TCR-engaging scaffolds selectively expand antigen-specific T-cells with a favorable phenotype for adoptive cell therapy.

BACKGROUND: Adoptive cell therapy (ACT) has shown promising results for the treatment of cancer and viral infections. Successful ACT relies on ex vivo expansion of large numbers of desired T-cells with strong cytotoxic capacity and in vivo persistence, which constitutes the greatest challenge to current ACT strategies. Here, in this study, we present a novel technology for ex vivo expansion of antigen-specific T-cells; artificial antigen-presenting scaffolds (Ag-scaffolds) consisting of a dextran-polysaccharide backbone, decorated with combinations of peptide-Major Histocompatibility Complex (pMHC), cytokines and co-stimulatory molecules, enabling coordinated stimulation of antigen-specific T-cells. METHODS: The capacity of Ag-scaffolds to expand antigen-specific T-cells was explored in ex vivo cultures with peripheral blood mononuclear cells from healthy donors and patients with metastatic melanoma. The resulting T-cell products were assessed for phenotypic and functional characteristics. RESULTS: We identified an optimal Ag-scaffold for expansion of T-cells for ACT, carrying pMHC and interleukin-2 (IL-2) and IL-21, with which we efficiently expanded both virus-specific and tumor-specific CD8+ T cells from peripheral blood of healthy donors and patients, respectively. The resulting T-cell products were characterized by a high frequency of antigen-specific cells with high self-renewal capacity, low exhaustion, a multifunctional cytokine profile upon antigen-challenge and superior tumor killing capacity. This demonstrates that the coordinated stimuli provided by an optimized stoichiometry of TCR engaging (pMHC) and stimulatory (cytokine) moieties is essential to obtain desired T-cell characteristics. To generate an 'off-the-shelf' multitargeting Ag-scaffold product of relevance to patients with metastatic melanoma, we identified the 30 most frequently recognized shared HLA-A0201-restricted melanoma epitopes in a cohort of 87 patients. By combining these in an Ag-scaffold product, we were able to expand tumor-specific T-cells from 60-70% of patients with melanoma, yielding a multitargeted T-cell product with up to 25% specific and phenotypically and functionally improved T cells. CONCLUSIONS: Taken together, the Ag-scaffold represents a promising new technology for selective expansion of antigen-specific CD8+ T cells directly from blood, yielding a highly specific and functionally enhanced T-cell product for ACT.

Highly efficient PD-1-targeted CRISPR-Cas9 for tumor-infiltrating lymphocyte-based adoptive T cell therapy.

Adoptive T cell therapy (ACT) with expanded tumor-infiltrating lymphocytes (TIL) can induce durable responses in cancer patients from multiple histologies, with response rates of up to 50%. Antibodies blocking the engagement of the inhibitory receptor programmed cell death protein 1 (PD-1) have been successful across a variety of cancer diagnoses. We hypothesized that these approaches could be combined by using CRISPR-Cas9 gene editing to knock out PD-1 in TILs from metastatic melanoma and head-and-neck, thyroid, and colorectal cancer. Non-viral, non-plasmid-based PD-1 knockout was carried out immediately prior to the traditional 14-day TIL-based ACT rapid-expansion protocol. A median 87.53% reduction in cell surface PD-1 expression was observed post-expansion and confirmed at the genomic level. No off-target editing was detected, and PD-1 knockout had no effect on final fold expansion. Edited cells exhibited few phenotypic differences and matched control functionality. Pre-clinical-scale results were confirmed at a clinical scale by generating a PD-1-deficient TIL product using the good manufacturing practice facilities, equipment, procedures, and starting material used for standard patient treatment. Our results demonstrate that simple, non-viral, non-plasmid-based CRISPR-Cas9 methods can be feasibly adopted into a TIL-based ACT protocol to produce treatment products deficient in molecules such as PD-1, without any evident negative effects.

Adoptive cell therapy with tumor-infiltrating lymphocytes supported by checkpoint inhibition across multiple solid cancer types.

BACKGROUND: Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) has shown remarkable results in malignant melanoma (MM), while studies on the potential in other cancer diagnoses are sparse. Further, the prospect of using checkpoint inhibitors (CPIs) to support TIL production and therapy remains to be explored. STUDY DESIGN: TIL-based ACT with CPIs was evaluated in a clinical phase I/II trial. Ipilimumab (3 mg/kg) was administered prior to tumor resection and nivolumab (3 mg/kg, every 2 weeks ×4) in relation to TIL infusion. Preconditioning chemotherapy was given before TIL infusion and followed by low-dose (2 10e6 international units (UI) ×1 subcutaneous for 14 days) interleukin-2 stimulation. RESULTS: Twenty-five patients covering 10 different cancer diagnoses were treated with in vitro expanded TILs. Expansion of TILs was successful in 97% of recruited patients. Five patients had sizeable tumor regressions of 30%-63%, including two confirmed partial responses in patients with head-and-neck cancer and cholangiocarcinoma. Safety and feasibility were comparable to MM trials of ACT with the addition of expected CPI toxicity. In an exploratory analysis, tumor mutational burden and expression of the alpha-integrin CD103 (p=0.025) were associated with increased disease control. In vitro tumor reactivity was seen in both patients with an objective response and was associated with regressions in tumor size (p=0.028). CONCLUSION: High success rates of TIL expansion were demonstrated across multiple solid cancers. TIL ACTs were found feasible, independent of previous therapy. Tumor regressions after ACT combined with CPIs were demonstrated in several cancer types supported by in vitro antitumor reactivity of the TILs. TRIAL REGISTRATION NUMBERS: NCT03296137, and EudraCT No. 2017-002323-25.

Clinical efficacy of T-cell therapy after short-term BRAF-inhibitor priming in patients with checkpoint inhibitor-resistant metastatic melanoma.

PURPOSE: Despite impressive response rates following adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs) in patients with metastatic melanoma, improvement is needed to increase the efficacy and broaden the applicability of this treatment. We evaluated the use of vemurafenib, a small-molecule BRAF inhibitor with immunomodulatory properties, as priming before TIL harvest and adoptive T cell therapy in a phase I/II clinical trial. METHODS: 12 patients were treated with vemurafenib for 7 days before tumor excision and during the following weeks until TIL infusion. TILs were grown from tumor fragments, expanded in vitro and reinfused to the patient preceded by a lymphodepleting chemotherapy regimen and followed by interleukin-2 infusion. Extensive immune monitoring, tumor profiling and T cell receptor sequencing were performed. RESULTS: No unexpected toxicity was observed, and treatment was well tolerated. Of 12 patients, 1 achieved a complete response, 8 achieved partial response and 3 achieved stable disease. A PR and the CR are ongoing for 23 and 43 months, respectively. In vitro anti-tumor reactivity was found in TILs from 10 patients, including all patients achieving objective response. Serum and tumor biomarker analyses indicate that baseline cytokine levels and the number of T cell clones may predict response to TIL therapy. Further, TCR sequencing suggested skewing of TCR repertoire during in vitro expansion, promoting certain low frequency clonotypes. CONCLUSIONS: Priming with vemurafenib before infusion of TILs was safe and feasible, and induced objective clinical responses in this cohort of patients with checkpoint inhibitor-resistant metastatic melanoma. In this trial, vemurafenib treatment seemed to decrease attrition and could be considered to bridge the waiting time while TILs are prepared.

Adoptive cell therapy in combination with checkpoint inhibitors in ovarian cancer.

Immune therapy is a promising field within oncology but has been unsuccessful in ovarian cancer (OC). Still, there is rationale and evidence supporting immune therapy in OC. We investigated the potential for adoptive cell therapy (ACT) from in vitro expanded tumor-infiltrating lymphocytes (TILs) in combination with checkpoint inhibitors (ICI) and conducted immunological testing of ex vivo expanded TILs (REP-TILs). Six patients with late-stage metastatic high-grade serous OC were treated with immune therapy consisting of ipilimumab followed by surgery to obtain TILs and infusion of REP-TILs, low-dose IL-2 and nivolumab. One patient achieved a partial response and 5 others experienced disease stabilization for up to 12 months. Analysis of the REP-TILs with flow- and mass-cytometry show primarily activated and differentiated effector memory T cells. REP-TILs showed in vitro reactivity and expression of inhibitory receptors, such as LAG-3 and PD-1. Furthermore, our data indicate that addition of ipilimumab therapy improves the T cell fold expansion during production, increase the level of CD8 T cell tumor reactivity, and favorably affect the T cell phenotype. We show that the combination of ICI and ACT is feasible and safe. With one partial response and one long-lasting SD, we demonstrated the potential of ACT in OC.

Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study.

BACKGROUND: Immune monitoring by flow cytometry is a fast and highly informative way of studying the effects of novel therapeutics aimed at reducing transplant rejection or treating autoimmune diseases. The ONE Study consortium has recently initiated a series of clinical trials aimed at using different cell therapies to promote tolerance to renal allografts. To compare the effectiveness of different cell therapies, the consortium developed a robust immune monitoring strategy, including procedures for whole blood (WB) leukocyte subset profiling by flow cytometry. METHODS: Six leukocyte profiling panels computing 7- to 9-surface marker antigens for monitoring the major leukocyte subsets as well as characteristics of T cell, B cell, and dendritic cell (DC) subsets were designed. The precision and variability of these panels were estimated. The assay was standardized within eight international laboratories using Flow-Set Pro beads for mean fluorescence intensity target definition and the flow cytometer setup procedure. Standardization was demonstrated by performing inter-site comparisons. RESULTS: Optimized methods for sample collection, storage, preparation, and analysis were established, including protocols for gating target subsets. WB specimen age testing demonstrated that staining must be performed within 4 hours of sample collection to keep variability low, meaning less than or equal to 10% for the majority of defined leukocyte subsets. Inter-site comparisons between all participating centers testing shipped normal WB revealed good precision, with a variability of 0.05% to 30% between sites. Intra-assay analyses revealed a variability of 0.05% to 20% for the majority of subpopulations. This was dependent on the frequency of the particular subset, with smaller subsets showing higher variability. The intra-assay variability performance defined limits of quantitation (LoQ) for subsets, which will be the basis for assessing statistically significant differences achieved by the different cell therapies. CONCLUSIONS: Local performance and central analysis of the ONE Study flow cytometry panel yields acceptable variability in a standardized assay at multiple international sites. These panels and procedures with WB allow unmanipulated analysis of changes in absolute cell numbers of leukocyte subsets in single- or multicenter clinical trials. Accordingly, we propose the ONE Study panel may be adopted as a standardized method for monitoring patients in clinical trials enrolling transplant patients, particularly trials of novel tolerance promoting therapies, to facilitate fair and meaningful comparisons between trials.