Cells In Tumor-infiltrating Lymphocytes Research Articles

Abstract CT130: Trial in progress: A phase 2 multicenter study (IOV-LUN-202) of autologous tumor-infiltrating lymphocyte (TIL) cell therapy (LN-145) in patients with metastatic non-small cell lung cancer (mNSCLC)

Abstract Background: Patients with mNSCLC without actionable driver mutations have limited second-line (2L) options after progression on concurrent or sequential front-line immune checkpoint inhibitors (ICI) and platinum-based chemotherapy ± bevacizumab. Early clinical experience with LN-145, an autologous TIL cell therapy, in heavily pretreated patients with mNSCLC has demonstrated feasibility, safety, and a 21.4% objective response rate (ORR), including 2 of 6 responders with PD-L1-negative tumors (Schoenfeld AJ, SITC 2021 [abs 458]). Based on the results of this signal-finding study and to address an unmet medical need in 2L, we amended the ongoing IOV-LUN-202 study, evaluating LN-145 in patients with stage IV mNSCLC after confirmed disease progression on front-line standard-of-care treatment, including concurrent or sequential ICI + chemotherapy ± bevacizumab. Methods: IOV-LUN-202 (NCT04614103) is an open-label, nonrandomized, phase 2 study. Cohorts 1 (tumor proportion score [TPS] <1% prior to ICI use or no historical TPS) and 2 (TPS ≥1% prior to ICI use) are actively enrolling patients. Cohort 3 (any TPS) uses core biopsies for tumor procurement and a 16-day Gen 3 manufacturing process for patients unable to undergo surgical resection. A retreatment cohort (n not prespecified) will enroll prior responders to LN-145 or patients with unconfirmed progressive disease from cohorts 1-3. A total of approximately 95 patients is planned for cohorts 1-3. Key eligibility criteria include mNSCLC with no EGFR, ALK, or ROS1 genomic alterations, progressing after ≤2 prior lines of therapy (if concurrent ICI + chemotherapy; ≤3 prior lines if sequential), including targeted therapy in those with actionable mutations (eg, MET, HER2, RET, BRAF, KRAS); in those without actionable mutations, progression after 1 prior concurrent ICI + chemotherapy (≤2 prior lines if sequential); ≥1.5-cm lesion(s) for TIL generation; ≥1 remaining RECIST-measurable lesion(s) after tumor harvest; and ECOG PS of 0 or 1. Tumor harvest and TIL manufacturing can occur before or after confirmed progression. LN-145 is generated at centralized GMP facilities, and the final cryopreserved infusion product is shipped to the sites. Upon progression, patients receive a conditioning regimen of nonmyeloablative lymphodepletion with 2 doses of cyclophosphamide (60 mg/kg) and 5 doses of fludarabine (25 mg/m2), followed by one-time infusion of LN-145 (1-150 × 109 cells), and ≤6 doses of IL-2 (600,000 IU/kg). The primary endpoint is ORR per RECIST v1.1. Secondary endpoints include complete response rate, duration of response, disease control rate, progression-free survival, overall survival, and safety (incidence of Grade ≥3 treatment-emergent adverse events [AEs] and serious AEs). Citation Format: Jason Alan Chesney, Adam J. Schoenfeld, Trisha Wise-Draper, Ammar Sukari, Kai He, Friedrich Graf Finckenstein, Parameswaran Hari, Madan Jagasia, Selda Samakoglu, Ann Leighton-Swayze, Guang Chen, Yong Ki Hong. Trial in progress: A phase 2 multicenter study (IOV-LUN-202) of autologous tumor-infiltrating lymphocyte (TIL) cell therapy (LN-145) in patients with metastatic non-small cell lung cancer (mNSCLC) [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 CT130.

Abstract 5609: Investigating the undifferentiated pleomorphic sarcoma microenvironment through cytokine profiles of primary tumor and immune infiltrates

Abstract Sarcoma is a heterogeneous group of connective tissue cancers with over 60 subtypes. Curative treatment relies on surgical resection combined with radiation. In the event of metastasis, chemotherapy is used but generally only with palliative intent. Given the heterogeneity of sarcomas, targeted therapies are limited, which has contributed to the relatively stagnant survival rates seen in the past decades. Objective: Personalized treatments, such as adoptive cell therapy (ACT), may be promising for select sarcoma patients, specifically those with Undifferentiated Pleomorphic Sarcoma (UPS). To better understand ACT’s feasibility for UPS, we aim to investigate the intersection of tumor-infiltrating lymphocytes (TILs) and tumor cells by examining their cytokine output, whose role remains largely unstudied. Methods: Tissue samples from untreated sarcoma patients have been collected and viably preserved by our group. The samples were processed for in vitro expansion of tumor or TIL cell lines. Tumor cultures were validated with Whole Exome Sequencing (WES) to ensure tumor-specific variations were retained. TILs were isolated with tumor fragment (TF) processing and magnetic bead coated anti-CD3/CD28 Rapid Expansion Protocol (REP). After validating tumor cultures, both tumor and TIL-secreted cytokines were evaluated using Luminex assays. Results: Of the 11 primary UPS samples cultured, 10 generated viable cell cultures as defined by the presentation of stable growth beyond passage 5. A total of 3 cell lines were selected for further validation, and through WES, retained tumor specific variations beyond passage 5. On the other hand, the sarcoma-optimized TF and REP protocols expanded TILs from 7 of 8 UPS cases and yielded up to 6 x 10^7 cells. Although IL-2 is commonly used to facilitate TIL growth, it can also cause activation-induced cell death which is problematic for therapy. We demonstrated that an alternative γ-chain interleukin, IL-15, can support UPS TIL growth comparable to IL-2. Unbiased cytokine screening detected anti-tumoral CXCL9/IFN-γ and pro-tumoral IL-10/TGF-Beta as highly secreted by TIL cultures while some tumor cultures secreted relatively high concentrations of IL-6 and CCL2. Conclusion: We developed a robust in vitro pipeline to expand sarcoma tumor cultures and TILs for downstream characterization. Functional assays aimed at dissecting the consequences of tumor-mediated IL-6 secretion are underway. These include siRNA-induced knock downs of genes hypothesized to augment the cytokine profile of tumor cells. Future evaluation of TILs will identify their clinical efficacy via flow cytometry and intracellular cytokine staining. Understanding cytokine secretion from both the tumor and immune components of sarcoma will aid in elucidating interactions within the microenvironment that may be therapeutically relevant. Citation Format: Victoria Coward, Jacky Chen, Nalan Gokgoz, Kim Tsoi, Jay S. Wunder, Irene L. Andrulis. Investigating the undifferentiated pleomorphic sarcoma microenvironment through cytokine profiles of primary tumor and immune infiltrates [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 5609.

Tumor mutational burden (TMB) in immune checkpoint inhibitor (ICI)-naïve and -experienced patients with metastatic melanoma treated with lifileucel, a tumor-infiltrating lymphocyte (TIL) cell therapy.

9524 Background: Cutaneous melanoma is characterized by high TMB, which is associated with increased tumor-specific neoantigen expression (Schumacher Science 2015) and an increased response rate to ICI (Yarchoan NEJM 2019). The TMB in tumors that recur/progress after ICI is not well defined. Lifileucel is a one-time, autologous TIL cell therapy under investigation for treatment of patients (pts) with advanced melanoma. We conducted a matched case-control study of prospectively enrolled pts with advanced melanoma treated with lifileucel in the ICI-naïve (IOV-COM-202 trial, Cohort 1A [C1A]) and post-ICI (C-144-01 trial, Cohort 2 [C2]) setting to investigate the potential association between prior ICI therapy, TMB, and response to lifileucel. Methods: All pts had unresectable or metastatic melanoma. Available cases from C1A (ICI-naïve pts receiving lifileucel + pembrolizumab [pembro]) were matched to controls from C2 (pts receiving lifileucel alone after progression on anti–PD-1/PD-L1 therapy); 3 controls per case were matched at least for BRAF status and disease stage at study entry, and if possible, for anatomic site of tumor harvest. Lifileucel regimen was similar in C1A and C2. In C1A, 1 dose of pembro was given after tumor harvest and before nonmyeloablative lymphodepletion and resumed after lifileucel per standard treatment, for up to 2 y. Objective response rate (ORR) was assessed by investigators per RECIST v1.1. TMB of the resected tumor was measured using the ImmunoID NeXT (C1A) or PGDx elio (C2) platform; a validated conversion factor was used to compare TMB between platforms (Vega Ann Oncol 2021). High TMB was defined as ≥10 mut/MB. Results: Seven pts in C1A and 21 in C2 were included in the case-control study and had ORR of 71.4% and 38.1%, respectively. The percentage of pts with high TMB was 57.1% in C1A and 19.0% in C2 ( P = 0.1). ORR in the low and high TMB groups was 66.7% and 75.0%, respectively, in C1A and 41.1% and 25.0% in C2; 60% of responders in C1A and 12.5% in C2 had high TMB. In logistic regression analysis adjusted for cohort, TMB was not associated with response to lifileucel (odds ratio, 1.0; 95% CI, 0.9–1.1; P = 0.8). Data on tumor mutations and neoantigens, T-cell receptor repertoire, and tumor microenvironment profile will be presented. Conclusions: Our preliminary data indicate that the efficacy (ORR) of lifileucel may be independent of TMB, regardless of treatment setting, consistent with its proposed immune checkpoint pathway-independent mechanism of action. The percentage of patients with high TMB tended to be lower in tumors with prior ICI exposure than in those that were ICI-naïve. Clinical trial information: NCT03645928; NCT02360579.

DELTA-1: A global, multicenter, phase 2 study of ITIL-168, an unrestricted autologous tumor-infiltrating lymphocyte (TIL) cell therapy, in adult patients with advanced cutaneous melanoma.

TPS9594 Background: Patients (pts) with advanced (unresectable or metastatic) cutaneous melanoma and persistent disease after checkpoint inhibitor therapy have poor outcomes and limited treatment options, highlighting a significant unmet medical need (Schadendorf D et al. Lancet. 2018;392:971-984). Investigational autologous TIL cell therapies have shown promise in this population, partly attributable to their intrinsic and patient-specific antitumor activity (Borch TH et al. J Immunother Cancer. 2020;8:e000668). Made from each patient’s digested and cryopreserved tumor, ITIL-168 is an autologous TIL cell therapy manufactured to offer an unrestricted T-cell receptor repertoire. A single-center, compassionate use clinical series demonstrated the feasibility and clinical utility of an earlier version of ITIL-168, with a high overall response rate among pts previously treated with PD-1 inhibitor (PD-1i) therapy (58%, n = 12; Pillai M et al. Ann Oncol. 2021;32[suppl 5]:S882). DELTA-1 (NCT05050006) is a global, multicenter phase 2 study to evaluate efficacy and safety of ITIL-168 in pts with cutaneous melanoma relapsed or refractory to a PD-1i, pts intolerant to a PD-1i, and pts whose current best response to a PD-1i is stable disease. Methods: Pts aged ≥18 years with histologically confirmed advanced cutaneous melanoma, ECOG performance status 0-1, and adequate organ function will be enrolled in 1 of 3 cohorts. Cohort 1 (n≈80) will include pts who relapsed after or were refractory to ≥1 prior line of systemic therapy, including a PD-1i and, if BRAF-mutated, a BRAFi ± MEKi. Cohorts 2 and 3 (n≈25 each) will include pts intolerant to PD-1i and those with stable disease after ≥4 doses of PD-1i, respectively. After tumor resection for TIL harvest, pts must have ≥1 remaining measurable lesion per RECIST 1.1. Pts with uveal, acral, or mucosal melanoma, prior allogeneic transplant or cell therapy, and with central nervous system (CNS) disorder or symptomatic and/or untreated CNS metastases are ineligible. Pts will receive 5 days of lymphodepleting chemotherapy (cyclophosphamide ×2 days overlapping with fludarabine ×5 days) followed by a single ITIL-168 infusion (≥5×109 cells) and supportive short-course, high-dose IL-2. The primary endpoint is objective response rate (ORR) per central review. Secondary endpoints include duration of response, progression-free survival, overall survival, disease control rate, TIL persistence, and safety. Hypothesis testing of ORR will be performed for cohort 1. The primary analysis will occur when all pts in the cohort 1 modified intent-to-treat population have been followed for ≥6 months after the first posttreatment disease assessment. DELTA-1 opened for enrollment in September 2021. Updated site information will be given at the time of presentation. Clinical trial information: NCT05050006.

Targeting of Nrf2 improves antitumoral responses by human NK cells, TIL and CAR T cells during oxidative stress

BackgroundAdoptive cell therapy using cytotoxic lymphocytes is an efficient immunotherapy against solid and hematological cancers. However, elevated levels of reactive oxygen species (ROS) in the hostile tumor microenvironment can impair...

Artificial intelligence-powered pathology image analysis merged with spatial transcriptomics reveals distinct TIGIT expression in the immune-excluded tumor-infiltrating lymphocytes.

2570 Background: TIGIT is a promising emerging immunotherapeutic target. However, the specific sources of TIGIT expression within the tumor microenvironment are largely unknown. Here, we present an AI-powered spatial tumor-infiltrating lymphocyte (TIL) analyzer, Lunit SCOPE IO, to integrate image analysis from whole slide images with single-cell molecular profiling. Methods: We used The Cancer Genome Atlas (TCGA) RNA expression data across 23 cancer types (n=6,930). Lunit SCOPE IO was developed, trained, and validated based on >17k H&E whole-slide images, to segment cancer area (CA) and cancer-associated stroma (CS) and to detect tumor cells and TILs. The intra-tumoral TIL, stromal TIL, and tumor cell purity (TCP) in the CA+CS area were calculated. The public spatial transcriptomics (ST) dataset for breast cancer was downloaded from the 10X Visium web page. Lunit SCOPE IO was applied to the associated H&E WSIs to match distinct TIGIT expression to single cells identified in the WSIs. Results: TIGIT was highly expressed in TGCT (3.45±0.11; median±SEM), LUAD (3.07±0.05), and HNSC (2.89±0.06), and was highly enriched in samples with microsatellite instability-high or tumor mutational burden-high (≥ 10/Mb) compared to those without them (fold change = 1.30, p < 0.001). At a macroscopic, bulk-level in the TCGA dataset, TIGIT expression was positively correlated with intra-tumoral TIL density (R=0.37, p<0.001) and stromal TIL density (R=0.42, p<0.001), but it was negatively correlated with TCP (R=-0.27, p<0.001). Lunit SCOPE IO analyzed the images from ST analysis and calculated intra-tumoral TIL, stromal TIL, and TCP of each region of interest, containing 2 (IQR 0-7) cells. Interestingly, at a microscopic, cell-level, TIGIT expression was still higher in areas of enriched stromal TIL (P < 0.001) and lower in tumor cell-dense areas, but it was not significantly correlated with enriched intra-tumoral TIL areas, meaning that TIGIT expression is likely derived from the excluded TILs in the CS area. Conclusions: Interactive analysis of spatial transcriptomics with AI-powered pathology image analysis revealed that TIGIT expression in the tumor microenvironment is exclusive to confined areas with stromal TIL enrichment, reflecting the exclusion of TIL from the tumor nest. [Table: see text]

Efficacy and safety of autologous expanded tumor infiltrating lymphocytes (TILs) in multiple solid tumors.

2536 Background: TIL therapy has been used extensively in metastatic melanoma patients for many years, now with ongoing efforts to commercialize treatment. The efficacy of TIL outside of melanoma is largely unknown thus we designed and implemented a trial using TIL manufactured at a single academic center for treatment refractory metastatic colorectal (CRC), pancreas (PDAC) and ovarian (OVA) cancers. Methods: Patients with CRC, PDAC and OVA refractory to standard therapies with ECOG PS 0-1 and normal organ function were eligible for TIL harvest. Ex vivo TIL expansion and manufacturing was conducted at the MD Anderson TIL lab under conditions that included IL2 and 41BB stimulation (using urelumab). All patients received a lymphodepletion regimen consisting of cyclophosphamide 60mg/kg days -7 and -6 and fludarabine 25mg/m2 days -5 through day -1, followed by infusion of pooled ex-vivo expanded TIL. Patients received up to 6 doses of high dose IL-2 (600,000 IU/kg) after TIL infusion. The primary endpoint was evaluation of the objective response rate (ORR) using RECIST 1.1 criteria with secondary endpoints including disease control rate, duration of response, PFS, OS and safety. Results: A total of 17 patients underwent TIL harvest and 16 were treated on protocol; including 8 CRC, 5 PDAC and 3 OVA. Median age was 57.5 (range 33-70) and 50% were females. Median number of lines of prior therapy was 2 (range 1-8). Median number of TIL infused was 76 X 109 (range 20.3 x 109-150 x 109). Median doses of cyclophosphamide and fludarabine administered were 2 (range, 2-2) and 3 (range, 1-5), respectively. Median doses of IL-2 administered was 6 (range, 1-6). There were no responders. Best response included prolonged SD in a patient with PDAC lasting until 18 months. Grade 3 or higher toxicities attributable to therapy was seen in 14 subjects (87.5%; 95% CI: 61.7 – 98.4) with the majority of toxicities representing expected pancytopenia from lymphodepletion. Infusion product analysis showed the presence of effector memory cells with high expression of CD39 irrespective of tumor type. Early on-treatment biopsy of PDAC patient with prolonged SD showed presence of proliferating (KI67+) CD4+ and CD8+ TIL. Conclusions: Generation of TIL at a single academic center for CRC, PDAC and OVA is feasible and treatment is associated with no new safety signals. For these tumor types, further research is required to identify host factors associated with resistance to TIL therapy and optimize manufacturing processes to create more effective TIL cell therapy. Clinical trial information: NCT03610490.

Distribution and Clinical Significance of IL-17A in Tumor-Infiltrating Lymphocytes of Non-Small Cell Lung Cancer Patients.

Objective: To investigate the distribution of IL-17A and its clinical significance in tumor infiltrating lymphocytes (TILs) of patients with non-small cell lung cancer (NSCLC). Methods: Expression level of IL-17A in TILs of 3 paired NSCLC and paracancerous specimens was measured by qRT-PCR. The distribution of IL-17A in immune cell subsets of 15 paired NSCLC and paracancerous specimens was examined by flow cytometry. The correlation between IL-17A and clinical features of NSCLC was identified. Results: IL-17A was significantly upregulated in TILs of NSCLC specimens than those of paracancerous ones (p < 0.0001). Meanwhile, T helper 17 cells (Th17 cells, p < 0.001), IL-17-secreting CD8+ T cells (Tc17 cells, p < 0.001) and IL-17-producing cells (γδT17 cells, p < 0.0001) were significantly abundant in TILs of NSCLC specimens than those of controls, and the higher abundance of the latter was much pronounced than that of the former two. Moreover, γδT17 cells in TILs were significantly correlated with lymphatic metastasis and CYFRA 21-1 level of NSCLC patients (p < 0.05). Conclusion: Tumor infiltrated γδT cells are the main source of IL-17 in early-stage NSCLC, and IL-17 may be a vital regulator involved in the development of NSCLC.

MP06-12 A WT1 ORAL CANCER VACCINE USING A BIFIDOBACTRERIUM VECTOR SUPPRESSES GROWTH OF ANTI-PD-1 ANTIBODY-RESISTANT MOUSE BLADDER TUMOR

You have accessJournal of UrologyCME1 May 2022MP06-12 A WT1 ORAL CANCER VACCINE USING A BIFIDOBACTRERIUM VECTOR SUPPRESSES GROWTH OF ANTI-PD-1 ANTIBODY-RESISTANT MOUSE BLADDER TUMOR Toshiro Shirakawa, Koichi Kitagawa, Hideto Ueki, Junya Furukawa, Nobuyuki Hinata, and Masato Fujisawa Toshiro ShirakawaToshiro Shirakawa More articles by this author , Koichi KitagawaKoichi Kitagawa More articles by this author , Hideto UekiHideto Ueki More articles by this author , Junya FurukawaJunya Furukawa More articles by this author , Nobuyuki HinataNobuyuki Hinata More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cancer immunotherapy with immune-checkpoint inhibitors (ICIs) including PD-1/PD-L1 inhibitors has been well established for various types of cancer including bladder cancer. Monotherapy with ICIs, however, can achieve a durable response in only a subset of patients. There is a great unmet need for ICI-resistant tumors. Although combining ICIs with cancer vaccines which forcibly induce an antitumor T cell response is a reasonable strategy, the preferred administration sequence is unknow. In our previous study, we demonstrated that an oral cancer vaccine, a recombinant Bifidobactrerium (B.) longum displaying a WT1 tumor associated antigen, inhibited the tumor growth in a TRAMP-C2 mouse prostate cancer tumor model and that its anti-tumor activity could be augmented by an anti-PD-1 antibody (Molecular Cancer Therapeutics, 2019;18:980-). In the present study we explored the feasibility of this oral vaccine in a mouse syngeneic bladder cancer model. METHODS: A recombinant B. longum 420 strain that expresses partial murine-WT1 protein fused to galacto-N-biose/lacto-N-biose I binding protein, which we used as an anchor to display antigen on the bacterial cell surface, was constructed. MBT-2, which is a bladder cancer cell line originated from C3H/He mice and endogenously expresses WT1 protein, was used to generate a syngeneic subcutaneous tumor model. Mice were orally administered 1.0×109 colony-forming units of B. longum 420, 5 times a week for 5 weeks, using a feeding needle. For an anti-PD-1 antibody treatment, 200μg of anti-PD-1 antibody was intraperitoneally injected into mice twice a week for 2 weeks. Tumors continuously grew despite the initial anti-PD-1 treatment were selected as the anti-PD-1 antibody-resistant tumor. In addition, immunological responses including tumor-infiltrating lymphocytes (TILs) were thoroughly analyzed. RESULTS: Combining B. longum 420 and following anti-PD-1 antibody treatment completely suppressed the MBT-2 tumor growth and cured all mice tested, while all anti-PD-1 antibody treated mice died. The significantly increased CD4 and CD8 positive T cells were observed in the TILs of combination treatment group. Moreover, in the anti-PD-1 antibody-resistant tumor model, this vaccine alone significantly inhibited the tumor growth, while combination with continuous anti-PD-1 antibody could not inhibit the tumor growth. Interestingly, the number of regulatory T cells in TILs of the vaccine alone group was significantly smaller than the combination treatment group. CONCLUSIONS: These results suggest that this oral cancer vaccine alone or as an adjunct to anti-PD-1 antibody could provide a novel treatment option for patients with advanced bladder cancer. Source of Funding: Japan Agency for Medical Research and Development (AMED): 20lm0203091h0002 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e81 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Toshiro Shirakawa More articles by this author Koichi Kitagawa More articles by this author Hideto Ueki More articles by this author Junya Furukawa More articles by this author Nobuyuki Hinata More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement PDF DownloadLoading ...

Decrease of Tumor-infiltrating Regulatory T Cells Using Pentoxifylline: An Ex Vivo Analysis in Triple-negative Breast Cancer Mouse Model.

Triple-negative breast cancer (TNBC) is the most aggressive type of BC with the highest percentage of tumor-infiltrating lymphocytes (TILs). Hence, TIL therapy is considered a promising approach to target TNBC. Depletion of regulatory T cells (Tregs) in TILs can improve the antitumor function of TIL therapy. Pentoxifylline (PTXF) is a xanthine derivative that can modulate the nuclear factor kappa B (NF-κB) signaling and probably affect the Treg proportion in TILs. We aimed to evaluate the ex vivo effect of PTXF on the proportion of Treg cells in the TILs derived from a mouse model of TNBC. The 4T1 cells were inoculated subcutaneously to BALB/c mice to induce TNBC. TILs were isolated from tumor tissue by enzymatic digestion and cultured alone or with 4T1 cells for 24, 48, and 72 h in the presence of interleukin (IL)-2 and different concentrations of PTXF. The toxicity of PTXF and its effects on Tregs proportion as well as cytokine production was evaluated using MTT assay, flow cytometry, and ELISA, respectively. PTXF had no significant impact on the viability of TILs. Both 500 and 1000 mg/mL of PTXF decreased the proportion of Tregs in a dose-dependent manner. The level of interferon-g and tumor growth factor-b in TILs supernatant was increased and decreased, respectively. Our data suggest that ex vivo treatment of TILs with pentoxifylline could decrease the proportion of Tregs in the conventional IL-2-mediated TIL expansion and change the cytokine balance of TILs in favor of antitumor immune response.

Abstract P039: The immune profile of colorectal and pancreas adenocarcinomas: Differences between central and peripheric tumor regions

Abstract Introduction The tumor microenvironment(TME) is a complex system, where malignant cells co-exist and communicate with immune and non-immune cells(1). This interaction can induce an immune response by releasing cytokines that will regulate and recruit immune cells which may promote tumor shrinkage (2). High tumor-infiltrating lymphocytes (TILs) in the TME have been correlated with favorable prognosis in colorectal and pancreatic adenocarcinomas (CRC and PDAC) (3,4). Also, high number of NK cells has been associated with a better prognosis in different solid tumors (5). Therefore, a detailed analysis of immune cells' distribution in the TME could help understand which of these cells have higher tumor-infiltrating capacities and may be used as cell product of immunotherapy. Experimental Procedure Fresh CRC (n=6) and PDAC (n=6) tumor specimens were obtained within 20 minutes after surgery. A specialized pathologist collected the central and peripheric regions (CR and PR, respectively) of the tumor. From each region, half of the specimen was used for immunophenotyping analysis and the other half was cultured with medium enriched in IL-2 (1000 IU/mL) for 12 days. Immunophenotyping was performed using CD45, CD19, CD3, CD4, CD8, CD56, CD16 and LiveDead marker through flow cytometry at days 0, 6 and 12 (% correspond to mean). Formalin-fixed paraffin-embedded blocks were generated with the ‘mirror' sample of the fresh collected specimens and immunohistochemistry (IHC) for CD8 and CD56 was performed. Results In the PR of CRC we identified 12% of B-cells, 66% of T-cells (31% CD4+ and 23% CD8+ T-cells) and 5% of NK cells (17% CD56brightCD16− cells and 24% CD56+CD16+ cells). In the CR of CRC we observed 7% of B-cells, 70% of T-cells (40% CD4+ and 23% CD8+ T-cells), and 6% of NK cells (15% CD56brightCD16− cells and 44% CD56+CD16+ cells). In the PR of PDAC we observed 13% of B-cells, 76% of T-cells (38% CD4+ and 35% CD8+ T-cells) and 3% of NK cells (5% CD56brightCD16− cells and 40% CD56+CD16+ cells). In the CR of PDAC we identified 16% of B-cells, 66% of T-cells (31% CD4+ and 32% CD8+ T-cells) and 10% of NK cells (2% CD56brightCD16− cells and 55% CD56+CD16+ cells). During culture, the % of B-cells decreased while the % of T-cells and NK cells increased. IHC results show that CD8+ T-cells are more abundant in the TME and formed larger clusters than NK cells. Furthermore, although the majority of NK cells were found in the stroma, in some cases NK cells were located inside the malignant epithelium. Conclusions Our data in this small set of specimens show that TME immune cells in PRs and CRs present different phenotypes for both CRC and PDAC. Indeed, the PRs show higher TIL and NK cells' infiltration than the central ones. Among different patients, the distribution and quantity of CD8+ T-cells and NK cells were different, which may have clinical significance. We also observed that immune cells may communicate with tumor cells both in CRC and PDAC since these cells were identified inside the malignant epithelium in some tumor specimens. Citation Format: Andreia Maia, Joana R. Lérias, Luis Miguel Borrego, Mireia Castillo-Martin, Markus Maeurer. The immune profile of colorectal and pancreas adenocarcinomas: Differences between central and peripheric tumor regions [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P039.

Isolation of TCR genes with tumor-killing activity from tumor-infiltrating and circulating lymphocytes in a tumor rejection cynomolgus macaque model

To develop effective adoptive cell transfer therapy using T cell receptor (TCR)-engineered T cells, it is critical to isolate tumor-reactive TCRs that have potent anti-tumor activity. In humans, tumor-infiltrating lymphocytes (TILs) have been reported to contain CD8+PD-1+ T cells that express tumor-reactive TCRs. Characterization of tumor reactivity of TILs from non-human primate tumors could improve anti-tumor activity of TCR-engineered T cells in preclinical research. In this study, we sought to isolate TCR genes from CD8+PD-1+ T cells among TILs in a cynomolgus macaque model of tumor transplantation in which the tumors were infiltrated with CD8+ T cells and were eventually rejected. We analyzed the repertoire of TCRα and β pairs obtained from single CD8+PD-1+ T cells in TILs and circulating lymphocytes and identified multiple TCR pairs with high frequency, suggesting that T cells expressing these recurrent TCRs were clonally expanded in response to tumor cells. We further showed that the recurrent TCRs exhibited cytotoxic activity to tumor cells in vitro and potent anti-tumor activity in mice transplanted with tumor cells. These results imply that this tumor transplantation macaque model recapitulates key features of human TILs and can serve as a platform toward preclinical studies of non-human primate tumor models.

Distinct mutational profile and immune microenvironment in microsatellite-unstable and POLE-mutated tumors

IntroductionMismatch repair (MMR)-deficient and DNA polymerase epsilon (POLE)-mutated tumors exhibit a high tumor mutation burden (TMB) and have been proven to be associated with good responses to immune checkpoint inhibitor...

YAP1/MMP7/CXCL16 axis affects efficacy of neoadjuvant chemotherapy via tumor environment immunosuppression in triple-negative breast cancer.

To evaluate the association of potential YAP1/MMP7/CXCL16 axis and tumor infiltrating lymphocytes (TILs) related chemo-response in triple-negative breast cancer (TNBC) patients. We estimated the messenger RNA (mRNA) expression levels of Yes-associated protein 1 (YAP1), MMP7, and CXCL16 in paired TNBC tumor/para-tumor tissues by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and performed statistical analysis according to neoadjuvant chemotherapy (NAC) response. Based on The Cancer Genome Atlas (TCGA) data, we noticed outstanding expression of MMP7/CXCL16 in TNBC cases, as well as associations between MMP7/CXCL16 and HIPPO-YAP1-relevant kinases. We also performed gene set enrichment analysis (GSEA) between MMP7/CXCL16 and YAP1-associated pathways. Western blotting assay was employed to evaluate YAP1/MMP7/CXCL16 expression in vitro and their modulation sequence. Logistic model stepwise regression analysis was used to assess YAP1, MMP7, CXCL16, and TILs as therapeutic predictors. Residual cancer burden (RCB) score was calculated and statistically analyzed according to intensity of these variables, and receiver operating characteristic (ROC) curve also showed their predictive value in NAC response. Recruitment efficacy for CD4+/CD8+ TIL cells (TCGA data) as well as quantified TIL cells density were both explored according to YAP1, MMP7, and CXCL16 expression level. Up-regulation of YAP1/MMP7 and down-regulation of CXCL16 were both significant in TNBC cases with poor NAC response. Inhibition of YAP1 induced down-regulation of MMP7 and up-regulation of CXCL16, whereas inhibition of MMP7 also induced up-regulation of CXCL16. It was also shown that MMP7/CXCL16 was enriched in the YAP1-related pathway. Activation of the YAP1/MMP7/CXCL16 axis obviously affected RCB of TNBC cases. The ROC curve also supported the predictive value of YAP1/MMP7/CXCL16 axis and TILs density in NAC response prospect. The density of TILs, meanwhile, demonstrated a strong link with the YAP1/MMP7/CXCL16 axis. Over expression of YAP1/MMP7 significantly suppressed recruitment of CD4+/CD8+ TILs, while CXCL16 over expression had a beneficial impact on anti-tumor immune. Over expression of causes up-regulation of MMP7 and down-regulation of CXCL16, which suppressed CD4+/CD8+ TILs recruitment and indirectly affected NAC response of TNBC patients.

Ultrafine Jujube Powder Enhances the Infiltration of Immune Cells during Anti-PD-L1 Treatment against Murine Colon Adenocarcinoma.

Simple SummaryWhile modulating gut microbiota using dietary intervention with natural nutrients has proven to be effective in improving the response rate of immune checkpoint inhibitors (ICIs), the underpinning mechanism is poorly understood. This work demonstrates that the oral administration of ultrafine jujube powder (JP) let to a significant alteration of gut microbiota, an increased abundance of Clostridiales, including Ruminococcaceae and Lachnospiraceae, an elevated SCFA production, an intensified infiltration of CD8+ T cells to the tumor microenvironment, and a greatly improved response of anti-PD-L1 treatment against murine colon adenocarcinoma. Moreover, the size of the JP particles had a significant impact on the abovementioned attributes. The present study demonstrates that dietary intervention with nutrients is highly effective in modulating the gut microbiota for an improved immune checkpoint blockage therapy.Whereas dietary intervention with natural nutrients plays an important role in activating the immune response and holds unprecedented application potential, the underpinning mechanism is poorly understood. The present work was dedicated to comprehensively examine the effects of ultrafine jujube powder (JP) on the gut microbiota and, consequentially, the effects associated with the response rate to anti-PD-L1 treatment against murine colon adenocarcinoma. A murine colon adenocarcinoma model with anti-PD-L1 immunotherapy was established to evaluate how dietary interventions affect the microbiota. In vitro and in vivo experiments confirmed the role of SCFAs in the immune response. Oral administration of JP greatly improves the response of anti-PD-L1 treatment against murine colon adenocarcinoma. Such an improvement is associated with the alteration of gut microbiota which leads to an increased abundance of Clostridiales, including Ruminococcaceae and Lachnospiraceae, an elevated SCFA production, and an intensified infiltration of CD8+ T cells to the tumor microenvironment. This work demonstrates that JP is particularly effective in modulating the gut microbiota for an improved immune checkpoint blockage therapy by boosting cytotoxic CD8+ T cells in tumor-infiltrating lymphocytes. The experimental findings of the present study are helpful for the development of dietary intervention methods for cancer immunotherapy using natural nutrients.

TCR function analysis using a novel system reveals the multiple unconventional tumor-reactive T cells in human breast cancer-infiltrating lymphocytes.

Tumor-infiltrating lymphocytes (TILs) are a potent source for obtaining tumor-reactive T cell receptors (TCRs). Although comprehensive methods to analyze the TCR repertoire in TILs have been reported, the evaluation system for TCR-reactivity to endogenously expressed antigen in tumor cells remains laborious and time consuming. Consequently, very limited numbers of TCRs in TILs have been analyzed for their reactivity to tumor cells. In this study, we developed an efficient evaluation system for TCR function designated c-FIT (comprehensive functional investigation of TCRs) to analyze TCR reactivity. The c-FIT system enabled us to analyze up to 90 TCRs for their reactivity to tumor cells by a single assay within a month. Using c-FIT, we analyzed 70 TCRs of CD8+ TILs derived from two breast cancer patients and obtained 23 TCRs that reacted to tumor cells. Surprisingly, although two TCRs were HLA class I-restricted, the remaining 21 TCRs were non-HLA-restricted. Thus, c-FIT can be applied for monitoring multiple conventional and unconventional antigen-specific killer T cells in TILs, leading to the development of new designs for more effective T-cell-based immunotherapies.

Abstract 1770: Ectopic expression of ULBP2 promotes tumor progression in syngeneic mice

Abstract ULBP2 is one of the ligands for NKG2D (NKG2DLs). ULBP2 expression is induced in transformed cells and is recognized by immune effector cells via the activating NKG2D immunoreceptor. Previously, we reported high concentrations of serum-soluble ULBP2 (sULBP2) were correlated with poor prognosis in NSCLC patients. As a possible mechanism, we demonstrated that sULBP2 directly suppressed the cytolytic activity of peripheral blood mononuclear cells (PBMCs). However, little is known about the in vivo function of human NKG2DLs on tumor cells. The present study investigates the function of human NKG2DLs in the syngeneic C57BL/6 mice. We generated ULBP2 stably expressing B16 melanoma cell lines (ULBP2-B16). As previously reported, 4-hour 51Cr releasing assay showed NK cell cytotoxicity against the tumor cells was enhanced by ectopic expression of ULBP2 on the tumor cells. IFN-gamma production in NK cells was decreased by non-contact co-culture with ULBP2-B16. To investigate the in vivo function of ULBP2, C57BL/6 mice were inoculated s.c. with mock-B16 cells or ULBP2-B16 cells. ULBP2-B16 tumors grew rapidly compared to mock-B16 tumors. NKG2D blockade by anti-NKG2D antibody injected i.p. suppressed tumor growth of ULBP2-B16 tumors. Spleen cells and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. NKG2D was expressed on splenic NK cells, while it was rarely expressed on splenic CD8+ T cells. NKG2D was downregulated on NK cells in TILs (NK-TILs) from ULBP2-B16. NKG2D was highly expressed on CD8+ T cells in TILs (CD8+-TILs) from mock-B16 tumors, while its expression was suppressed on these from ULBP2-B16 tumors. Degranulation analysis revealed that ULBP2-B16 reduced activity of NK-TILs and CD8+-TILs compared to mock-B16. In conclusion, ectopic expression of ULBP2 on tumor cells promotes tumor progression via suppression of the activity of NK cells and CD8+ T cells. These findings suggest that neutralization of NKG2DLs may be a promising approach for the treatment of NKG2DLs-expressing tumors. Citation Format: Kosuke Yamaguchi, Naomi Miyake, Yasuhiko Teruya, Kohei Yamane, Naoki Kinoshita, Tomohiro Sakamoto, Masahiro Kodani, Hiroki Chikumi, Akira Yamasaki. Ectopic expression of ULBP2 promotes tumor progression in syngeneic mice [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 1770.

Cellular based treatment modalities for unresectable hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and is unfortunately associated with an overall poor prognosis and high mortality. Early and intermediate stages of HCC allow for treatment with surgical resection, ablation and even liver transplantation, however disease progression warrants conventional systemic therapy. For years treatment options were limited to molecular-targeting medications, of which sorafenib remains the standard of care. The recent development and success of immune checkpoint inhibitors has proven to be a breakthrough in the treatment of HCC, but there is an urgent need for the development of further novel therapeutic treatments that prolong overall survival and minimize recurrence. Current investigation is focused on adoptive cell therapy including chimeric antigen receptor-T cells (CAR-T cells), T cell receptor (TCR) engineered T cells, dendritic cells, natural killer cells, and tumor infiltrating lymphocyte cells, which have shown remarkable success in the treatment of hematological and solid tumor malignancies. In this review we briefly introduce readers to the currently approved systemic treatment options and present clinical and experimental evidence of HCC immunotherapeutic treatments that will hopefully one day allow for revolutionary change in the treatment modalities used for unresectable HCC. We also provide an up-to-date compilation of ongoing clinical trials investigating CAR-T cells, TCR engineered T cells, cancer vaccines and oncolytic viruses, while discussing strategies that can help overcome commonly faced challenges when utilizing cellular based treatments.

Safety and efficacy of lifileucel (LN-144), an autologous, tumor infiltrating lymphocyte cell therapy in combination with pembrolizumab for immune checkpoint inhibitor naïve patients with advanced melanoma.

9537 Background: Tumor infiltrating lymphocyte (TIL) cell therapy has demonstrated safety and efficacy in advanced melanoma, both in the pre-immune checkpoint inhibitor (ICI) setting (Goff, JCO 2016) and in patients who have failed anti-PD-1/PD-L1 therapy (Sarnaik, 2020). Combination of TIL and pembrolizumab (pembro) in ICI-naïve patients has demonstrated encouraging efficacy data with acceptable safety in head and neck squamous cell carcinoma (Jimeno, 2020). To improve treatment options in early lines, we explore a combination of LN-144 and pembro in patients with ICI-naïve advanced melanoma. Methods: IOV-COM-202 is a Phase 2 multicenter, multi-cohort, open-label study evaluating TIL cell therapy in multiple settings and indications. We report on Cohort 1A enrolling ICI-naïve advanced melanoma (unresectable or metastatic) patients for treatment with a combination of LN-144 and pembro. Key eligibility criteria include ≤ 3 lines of prior therapy, ECOG &lt; 2, one resectable lesion for lifileucel manufacturing, and ≥ 1 measurable lesion for response assessment. Primary endpoints are objective response rate (ORR) per RECIST 1.1 and safety as measured by incidence of Grade ≥3 treatment-emergent adverse events (TEAE). LN-144 is generated at centralized GMP facilities in a 22-day process. A nonmyeloablative lymphodepletion (NMA-LD) using cyclophosphamide and fludarabine is administered preceding a single LN-144 infusion, followed by &lt; 6 doses of IL-2 (600,000 IU/kg). Pembro is administered after tumor harvest but prior to NMA-LD and continues after lifileucel per label. Results: Seven patients have received lifileucel in combination with pembro as of data extraction date (Feb 14, 2021). Five of the 7 treated patients were treatment-naïve, 1 patient had prior BRAFi + MEKi and 1 had received prior chemotherapy; 71% had liver/brain lesions, 43% had LDH &gt; ULN. Mean SOD for the target lesions was 111 mm, with 86% of patients with &gt; 3 target lesions, representing advanced disease at baseline for this patient group. The TEAE profile was consistent with the underlying disease and known AE profiles of pembro, NMA-LD and IL-2. Six patients had a confirmed objective response with an ORR of 86% (1 CR, 5 PR) and 1 best response of SD. Three of the responding patients have remained off pembro due to pembro related AEs for 3, 4 and 13 months (mos), yet maintaining response. All responding patients remain in response with the longest duration of response being 16.8 mos. Conclusions: Lifileucel can be safely combined with pembro in patients with ICI-naïve advanced melanoma. The ORR of 86% is encouraging when compared to pembro alone in a similar patient population, especially considering the disease burden at baseline and persistence of responses in patients off therapy. Enrollment is ongoing and updated data to be presented. Clinical trial information: NCT03645928.

CD3D has the Potential to be a Prognostic Factor for Endometrial Carcinoma and an Indicator of Tumor Immune Microenvironment Regulation: a Study based on TCGA Data Mining

Accumulating evidence shows that tumor immune microenvironment infiltration plays a crucial role in the occurrence and development of endometrial carcinoma (EC). At present, studying the regulation mechanism of the immune microenvironment in EC remains a challenge. We calculated the amount of immune and stromal components and the proportion of tumor-infiltrating immune lymphocytes via ESTIMATE, single-sample gene enrichment analysis (ssGSEA), and CIBERSORT computational methods. Differentially expressed genes were analyzed by protein–protein interaction network construction and univariate survival analysis. CD3D expression was negatively correlated with clinicopathological features, such as age, histological type, clinical grade, and pathological stage, but positively correlated with the survival of patients with EC. Logistic regression analysis suggested that CD3D expression could be used as a biomarker to predict EC occurrence. Gene set enrichment analysis revealed that CD3D was upregulated in the immune effect pathway. Interestingly, CD3D was downregulated in the EC pathway. EC was divided into three immune cell subsets by ssGSEA. Correlation analysis between ssGSEA and CIBERSORT showed that the infiltration level of CD8 + T cells and plasma cells in tumor-infiltrating lymphocytes was lower than that in the high-immune-score group (Immunity_H). Moreover, CD3D expression in Immunity_H was low, and the prognosis was even worse. This study demonstrated that low CD3D expression is an index of poor prognosis in patients with EC. Levels of CD3D in tumor microenvironment (TME) might be a useful indicator for predicting the prognosis of patients with EC patients.