Tumor-infiltrating Lymphocytes Production Research Articles

Adoptive transfer of tumor-infiltrating lymphocytes in patients with sarcomas, ovarian, and pancreatic cancers.

TPS2650 Background: Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TIL) has a long history of efficacy in metastatic melanoma, and is being increasingly considered across other solid tumors. Preclinical data generated at MD Anderson Cancer Center has demonstrated the ability to grow TIL from a variety of tumor types including various types of sarcomas, ovarian and pancreas cancers. We are testing the efficacy of TIL across multiple tumor types using two different manufacturing protocols. Methods: We are conducting two ongoing investigator initiated basket TIL therapy trials. The first (NCT03449108) includes cohorts with poorly differentiated soft tissue and bone sarcomas, osteosarcoma, and platinum resistant ovarian cancer. The TIL product used in this trial is an investigational cell product (LN-145, Iovance Biotherapeutics, Inc.). The second trial (NCT03610490) includes cohorts of osteosarcoma, platinum resistant ovarian cancer, and pancreatic cancer (who have progressed on, or received maximal benefit from, front-line therapy). For this trial, TIL are manufactured at MD Anderson Cancer Center using a protocol that includes the use of urelumab (an agonistic anti-CD137 antibody) combined with T cell receptor activation during TIL expansion. In both trials eligible subjects undergo tumor harvest using a surgical excisional biopsy of the tumor for TIL manufacturing, receive a modified cyclophosphamide and fludarabaine lymphodepletion regimen and up to six doses of IL-2 (600,000 IU/kg) following TIL infusion. No intervening therapy is allowed between tumor harvest and initiation of lymphodepletion. The primary endpoint for each cohort is ORR as assessed by investigators using RECIST 1.1 criteria. The Simon’s two stage design is used to monitor the efficacy of each cohort independently. In the first stage, 10 patients will be treated per cohort. If there is no confirmed response in these 10 evaluable patients, the cohort will be terminated. If the cohort moves forward to Stage II, an additional 8 patients will be treated leading to a total of 18 patients. Three or more responders out of 18 treated patients for the cohort will be considered clinically relevant to justify further investigation. Enrollment is ongoing in all cohorts in both trials. An accrual update will be provided at the annual meeting. Clinical trial information: NCT03449108, NCT03610490.

Safety and efficacy of adoptive cell transfer using autologous tumor infiltrating lymphocytes (LN-145) for treatment of recurrent, metastatic, or persistent cervical carcinoma.

2538 Background: There is a high unmet medical need for effective treatments for patients with recurrent, metastatic, or persistent cervical cancer. Most patients are young and survival rates are poor. ORR for second line therapies is between 4 and 14% for chemotherapy and recently approved immunotherapy. Adoptive cell transfer using tumor infiltrating lymphocytes (TIL) have demonstrated durable responses in some patients with recurrent cervical cancer thus offering the potential for long-term disease control. Methods: Study C-145-04 is an ongoing, open-label, multicenter Phase 2 clinical trial evaluating the safety and efficacy of LN-145 TIL therapy in patients with advanced cervical cancer who have undergone at least one prior line of chemotherapy. Prior checkpoint inhibitor therapy is an exclusion criterion. The primary endpoint is ORR per RECIST 1.1; secondary endpoints include duration of response (DOR), disease control rate (DCR), and LN-145 safety. Tumors surgically harvested at local institutions are shipped to central GMP facilities for TIL generation in a 22-day manufacturing process. Final LN-145 TIL product is cryopreserved and shipped to sites. Patients receive one week of preconditioning lymphodepletion (cyclophosphamide, fludarabine), a single LN-145 infusion, followed by up to 6 doses of IL-2 (600,000 IU/kg). Results: As of 4 Feb 2019, 27 efficacy-c patients have received Gen 2 of LN-145, with a mean age of 47 years and 2.6 mean prior lines of therapy. Preliminary efficacy results: ORR was 44% (1 CR, 9 PR, 2 uPR), DCR was 89% at 3.5-month median study follow-up with 11/12 patients maintaining their response. Improved responses were observed in 4 patients with longer follow-up. Mean TIL cells infused was 28x109. Median IL-2 doses administered was 6.0. The adverse event profile was generally consistent with the underlying advanced disease and the profile of the lymphodepletion and IL-2 regimens. Conclusions: LN-145 results in 44% ORR in previously treated cervical cancer patients with acceptable safety and efficacy profile. LN-145 offers patients a viable therapeutic option warranting further investigation. Clinical trial information: NCT03108495.

Safety and efficacy of cryopreserved autologous tumor infiltrating lymphocyte therapy (LN-144, lifileucel) in advanced metastatic melanoma patients who progressed on multiple prior therapies including anti-PD-1.

2518 Background: Treatment options are limited for patients with advanced melanoma who have progressed on checkpoint inhibitors and targeted therapies such as BRAF/MEK inhibitors (if BRAF-V600E mutated). Adoptive cell therapy utilizing tumor-infiltrating lymphocytes (TIL) has shown antitumor efficacy with durable long-term responses in heavily pretreated melanoma patients. Safety and efficacy of lifileucel (LN-144), a centrally manufactured autologous TIL therapy are presented. Methods: C-144-01 is a global Phase 2 open-label, multicenter study of the efficacy and safety of lifileucel in patients with unresectable metastatic melanoma. We report on Cohort 2 (N = 55) patients who received cryopreserved lifileucel. Tumors resected at local institutions were processed in central GMP facilities for TIL production in a 22-day process. Final TIL infusion product was cryopreserved and shipped to sites. Patients received one week of cyclophosphamide/fludarabine preconditioning lymphodepletion, a single lifileucel infusion, followed by up to 6 doses of IL-2. Results: In 55 patients with Stage IIIC/IV unresectable melanoma, 3.1 mean prior therapies (anti-PD1 100%; anti-CTLA-4 80%; BRAF/MEK inhibitor 24%), high baseline tumor burden (110 mm mean target lesion sum of diameters), ORR was 38% (2 CR, 18 PR, 1 uPR). Of 21 responders, 4 have progressed to date with median follow up of 7.4 months. Overall disease control was 76%. Improved responses in some patients were observed with longer follow up. Most (54) patients progressed on prior anti-PD1 and those with PD-L1 negative status (TPS < 5%) were among responders. Mean cells infused was 28 x109. Median IL-2 doses administered was 6.0. Adverse events resolved to baseline, 2 weeks post TIL infusion, a potentially important benefit of one-time TIL therapy. Conclusions: Lifileucel treatment results in 38% ORR in heavily pretreated metastatic melanoma patients with high baseline disease burden who received prior anti-PD1 and BRAF/MEK inhibitor if BRAF mutated. Based on these data, a new Cohort 4 in C-144-01 has been initiated to support lifileucel registration. Clinical trial information: NCT02360579.

PPM6 USING SCENARIO DRAFTING TO EVALUATE AND FACILITATE FUTURE ADOPTION OF TUMOR-INFILTRATING LYMPHOCYTES (TIL) THERAPY IN ADVANCED MELANOMA

Tumor-Infiltrating Lymphocytes (TIL) therapy is a promising but complex therapy for advanced melanoma which is despite promising results (phase I/II studies) and its expected cost-effectiveness not yet implemented as a standard therapy. Scenario drafting can be used to systematically evaluate future dynamics surrounding an innovative treatment and thereby facilitate its adoption. This study aims to evaluate future perspectives related to the adoption of TIL-therapy in advanced melanoma in terms of various scenarios of cost-effectiveness. Using a Delphi approach, 14 adoption scenarios for TIL-therapy were drafted, incorporating factors related to e.g.: patients perspective, pharmaceutical influence, and research developments. The likelihood of the scenarios taking place within 5 years was evaluated among international experts using a web-based questionnaire. Based on the questionnaire, scenarios were labeled as “likely” and “unlikely”. The likely scenarios were incorporated in an existing cost-effectiveness model comparing TIL-therapy to ipilimumab. Twenty-nine experts from 12 countries completed the questionnaire. The scenarios showed an average likelihood ranging from 29% to 58%, indicating that future developments of TIL-therapy are uncertain. Seven of the scenarios were labeled as “likely”, the other 7 were “unlikely”. Four of the 7 likely scenarios showed a high probability for TIL-therapy to be cost-effective (89%-99%). The three other likely scenarios: “commercialization of TIL production”, “pharmaceutical companies lowering the prices of ipilimumab” and “using TIL-therapy combined with ipilimumab” showed a smaller chance for TIL-therapy to become cost-effective (0%-56%). Scenario drafting can be a useful tool to minimize uncertainty surrounding decisions on adoption and coverage. Moreover, when scenarios are not cost-effective, it can facilitate adoption by identifying crucial contextual factors influencing the adoption. In this analysis, TIL-therapy was cost-effective compared to ipilimumab in most of the “likely” future scenarios. However, the factors involved in the “likely” scenarios resulting in TIL-therapy not being cost-effective should be considered in the adoption process.

Abstract A185: Effective expansion of poly-functional tumor-reactive TILs from NSCLC correlates with an immune-engaged T-cell profile in tumor tissues

Abstract Non-small cell lung cancer (NSCLC) is the second most frequently occurring type of cancer. Because of the high mortality rates with the current treatment regimens, novel therapeutic approaches are warranted. The high mutational rate of the tumors, and the high level of T-cell infiltrates should render NSCLC patients eligible for autologous T-cell therapy. Here we show that tumor-infiltrating lymphocytes (TILs) from treatment naive, stage I-IIIa NSCLC tumors can be effectively expanded and reprogrammed. With an optimized detection essay for tumor-reactive T-cells, we observed that 13/17 tested TIL products (76.5%) from primary NSCLC tumor tissues contained cytokine-producing CD4+ and/or CD8+ T-cells in response to primary tumor digest. Tumor responses ranged from 0.5%-30%, and correlated well with activation markers on CD4+ and CD8+ T-cells. Importantly, 29.4% of the TIL products contained poly-functional T-cells that produced TNF-alfa and/or IL-2 in addition to IFN-gamma, and poly-functional T-cells correlated with high levels of tumor reactivity. Furthermore, high percentages of CD103+CD69+CD8+ T-cells, PD-1+CD4+ T-cells and FoxP3+CD25+CD4+ T-cells in the tumors were good predictors for the generation of highly tumor-reactive TIL products. In conclusion, we show that most NSCLCs contain tumor-specific T-cells that can be efficiently expanded and become highly poly-functional, depending on the initial T-cell profile of TILs. Our findings show the feasibility of culturing tumor-reactive TILs from NSCLC and provide keys to individualize adoptive T-cell therapy. Therefore, autologous T-cell therapy should be reconsidered as treatment for NSCLC patients. Citation Format: Rosa De Groot, Marleen M. van Loenen, Aurélie Guislain, Derk Amsen, John B.A.G. Haanen, Kim Monkhorst, Koen J. Hartemink, Monika C. Wolkers. Effective expansion of poly-functional tumor-reactive TILs from NSCLC correlates with an immune-engaged T-cell profile in tumor tissues [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A185.

Abstract B015: T-cell recognition profiling of CD8+ T-cells in tumor-infiltrating lymphocytes expanded for adoptive cell transfer

Abstract Adoptive T-cell therapy (ACT) has proven to be a highly effective therapy option for melanoma and other immunogenic cancer types, however only a subset of 40% of the patients respond. Due to the intensive and costly regimen, it is important to find predictive biomarkers to identify patients that more likely respond to therapy.Melanoma-associated tissue antigens play an important role in melanoma immunotherapy, as they are expressed in the majority of tumors across different patients and can elicit T-cell recognition. Furthermore, such melanoma-associated T-cell responses may serve as a surrogate marker for T-cell reactivity in general and hence potentially also reflect the level of neoepitope reactivity. Several recent reports point towards the importance of mutation-derived neo-epitopes for cancer immunotherapy, as the mutational and putative neoantigen load correlates with therapeutically benefit of immune checkpoint blockade and ACT. In this study, we analyzed the impact of the number and size of T-cell responses against a library of shared antigens and predicted neoantigens to correlate the number of responses with the outcome of the ACT. We screened expanded tumor-infiltrating lymphocytes (TILs) from stage IV melanoma patients within a phase I/II clinical trial of ACT for CD8+ T-cell reactivity. A library of classical melanoma-associated shared antigens as well as a personalized library of predicted neoantigens for each patient was selected. CD8+ T-cell recognition in the TIL product was investigated by use of a novel technology based on DNA-barcode labeled MHC multimers, enabling high-throughput screening for >1,000 specific T-cell-populations in a single sample. Specific T-cell responses against shared antigens could be found and verified among the TIL samples. The number of CD8+ T-cells responses against tumor-associated shared antigens does not seem to correlate with progression-free or overall survival in this patient cohort, but further screenings have to be conducted. The analysis of neoantigen-reactive CD8+ T-cells within the TILs is ongoing.Identification of predictive biomarkers is an important step towards higher effectiveness of adoptive cell transfer as immunotherapeutic approach and the recognition pattern of melanoma by CD8+ T-cells associated with favorable clinical outcome provides mechanistic insights important for future developments. Citation Format: Christina Heeke, Anne-Mette Bjerregaard, Amalie Kai Bentzen, Marco Donia, Rikke Andersen, Marie Stentoft Svane, Sine Reker Hadrup. T-cell recognition profiling of CD8+ T-cells in tumor-infiltrating lymphocytes expanded for adoptive cell transfer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B015.

Expansion and Characterization of Iovance Marrow Infiltrating Lymphocytes: A Potential Novel Therapeutic Strategy for the Treatment of Acute Myeloid Leukemia

▪Background: Acute myeloid leukemia (AML) is a poor-prognosis malignancy arising from hematopoietic stem/progenitor cells. To date, novel immunotherapies such as checkpoint inhibitors, vaccines and adoptive cell therapy (ACT) using CAR T cells have demonstrated only modest success for the treatment of patients who are ineligible for marrow transplantation and have minimal residual disease; additional approaches are warranted (Beyar-Katz O and Gill S, Clin Cancer Res 2018). ACT with tumor infiltrating lymphocytes (TIL) has emerged as an effective treatment for patients with metastatic melanoma (Goff SL et al, J Clin Oncol 2016), likely owing to the heterogeneous population of tumor-reactive T cells that comprises the TIL products. As demonstrated for solid cancers, such tumor-reactive T cells are preferentially found in the tumor microenvironment (Gros A et al JCI 2014; Thommen DS et al Nat Med 2018). By avoiding the highly immunosuppressive tumor microenvironment, ex vivo activation of those cells rescues them from tolerance and anergic status. We hypothesized that, in the case of AML for which the bone marrow represents the tumor microenvironment, tumor antigen-specific T cells could be recovered from the patient bone marrow to produce a highly effective therapeutic product that is cytotoxic to AML tumor cells. We present findings related to the ex vivo expansion of Iovance marrow infiltrating lymphocytes (MIL) for the treatment of AML patients.Methods: Immune cell and non-immune cell fractions were sorted from bone marrow mononuclear cells. Immune cell fractions loaded with sonicated non-immune cell fractions were expanded for 14 days in the presence of αCD3/αCD28 beads and interleukin-2 (IL-2) to generate MIL products. Phenotypic and functional characteristics of the cells were determined by flow cytometry and enzyme-linked immunospot assay (ELISpot).Results: MIL were generated from isolated bone marrow mononuclear cells (n=2) with a mean expansion fold of 86 (range 78-93). Equal percentage of CD4+ and CD8+ T cell subsets constituted the MIL products. Phenotypic analysis of the cells showed that the majority of T cell subsets are effector memory and CD28 positive. Low percentages of the T cell subsets were positive for immunosuppressive markers PD-1 and LAG3. ELISpot analysis demonstrated that MIL were readily activatable and produced normal levels of IFNγ in response to CD3/CD28 stimulation. Antigen specificity of MIL is being investigated.Conclusion: We demonstrated the feasibility of MIL expansion from bone marrow mononuclear cells from AML patients. MIL are functionally active and mostly comprised of effector memory T cells. Confirmation of tumor cell antigen specificity will determine whether MIL may deploy a robust anti-tumor activity in vivo. DisclosuresKaryampudi:Iovance Biotherapeutics: Employment, Equity Ownership. Frank:Iovance Biotherapeutics: Employment, Equity Ownership. Blaskovich:Iovance Biotherapeutics: Equity Ownership. Chartier:Iovance Biotherapeutics: Equity Ownership.

Effects of Glycogen Synthase Kinase-3β Inhibitor TWS119 on Proliferation and Cytokine Production of TILs From Human Lung Cancer.

The canonical Wnt-β-catenin signaling pathway arrests the differentiation of T cells and plays an important role in phenotypic maintenance of naive T cells and stem cell-like memory T cells in human peripheral blood, but its effect on tumor-infiltrating lymphocytes (TILs) from non–small cell lung cancer is little known. In this study, we showed that glycogen synthase kinase-3β inhibitor TWS119 has different effects on CD4+ and CD8+ T cells in TILs. TWS119 preserved the expansion of naive T cell and CD8+ stem cell-like memory T cells, and induced CD8+ effector T-cell proliferation in TILs. To further determine whether TWS119 impaired the effector function of TILs, TILs were stimulated with polyclonal stimulation, IL-2 and IFN-γ production were detected. Our data showed that TWS119 does not affect the production of IFN-γ in TILs compared with the control group; whereas TWS119 inhibited IFN-γ secretion of T cells from healthy donor. IL-2 production in CD4+ central memory T cells and CD4+ effector memory T cells from TILs was significantly increased with the TWS119 treatment; TWS119 also promoted the secretion of IL-2 in all cell subsets of CD8+ TILs. These findings reveal that TWS119 has a distinct effect on the proliferation and cytokine production of TILs, and provide new insights into the clinical application of TILs with TWS119 treatment for the adoptive immunotherapy.

Abstract LB-110: Anti-OX40 agonistic antibody enhances ex vivo CD8+ TIL expansion with increased T-cell effector function

Abstract Adoptive T cell therapy (ACT) using tumor infiltrating lymphocytes (TILs) has demonstrated efficacy in metastatic melanoma patients, with ~50% objective responses. Amongst parameters recognized as important for the efficacy of ACT is a high proportion of CD8+ T cells in the infusion product. OX40 (CD134) belongs to the tumor necrosis factor receptor super family and is mainly expressed by activated T lymphocytes. Activation of OX40 signaling promotes proliferation and survival of T-cells via the NF-kB pathway. Agonistic anti-OX40 antibodies have been developed as immunotherapy drugs for the treatment of cancer, one of which was recently shown to increase reactivity of tumor antigen-specific CD8+ T cells in patient's peripheral blood. In this work, we studied the expression of the OX40 receptor on TILs and investigated the impact of an anti-OX40 agonistic antibody on the ex vivo expansion and effector function of TILs. We found that OX40 was enriched in the CD4+ T cell subset of TILs. Its expression was up-regulated in response to anti-CD3 stimulation during the rapid expansion protocol (REP) of TIL production. An anti-OX40 monoclonal antibody was obtained from commercial sources and characterized in vitro. It was shown to bind OX40 specifically and to mediate NF-κB activation in a dose-dependent manner upon clustering. Consistent with its agonistic activity in the reporter assay, the anti-OX40 antibody also heightened IFN-γ production of OKT3-stimulated TILs, suggesting improved T cell effector function (N=6; p=0.03) and supporting its use during TIL expansion. When TILs were propagated by REP in the presence of anti-OX40, the frequency of CD8+ T cells increased significantly in the final product relative to control (N=21, p=0.001). The effect was dose-dependent. Interestingly, cell surface expression of the OX40 receptor was specifically down-regulated in the responding anti-OX40-treated TILs, suggesting a link between regulation of the OX40 signaling axis and the antibody-mediated effect on the TIL subsets. The TCR Vβ repertoire of TILs expanded with anti-OX40 was not changed significantly (N=3). However, expression of T cell surface markers was altered by the antibody, which skewed the T cell phenotype towards a differentiated and activated state. Taken together, maintenance of T cell diversity and differentiation state of the T cells suggest that anti-OX40 acted by selectively promoting the proliferation of CD8+ T cells. When assessed functionally in an IFNγ assay, the potency of the TILs produced in the presence of anti-OX40 was shown to be comparable to that of control TILs. In conclusion, our work shows that activation of OX40 signaling using an agonistic antibody could significantly improve expansion of CD8+ TILs. Implications on in vivo anti-tumor activity remain to be assessed. Citation Format: Krit Ritthipichai, Marcus Machin, Maria Fardis, Cecile Chartier. Anti-OX40 agonistic antibody enhances ex vivo CD8+ TIL expansion with increased T-cell effector function [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 LB-110.

Abstract CT169: A phase II, multicenter study to assess the efficacy and safety of autologous tumor infiltrating lymphocytes (LN-144) for treatment of patients with metastatic melanoma

Abstract Adoptive cell therapy (ACT) utilizing tumor-infiltrating lymphocytes (TIL) has been employed for years in hundreds of patients with metastatic melanoma and other solid tumors, demonstrating durable and complete responses, even in heavily pretreated patients. Despite the approvals of checkpoint-directed therapies, metastatic melanoma remains an unmet need due to progression subsequent to administration of checkpoints, as well as due to intolerance. The C-144-01 study will enroll metastatic melanoma patients who progressed on anti-PD-1 and BRAF inhibitors if BRAF mutation positive. This phase II trial utilizes a central GMP facility for the manufacture of LN-144 in either a non-cryopreserved generation-1 (Gen-1), or cryopreserved generation-2 (Gen-2) investigational TIL infusion product. The Gen-2 manufacturing process reduces the time required for manufacture of TIL product to 22 days. The process development of cryopreservation for the final LN-144 infusion product incorporates dramatic improvements in the flexibility of scheduling, distribution and delivery required for commercial use. C-144-01 (NCT02360579) is a global phase 2 multicenter, open-label study evaluating the efficacy and safety of autologous TIL (LN-144) therapy for the treatment of patients with advanced metastatic melanoma. The study consists of three treatment cohorts: 30 patients in Cohort 1 will receive a single dose of Gen-1, non-cryopreserved LN-144; 30 patients in Cohort 2 will receive a single dose of Gen-2, cryopreserved LN-144; and up to 10 eligible patients from either Cohort 1 or Cohort 2 may enter a third cohort (Cohort 3) for re-treatment with a second dose of LN-144. The investigational LN-144 TIL infusion product for all cohorts is prepared at the central GMP facility using lymphocytes that are extracted from a surgically-resected sample of patient tumor. LN-144 infusion is preceded by a non-myeloablative lymphodepletion regimen of cyclophosphamide and fludarabine and followed by IL-2 for up to 6 doses. Patients ≥ 18 years of age must have progressive, unresectable metastatic melanoma (Stage IIIc or Stage IV) following ≥1 line of prior systemic therapy including immune checkpoint inhibitors and BRAF-targeted therapy (if BRAF mutation-positive). A minimum of 2 tumor lesions are required: 1 for resection and TIL manufacture and 1 for assessment of response by RECIST 1.1 criteria. Other major eligibility criteria include: adequate bone marrow, cardiac, liver, pulmonary, and renal function; ECOG PS of 0 or 1. Efficacy is being assessed as a function of ORR, CR rate, DOR, DCR, and PFS per RECIST 1.1 and OS. Assessment of safety data will be descriptive and based on the summarization of TEAEs, SAEs, AEs leading to discontinuation from treatment and the study, vital signs, physical examinations, and clinical laboratory tests. Citation Format: Amod Sarnaik, Brendan Curti, Diwakar Davar, Omid Hamid, Jose Lutzky, Melissa Wilson, Harriet Kluger, Jason Chesney, Kevin Kim, Giao Phan, Sajeve Thomas, Eric Whitman, Bente Larsen, Sam Suzuki, Nancy Samberg, Igor Gorbatchevsky, Maria Fardis, John M. Kirkwood. A phase II, multicenter study to assess the efficacy and safety of autologous tumor infiltrating lymphocytes (LN-144) for treatment of patients with metastatic melanoma [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 CT169.

Activation and propagation of tumor-infiltrating lymphocytes from malignant pleural effusion and ascites with engineered cells for costimulatory enhancement

Adoptive cell therapy (ACT) of autologous tumor-infiltrating lymphocytes (TILs) has shown an effect on mediating tumor regression in some patients with highly advanced, refractory metastatic malignancy. Here, the in vitro generation of TILs isolated from malignant pleural effusion and ascites was compared with which using engineered cells for costimulatory enhancement (ECCE) and 3 common γ-chain cytokines, interleukin (IL)-2, IL-7, and IL-15, alone or in combination. We showed the robust clinical-scale production of TILs with a less differentiated 'young' phenotype by expansion in the presence of ECCE combined with IL-2/7/15. Furthermore, a major fraction of the TILs generated in this fashion was shown to produce much more IFN-γ and TNF-α, and displayed cytolytic activity against target cells expressing the relevant antigens. To our knowledge, this is the first time that the combination of ECCE and IL-2/7/15 has been applied for the generation of TILs isolated from malignant pleural effusion and ascites.

Self-Delivering RNAi Targeting PD-1 Improves Tumor-Specific T Cell Functionality for Adoptive Cell Therapy of Malignant Melanoma.

Adoptive cell therapy (ACT) is becoming a prominent alternative therapeutic treatment for cancer patients relapsing on traditional therapies. In parallel, antibodies targeting immune checkpoint molecules, such as cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4) and cell death protein 1 pathway (PD-1), are rapidly being approved for multiple cancer types, including as first line therapy for PD-L1-expressing non-small-cell lung cancer. The combination of ACT and checkpoint blockade could substantially boost the efficacy of ACT. In this study, we generated a novel self-delivering small interfering RNA (siRNA) (sdRNA) that knocked down PD-1 expression on healthy donor T cells as well as patient-derived tumor-infiltrating lymphocytes (TIL). We have developed an alternative chemical modification of RNA backbone for improved stability and increased efficacy. Our results show that T cells treated with sdRNA specific for PD-1 had increased interferon γ (IFN-γ) secreting capacity and that this modality of gene expression interference could be utilized in our rapid expansion protocol for production of TIL for therapy. TIL expanded in the presence of PD-1-specific sdRNA performed with increased functionality against autologous tumor as compared to control TIL. This method of introducing RNAi into T cells to modify the expression of proteins could easily be adopted into any ACT protocol and will lead to the exploration of new combination therapies.

A preclinical study of tumor-infiltrating lymphocytes in NSCLC.

161 Background: Multiple clinical studies have shown that adoptive cell transfer (ACT) of autologous tumor-infiltrating lymphocytes (TIL) is remarkably effective in melanoma patients. Non-small cell lung cancer (NSCLC) shares similarities with melanoma in terms of mutational burden and sensitivity to immune checkpoint inhibitors. We therefore sought to test whether TIL ACT may represent a viable option for the treatment of NSCLC patients. We utilized tissue collected from patients enrolled on the prospective ImmunogenomiC prOfiling of Non-small cell lung cancer (ICON) study. Methods: TIL and tissue-infiltrating lymphocytes were expanded ex vivo from 97 freshly resected early-stage localized NSCLC tumors and 39 matched uninvolved lung tissues. Growth and functional characteristics of TIL were assessed via flow cytometry, TIL-tumor reactivity assays, and analysis of TCRβ sequencing data. Results: NSCLC showed an increased proportion of CD3+ lymphocytes within the tumor-infiltrating leukocyte component as compared to matched normal lung tissue. The TIL compartment included a suppressed CD8+ T cell subset expressing significantly higher levels of PD-1 and lacking cytolytic potential compared to T cells expanded from normal tissue. TIL contained a higher proportion of proliferating (Ki67+) CD8+CD103+ tissue-resident memory (TRM) cells expressing activation markers such as CTLA4, LAG3, PD1 and ICOS, and increased CD4+ Tregs. Despite a highly immunosuppressive environment, TIL expansion was achieved with a success rate of 68% (n = 97) but appeared hindered in patients undergoing neoadjuvant chemotherapy treatment prior to surgery (56.2%, n = 16 vs 72.5% success rate in therapy-naïve patients). In addition, expansion efficiency (number expanded and time of culture) of TIL and matched lung residing lymphocytes were significantly associated (r = 0.379, p = 0.017, n = 39). Importantly, expanded CD8+ TIL products were oligoclonal and showed reactivity toward autologous tumors. Conclusions: Although NSCLC TIL are functionally inhibited in vivo they can be successfully expanded ex vivo and demonstrate recognition of autologous tumor cells. These data suggest that TIL can potentially be used for adoptive T cell-based immunotherapy in NSCLC.

Selection of Shared and Neoantigen-Reactive T Cells for Adoptive Cell Therapy Based on CD137 Separation.

Adoptive cell therapy (ACT) of autologous tumor infiltrating lymphocytes (TIL) is an effective immunotherapy for patients with solid tumors, yielding objective response rates of around 40% in refractory patients with metastatic melanoma. Most clinical centers utilize bulk, randomly isolated TIL from the tumor tissue for ex vivo expansion and infusion. Only a minor fraction of the administered T cells recognizes tumor antigens, such as shared and mutation-derived neoantigens, and consequently eliminates the tumor. Thus, there are many ongoing effects to identify and select tumor-specific TIL for therapy; however, those approaches are very costly and require months, which is unreasonable for most metastatic patients. CD137 (4-1BB) has been identified as a co-stimulatory marker, which is induced upon the specific interaction of T cells with their target cell. Therefore, CD137 can be a useful biomarker and an important tool for the selection of tumor-reactive T cells. Here, we developed and validated a simple and time efficient method for the selection of CD137-expressing T cells for therapy based on magnetic bead separation. CD137 selection was performed with clinical grade compliant reagents, and TIL were expanded in a large-scale manner to meet cell numbers required for the patient setting in a GMP facility. For the first time, the methodology was designed to comply with both clinical needs and limitations, and its feasibility was assessed. CD137-selected TIL demonstrated significantly increased antitumor reactivity and were enriched for T cells recognizing neoantigens as well as shared tumor antigens. CD137-based selection enabled the enrichment of tumor-reactive T cells without the necessity of knowing the epitope specificity or the antigen type. The direct implementation of the CD137 separation method to the cell production of TIL may provide a simple way to improve the clinical efficiency of TIL ACT.

1017P - Phenotypic and functional characterization of tumor infiltrating lymphocytes (TIL) grown from non-hodgkin lymphoma tumors: Implications for the development of novel therapies for lymphoma

Background: Cell therapy with TIL is an effective treatment with acceptable safety profile for advanced metastatic melanoma patients. TIL products can be centrally manufactured for broad clinical application. Adoptive cell therapy with TIL involves collection of autologous lymphocytes from the tumor via surgical resection, ex vivo expansion of TIL, lymphodepletion of the patient prior to infusion of TIL, followed by infusion of TIL and treatment with IL-2. Here, we present findings related to expanding TIL directly from lymphoma. Methods: Using methods for TIL isolation and growth developed at Lion, we expanded TIL from 5/5 lymphoma (1 MCL, 3 follicular, 1 DLBCL) with Interleukin 2 for 11-14 days and subsequent rapid expansion for 14 days using mitogenic anti-CD3 antibody and irradiated allogeneic PBMC. Results: TIL were generated from all 5 lymphoma tumors with a maximum expansion index of 680-fold, significantly higher than previously reported. Mean CD3+ T cell population was 95% (vs 75% previously reported). As with TIL expansion from melanoma, we observed a marked relative increase in effector memory cells in lymphoma TIL. A significant increase in TEMRA (p = 0.0013; CD4, CD8) and CD28+CD4 + (p = 0.008) subsets was observed in lymphoma compared with melanoma TIL cultures. Bioluminescent Redirected Lysis Assay (BRLA) to assess TIL cytolytic activity at 4 hrs ranged from <1-6 LU50 in lymphoma TIL compared to melanoma TIL (11-75 LU50, 4hrs). ELISpot and ELISA analysis revealed IFN-γ production by lymphoma TIL comparable to that of melanoma TIL. Consistent with the observation that lymphoma-reactive T-cells are primarily TH2 and TH17, nanostring analysis revealed that lymphoma TIL expressed higher levels of RORC and IL17A than melanoma TIL. Conclusions: We demonstrate here the feasibility of growing TIL from lymphoma that have effector functions comparable to that of melanoma TIL. These findings serve as a rationale for considering TIL cell therapy for patients with lymphoma. Legal entity responsible for the study: Lion Biotechnologies, Inc Funding: Lion Biotechnologies Inc Disclosure: L. Karyampudi, A. Gokuldass, M. Blaskovich, M.T. Lotze: Employee of Lion Biotechnologies, Inc, receive compensation from Lion. Hold stock and/or stock options in Lion Biotechnologies, Inc. M. Fardis: Employee and director of Lion Biotechnologies, Inc, receive compensation from Lion. Hold stock and/or stock options in Lion Biotechnologies, Inc. Member of the Board of Directors of Lion Biotechnologies, Inc.

A Novel Method to Generate and Expand Clinical-Grade, Genetically Modified, Tumor-Infiltrating Lymphocytes.

Following the clinical success achieved with the first generation of adoptive cell therapy (ACT) utilizing in vitro expanded tumor-infiltrating lymphocytes (TILs), the second and third generations of TIL ACT are evolving toward the use of genetically modified TIL. TIL therapy generally involves the transfer of a high number of TIL, ranging from 109 to 1011 cells. One of the technical difficulties in genetically modifying TIL, using a retroviral vector, is the ability to achieve large expansion of transduced TIL, while keeping the technique suitable to a Good Manufacturing Practices (GMP) environment. Consequently, we developed and optimized a novel method for the efficient production of large numbers of GMP-grade, gene-modified TIL for the treatment of patients with ACT. The chemokine receptor CXCR2 was used as the gene of interest for methodology development. The optimized procedure is currently used in the production of gene-modified TIL for two clinical trials for the treatment of metastatic melanoma at MD Anderson Cancer Center.

Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-Infiltrating T Cells in Hepatocellular Carcinomas

Ligand binding to inhibitory receptors on immune cells, such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), down-regulates the T-cell-mediated immune response (called immune checkpoints). Antibodies that block these receptors increase antitumor immunity in patients with melanoma, non-small-cell lung cancer, and renal cell cancer. Tumor-infiltrating CD4+ and CD8+ T cells in patients with hepatocellular carcinoma (HCC) have been found to be functionally compromised. We analyzed HCC samples from patients to determine if these inhibitory pathways prevent T-cell responses in HCCs and to find ways to restore their antitumor functions. We collected HCC samples from 59 patients who underwent surgical resection from November 2013 through May 2017, along with tumor-free liver tissues (control tissues) and peripheral blood samples. We isolated tumor-infiltrating lymphocytes (TIL) and intra-hepatic lymphocytes. We used flow cytometry to quantify expression of the inhibitory receptors PD-1, hepatitis A virus cellular receptor 2 (TIM3), lymphocyte activating 3 (LAG3), and CTLA4 on CD8+ and CD4+ T cells from tumor, control tissue, and blood; we studied the effects of antibodies that block these pathways in T-cell activation assays. Expression of PD-1, TIM3, LAG3, and CTLA4 was significantly higher on CD8+ and CD4+ T cells isolated from HCC tissue than control tissue or blood. Dendritic cells, monocytes, and B cells in HCC tumors expressed ligands for these receptors. Expression of PD-1, TIM3, and LAG3 was higher on tumor-associated antigen (TAA)-specific CD8+ TIL, compared with other CD8+ TIL. Compared with TIL that did not express these inhibitory receptors, CD8+ and CD4+ TIL that did express these receptors had higher levels of markers of activation, but similar or decreased levels of granzyme B and effector cytokines. Antibodies against CD274 (PD-ligand1 [PD-L1]), TIM3, or LAG3 increased proliferation of CD8+ and CD4+ TIL and cytokine production in response to stimulation with polyclonal antigens or TAA. Importantly, combining antibody against PD-L1 with antibodies against TIM3, LAG3, or CTLA4 further increased TILfunctions. The immune checkpoint inhibitory molecules PD-1, TIM3, and LAG3 are up-regulated on TAA-specific T cells isolated from human HCC tissues, compared with T cells from tumor-free liver tissues or blood. Antibodies against PD-L1, TIM3, or LAG3 restore responses of HCC-derived T cells to tumor antigens, and combinations of the antibodies have additive effects. Strategies to block PD-L1, TIM3, and LAG3 might be developed for treatment of primary liver cancer.

Efficacy of single administration of tumor-infiltrating lymphocytes (TIL) in heavily pretreated patients with metastatic melanoma following checkpoint therapy.

3045 Background: Adoptive cell therapy with TIL involves collection of autologous lymphocytes from the tumor via surgical resection, ex vivo expansion of TIL, lymphodepletion of the patient prior to infusion of TIL using Fludarabine and Cyclophosphamide, followed by infusion of TIL. Up to 6 doses of IL-2 (600,000 IU/kg) is administered to support multiplication of TIL and engraftment. Here, we present the preliminary results from an ongoing, multi-site Phase 2 study of TIL for advanced metastatic melanoma. Methods: Patients with advanced metastatic melanoma who have failed at least one prior systemic therapy were enrolled. Primary objective of the study was to characterize safety profile of LN-144. At baseline, patients had a median age of 56 (41-72) years; 44% were ≥ 60 years old. Median sum of tumor diameters for the target lesions was 10.4 cm, and median of 3 prior therapies. All enlisted patients had prior anti-PD1 as well as anti-CTLA4 and 67% had received ≥ 3 prior therapies. Responses were assessed by RECIST 1.1. TIL products were centrally manufactured. No complications arose from shipment of tumors or TIL. Results: Results are presented through 31 Jan 2017 for the first 9 infused patients evaluable by two assessments. Eight of 9 patients received all 6 doses of IL-2 per protocol. The most common (≥3 patients) non-hematologic grade 3-4 TEAE was hypophosphatemia. No neurotoxicity of grade ≥ 3 was reported. There were no deaths or discontinuations due to SAEs related to study treatment. ORR was 33% (CR = 11%, PR = 22%, SD = 22%, PD = 33%, NE = 11%). Mean time to best response was 3.0 months and median duration of follow up was 3.6 months (1.1+, 12.1). Responses were observed in patients with tumors carrying wild type or BRAF mutations. All patients demonstrated persistence of TIL on day 14 post-infusion. Conclusions: Cell therapy with TIL is an effective treatment with acceptable safety profile for advanced metastatic melanoma patients who are refractory to anti-PD1. TIL products can be centrally manufactured for broad clinical application. This study will be expanded to enroll patients with a shorter manufacturing process as well as offering retreatment for study patients. Clinical trial information: NCT02360579.

Antigen-specific TIL therapy for melanoma: A flexible platform for personalized cancer immunotherapy.

Tumor infiltrating lymphocyte (TIL) therapy has shown objective clinical response rates of 50% in stage IV melanoma patients in a number of clinical trials. Nevertheless, the majority of patients progress either directly upon therapy or after an initial period of tumor control. Recent data have shown that most TIL products that are used for therapy contain only low frequencies of T cells reactive against known melanoma-associated epitopes. Because of this, the development of a technology to create T-cell products that are enriched for reactivity against defined melanoma-associated antigens would seem valuable, both to evaluate the tumoricidal potential of T cells directed against different antigen classes and to potentially increase response rates. Here, we developed and validated a conditional MHC streptamer-based platform for the creation of TIL products with defined antigen reactivities. We have used this platform to successfully enrich both high-frequency (≥1%) and low-frequency (<1%) tumor-specific CD8(+) T-cell populations, and thereby created T-cell products with enhanced tumor recognition potential. Collectively, these data demonstrate that selection of antigen-specific T-cell populations can be used to create defined T-cell products for clinical use. This strategy thus forms a highly flexible platform for the development of antigen-specific cell products for personalized cancer immunotherapy.

Selective inhibition of the p38 alternative activation pathway in infiltrating T cells inhibits pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive neoplasm characterized by a marked fibro-inflammatory microenvironment1, the presence of which can promote both cancer induction and growth2–4. Therefore, selective manipulation of local cytokines is an attractive if unrealized therapeutic approach. T cells possess a unique mechanism of activation of p38 MAPK downstream of T cell receptor (TCR) engagement by phosphorylation of Tyr-323 (pY323). This alternative p38 activation pathway is required for pro-inflammatory cytokine production5,6. Here we show in human PDAC that a high percentage of infiltrating pY323+ T cells was associated with large numbers of TNFα and IL-17-producing CD4+ tumor-infiltrating lymphocytes (TIL) and aggressive disease. The growth of murine pancreatic tumors was inhibited by genetic ablation of the alternative p38 pathway, and transfer of wild type CD4+ T cells but not those lacking the alternative pathway enhanced tumor growth in T cell-deficient mice. Strikingly, a plasma membrane-permeable peptide derived from Gadd45α, the naturally-occurring inhibitor of p38 pY323+ (ref. 7), reduced CD4+ TIL production of TNFα, IL-17A, IL-10, and secondary cytokines, halted growth of implanted tumors, and inhibited progression of spontaneous K-ras-driven adenocarcinoma in mice. Thus, TCR-mediated activation of CD4+ TIL results in alternative p38 activation and production of pro-tumorigenic factors, and can be targeted for therapeutic benefit.