Tumor-infiltrating Lymphocytes Clones Research Articles

Pelareorep driven blood TIL expansion in patients with pancreatic, breast and colon cancer.

e14625 Background: Tumor infiltrating lymphocytes (TILs) represent a major immunological tumor control mechanism that is associated with better prognosis in cancer. One emerging cancer treatment approach to leverage the therapeutic potential of TILs is the expansion and adoptive cell transfer of autologous TILs, commonly referred to as autologous adoptive therapy (ACT). ACT clinical studies have shown encouraging results. While the majority of these trials have targeted melanoma, impressive clinical benefit has also been observed in cervical cancer, with preliminary efficacy in colorectal cancer (CRC), cholangiocarcinoma, non-small cell lung cancer, and breast cancer. Pelareorep (pela) is a live, replication competent reovirus (T3D) that selectively replicates in cancer cells and represents a new class of immunotherapy currently being explored in multiple clinical settings. To examine the effect of pela therapy on TIL expansion we applied T cell receptor sequencing of matched tumor tissue and whole blood pre- and post-treatment in a subset of breast, pancreatic, and (CRC) subjects receiving chemotherapy and atezolizumab, an anti-PD-L1 therapy, and intravenous treatment with pela. Methods: Identification of TIL clones was performed by immunosequencing of the CDR3 regions of human T-cell receptor-β (TCRβ) chains (ImmunoSEQ Assay, Adaptive Biotechnologies). DNA was isolated from tissue and blood at baseline and from blood collected post-treatment. TCRβ CDR3 regions were amplified by a multiplex, bias-controlled PCR with primers targeting the V and J genes of T cells as well as primers targeting housekeeping genes to quantitate the total nucleated cells in each sample. PCR products were sequenced on an Illumina NextSeq. Results: Pela treatment was observed to increase the expansion of pre-existing and new TIL clones in the blood in from all tumor samples after one cycle of treatment. We also observed that pre-existing TIL clonal expansion in the blood seemed to correlate with reductions in tumor volume in pancreatic cancer and to a lesser extent in CRC patients who received multiple cycles of therapy. Interestingly, unlike atezolizumab, pela’s clinical activity was lost when combined with avelumab, a PD-LI inhibitor capable of binding Fc receptors and inducing ADCC. The addition of avelumab eliminated pre-existing TIL expansion in the blood highlighting the importance of TIL expansion following pela therapy. Conclusions: These findings, while preliminary, suggest that the clinical benefits observed following pela immunotherapy may be mechanistically analogous to ACT. Accordingly, pela therapy may offer a means to directly expand TILs without the need for tumor resection, ex vivo TIL expansion, T cell ablation and IL-2 therapy. Clinical trial information: Eudra-CT: 2020-003996-16 .

Phase 1/2 randomized, open-label, multicenter, Simon two-stage study of pelareorep combined with modified FOLFIRINOX +/- atezolizumab in patients with metastatic pancreatic ductal adenocarcinoma.

TPS4203 Background: Immune therapies benefit only the small subset of metastatic pancreatic ductal adenocarcinoma (mPDAC) patients who have microsatellite instability-high (MSI-H) tumors (approximately 1%). Most PDAC tumors are microsatellite stable (MSS) and have an immunologically “cold” phenotype with fewer genetic mutations, reduced immune cell infiltration, and downregulated immune checkpoint proteins. These attributes make most mPDAC tumors resistant to conventional immunotherapy including checkpoint inhibitors. Pelareorep is a naturally occurring, non-genetically modified reovirus that selectively infects cancer cells following intravenous administration. Upon infection, pelareorep’s double-stranded RNA genome stimulates a pro-inflammatory response that primes tumors for immunologic killing. This includes increased T cell infiltration into tumors, expansion of tumor-infiltrating lymphocyte clones, increased PD-L1 expression, and stimulation of tumor-directed innate and adaptive immune responses. Pelareorep combined with chemotherapy, checkpoint inhibitors, or both has shown promising efficacy in several malignancies, including mPDAC. This study is designed to evaluate pelareorep plus modified FOLFIRINOX with or without the PD-L1 inhibitor atezolizumab as first-line therapy in patients with mPDAC. Methods: This trial represents a new cohort within the ongoing GOBLET signal finding platform study in gastrointestinal cancers. This is a phase 1/2 randomized, open-label study that uses a screened selection design within a Simon two-stage approach. Eligible patients must be 18 years or older and have newly diagnosed, radiologically evaluable mPDAC with no prior chemotherapy and an ECOG performance status of 0 or 1. Patients are randomized 1:1 to receive either pelareorep + mFOLFIRINOX orpelareorep + mFOLFIRINOX + atezolizumab. The first stage enrolls 15 evaluable patients into each arm. If pre-specified efficacy criteria are met in Stage 1, one or both arms may be expanded to Stage 2 and enroll 17 additional evaluable patients per arm. The first 3-6 patients enrolled into both arms comprise a safety run-in that must be successfully concluded prior to enrolling additional patients. The treatment period consists of 4-week treatment cycles. The primary objectives are 1) safety and tolerability of pelareorep + mFOLFIRINOX with or without atezolizumab and 2) efficacy based on objective response rate per RECIST v1.1. Secondary objectives include duration of response, progression-free survival, and overall survival. In addition, tumor and blood samples are being collected to evaluate the immunological effects of treatment and to explore potential biomarkers of response. Clinical trial information: Eudra-CT: 2020-003996-16.

Abstract PO1-02-01: T Cell Receptor Sequencing to Monitor Pelareorep-Induced Expansion of Tumor Infiltrating Lymphocytes

Abstract Background: Tumor infiltrating lymphocytes (TILs) represent a major immunological tumor control mechanism that is associated with better prognosis in breast cancer. We have previously reported the effect of pelareorep (pela), an intravenously delivered, non-engineered oncolytic reovirus, on a composite measurement of TILs and tumor cellularity (CelTIL) from the AWARE-1 window-of-opportunity study in patients with early breast cancer (eBC). These results showed treatment increased in CelTIL scores especially in the atezolizumab group. To confirm and extend these findings we applied T cell receptor sequencing of matched tumor tissue and whole blood pre and post-treatment from the AWARE-1 study to further explore the effects of pela therapy on TILs. Methods: Newly diagnosed HR+/HER2- eBC patients were enrolled into two cohorts: Cohort 1: pela + letrozole (n=10); and Cohort 2: pela + letrozole + atezolizumab (n=10). Pela was administered on days 1, 2 and 8, 9, and atezolizumab was given on day 3. Tumor biopsies (FFPE samples) collected pre-treatment (~D-23) and on day ~21 when tumors were surgically removed. T cell fraction analysis and T cell receptor sequencing were performed by Adaptive Biotechnology (Seattle, Washington) Immunoseq protocol. Results: As was shown for the CelTIL scores, an increase in the tumor T cell fraction, a direct measurement of TILs, was observed at 1-month post-treatment in both cohorts with a mean percent increase of 21% for Cohort 1 and 67% for Cohort 2. Through TCR-sequencing of tumor tissue, we identified TIL clones and tracked their differential abundance in tumor tissue and blood post-treatment. The results of this analysis at Day 21 showed a mean post-treatment expansion of 10 tumor specific clones in the blood for Cohort 1, and a mean post-treatment increase of 40 tumor specific clones for Cohort 2. Conclusions: These results confirm the previously reported CelTIL results from AWARE-1 on pela-induced increases in TILs and demonstrate the expansion of these clones in both tumor and blood as a consequence of pela therapy for HR+/HER2- breast cancer. Citation Format: Richard Trauger, Houra Loghmani, Matt Coffey, Fernando Salvador, Joaquín Gavilá, Luis Manso, Tomás Pascual, Aleix Prat, Thomas Heineman. T Cell Receptor Sequencing to Monitor Pelareorep-Induced Expansion of Tumor Infiltrating Lymphocytes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-02-01.

Neoantigen-specific CD4+ tumor-infiltrating lymphocytes are potent effectors identified within adoptive cell therapy products for metastatic melanoma patients

BackgroundAdoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) is a promising immunotherapeutic approach for patients with advanced solid tumors. While numerous advances have been made, the contribution of neoantigen-specific CD4+T...

A phase 1, first-in-human (FIH) study of the anti-HER2 CAR macrophage CT-0508 in subjects with HER2 overexpressing solid tumors.

2533 Background: Most solid tumors are resistant to immunotherapy. In pre-clinical studies, CAR-M infiltrate tumors, phagocytose tumor cells, activate the tumor micro environment (TME), recruit T cells, and present tumor antigens to T cells leading to robust anti-tumor immunity. CT-0508 is a first in class CAR-M, comprised of autologous monocyte derived macrophages expressing an anti-HER2 CAR. Here we present preliminary clinical results from the CT-0508 Phase 1 FIH study. Methods: This multi-center, open-label study is evaluating CT-0508’s safety, tolerability, and manufacturing feasibility in 18 participants (pts) with advanced solid tumors overexpressing HER2 who have progressed on prior therapies. Monocytes are isolated from mobilized apheresis products, differentiated into macrophages and engineered with an anti-HER2 CAR. Group 1 pts (n = 9) receive a fractionated dose (D1, D3, D5) and Group 2 pts (n = 9) receive the full dose on D1. CT-0508 is administered without preparative chemotherapy (bridging is permitted). Serial blood samples, 1 pre and 2 post-treatment biopsies are collected to investigate safety, pharmacokinetics and mechanism of action. Results: Seven pts (4F/3M), median age 64 (49-73), have been treated [breast (2), esophageal (2), cholangiocarcinoma, ovarian and parotid gland cancers]. A median of 3 (2-10) prior lines of therapy have been administered, most pts (85.7%) received prior anti HER2 therapy. CT-0508 was successfully manufactured with high viability, purity and CAR expression, and was well tolerated with no dose limiting toxicities or AEs leading to discontinuation or dose modification. Two related SAEs were reported in the same pt (Grade 1 CRS, hospitalized for monitoring and Grade 2 infusion reaction, resolved within 1h). Three other pts had Grade 1-2 CRS; resolved within 4d with no use of tocilizumab needed. There were no major organ toxicities and no on-target off-tumor toxicities. Post-infusion cytokines were transiently elevated in most pts and were self-limiting. Among the 4 pts who reached week 8, the best overall response was stable disease (n = 3) and 1 pt progressed, with a median follow up of 8w. CT-0508 rapidly egressed from peripheral blood. CT-0508 CAR mRNA was detected in all tumor biopsies of the first 2 pts. CT-0508 activated the TME, with increased myeloid cell activation, T cell infiltration, activation and proliferation. TCR sequencing demonstrated peripherally expanding T cells enriched for tumor infiltrating lymphocyte clones, suggesting expansion of tumor reactive T cells. Correlative data from additional pts will be presented. Conclusions: In 7 pts, CT-0508 was safe and feasible to manufacture. Early correlative data demonstrate trafficking, TME modulation, and potential induction of anti-tumor T cell immunity. The study is actively enrolling. Clinical trial information: NCT04660929.

Combined IL-2, agonistic CD3 and 4-1BB stimulation preserve clonotype hierarchy in propagated non-small cell lung cancer tumor-infiltrating lymphocytes

BackgroundAdoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) yielded clinical benefit in patients with checkpoint blockade immunotherapy-refractory non-small cell lung cancer (NSCLC) prompting a renewed interest in TIL-ACT. This preclinical...

EX VIVO EXPANSION OF TUMOR INFILTRATING LYMPHOCYTES (TILS) AND CANCER STEM CELLS FROM MALIGNANT GLIOMAS

<h3>Background</h3> Gliomas are the most common type of primary malignant tumors of the Central Nervous System (CNS). Conventional therapies show low effectiveness and are frequently associated with important side effects. Glioblastoma (GBM) is the most lethal and frequent type of glioma, endowed with a subpopulation of cancer stem cells (CSC) responsible for resistance to conventional treatment and ensuing tumor relapse. Different forms of immunotherapy are increasingly recognized as promising therapeutic approach against aggressive tumors, including adoptive cell therapy with tumor infiltrating lymphocytes (TILs). However, studies accessing the potential of TILs in aggressive CNS tumors are limited. In this work, we describe the feasibility of isolating TILs and CSC from the same tumor specimen for ex vivo expansion and downstream immunotherapy application. <h3>Methods</h3> Fresh tumor samples were obtained from 10 patients (mean age: 60 years; 4 men and 6 women), diagnosed with malignant glioma and subjected to tumor resection in the Instituto do Câncer do Estado de São Paulo (ICESP), after informed consent approved by the Institutional Review Board. Brain tumor tissue was mechanically dissociated and enzymatically digested to generate a cell suspension. The CSC population was cultured in neurosphere formation media supplemented with EGF and FGF. TILs were expanded in culture medium supplemented with IL-2. <h3>Results</h3> Anatomopathological analyses of resected tumors confirmed diagnosis of 9 GBM and a grade I glioneural tumor. Tumorspheres could be generated in 8 out of 10 tumor samples, all of them GBM. No tumorspheres were formed in the respective cultures derived from the grade I glioneural tumor and from a GBM containing mutated ATRX. TILs could be expanded from all tumor samples tested, reaching a mean of 1,1 ± 0,7 × 10e6 total cells and mean cell viability of 60% ± 4%, after 5 days of in vitro expansion. <h3>Conclusion</h3> CSC could be expanded from all fresh glioma samples, except from a grade I glioma and a GBM with ATRX mutation, indicating a possible positive correlation with unfavorable clinical prognosis. Viable TILs could also be initially expanded from these glioma samples, indicating the possibility of using them to seed larger cultures subjected to typical rapid expansion protocols to attain clinical doses. Altogether, this procedure could help identify targetable CSC antigens, as well as screen for CSC-reactive TIL clones for possible adoptive cell therapy.

T Cells Expanded from PD-1+ Peripheral Blood Lymphocytes Share More Clones with Paired Tumor-Infiltrating Lymphocytes

Both tumor-infiltrating lymphocytes (TIL) and PD-1+ peripheral blood lymphocytes (PBL) are enriched for tumor-reactive clones recognizing known and unknown tumor antigens. However, the relationship between the T-cell receptor (TCR)-β repertoires of the TIL and T cells expanded from paired PD-1+ PBL and whether T cells expanded from PD-1+ PBL can be used to treat patients with cancer as TIL substitutes remains unclear. Here we established a highly efficient protocol to prepare polyclonal T cells from PD-1+ PBL. A functional T-cell assay and tetramer staining revealed that cells from PD-1+ PBL were relatively enriched for tumor-reactive T cells. Furthermore, deep TCR-β sequencing data revealed that an average of 11.29% (1.32%-29.06%, p=0.015, n=8) tumor-resident clonotypes were found in T cells expanded from paired PD-1+ PBL, and the mean accumulated frequency of TIL clones found in T cells expanded from PD-1+ PBL was 35.11% (7.23%-78.02%, p=0.017, n=8). Moreover, treatment of four patients, who failed multi-line therapy and developed acquired resistance to anti-PD-1, with autologous T cells expanded from PD-1+ PBL combined with anti-PD-1 antibody elicited objective responses from three of them. These results indicate that T cells expanded from PD-1+ PBL share more clones with paired TIL and could be used to treat patients with cancer as TIL substitutes.

The importance of CD4+ Tumor-Infiltrating Lymphocytes (TIL) in Adoptive Cell Transfer

Abstract Immunotherapy, including adoptive cell transfer (ACT) with tumor-infiltrating lymphocytes (TIL) predominantly targets improvement in MHC Class I-mediated anti-tumor immune responses. The primary objective of this study is to better understand the role of CD4+ TIL in ACT as a complementary avenue for therapeutic efficacy. Briefly, CD4+ TIL were isolated by negative selection from metastatic melanoma patients who received TIL therapy at Moffitt Cancer Center on IRB approved protocols. Individual T cell clones were tracked by TCRbeta sequencing to quantify clonal persistence in patients. CD4+ TIL clones were found to be decidedly persistent in a candidate patient who achieved a complete response (CR) after infusion of 88% CD4+ T cells. CD4+ TIL from additional patients were stimulated with anti-CD3/CD28 in vitro and those who were clinical responders demonstrated a pleiotropic cytokine profile marked by an elevated ratio of Th1:Th2 cytokines (p=0.07, n=13). When cultured with APCs loaded with autologous tumor (AT), CD4+ TIL produced high levels of IFN-gamma in an MHC Class II-dependent manner. Induction of MHC Class II on melanoma cell lines and AT determined that CD4+ TIL secreted IFNg and TNFa directly in response to AT. ACT of tumor-reactive CD4+ TIL in immunodeficient (NSG) mice provided significant control of AT growth when compared to non-reactive CD4+ TIL. Preliminary data in syngeneic mouse models also suggests that antigen-specific CD4+ T cells aid in initial tumor rejection, memory formation and epitope spreading, resulting in an overall increased therapeutic efficacy during ACT. This data supports the conclusion that CD4+ TIL are tumor-reactive and instrumental to an effective anti-tumor immune response in cancer patients.

Abstract LB-069: Persistence of cryopreserved tumor-infiltrating lymphocyte product lifileucel (LN-144) in C-144-01 study of advanced metastatic melanoma

Abstract Adoptive cell transfer utilizing tumor-infiltrating lymphocytes (TIL) is recognized as an effective treatment in metastatic melanoma and other solid tumors eliciting durable and complete responses in heavily pretreated patients, presumably by targeting somatic mutations specific to each tumor1. We have treated patients with anti-PD-1-refractory advanced melanoma with lifileucel and reported an overall response rate of 38%2. Here, we analyzed the composition of the initial TIL products and the T cells circulating 42 days post-infusion (D42) to uncover a potential link between clonal diversity, TIL in vivo persistence, and anti-tumor activity. Since TIL products are preparations of polyclonal autologous T cells, each T cell clone expresses a unique T cell receptor (TCR) that can be identified by its complementary determining region 3 (CDR3). CDR3 of TIL products and corresponding post-infusion peripheral blood samples were subjected to RNA-seq, using iRepertoire technology (Huntsville, AL). The average number of unique TCR CDR3 sequences (uCDR3) was 17511 [3574-110797] across TIL products, with Shannon diversity indexes varying from 2.7 to 10.8. Correlation analyses revealed no association between either parameter and clinical response, suggesting that tumor-reactive T cells may be present in low and high diversity bulk TIL products. At D42, TIL clones could be detected in the circulation of all treated patients. Numbers of Shared uCDR3 ranged from 28 to 6964 unique clonotypes and represented highly variable fractions of both the TIL product and the D42 circulating T cells. A link between Shared T cell clones and clinical response was previously hypothesized3. In this study, comparable percentages of Shared uCDR3 were detected in responders and non-responders. Importantly, the majority of Shared uCDR3 were not detected in the patients’ peripheral blood at the time of enrollment, indicating that they represented intratumoral clonotypes that persisted after TIL administration. In addition, Shared uCDR3 clones represented at either high or low frequencies in the TIL product could persist for at least 6 weeks post-infusion. Finally, more than 97% of persisting clones were uniquely present in individual responding and non-responding patients, indicating a unique repertoire in each TIL preparation. Overall, the data demonstrate that a fraction of TIL clones persisted in all patients. The uniqueness of the clonal profiles associated with response highlights the challenge of identifying a single TCR as mediator of activity and supports using a polyclonal product such as bulk TILs to treat solid tumors with their associated unique, patient-specific, mutational and neoantigen spectra. 1 Rosenberg et al. CCR 2011; 2 SITC Nov2018; 3 Robbins et al. J Immunol 2004 Citation Format: Viktoria Gontcharova, Sam Suzuki, Michelle R. Simpson-Abelson, Michelle Blaskovich, Cécile Chartier. Persistence of cryopreserved tumor-infiltrating lymphocyte product lifileucel (LN-144) in C-144-01 study of advanced metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-069.

Similar T-cell repertoires of tumor infiltrating lymphocytes and Crohn’s-like lymphoid reaction in colorectal cancer.

e15133 Background: Tumor infiltrating lymphocytes (TILs) and Crohn’s-like Lymphoid Reaction (CLR) are independently associated with improved survival in colorectal cancer (CRC). Whereas TILs are localized within tumors, CLR are extra-tumoral lymphocytic aggregates. The origins and relationships of the T-cell repertoire of TILs and the T cells within CLR of the colorectal cancer tumor microenvironment are unknown. Methods: Expert pathology review identified and circled areas of invasive adenocarcinoma and areas containing CLR from 13 CRC patients for macrodissection from formalin fixed paraffin embedded (FFPE) slides. DNA was extracted from matched tumor and CLR areas for multiplex PCR sequencing of the CDR3 region of the T-cell receptor beta chain (TCRβ), using the immunoSEQ platform. This approach permits 1) estimating the T-cell content of each sample, 2) measuring the clonality of the T-cell repertoire as a measure of diversity, and 3) quantifying the overlap and similarity of T-cell repertoires across samples. Results: The T-cell content (Spearman’s rs = 0.56, p = 0.046) and clonality (Spearman’s rs = 0.66, p = 0.014) were highly correlated among matched tumor and CLR samples. The ten most frequently identified TIL clones were found at similar frequencies in matched CLR enriched tissues. Comparisons of all the clones detected in tumor and matched CLR tissue demonstrated substantial similarity of these immune repertoires, with an average of 186 shared clones between samples. This degree of similarity was significantly greater than published reports of the similarity of the T-cell repertoire of colorectal tumors and adjacent normal tissue (p = 1.1e-5). Conclusions: The T-cell repertoire of CLR is highly similar to the tumor infiltrating T-cell repertoire, providing supporting evidence for the hypothesis that tumor-specific antigen presentation and lymphocyte maturation occur within CLR.

Intratumoral G100 to induce systemic immune responses and abscopal tumor regression in patients with follicular lymphoma.

7537 Background: Follicular lymphoma (FL) is an incurable malignancy with patients (pts) ultimately relapsing following standard therapies. Active immunotherapy has the potential to induce life-long host anti-tumor immunity and disease control. G100 consists of glucopyranosyl lipid-A (GLA), a TLR-4 agonist in a specific formulation. Preclinically, G100 activates dendritic cells, T cells and NK cells, and triggers systemic anti-tumor immunity. In Merkel Cell carcinoma pts, G100 administered intratumorally (IT) induced tumor inflammation and responses including a CR after G100 alone. This is the first study of G100 IT in pts with NHL. Methods: Previously treated or naïve pts with FL with an injectable tumor site and distal sites of disease were eligible. In Part 1, G100 cohorts of 5 or 10µg were enrolled in a 3+3 design, followed by a large tumor ( &gt; 4cm) cohort at 20µg. Pts received 6-9 doses of G100 IT ~qwk after radiation (RT, 2 Gy x2 doses) to the lesion. A 2nd course of G100 could be given without RT to an additional site. Results: As of 31Dec16, all 9 pts in Part 1 dose escalation (3 pts each at 5, 10, or 20 µg/dose) were evaluable for safety and efficacy. An additional 13 pts at 10µg/dose were included in the safety analysis only. No G100-related DLTs or SAEs were observed at any dose level. Of 22 safety pts, all G100 related AEs were grade 1/2 and none occurred in &gt; 2 pts. Tumor biopsies following G100 demonstrated diffuse infiltration of CD8+ T cells in 5/5 pts and T cell repertoire analyses indicated an increased frequency of clonal tumor infiltrating lymphocytes (TILs). Best responses include: 4 PRs (45%), 3 SDs (33%) and 2 pending (22%). Of the 4 PR pts, tumor regression ranged 58-89% including up to 56% shrinkage of abscopal (distal) sites. Conclusions: G100 IT was safe, well-tolerated, induced CD8+ T cell infiltration and expansion of TIL clones. G100/RT treated and abscopal lesion regressions were observed signifying the induction or boosting of systemic anti-tumor immunity. The induction of immune responses, favorable safety profile and clinical activity indicate that G100 IT is an active agent that warrants further investigation. Part 2 enrollment continues with randomization to G100/RT ± pembrolizumab. Clinical trial information: NCT02501473.

Multi-omics study revealing the complexity and spatial heterogeneity of tumor-infiltrating lymphocytes in primary liver carcinoma.

Intratumoral heterogeneity has been revealed in primary liver carcinoma (PLC). However, spatial heterogeneity of tumor-infiltrating lymphocytes (TILs), which reflects one dimension of a tumor's spatial heterogeneity, and the relationship between TIL diversity, local immune response and mutation burden remain unexplored in PLC. Therefore, we performed immune repertoire sequencing, gene expression profiling analysis and whole-exome sequencing in parallel on five regions of each tumor and on matched adjacent normal tissues and peripheral blood from five PLC patients. A significantly higher cumulative frequency of the top 250 most abundant TIL clones was observed in tumors than in peripheral blood. Besides, overlap rates of T cell receptor (TCR) repertoire for intratumor comparisons, significant higher than those for tumor-adjacent normal tissue comparisons and tumor-blood comparisons, which provide evidence for antigen-driven clonal expansion in PLC. Analysis of the percentage of ubiquitous TCR sequences, regional frequencies of each clone and TIL diversity suggested TIL clones varying between distinct regions of the same tumor, which indicated weak TCR repertoire similarity within a single tumor. Furthermore, correlation analysis revealed that TIL diversity significantly correlated with the expression of immune response genes rather than the mutation load. We conclude that intratumoural T-cell clones are spatially heterogeneous, which can lead to underestimate the immune profile of PLC from a single biopsy sample and may present challenge to adoptive cell therapy using autologous TILs. TIL diversity provides a reasonable explanation for the degree of immune response, implied TIL diversity can serve as a surrogate marker to monitor the effect of immunotherapy.

Abstract B074: Tumor-reactive CD4+ T cells in metastatic gastrointestinal cancer refractory to chemotherapy

Abstract Purpose: To evaluate whether patients with metastatic gastrointestinal adenocarcinomas refractory to chemotherapy harbor tumor-reactive CD4+ T cells. Experimental Design: Expansion of CD4+ tumor-infiltrating lymphocytes (TIL) and cancer cell lines was performed from gastrointestinal cancer metastases in 5 patients for the study of antitumor immune recognition. Retroviral transduction of genes encoding the class II, major histocompatibility complex, transactivator (CIITA) was used to induce the expression of major histocompatibility complex (MHC) class II in tumor cell lines. Recognition of autologous tumor cell lines by TIL was evaluated by up-regulation of 4-1BB and OX40 by flow cytometry, and/or secretion of IFNg by ELISA. Results: TIL were expanded from metastases, and new tumor cell lines were generated in 5 patients. Retroviral transduction of CIITA in tumor cell lines effectively induced expression of MHC class II in &amp;gt;80% of cells. Autologous tumor recognition was found in CD4+ TIL from 2 of these 5 patients. In a patient with gastric cancer liver metastases, tumor-reactive CD4+OX40+ TIL were cell-sorted from a TIL cell line. These cells up-regulated OX40 in the presence of all 4 autologous cancer cell lines albeit at different levels, but they did not produce IFNg. This recognition was specifically abolished by pan-MHC class II blocking antibodies. CD4+ TIL clones have been isolated and are being further characterised. Interestingly, tumor-reactive CD8+ TIL were previously identified and characterized in this patient. In a second patient with colon cancer abdominal wall metastases, tumor-reactive CD4+OX40+ TIL were cell-sorted from a TIL cell line. These cells were reactive to all 4 autologous cancer cell lines as they up-regulated OX40 and secreted IFNg. Recognition was blocked by anti-HLA-DR blocking antibodies. CD4+ TIL clones have been isolated and are being further characterized. In the near future, we expect to identify the TCR and HLA restriction element for both patients. We will also determine if CD4+ TIL clones have cross-reactivity against allogeneic HLA-matched gastrointestinal tumor cell lines. Conclusions: This study provides a basis for the development of immunotherapy for patients with advanced gastrointestinal malignancies by first establishing the presence of naturally occurring tumor-reactive CD4+ TIL at the molecular level. Citation Format: Sandy Pelletier, Simon Turcotte. Tumor-reactive CD4+ T cells in metastatic gastrointestinal cancer refractory to chemotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B074.

PD-1 expression conditions T cell avidity within an antigen-specific repertoire

ABSTRACTDespite its negative regulatory role on tumor-specific T cells, Programmed cell death 1 (PD-1) is also a marker of activated tumor-infiltrating T cells. In cancer, PD-1 blockade partially reverses T cell dysfunction allowing the amplification of tumor reactive T cells. Here, we investigated the role of PD-1 signaling on effector/memory human T cells specific for shared melanoma antigens, derived from blood. We documented for the first time the existence of melanoma-specific T cell clones unable to express PD-1. This stable feature was due to the persistent methylation of the PDCD1 promoter. These PD-1neg clones were of lower avidity than their PD-1pos counterparts, suggesting that high-affinity-specific T cell clones unable to express PD-1 are not or rarely present in peripheral blood, as they are probably eliminated by negative selection, due to their high reactivity. We also documented the existence of such PD-1neg T cell clones in melanoma tumor-infiltrating lymphocytes (TIL), which also exhibited a lower functional avidity than PD-1pos TIL clones. This clearly shows that PD-1 expression identifies antigen-specific T cell clonotypes of high functional avidity. Finally, we demonstrated that PD-1 blockade during the in vitro selection process of Melan-A-specific T cells favored the amplification of higher avidity T cell clonotypes. This preferential amplification of high-avidity memory T cells upon PD-1 blockade resonates with the expansion of reactive T cells, including neo-antigen-specific T cells observed in anti-PD-1-treated patients. This feature should also be a useful biomarker of clinical efficiency, while providing new insights for adoptive transfer treatments.

Study of tumor-infiltrating T-cell reactivity to metastatic gastrointestinal cancers.

e14179 Background: A high density of tumor-infiltrating lymphocytes (TIL) found in gastrointestinal cancers has been associated with improved prognosis. Evidence supporting specific autologous tumor recognition by TIL remains sparse and limits the development of innovative immunotherapy approaches. Methods: Metastases from 15 GI cancer patients (13 colorectal, 1 gastric, 1 cholangiocarcinoma) processed into fragments and fresh tumor cell suspensions (FrTu) were used to grow TIL and cancer cell lines. TIL phenotype was assessed by flowcytometry and reactivity by coculture with available cryopreserved FrTu and cancer cell lines. Limiting dilution techniques were used to isolate reactive CD8+ TIL clones. T-cell receptors (TCR) were sequenced using PCR amplification. Results: TIL were expanded successfully in all 15 cases. TIL phenotype studied from 7 freshly resected colorectal cancer metastases revealed a higher percentage of immunosuppressive T cells (CD4+CD25HIFoxP3+) compared to adjacent normal tissue and blood (respectively 5.4 ± 1.0%, 1.3 ± 0.5%, and 1.0 ± 0.4% of CD3+ cells). Interferon-gamma release by TIL after their first outgrowth upon coculture with cryopreserved FrTu was significant in all 6 cases studied, with positive results ranging from 7% (3/42) to 89% (8/9) of patient’s distinct cultures. Two newly established cancer cell lines allowed extensive investigation of TIL reactivity. In a case of gastric cancer, 65% of the CD3+CD8+ TIL expanded from liver metastases expressed the same TCR. TIL clones expressing this TCR could recognize the autologous cancer cell line with a high degree of specificity, suggesting that within this tumor a productive immune response occurred. In a case of metastatic cholangiocarcinoma, two TIL clones with distinct TCR represented less than 1% of the CD3+CD8+ cells grown from an omental metastasis and the malignant ascites, but could nonetheless lyse the autologous tumor with specificity. Conclusions: Despite the immunosuppressive microenvironment of GI cancer metastases, CD8+ TIL that recognize autologous tumor with specificity can be identified and expanded in vitro. The adoptive transfer of TIL for the therapy of metastatic GI cancer patients is currently being studied.

Intratumoral Induction of CD103 Triggers Tumor-Specific CTL Function and CCR5-Dependent T-Cell Retention

We have reported previously that the interaction of alpha(E)(CD103)beta(7) integrin, expressed on a CD8(+) tumor-infiltrating lymphocyte (TIL) clone but not on a peripheral blood lymphocyte (PBL) counterpart, with the epithelial marker E-cadherin on human lung tumor cells plays a crucial role in T-cell receptor-mediated cytotoxicity. We show here that both TIL and PBL clones are able to migrate toward autologous tumor cells and that chemokine receptor CCR5 is involved in this process. Adoptive transfer of the PBL clone in the cognate tumor engrafted in nonobese diabetic/severe combined immunodeficient mice and subsequent coengagement of T-cell receptor and transforming growth factor-beta1 receptor triggers CD103 expression on T-cell surface resulting in strong potentiation of antitumor lytic function. Moreover, interaction of alpha(E)beta(7) integrin with E-cadherin, but not lymphocyte function-associated antigen-1 with intercellular adhesion molecule-1, promotes CCR5 recruitment at the immunologic synapse formed between TIL and tumor cells, leading to inhibition of T-cell sensitivity to CCL5 chemotactic gradient. These results provide evidence for a role of tumor microenvironment, namely MHC class I-restricted antigen presentation and transforming growth factor-beta1 secretion, in regulating the effector phase of tumor-specific CTL response. They also suggest a unique role of CD103 in T-cell retention at the tumor site by a CCR5-dependent mechanism.

Interaction of CD103 with E-cadherin triggers CCR5 recruitment at the immunological synapse resulting in intratumoral retention of tumor-specific CTL (40.37)

Abstract We previously characterized two tumor-specific T-cell clones from PBL and tumor-infiltrating lymphocytes (TIL) of a lung cancer patient with identical TCR and similar lytic potential, but with different anti-tumor reactivity. We demonstrated that the interaction of alphaE(CD103)beta7 integrin, expressed on the TIL clone but not on the PBL counterpart, with the epithelial marker E-cadherin on human lung tumor cells, plays a crucial role in TCR-mediated cytotoxicity. In the present study we show that both TIL and PBL clones are able to migrate towards autologous tumor cells and that chemokine receptor CCR5 is involved in this process. Interestingly, adoptive transfer of the PBL clone in the cognate tumor engrafted in SCID-NOD mice and subsequent TCR engagement and TGF-β1 secretion triggers CD103 expression on T-cell surface and thus potentiation of anti-tumor cytotoxic activity. Furthermore, confocal microscopy analyses indicated that the interaction of αEβ7 integrin with E-cadherin, but not LFA-1 with ICAM-1, promotes CCR5 recruitment at the immunological synapse formed between TIL and tumor cells, leading to inhibition of T-cell sensitivity to CCL5 chemotactic gradient. Our results provide evidence for a role of tumor microenvironment in regulating anti-tumor CTL function and suggest a role of CD103 in intratumoral retention of tumor-specific T cells by a mechanism involving CCR5.

Tumor‐infiltrating lymphocytes derived from human renal cell carcinoma: Clonal analysis of its characteristics

To assess the characteristics of activated tumor-infiltrating lymphocytes (TIL), we report the isolation, growth response, and functional analysis of a CD4(-) CD8(+) TIL-clone derived from human renal cell carcinoma (RCC). Bulk TILs were expanded from a human RCC and the lymphocytes were separated into a CD8(+) enriched population. Subsequently, using the limiting dilution technique, a TIL clone was established and its growth response, phenotype and cytotoxic activity were analyzed. A clone, T16-13, by day 94 numbering 1 x 10(7) cells, was harvested and characterized as a CD4(-) CD8(+) clone. On day 144, the cytotoxic activity of this clone against the autologous tumor was relatively high (2.3 +/- 0.7 LU(30)/10(6) cells). Meanwhile, against allogeneic renal tumors, there was no cytotoxic activity (-0.1 LU(30)/10(6) cells). A TIL clone possessing modest autologous tumor-specific cytotoxicity can be isolated from human RCC. The characteristics analysis of various TIL clones may provide a better understanding of an RCC tumor microenvironment and may help to establish new modalities for the treatment of patients with metastatic kidney cancer.

A HLA-Cw*0701 restricted Melan-A/MART1 epitope presented by melanoma tumor cells to CD8+ tumor infiltrating lymphocytes.

Melan-A/MART1 is a melanocytic differentiation antigen recognized on melanoma tumor cells by CD8+ and CD4+ T cells. In this study, we describe a new epitope of this protein recognized in the context of HLA-Cw*0701 molecules by a CD8+ tumor infiltrating lymphocyte (TIL) clone. This CD8+ TIL clone specifically recognized and killed a fraction of melanoma cells lines expressing Melan-A/MART1 and HLA-Cw*0701. We further show that the Melan-A/MART1(51-61) peptide is the optimal peptide recognized by this clone. Together, these data significantly enlarge the fraction of melanoma patients susceptible to benefit from a Melan-A/MART1 vaccine approach.