introduced into patient CD8 T cells with a lentiviral vector and the transduced cells are being infused into the patient. The early results from this trial appear promising in terms of both evidence of antileukemic activity and the capacity for the transferred cells to persist in patients, and we plan to begin very shortly another trial in patients with non-small cell lung cancer (NSCLC) utilizing this same TCR, as WT1 is also commonly overexpressed in NSCLC as well as many other malignancies. For many candidate target antigens that are also normal self-antigens, isolating high affinity TCRs may not be readily achieved from normal repertoires. Therefore, we have developed strategies to enhance the affinity of isolated TCRs with retention of specificity, including saturation mutagenesis of CDR3 regions and an in vitro thymic selection system that allows for capture of a more diverse set of high affinity specific TCR genes during TCR gene rearrangement. These approaches induce modifications to the TCR region that predominantly makes contacts with the peptide epitope rather than MHC, which is necessary to minimize the risk of off-target toxicity from promiscuous peptide/MHC recognition. However, it remains essential that such modified TCRs do not induce unanticipated tissue damage, and we are using bioinformatics as well as modeling in the mouse to uncover any potential for off-target toxicity. Unfortunately, providing a high avidity T cell response does not necessarily result in tumor eradication, as there are other substantive obstacles that can preclude even a T cell expressing a high affinity TCR from being effective. These impediments include the development of T cell dysfunction, particularly within the microenvironment of solid tumors, and we are using genetically engineered mouse models to elucidate the cellular and molecular pathways that need to be modulated to achieve meaningful therapeutic benefit in a variety of solid tumor settings, including pancreatic and ovarian cancer. Our preclinical therapy studies, particularly in a pancreatic ductal adenocarcinoma (PDA) model, already appear very promising, as we have demonstrated that T cells expressing a high affinity TCR targeting a tumor antigen expressed by PDA cells can infiltrate the tumor, mediate tumor lysis, modify the tumor stroma, and provide therapeutic benefit. We have already identified high affinity human TCRs specific for this tumor antigen, and plan to use the insights derived from these studies to initiate within the next 1-2 years clinical trials in human pancreatic and ovarian cancers. The genetically-engineered mouse models of spontaneously developing tumors we are using, which recapitulate many aspects of the analogous human cancer, are also making it possible to assess strategies to improve the efficacy of T cell therapy. These models have helped elucidate the importance of not only cell extrinsic mechanisms of regulation and dysfunction that render T cells unresponsive, particularly via inhibitory cells commonly present in the tumor microenvironment that interfere with an effector response such as the accumulation of regulatory CD4 T cells (Treg), myeloid derived suppressor cells (MDSC), and tumor-associated macrophages (TAM), but also the cell intrinsic mechanisms that derive in large part from persistent stimulation by the tumor antigen and ultimately can render T cells progressively dysfunctional, leading to epigenetic modifications that eventually result in non-responsive cells that cannot be readily rescued. These cumulative mechanisms highlight the difficulties eliciting and/or sustaining responses to tumor antigens. Strategies to disrupt inhibitory pathways by systemic administration of mAbs or cytokines are currently being pursued clinically, but such reagents globally disrupt inhibitory pathways which can have significant toxicity to the host. Therefore, we are evaluating strategies to sustain function and anti-tumor activity by genetically modifying T cells to enhance function and to be resistant to obstacles that prevent tumor eradication. As different tumor types exhibit unique characteristics and are capable of engaging distinct inhibitory pathways, improved understanding of the immunobiology of the tumor type to be treated will likely prove essential for designing effective therapies. However, the relatively straightforward means to use synthetic biology to genetically engineer T cells to acquire novel capacities to overcome inhibitory signals and function in the tumor microenvironment suggests that cancer therapy with engineered T cells will likely find an increasing role in the treatment of human cancers. Citation Format: Philip D. Greenberg, Tom M. Schmitt, Andrea Schietinger, Ingunn M. Stromnes, Sunil R. Hingorani, Shannon K. Oda, Rachel Perret, Kristin G. Anderson, Merav Bar, Aude G. Chapuis. Employing TCRs in engineered T cells to develop therapeutic reagents for effectively targeting malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY31-03. doi:10.1158/1538-7445.AM2015-SY31-03

T cell expression of inhibitory proteins can be a critical component for the regulation of immunopathology owing to self-reactivity or potentially exuberant responses to pathogens, but it may also limit T cell responses to some malignancies, particularly if the tumor Ag being targeted is a self-protein. We found that the abrogation of Src homology region 2 domain-containing phosphatase-1 (SHP-1) in tumor-reactive CD8+ T cells improves the therapeutic outcome of adoptive immunotherapy in a mouse model of disseminated leukemia, with benefit observed in therapy employing transfer of CD8+ T cells alone or in the context of also providing supplemental IL-2. SHP-1−/− and SHP-1+/+ effector T cells were expanded in vitro for immunotherapy. Following transfer in vivo, the SHP-1−/− effector T cells exhibited enhanced short-term accumulation, followed by greater contraction, and they ultimately formed similar numbers of long-lived, functional memory cells. The increased therapeutic effectiveness of SHP-1−/− effector cells was also observed in recipients that expressed the tumor Ag as a self-antigen in the liver, without evidence of inducing autoimmune toxicity. SHP-1−/− effector CD8+ T cells expressed higher levels of eomesodermin, which correlated with enhanced lysis of tumor cells. Furthermore, reduction of SHP-1 expression in tumor-reactive effector T cells by retroviral transduction with vectors that express SHP-1–specific small interfering RNA, a translatable strategy, also exhibited enhanced antitumor activity in vivo. These studies suggest that abrogating SHP-1 in effector T cells may improve the efficacy of tumor elimination by T cell therapy without affecting the ability of the effector cells to persist and provide a long-term response.

Unlike cancer vaccines and immune modulators such as checkpoint inhibitors that seek to harness patient immune responses, adoptive therapy with genetically engineered T cells seeks to create responses that don’t exist in the patient’s immune system. Molecular technologies now make it feasible to not only create T cells with specificity for the tumor by introduction of a selected antigen-specific receptor, but also with qualities not naturally found, including improved function and resistance to immunosuppression. We have been exploring in preclinical models and clinical trials methods to reproducibly provide therapeutic T cell responses by transfer of genetically engineered T cells. For human acute myelogenous leukemia (AML), we have pursued targeting WT1, a gene overexpressed in human leukemic stem cells that is associated with promoting leukemic transformation. Preclinical studies performed in a mouse model demonstrated that CD8 T cells expressing a high affinity TCR specific for this oncogene can be safely administered, with no evidence of toxicity to the normal tissues known to express low but detectable levels of WT1. We have advanced this approach to a clinical trial in leukemia patients with poor prognostic factors that place them at high risk of relapse after hematopoietic cell transplant (HCT), using a high-affinity human TCR specific for WT1 to transduce CD8 cells and reproducibly create high-avidity T cells that recognize leukemic cells. Our clinical results demonstrate that such T cells can prevent leukemic relapse and sustain long-term remissions, and can mediate antileukemic activity in patients who have relapsed. This therapy is now being tested in AML patients who have minimal residual disease after induction therapy and are not candidates for HCT, as well as in solid tumors that similarly overexpress WT1. Unfortunately, there are substantive obstacles in targeting established tumors that can preclude even a T cell expressing a high-affinity TCR from being effective. These impediments include the development of T cell dysfunction, particularly within the microenvironment of solid tumors, and we are using genetically engineered mouse models to elucidate the cellular and molecular pathways that need to be modulated to achieve meaningful therapeutic benefit in a variety of hematologic and solid tumor settings, including pancreatic and ovarian cancer. Our preclinical therapy studies reveal promising antitumor activity, but demonstrate that repeated infusions of functional T cells are required to sustain a therapeutic response in the context of the immunosuppressive tumor microenvironment, and we are engineering T cells to overcome these inhibitory signals and enhance efficacy. In place of current strategies that disrupt inhibitory pathways by systemic administration of blocking mAbs, which globally disrupt immune regulation and thus can have significant toxicity to the host, we are creating synthetic immunomodulatory fusion proteins that take advantage of the expression of inhibitory ligands by tumors by still binding the inhibitory ligand but alternatively delivering a costimulatory rather than inhibitory signal. Additionally, as the antitumor activity of CD8 T cells is enhanced by a concurrent CD4 T cell response, we are engineering CD4 T cells as well as CD8 T cells to create an orchestrated antitumor response. The results suggest that cancer therapy with engineered T cells can provide effective antitumor responses and will likely find an increasing role in the treatment of human cancers. Citation Format: Philip D. Greenberg, Kristin G. Anderson, Dan Egan, Sunil R. Hingorani, Shannon K. Oda, Rachel Perret, Tom M. Schmitt, Ingunn M. Stromnes, Leah Schmidt, Aude G. Chapuis. Engineering T cells to eradicate tumors in the age of synthetic biology [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA02.

The immune system, T cells in particular, have the ability to target and destroy malignant cells. However, antitumor immune responses induced from the endogenous T-cell repertoire are often insufficient for the eradication of established tumors, as illustrated by the failure of cancer vaccination strategies or checkpoint blockade for most tumors. Genetic modification of T cells to express a defined T-cell receptor (TCR) can provide the means to rapidly generate large numbers of tumor-reactive T cells capable of targeting tumor cells in vivo. However, cell-intrinsic factors as well as immunosuppressive factors in the tumor microenvironment can limit the function of such gene-modified T cells. New strategies currently being developed are refining and enhancing this approach, resulting in cellular therapies that more effectively target tumors and that are less susceptible to tumor immune evasion. Clin Cancer Res; 21(23); 5191–7. ©2015 AACR.

SummaryPancreatic ductal adenocarcinomas (PDAs) erect physical barriers to chemotherapy and induce multiple mechanisms of immune suppression, creating a sanctuary for unimpeded growth. We tested the ability of T cells engineered to express an affinity-enhanced T cell receptor (TCR) against a native antigen to overcome these barriers in a genetically engineered model of autochthonous PDA. Engineered T cells preferentially accumulate in PDA and induce tumor cell death and stromal remodeling. However, tumor-infiltrating T cells become progressively dysfunctional, a limitation successfully overcome by serial T cell infusions that resulted in a near-doubling of survival without overt toxicities. Similarly engineered human T cells lyse PDA cells in vitro, further supporting clinical advancement of this TCR-based strategy for the treatment of PDA.

Experimental allergic encephalomyelitis (EAE) is a widely used animal model of the human demyelinating disease multiple sclerosis. EAE is initiated by immunization with myelin antigens in adjuvant or by adoptive transfer of myelin-specific T cells, resulting in inflammatory infiltrates and demyelination in the central nervous system. Induction of EAE in rodents typically results in ascending flaccid paralysis with inflammation primarily targeting the spinal cord. This protocol describes passive induction of EAE by adoptive transfer of T cells isolated from mice primed with myelin antigens into naïve mice. The advantages of using this method versus active induction of EAE are discussed.

This protocol details a method to actively induce experimental allergic encephalomyelitis (EAE), a widely used animal model for studies of multiple sclerosis. EAE is induced by stimulating T-cell-mediated immunity to myelin antigens. Active induction of EAE is accomplished by immunization with myelin antigens emulsified in adjuvant. This protocol focuses on induction of EAE in mice; however, the same principles apply to EAE induction in other species. EAE in rodents is manifested typically as ascending flaccid paralysis with inflammation targeting the spinal cord. However, more diverse clinical signs can occur in certain strain/antigen combinations in rodents and in other species, reflecting increased inflammation in the brain.

Myelin basic protein-specific CD8(+) T cells can induce central nervous system autoimmunity; however, immune tolerance prevents these autoreactive cells from causing disease. To define the mechanisms that mediate tolerance, we developed two T cell receptor-transgenic mouse lines with different affinities for the H-2K(k)-restricted myelin basic protein epitope consisting of amino acids 79-87 (MBP(79-87)). We observed both thymic deletion and peripheral tolerance in the lower-affinity T cells. The higher-affinity T cells, however, showed no evidence of tolerance induction and were able to prevent tolerance of the lower-affinity T cells by removing H-2K(k)-MBP(79-87) complexes from antigen-presenting cells without proliferating. This form of immune regulation could limit responses of self-reactive T cells that escape other tolerance mechanisms.

The establishment of viral persistence generally requires evasion of the host CD8(+) T cell response. Here we describe a form of evasion wherein the CD8(+) T cells are fully capable of recognizing their cognate antigen but their effector functions are suppressed by regulatory T cells. Virus-specific CD8(+) T cells adoptively transferred into mice persistently infected with Friend virus proliferated and appeared activated, but failed to produce IFNgamma or reduce virus loads. Cotransfer experiments revealed that a subpopulation of CD4(+) T cells from persistently infected mice suppressed IFNgamma production by the CD8(+) T cells. Treatment of persistently infected mice with anti-GITR antibody to ameliorate suppression by regulatory T cells significantly improved IFNgamma production by transferred CD8(+) T cells and allowed a significant reduction in viral loads. The results indicate that CD4(+) regulatory T cells contribute to viral persistence and demonstrate an immunotherapy for treating chronic retroviral infections.

The current studies demonstrate complex and seemingly contradictory effects by gamma interferon (IFN-γ) on Friend virus (FV) infection. Both temporal and tissue-specific effects were observed. During the first week of infection, IFN-γ-deficiency caused increased levels of FV infection in multiple tissues. Surprisingly, however, by 2 weeks postinfection, IFN-γ-deficient mice had significantly lower levels of infection in both the spleen and bone marrow compared to wild-type mice. The rapid reduction of virus in the IFN-γ-deficient mice correlated with a more rapid virus-neutralizing antibody response than was observed in the wild-type mice. Furthermore, the virus-neutralizing antibody response in wild-type mice could be accelerated by ablation of their IFN-γ response. Although the IFN-γ-deficient mice developed an accelerated virus-neutralizing antibody response, they did not class-switch to immunoglobulin G class immunoglobulins nor could they maintain long-term virus-neutralizing antibody titers. Eventually, all of the IFN-γ-deficient mice failed to keep persistent virus in check and developed fatal FV-induced erythroleukemia.

Effective cellular therapy for human malignancies requires first identifying and validating an appropriate antigenic target, and then establishing in each patient a tumor-reactive T cell response of high avidity and high magnitude that is not only safe but can infiltrate and retain function in the tumor microenvironment. We have used molecular expression profiling to detect antigens selectively or markedly over-expressed by tumors, and then used these antigens as stimuli to generate T cells from normal repertoires. We have developed a high throughput technology to identify those T cells that express high affinity TCRs, and to then isolate from these T cells the TCR genes, place them either directly or after affinity enhancement into shuttle vectors, and use these reagents to create recipient T cells with high avidity for tumor targets that can be administered in vivo. We have utilized in silico, in vitro, and preclinical mouse models to assess the safety and potential efficacy of T cells expressing such TCRs. We are currently pursuing targeting of 3 antigens that are expressed in both murine and human tumors and are pro-oncogenic, contributing to the malignant phenotype. This includes ongoing clinical trials that will be discussed in acute myelogenous leukemia and in non-small cell lung cancer or mesothelioma targeting WT1 with T cells transduced to express a high affinity TCR specific for WT1, as well as trials being designed to target Mesothelin (MSLN) with T cells transduced to express a high affinity TCR specific for MSLN in pancreatic and ovarian cancer that are anticipated to begin within 8-12 months. Our clinical results in treatment of AML and preclinical results in mouse models of pancreatic and ovarian cancer appear very promising. However, the composite clinical data, as well as the results in the preclinical mouse models that drive our clinical trials, demonstrate substantial obstacles to sustaining T cell function in vivo after transfer, particularly in the context of solid tumors. Engineered T cells with specificity for tumor antigens appear to have the capacity to infiltrate and accumulate in solid tumors, and to initially mediate anti-tumor activity, but frequently become dysfunctional in the tumor microenvironment. Studies are being pursued to identify the critical obstacles to maintaining T cell function and achieving more reproducible tumor eradication, including modulating the tumor microenvironment and engineering T cells to express immunomodulatory fusion proteins (IFP) that recognize ligands for inhibitory signals but deliver an activation/costimulatory signal. Our data suggest that engineering T cells to acquire novel properties not naturally found in unmanipulated T cells has the potential to create effective therapies for human cancers. Citation Format: Philip D. Greenberg, Aude Chapuis, Dan Egan, Ingunn Stromnes, Sunil Hingorani, Shannon Oda, Rachel Perret, Kristin Anderson, Tom Schmitt. Building better T cells for targeting and eliminating tumors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA18.

Cancer-induced inflammation results in accumulation of myeloid cells. These myeloid cells include progenitors and progeny of monocytes, granulocytes, macrophages, and dendritic cells. It has become increasingly evident that tumor-dependent factors can condition myeloid cells toward an immunosuppressive and protumorigenic phenotype. Thus, myeloid cells are not simply bystanders in malignancy or barometers of disease burden. Reflecting their dynamic and plastic nature, myeloid cells manifest a continuum of cellular differentiation and are intimately involved at all stages of neoplastic progression. They can promote tumorigenesis through both immune-dependent and -independent mechanisms and can dictate response to therapies. A greater understanding of the inherent plasticity and relationships among myeloid subsets is needed to inform therapeutic targeting. New clinical trials are being designed to modulate the activities of myeloid cells in cancer, which may be essential to maximize the efficacy of both conventional cytotoxic and immune-based therapies for solid tumors. Clin Cancer Res; 20(20); 5157–70. ©2014 AACR.

Pancreatic ductal adenocarcinoma co-opts multiple cellular and extracellular mechanisms to create a complex cancer organ with an unusual proclivity for metastasis and resistance to therapy. Cell-autonomous events are essential for the initiation and maintenance of pancreatic ductal adenocarcinoma, but recent studies have implicated critical non-cell autonomous processes within the robust desmoplastic stroma that promote disease pathogenesis and resistance. Thus, non-malignant cells and associated factors are culprits in tumor growth, immunosuppression and invasion. However, even this increasing awareness of non-cell autonomous contributions to disease progression is tempered by the conflicting roles stromal elements can play. A greater understanding of stromal complexity and complicity has been aided in part by studies in highly faithful genetically engineered mouse models of pancreatic ductal adenocarcinoma. Insights gleaned from such studies are spurring the development of therapies designed to reengineer the pancreas cancer stroma and render it permissive to agents targeting cell-autonomous events or to reinstate immunosurveillance. Integrating conventional and immunological treatments in the context of stromal targeting may provide the key to a durable clinical impact on this formidable disease.

Background Pancreatic ductal adenocarcinoma (PDA) is characterised by a robust desmoplasia, including the notable accumulation of immunosuppressive cells that shield neoplastic cells from immune detection. Immune evasion may be further enhanced if the malignant cells fail to express high levels of antigens that are sufficiently immunogenic to engender an effector T cell response. Objective To investigate the predominant subsets of immunosuppressive cancer-conditioned myeloid cells that chronicle and shape the progression of pancreas cancer. We show that selective depletion of one subset of myeloid-derived suppressor cells (MDSC) in an autochthonous, genetically engineered mouse model (GEMM) of PDA unmasks the ability of the adaptive immune response to engage and target tumour epithelial cells. Methods A combination of in vivo and in vitro studies were performed employing a GEMM that faithfully recapitulates the cardinal features of human PDA. The predominant cancer-conditioned myeloid cell subpopulation was specifically targeted in vivo and the biological outcomes determined. Results PDA orchestrates the induction of distinct subsets of cancer-associated myeloid cells through the production of factors known to influence myelopoiesis. These immature myeloid cells inhibit the proliferation and induce apoptosis of activated T cells. Targeted depletion of granulocytic MDSC (Gr-MDSC) in autochthonous PDA increases the intratumoral accumulation of activated CD8 T cells and apoptosis of tumour epithelial cells and also remodels the tumour stroma. Conclusions Neoplastic ductal cells of the pancreas induce distinct myeloid cell subsets that promote tumour cell survival and accumulation. Targeted depletion of a single myeloid subset, the Gr-MDSC, can unmask an endogenous T cell response, disclosing an unexpected latent immunity and invoking targeting of Gr-MDSC as a potential strategy to exploit for treating this highly lethal disease.

Normal levels of CD4+regulatory T cells are critical for the maintenance of immunological homeostasis and the prevention of autoimmune diseases. However, we now show that the expansion of CD4+regulatory T cells in response to a chronic viral infection can lead to immunosuppression. Mice persistently infected with Friend retrovirus develop approximately twice the normal percentage of splenic CD4+regulatory T cells and lose their ability to reject certain tumor transplants. The role of CD4+regulatory T cells was demonstrated by the transmission of immunosuppression to uninfected mice by adoptive transfers of CD4+T cells. CD4+T cells from chronically infected mice were also immunosuppressivein vitro, inhibiting the generation of cytolytic T lymphocytes in mixed lymphocyte cultures. Inhibition occurred at the level of blast-cell formation through a mechanism or mechanisms involving transforming growth factor-β and the cell surface molecule CTLA-4 (CD152). These results suggest a possible explanation for HIV- and human T cell leukemia virus-I-induced immunosuppression in the absence of T cell depletion.

Adoptive T-cell therapy involves the isolation, expansion, and reinfusion of T lymphocytes with a defined specificity and function as a means to eradicate cancer. Our research has focused on specifying the requirements for tumor eradication with antigen-specific T cells and T cells transduced to express a defined T-cell receptor (TCR) in mouse models and then translating these strategies to clinical trials. Our design of T-cell-based therapy for cancer has reflected efforts to identify the obstacles that limit sustained effector T-cell activity in mice and humans, design approaches to enhance T-cell persistence, develop methods to increase TCR affinity/T-cell functional avidity, and pursue strategies to overcome tolerance and immunosuppression. With the advent of genetic engineering, a highly functional population of T cells can now be rapidly generated and tailored for the targeted malignancy. Preclinical studies in faithful and informative mouse models, in concert with knowledge gained from analyses of successes and limitations in clinical trials, are shaping how we continue to develop, refine, and broaden the applicability of this approach for cancer therapy.

Many of the most promising tumor antigens for T-cell-based cancer immunotherapies are unmodified self-antigens. Unfortunately, the avidity of T cells specific for these antigens is limited by central tolerance during T-cell development in the thymus, resulting in decreased anti-tumor efficacy of these T cells. One approach to overcoming this obstacle is to mutate T-cell receptor (TCR) genes from naturally occurring T cells to enhance the affinity for the target antigen. These enhanced-affinity TCRs can then be developed for use in TCR gene therapy. Although TCRs with significantly enhanced affinity have been generated using this approach, it is not clear whether these TCRs, which bypass the affinity limits imposed by negative selection, remain unresponsive to the low levels of self-antigen generally expressed by some normal tissues. Here we show that 2 variants of a high-affinity WT1-specific TCR with enhanced affinity for WT1 are safe and do not mediate autoimmune tissue infiltration or damage when transduced into peripheral CD8 T cells and transferred in vivo. However, if expressed in developing T cells and subjected to thymic selection, the same enhanced-affinity TCRs signal tolerance mechanisms in the thymus, resulting in T cells with attenuated antigen sensitivity in the periphery.