Adoptive transfer of T cells engineered with tumor-specific T cell receptors (TCRs) has shown limited efficacy in solid tumors, hindered by insufficient persistence, tumor trafficking, and dependence on tumor-associated co-stimulatory ligands. In a phase I trial (NCT 04639245) for patients with metastatic MAGE-A1-expressing tumors and adequate organ function; one participant received treatment, which was well-tolerated. In this case and NSG murine models, infusion of CD4/CD8 T cells co-expressing a class-I MAGE-A1-specific TCR and CD8αβ, failed to control tumor progression. To enhance function downstream of TCR signaling, here we investigate the adaptability of TCR components to synthetic modification. Leveraging the obligate co-expression of CD8αβ required for class-I TCR function in CD4 T cells, we identify CD8β as a tractable site for engineering without loss of function. In vitro screening demonstrates incorporation of the CD28 intracellular tail, yielding a CD8/CD28 chimeric co-receptor, most effectively enhances cytokine production, T cell persistence, and tumor control in immunodeficient murine models while preserving stem-like transcriptional features compared to native CD8β. Further rational modification of the CD28 binding motifs improves tumor control in vivo with increased intratumoral accumulation and reduced exhaustion. This benefit also extends to PRAME and WT1-specific TCRs in vitro supporting generalizability. TCR-engineered T cells have shown limited efficacy in part due to the absence of co-stimulation leading to limited accumulation in solid tumors. The authors here show engineering the CD8β coreceptor with an intracellular CD28 domain enhances cytokine production, persistence, and tumor control in vivo independent of tumor-associated co-stimulatory ligand encounter.

Adoptive therapies using T cells genetically modified with T cell receptors (TCR)s have shown limited efficacy in the solid tumor setting. Although functional CD4 + and CD8 + T cells transduced with a TCR specific for HLA-A2-restricted melanoma-associated antigen A1 (MAGE-A1, T TCR−MA1−CD8αβ ) could be detected post-transfer and were safe in one patient who subsequently progressed, T TCR−MA1−CD8αβ were insufficient to sustain antitumor activity in “stress” mouse tumor models. Leveraging the obligate co-expression of CD8αβ required for engagement of CD4 + T cells expressing the TCR, we screened positive co-stimulatory signals tethered to the intracellular tail of CD8β and identified that CD28 reduced exhaustion, enhanced tumor infiltration and improved murine tumor control. Further modifications of the CD28 intracellular domain produced a mutant CD8β-CD28 construct that conferred superior therapeutic control across tumor models. Thus, integrating co-stimulatory signals downstream of the TCR signaling complex can enhance TCR-engineered T cell function, independent of tumor-associated co-stimulatory ligand expression.

Although immune check-point inhibitors (CPIs) revolutionized treatment of Merkel cell carcinoma (MCC), patients with CPI-refractory MCC lack effective therapy. More than 80% of MCC express T-antigens encoded by Merkel cell polyomavirus, which is an ideal target for T-cell receptor (TCR)-based immunotherapy. However, MCC often repress HLA expression, requiring additional strategies to reverse the downregulation for allowing T cells to recognize their targets. We identified TCRMCC1 that recognizes a T-antigen epitope restricted to human leukocyte antigen (HLA)-A*02:01. Seven CPI-refractory metastatic MCC patients received CD4 and CD8 T cells transduced with TCRMCC1 (TTCR-MCC1) preceded either by lymphodepleting chemotherapy or an HLA-upregulating regimen (single-fraction radiation therapy (SFRT) or systemic interferon gamma (IFNγ)) with concurrent avelumab. Two patients who received preceding SFRT and IFNγ respectively experienced tumor regression. One experienced regression of 13/14 subcutaneous lesions with 1 "escape" lesion and the other had delayed tumor regression in all lesions after initial progression. Although TTCR-MCC1 cells with an activated phenotype infiltrated tumors including the "escape" lesion, all progressing lesions transcriptionally lacked HLA expression. While SFRT/IFNγ did not immediately upregulate tumor HLA expression, a secondary endogenous antigen-specific T cell infiltrate was detected in one of the regressing tumors and associated with HLA upregulation, indicating in situ immune responses have the potential to reverse HLA downregulation. Indeed, supplying a strong co-stimulatory signal via a CD200R-CD28 switch receptor allows TTCR-MCC1 cells to control HLA-downregulated MCC cells in a xenograft mouse model, upregulating HLA expression. Our results demonstrate the potential of TCR gene therapy for metastatic MCC and propose a next strategy for overcoming epigenetic downregulation of HLA in MCC.

Supplementary Figure from B7-H3 Specific CAR T Cells for the Naturally Occurring, Spontaneous Canine Sarcoma Model

Supplementary Tables 1-3 and Supplementary Tables 1-6

The success of T cell immunotherapies in the solid tumor is impeded by challenges in part created by the secretion of metabolic byproducts from the tumor, which generate a hostile and immunosuppressive tumor microenvironment (TME) (Tan et al., 2021). Dysregulated tumor metabolism results in the accumulation of metabolites that are exported into the interstitial space in the tumor, where they act as signaling molecules promoting the immunosuppressive landscape, ultimately reducing anti-tumor responses to T cell therapies (Jiang et al., 2020; Joyce & Fearon, 2015). Tumor cells exhibit several advantages through their high metabolic plasticity, but metabolic aggression deprives surrounding immune cells of nutrients, leading to exhausted, non-functional, and suppressive phenotypes (Lim et al., 2020). Regulatory T cells (Tregs) are a central immunosuppressive component of the TME that can rewire their metabolism and obstruct anti-tumor immunity by suppressing proliferation and activation of CD8+ T cells and CD4+ helper T cells in the TME (Betts et al., 2012; McNally et al., 2011). High Treg accumulation is associated with reduced anti-tumor responses, reduced efficacy of TCR therapies and poor clinical outcomes (Onda et al., 2019; Preston et al., 2013; Tang et al., 2014). Therefore, there is an imminent clinical need for the development of more synergistic therapies that specifically target Tregs in the TME to successfully overcome the barriers of infiltration and function of antigen-specific T cells. Accumulation of succinate, a key mitochondrial metabolite, occurs in conditions of low oxygen and increased energy demand, such the TME of solid tumors, and mutations in the gene that encoding succinate dehydrogenase ( Sdh) have been identified in a wide variety of solid tumors (Killian et al., 2013; Roh et al., 2019),resulting further an elevation of succinate levels. Accumulated succinate is secreted into the interstitial space (Garrigue et al., 2017) where it can stimulate the membrane bound succinate receptor (SUCNR1) on neighboring cells, further polarizing their immunosuppressive phenotype, and skewing macrophages to an M2 anti-inflammatory phenotype (Trauelsen et al., 2021; Wu et al., 2020). However, the effect of succinate on Tregs has not been explored. We hypothesized that succinate drives immunosuppression in the solid tumor by promoting Treg proliferation and function, leading to reduced anti-tumor responses to antigen-specific T cells. Consistent with this hypothesis, we confirmed that CD4+ T cells express the SUCNR1 and demonstrated that succinate promotes Treg numbers in CD4+ T cells isolated from healthy donor PBMCs ( Figure 1A). This effect was blocked upon administration of the SUCNR1 antagonist, NF-56-EJ40 ( Figure 1A). Deletion of the Sdhb gene, which encodes a subunit of the succinate processing enzyme, leads to intracellular accumulation of succinate. We deleted Sdhb in the non-small cell lung carcinoma cell line (H1299) and a melanoma cells line (A375) and confirmed that Sdhb -/- tumors secreted higher levels of succinate than WT. We translated these findings to a pre-clinical immunodeficient mouse model and determined that high succinate producing H1299 and A375 tumors had higher numbers of infiltrated Tregs in the TME compared with WT tumors ( Figure 1B), indicating a central role for succinate in the immunosuppressive landscape of the TME and identifying the suppression of succinate signaling as a potential therapeutic strategy to decrease immunosuppressive Tregs. Targeting Tregs in the TME is an attractive therapeutic strategy and regulating the induction and function of Tregs has the potential to increase the efficacy of effector T cells, thus improving anti-tumor responses by removing a critical suppressive barrier. Based on these findings, we propose a novel mechanism of Treg-mediated immunosuppression driven by succinate, whereby succinate promotes a Treg-rich environment in the TME and pushes Tregs towards a Th1-suppressing phenotype. These studies identify a previously unknown molecular regulation of Tregs by extracellular succinate and provide a novel therapeutic target that has the potential to enhance the effectiveness of TCR therapy in solid tumors thereby laying a framework for translational therapeutic development and innovation, that will limit suppression of and enhance tumor-specific T cell cytotoxicity.

Abstract One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant animal models. In this study, we sought to establish a chimeric antigen receptor (CAR) T-cell treatment model for naturally occurring canine sarcomas as a model for human CAR T-cell therapy. Canine CARs specific for B7-H3 were constructed using a single-chain variable fragment derived from the human B7-H3–specific antibody MGA271, which we confirmed to be cross-reactive with canine B7-H3. After refining activation, transduction, and expansion methods, we confirmed target killing in a tumor spheroid three-dimensional assay. We designed a B7-H3 canine CAR T-cell and achieved consistently high levels of transduction efficacy, expansion, and in vitro tumor killing. Safety of the CAR T cells were confirmed in two purposely bred healthy canine subjects following lymphodepletion by cyclophosphamide and fludarabine. Immune response, clinical parameters, and manifestation were closely monitored after treatments and were shown to resemble that of humans. No severe adverse events were observed. In summary, we demonstrated that similar to human cancers, B7-H3 can serve as a target for canine solid tumors. We successfully generated highly functional canine B7-H3–specific CAR T-cell products using a production protocol that closely models human CAR T-cell production procedure. The treatment regimen that we designed was confirmed to be safe in vivo. Our research provides a promising direction to establish in vitro and in vivo models for immunotherapy for canine and human solid tumor treatment.

BackgroundT Cell Receptor (TCR)-T cell therapies have shown some promising results in cancer clinical trials, however the efficacy of treatment remains suboptimal. Outcomes could potentially be improved by utilizing highly functional TCRs for future trials. Current TCR discovery methods are relatively low throughput and rely on synthesis and screening of individual TCRs based on tetramer binding and peptide specificity, which is costly and labor intensive. We have developed and validated a pooled approach relying on directly cloned TCRs transduced into a fluorescent Jurkat reporter system (figure 1). This approach provides an unbiased, high-throughput method for TCR discovery.MethodsAs a model for POTS, T cells specific for a peptide derived adenovirus structural protein were sorted on tetramer and subjected to 10x single cell VDJ analysis. Pools of randomly paired TCR alpha and beta chains were cloned from the 10x cDNA into a lentiviral vector and transduced into a Jurkat reporter cells. Consecutive stimulations with cognate antigen followed by cell sorts were performed to enrich for functional TCRs. Full length TCRab pools were sequenced by Oxford Nanopore Technologies (ONT) and compared to a 10x dataset to find naturally paired TCRs.ResultsComparison between the ex vivo single cell VDJ sequencing and ONT sequencing of the transduced antigen specific TCRs showed more than 99% of the TCR pairs found in reporter positive Jurkat cells were naturally paired TCRs. The functionality of 8 TCR clonotypes discovered using POTS were compared and clone #2 showed the strongest response. Of the selected clonotypes, clone #2 showed a low frequency of 0.9% in the ex vivo single cell VDJ sequencing. After the first round of stimulation and sequencing, clone #2 takes up of 5% of all reporter-positive clones. The abundance of clone #2 further increased to 17% after another round of stimulation, sorting and sequencing, suggesting this method can retrieve and enrich for highly functional antigen specific TCRs.Abstract 192 Figure 1Outline of the POTS workflow.ConclusionsPOTS provides a high-throughput method for discovery of naturally paired, high-avidity T cell receptors. This method mitigates bias introduced by T cell differentiation state by screening TCRs in a clonal reporter system. Additionally, POTS allows for screening of low abundance clones when compared with traditional TCR discovery techniques. Pooled TCRs could also be screened in vivo with primary T cells in a mouse model to screen for the most functional and physiologically fit TCR for cancer treatment.

Background Synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) are ideal solid tumors for the development of adoptive cellular therapy (ACT) targeting NY-ESO-1, as a high frequency of tumors homogeneously express this cancer-testes antigen. Data from early phase clinical trials have shown antitumor activity after the adoptive transfer of NY-ESO-1–specific T cells. In these studies, persistence of NY-ESO-1 specific T cells is highly correlated with response to ACT, but patients often continue to have detectable transferred cells in their peripheral blood following progression. Method We performed a phase I clinical trial evaluating the safety of NY-ESO-1–specific endogenous T cells (ETC) following cyclophosphamide conditioning. Peripheral blood mononuclear cells (PBMCs) from treated patients were evaluated by flow cytometry and gene expression analysis as well as through ex vivo culture assays with and without IL-15. Results Four patients were treated in a cohort using ETC targeting NY-ESO-1 following cyclophosphamide conditioning. Treatment was well tolerated without significant toxicity, but all patients ultimately had disease progression. In two of four patients, we obtained post-treatment tumor tissue and in both, NY-ESO-1 antigen was retained despite clear detectable persisting NY-ESO-1–specific T cells in the peripheral blood. Despite a memory phenotype, these persisting cells lacked markers of proliferation or activation. However, in ex vivo culture assays, they could be induced to proliferate and kill tumor using IL-15. These results were also seen in PBMCs from two patients who received gene-engineered T-cell receptor–based products at other centers. Conclusions ETC targeting NY-ESO-1 with single-agent cyclophosphamide alone conditioning was well tolerated in patients with SS and those with MRCL. IL-15 can induce proliferation and activity in persisting NY-ESO-1–specific T cells even in patients with disease progression following ACT. These results support future work evaluating whether IL-15 could be incorporated into ACT trials post-infusion or at the time of progression.

Abstract Background: Multiple factors, including dense stroma and high infiltration of suppressive immune cells make pancreatic ductal adenocarcinoma (PDA) challenging to treat with immunotherapy. The presence of intra-tumoral T cells positively correlates with improved survival for PDA patients. Gamma chain cytokines (GCCs) can augment T cell mediated anti-tumor immunity and various GCC agonist drugs have been applied in clinical trials for cancer. Besides evaluating the effect of GCC derived agonists using recovered blood or biopsies of treated patients, it is challenging to mechanistically study the effect of GCCs on tumor-resident immune cells in human samples. Methods: Here we apply organotypic slice culture of surgically resected tumor tissues to study the effect of GCCs on intra-tumoral immune cells. We treated slices of PDAs with GCCs (IL-2, IL-7, IL-15 and IL-21) for 6 days. Immune cells, especially T cells emigrated from the tumor slices into the culture supernatant. On day 6 we fixed intact tumor slices for multiplex immunohistochemistry (mIHC) and analyzed emigrated cells using flow cytometry. Results: IL-15 and IL-7 potently induced T cell proliferation. The effect of IL-7 and IL-15 on T cell proliferation was higher for T cells that expressed markers of antigen experience such as CD39 and PD1 (Table 1). While IL-15 had the strongest effect on tumor-derived T cells, IL-15 and IL-7 were similar in their effect on T cells in the blood of the same patient, suggesting that IL-15 is especially effective in inducing the proliferation of tumor-infiltrating T cells. The effect of IL-15 on enhanced T cell proliferation was also seen within tumor slices, as revealed by mIHC. Conclusion: Our data suggest that IL-15 is the most potent GCC at inducing in situ expansion of tumor-resident T cells, including T cells that show signs of antigen experience. Table 1.IL-15 induces in situ T cell proliferation in PDA.AssayGroupsUntreatedIL-2IL-7IL-15IL-21ParameterFlow Cytometry%Ki67+CD8+cells of live cells0.090.040.360.595.001.3514.331.200.310.14%CD39+CD103+of CD8+0.130.150.110.040.290.140.620.180.130.08%PD1+of CD8+25.107.2223.233.1030.484.7446.483.5037.402.32mIHC%CD8+Ki67+ of all cells0.16 0.04(n=2)0.83 (n=1)0.550.38(n=2)2.200.04(n=2)0.490.41(n=2)n=4 slices for each group for flow cytometry. Data are from one experiment*. MeanSD is shown. Two doses (10-fold difference) for each cytokine were tested and results only for the low dose are shown.*Another experiment with groups IL-2 and IL-2+IL-15 was performed and had similar results for the effect of IL-15 Citation Format: Karan Kohli, Shihong Zhang, Xiuyun Jiang, Cynthia Hsu, Arezou Abbasi, Teresa S. Kim, Venu G. Pillarisetty. IL-15 is the most potent of tested gamma chain cytokines at inducing in situ proliferation of T cells in human pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 607.

BACKGROUND:Dedifferentiated liposarcoma (DDLPS) is one of the most common soft tissue sarcoma subtypes and is devastating in the advanced/metastatic stage. Despite the observation of clinical responses to PD-1 inhibitors, little is known about the immune microenvironment in relation to patient prognosis.METHODS:We performed a retrospective study of 61 patients with DDLPS. We completed deep sequencing of the T-cell receptor (TCR) β-chain and RNA sequencing for predictive modeling, evaluating both immune markers and tumor escape genes. Hierarchical clustering and recursive partitioning were employed to elucidate relationships of cellular infiltrates within the tumor microenvironment, while an immune score for single markers was created as a predictive tool.RESULTS:Although many DDLPS samples had low TCR clonality, high TCR clonality combined with low T-cell fraction predicted lower 3-year overall survival (p=0.05). Higher levels of CD14+ monocytes (p=0.02) inversely correlated with 3-year recurrence-free survival (RFS), while CD4+ T-cell infiltration (p=0.05) was associated with a higher RFS. Genes associated with longer RFS included PD-1 (p=0.003), ICOS (p=0.006), BTLA (p=0.033), and CTLA4 (p=0.02). In a composite immune score, CD4+ T cells had the strongest positive predictive value, while CD14+ monocytes and M2 macrophages had the strongest negative predictive values.CONCLUSIONS:Immune cell infiltration predicts clinical outcome in DDLPS, with CD4+ cells associated with better outcomes; CD14+ cells and M2 macrophages are associated with worse outcomes. Future checkpoint inhibitor studies in DDLPS should incorporate immunosequencing and gene expression profiling techniques that can generate immune landscape profiles.

IMPORTANCE Anthracycline-based therapy is standard first-line treatment for most patients with advanced and metastatic sarcomas. Although multiple trials have attempted to show improved outcomes in patients with soft-tissue sarcoma over doxorubicin monotherapy, each has fallen short of demonstrating improved outcomes. OBJECTIVE To evaluate the safety and efficacy of doxorubicin in combination with pembrolizumab in patients with advanced, anthracycline-naive sarcomas. DESIGN, SETTING, AND PARTICIPANTS This nonrandomized clinical trial used a 2-stage phase 2 design and was performed at a single, academic sarcoma specialty center. Patients were adults with good performance status and end-organ function. Patients with all sarcoma subtypes were allowed to enroll with the exception of osteosarcoma, Ewing sarcoma, and alveolar and embryonal rhabdomyosarcoma. INTERVENTIONS Two dose levels of doxorubicin (45 and 75mg/m2) were tested for safety in combination with pembrolizumab. MAIN OUTCOMES AND MEASURES Objective response rate (ORR) was the primary end point. Overall survival (OS) and progression-free survival (PFS) were secondary end points. Correlative studies included immunohistochemistry, gene expression, and serum cytokines. RESULTS A total of 37 patients (22 men; 15 women) were treated in the combined phase 1/2 trial. The median (range) patient age was 58.4 (25-80) years. The most common histologic subtype was leiomyosarcoma (11 patients). Doxorubicin plus pembrolizumab was well tolerated without significant unexpected toxic effects. The ORR was 13% for phase 2 patients and 19% overall. Median PFS was 8.1 (95% CI, 7.6-10.8) months. Median OS was 27.6 (95% CI, 18.7-not reached) months at the time of this analysis. Two of 3 patients with undifferentiated pleomorphic sarcoma and 2 of 4 patients with dedifferentiated liposarcoma had durable partial responses. Tumor-infiltrating lymphocytes were present in 21% of evaluable tumors and associated with inferior PFS (log-rank P=.03). No dose-limiting toxic effects were observed. CONCLUSIONS AND RELEVANCE In this nonrandomized clinical trial, doxorubicin plus pembrolizumab was well tolerated. Although the primary end point for ORR was not reached, the PFS and OS observed compared favorably with prior published studies. Further studies are warranted, especially those focusing on undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT02888665

Background Adoptive cellular therapy (ACT) is a promising treatment for synovial sarcoma (SS) with reported response rates of over 50%. However, more work is needed to obtain deeper and more durable responses. SS has a ‘cold’ tumor immune microenvironment with low levels of major histocompatibility complex (MHC) expression and few T-cell infiltrates, which could represent a barrier toward successful treatment with ACT. We previously demonstrated that both MHC expression and T-cell infiltration can be increased using systemic interferon gamma (IFN-γ), which could improve the efficacy of ACT for SS. Case presentation We launched a phase I trial incorporating four weekly doses of IFN-γ in an ACT regimen of high-dose cyclophosphamide (HD Cy), NY-ESO-1-specific T cells, and postinfusion low-dose interleukin (IL)-2. Two patients were treated. While one patient had significant tumor regression and resultant clinical benefit, the other patient suffered a fatal histiocytic myocarditis. Therefore, this cohort was terminated for safety concerns. Conclusion We describe a new and serious toxicity of immunotherapy from IFN-γ combined with HD Cy-based lymphodepletion and low-dose IL-2. While IFN-γ should not be used concurrently with HD Cy or with low dose IL-2, IFN-γ may still be important in sensitizing SS for ACT. Future studies should avoid using IFN-γ during the immediate period before/after cell infusion. Trial registration numbers NCT04177021, NCT01957709, and NCT03063632.

Background Chimeric antigen receptor (CAR) T cell therapy has transformed therapy for hematological malignancies but has not yet been established as standard of care for any solid tumors. One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant, immunocompetent animal models. In this study, we sought to establish CAR T cells for naturally occurring canine sarcomas in client owned animals as a model for human CAR T cell therapy. Methods Archived FFPE, freshly isolated canine solid tumor samples as well as tumor lines were tested for B7H3 expression by immunohistochemistry (IHC) and flow cytometry analysis. We designed CARs using the scFv from the human B7H3-specific antibody MGA271 and confirmed the cross-reactivity to canine B7H3 (construct information see figure 1A). A truncated EGFR (tEGFR) was included in the construct to allow for IHC and flow cytometry testing for the presence of CAR T cells. Killing efficiency was evaluated using 3D tumor spheroid killing assays to monitor dynamics. Safety of the CAR products following lymphodepletion was confirmed in two healthy dogs (figure 1B). Results Canine solid tumors were confirmed to be B7H3 positive in almost all cases. Using the GALV-pseudotyped retrovirus system, transduction was efficient with up to 70% CAR+ cells. Post-transduction expansion was over 100 folds. B7H3 CAR transduced canine T cells were able to eliminate B7H3+ canine tumor spheroids effectively (figure 2). Safety of the CAR T cells (dose: 1 × 109/m2) were confirmed in both healthy animals following cyclophosphamide lymphodepletion. After week 6, cetuximab was given to the subjects to deplete EGFR+ cells. Subject 2 experienced fever after CAR T cell administration. Both dogs showed elevated serum ALP and ALT levels and returned to normal (figure 3). No other treatment-related adverse events were observed. Information of the CAR T cell products can be found in table 1. Conclusions We demonstrated that, similar to human cancers, B7H3 is a target in canine solid tumors. We successfully generated canine B7H3 specific CAR T cell products that are highly efficient at killing canine 3D tumor spheroids using a production protocol that closely models human CAR T cell production procedure and confirmed the safety in vivo. We plan to test and optimize various approaches to enhance CAR T cell efficacy for solid tumor treatment both in vitro and in canine sarcoma patients. Ethics Approval The study was approved by Fred Hutchinson Cancer Research Center‘s Institutional Animal Care and Use Committee (IACUC), approval number PROTO201900860

Abstract Interferon-γ (IFNγ) has been studied as a cancer treatment with limited evidence of clinical benefit. However, it could play a role in cancer immunotherapy combination treatments. Despite high expression of immunogenic cancer–testis antigens, synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) have a cold tumor microenvironment (TME), with few infiltrating T cells and low expression of major histocompatibility complex class I (MHC-I). We hypothesized that IFNγ treatment could drive inflammation in a cold TME, facilitating further immunotherapy. We conducted a phase 0 clinical trial treating 8 SS or MRCL patients with weekly systemic IFNγ. We performed pre- and posttreatment biopsies. IFNγ changed the SS and MRCL TME, inducing tumor-surface MHC-I expression and significant T-cell infiltration (P < 0.05). Gene-expression analysis suggested increased tumor antigen presentation and less exhausted phenotypes of the tumor-infiltrating T cells. Newly emergent antigen-specific humoral and/or T-cell responses were found in 3 of 7 evaluable patients. However, increased expression of PD-L1 was observed on tumor-infiltrating myeloid cells and in some cases tumor cells. These findings suggest that systemic IFNγ used to convert SS and MRCL into “hot” tumors will work in concert with anti–PD-1 therapy to provide patient benefit.

T cell immunotherapy is a concept developed for the treatment of cancer and infectious diseases, based on cytotoxic T lymphocytes to target tumor- or pathogen-specific antigens. Antigen-specificity of the T cell receptors (TCRs) is an important selection criterion in the developmental design of immunotherapy. However, off-target specificity is a possible autoimmunity concern if the engineered antigen-specific T cells are cross-reacting to self-peptides in-vivo. In our recent work, we identified several hepatitis E virus (HEV)-specific TCRs as potential candidates to be developed into T cell therapy to treat chronic hepatitis E. One of the identified TCRs, targeting a HLA-A2-restricted epitope at the RNA-dependent RNA polymerase (HEV-1527: LLWNTVWNM), possessed a unique multiple glycine motif in the TCR-β CDR3, which might be a factor inducing cross-reactivity. The aim of our study was to explore if this TCR could cross-recognize self-peptides to underlay autoimmunity. Indeed, we found that this HEV-1527-specific TCR could also cross-recognize an apoptosis-related epitope, Nonmuscle Myosin Heavy Chain 9 (MYH9-478: QLFNHTMFI). While this TCR had dual specificities to both viral epitope and a self-antigen by double Dextramer binding, it was selectively functional against HEV-1527 but not activated against MYH9-478. The consecutive glycine motif in β chain may be the reason promoting TCR binding promiscuity to recognize a secondary target, thereby facilitating cross-recognition. In conclusion, candidate TCRs for immunotherapy development should be screened for autoimmune potential, especially when the TCRs exhibit unique sequence pattern.

Abstract Synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) cells homogeneously express high levels of NY-ESO-1 and other cancer/testis antigens, which makes these sarcoma types good candidates for T cell immunotherapy. However, these tumors demonstrate a “cold” microenvironment, with low cell surface expression of human leukocyte antigen (HLA) molecules and few infiltrating T cells. Interferon gamma (IFNγ) has been used both as a single agent and in combination with other immunotherapies in cancers with “hot”, highly inflamed tumor microenvironments such as melanoma. To date, little work has explored its impact on immunologically quiet tumors. In order to increase the expression of HLA on the tumor cell surface and improve the T cell recognition and cytotoxicity, we initiated a pilot “window of opportunity” clinical trial, 6 SS/MRCL patients were treated with subcutaneous systemic interferon gamma (IFNγ) weekly for 2 or 4 weeks at a dose of 100 mcg/m2. The primary objective of the study was to determine if HLA expression increased on post-treatment biopsies in comparison to pre-treatment biopsies. The treatment was relatively well-tolerated, patients experienced flu-like symptoms with no severe adverse effects. Patients' sera, PBMCs and tumor biopsies were collected pre- and post-IFNγ treatment for further analysis. Several cytokines including interleukin-16, microphage migration inhibitory factor and CXCL-10 etc. changed considerably in patients' periphery. Transcriptome profiling on the PBMCs showed significant changes in some immune regulatory genes in T cells and monocytes. Flow cytometry analysis of the tumor biopsies showed significant increase of both class I and class II HLA on the tumor surface in all treated patients. Tumor infiltrating immune cell frequencies increased by 3-10 times, of which, the majority were T cells. Functional assay of expanded tumor infiltrating lymphocytes (TIL) showed an increased and broadened tumor-associated peptide recognition capability. However, in all evaluable tumors there was an increase in PD-L1 either on tumor or on infiltrating macrophages. In conclusion, weekly IFNγ treatment is well tolerated in SS/MRCL patients and it can significantly enhance the expression of HLA on tumor surface, and increase T cell infiltration; however, increased PD-L1 expression on tumor cells and infiltrating immune cells may help tumors evade T cell elimination. Strategies combining PD-1 blockade and IFNγ should be explored. Citation Format: Shihong Zhang, Sara Cooper, Bailey Donahue, Venu G. Pillarisetty, Robin L. Jones, Stanley R. Riddell, Brian A. Van Tine, Seth M. Pollack. Interferon gamma induced transformation of the cold tumor microenvironment in patients with NY-ESO-1 expressing sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 616.

Influenza A virus remains a major threat to public health, and the inactivated split-virus vaccine is the most prevalent vaccine used worldwide. However, our knowledge about cellular immune responses to the inactivated influenza virus vaccine and its correlation with humoral responses are yet limited, which has restricted our understanding of the vaccine’s protective mechanisms. Herein, in two clinical trials, T-cell responses specific for both previously identified human leucocyte antigen (HLA)-I-restricted epitopes from influenza virus and hemagglutinin (HA) protein were longitudinally investigated before, during, and after a two-dose vaccination with the inactivated 2009 pandemic H1N1 (2009-pH1N1) vaccine. A robust antibody response in all of the donors after vaccination was observed. Though no CD8+ T-cell responses to known epitopes were detected, HA-specific T-cell responses were primed following vaccination, and the responses were found to be mainly CD4+ T-cell dependent. However, HA-specific T-cells circulating in peripheral blood dropped to baseline levels 6 weeks after vaccination, but humoral immune responses maintained a high level for 4 months post-vaccination. Significant correlations between the magnitude of the HA-specific T-cell responses and hemagglutination inhibition antibody titers were demonstrated, indicating a priming role of HA-specific T-cells for humoral immune responses. In conclusion, our study indicates that HA-specific CD4+ T-cell responses can be primed by the inactivated 2009-pH1N1 vaccine, which may coordinate with the elicitation of antibody protection. These findings would benefit a better understanding of the immune protective mechanisms of the widely used inactivated 2009-pH1N1 vaccine.