Combined targeting of soluble latent TGF-ß and a solid tumor-associated antigen with adapter CAR T cells.

Solid tumors consist of malignant and nonmalignant cells that together create the local tumor microenvironment (TME). Additionally, the TME is characterized by the expression of numerous soluble factors such as TGF-ß. TGF-ß plays an important role in the TME by suppressing T cell effector function and promoting tumor invasiveness. Up to now CAR T cells exclusively target tumor-associated antigens (TAA) located on the cell membrane. Thus, strategies to exploit soluble antigens as CAR targets within the TME are needed. This study demonstrates a novel approach using Adapter CAR (AdCAR) T cells for the detection of soluble latent TGF-ß within the TME of a pancreatic tumor model. We show that AdCARs in combination with the respective adapter can be used to sense soluble tumor-derived latent TGF-ß, both in vitro and in vivo. Sensing of the soluble antigen induced cellular activation and effector cytokine production in AdCAR T cells. Moreover, we evaluated AdCAR T cells for the combined targeting of soluble latent TGF-ß and tumor cell killing by targeting CD66c as TAA in vivo. In sum, our study broadens the spectrum of targetable moieties for AdCAR T cells by soluble latent TGF-ß.

Titratable Pharmacological Regulation of CAR T Cells Using Zinc Finger-Based Transcription Factors.

Chimeric antigen receptor (CAR) T cell therapy has emerged as an attractive strategy for cancer immunotherapy. Despite remarkable success for hematological malignancies, excessive activity and poor control of CAR T cells can result in severe adverse events requiring control strategies to improve safety. This work illustrates the feasibility of a zinc finger-based inducible switch system for transcriptional regulation of an anti-CD20 CAR in primary T cells providing small molecule-inducible control over therapeutic functions. We demonstrate time- and dose-dependent induction of anti-CD20 CAR expression and function with metabolites of the clinically-approved drug tamoxifen, and the absence of background CAR activity in the non-induced state. Inducible CAR T cells executed fine-tuned cytolytic activity against target cells both in vitro and in vivo, whereas CAR-related functions were lost upon drug discontinuation. This zinc finger-based transcriptional control system can be extended to other therapeutically important CARs, thus paving the way for safer cellular therapies.

CAR T-cell Entry into Tumor Islets Is a Two-Step Process Dependent on IFNγ and ICAM-1.

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies but not yet against solid tumors. Here, we used fluorescent imaging microscopy and ex vivo assays to compare the early functional responses (migration, Ca2+, and cytotoxicity) of CD20 and EGFR CAR T cells upon contact with malignant B cells and carcinoma cells. Our results indicated that CD20 CAR T cells rapidly form productive ICAM-1-dependent conjugates with their targets. By comparison, EGFR CAR T cells only initially interacted with a subset of carcinoma cells located at the periphery of tumor islets. After this initial peripheral activation, EGFR CAR T cells progressively relocated to the center of tumor cell regions. The analysis of this two-step entry process showed that activated CAR T cells triggered the upregulation of ICAM-1 on tumor cells in an IFNγ-dependent pathway. The ICAM-1/LFA-1 interaction interference, through antibody or shRNA blockade, prevented CAR T-cell enrichment in tumor islets. The requirement for IFNγ and ICAM-1 to enable CAR T-cell entry into tumor islets is of significance for improving CAR T-cell therapy in solid tumors.

Anti-CD19 CARs displayed at the surface of lentiviral vector particles promote transduction of target-expressing cells.

Recently, a rare type of relapse was reported upon treating a B cell acute lymphoblastic leukemia (B-ALL) patient with anti-CD19 chimeric antigen receptor (CAR)-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cells). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs), inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LVs containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner, and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMCs and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future.

Vectofusin-1 Improves Transduction of Primary Human Cells with Diverse Retroviral and Lentiviral Pseudotypes, Enabling Robust, Automated Closed-System Manufacturing.

Cell and gene therapies are finally becoming viable patient treatment options, with both T cell- and hematopoietic stem cell (HSC)-based therapies being approved to market in Europe. However, these therapies, which involve the use of viral vector to modify the target cells, are expensive and there is an urgent need to reduce manufacturing costs. One major cost factor is the viral vector production itself, therefore improving the gene modification efficiency could significantly reduce the amount of vector required per patient. This study describes the use of a transduction enhancing peptide, Vectofusin-1®, to improve the transduction efficiency of primary target cells using lentiviral and gammaretroviral vectors (LV and RV) pseudotyped with a variety of envelope proteins. Using Vectofusin-1 in combination with LV pseudotyped with viral glycoproteins derived from baboon endogenous retrovirus, feline endogenous virus (RD114), and measles virus (MV), a strongly improved transduction of HSCs, B cells and T cells, even when cultivated under low stimulation conditions, could be observed. The formation of Vectofusin-1 complexes with MV-LV retargeted to CD20 did not alter the selectivity in mixed cell culture populations, emphasizing the precision of this targeting technology. Functional, ErbB2-specific chimeric antigen receptor-expressing T cells could be generated using a gibbon ape leukemia virus (GALV)-pseudotyped RV. Using a variety of viral vectors and target cells, Vectofusin-1 performed in a comparable manner to the traditionally used surface-bound recombinant fibronectin. As Vectofusin-1 is a soluble peptide, it was possible to easily transfer the T cell transduction method to an automated closed manufacturing platform, where proof of concept studies demonstrated efficient genetic modification of T cells with GALV-RV and RD114-RV and the subsequent expansion of mainly central memory T cells to a clinically relevant dose.

Automated Enrichment, Transduction, and Expansion of Clinical-Scale CD62L+ T Cells for Manufacturing of Gene Therapy Medicinal Products.

Multiple clinical studies have demonstrated that adaptive immunotherapy using redirected T cells against advanced cancer has led to promising results with improved patient survival. The continuously increasing interest in those advanced gene therapy medicinal products (GTMPs) leads to a manufacturing challenge regarding automation, process robustness, and cell storage. Therefore, this study addresses the proof of principle in clinical-scale selection, stimulation, transduction, and expansion of T cells using the automated closed CliniMACS® Prodigy system. Naïve and central memory T cells from apheresis products were first immunomagnetically enriched using anti-CD62L magnetic beads and further processed freshly (n = 3) or split for cryopreservation and processed after thawing (n = 1). Starting with 0.5 × 108 purified CD3+ T cells, three mock runs and one run including transduction with green fluorescent protein (GFP)-containing vector resulted in a median final cell product of 16 × 108 T cells (32-fold expansion) up to harvesting after 2 weeks. Expression of CD62L was downregulated on T cells after thawing, which led to the decision to purify CD62L+CD3+ T cells freshly with cryopreservation thereafter. Most important in the split product, a very similar expansion curve was reached comparing the overall freshly CD62L selected cells with those after thawing, which could be demonstrated in the T cell subpopulations as well by showing a nearly identical conversion of the CD4/CD8 ratio. In the GFP run, the transduction efficacy was 83%. In-process control also demonstrated sufficient glucose levels during automated feeding and medium removal. The robustness of the process and the constant quality of the final product in a closed and automated system give rise to improve harmonized manufacturing protocols for engineered T cells in future gene therapy studies.

Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy.

Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability.

Toll-like receptor agonist therapy can profoundly augment the antitumor activity of adoptively transferred CD8(+) T cells without host preconditioning.

BACKGROUND: Lymphodepletion enhances adoptive T cell transfer (ACT) therapy by activating the innate immune system via microbes released from the radiation-injured gut. Microbial components, such as LPS, are key mediators of total body irradiation (TBI) enhancement, but our ability to strategically use these toll-like receptor (TLR) agonists to bolster the potency of T cell-based therapies for cancer remains elusive. Herein, we used TLR4 agonist LPS as a tool to address how and when to use TLR agonists to effectively improve cancer immunotherapy. METHODS: To determine the mechanisms of how innate immune activation via lymphodepletion potentiated antitumor T cell immunity, we utilized the pmel-1 melanoma mouse model. B16F10-bearing mice were preconditioned with 5Gy TBI and given a tripartite ACT therapy (consisting of transferred pmel-1 CD8(+) T cells, vaccination with fowlpox encoding gp100, and IL-2) along with TLR4 agonist LPS. The timing of LPS administration and the requirement of individual components of the tripartite therapy were evaluated based on tumor growth and the phenotype of recovered splenocytes by flow cytometry. We also evaluated the role of non-toxic and clinically used TLR4 and TLR9 agonists-monophosphoryl lipid A (MPL) and CpG Oligodeoxynucleotide (CpG ODN), respectively- for ACT therapy. RESULTS: Here we report that while exogenous administration of LPS was able to enhance adoptively transferred CD8(+) T cells' tumor destruction, LPS treatment alone did not replace individual components of the tripartite ACT regimen, or obviate TBI. Moreover, we found that sequentially administering LPS during or one day prior to ACT therapy compromised tumor regression. In contrast, administering LPS after ACT potentiated the antitumor effectiveness of the regimen, thereby supporting the expansion of transferred tumor-specific CD8(+) T cells over host CD4(+) T cells. We also found that non-toxic TLR agonists MPL and CpG potentiated the antitumor activity of infused CD8(+) T cells. Finally, TBI was no longer needed to regress tumors in mice who were depleted of host CD4(+) T cells, given a tripartite ACT regimen and then treated with low dose LPS. CONCLUSIONS: Collectively, our results identify how and when to administer TLR agonists to augment T cell-based immunotherapy in the absence or presence of host preconditioning for treatment of advanced malignancies. Our findings have clinical implications for the design of next generation immune-based therapies for patients with cancer.

Mimicking homeostatic proliferation in vitro generates T cells with high anti-tumor function in non-lymphopenic hosts.

CD8(+) T cells undergoing homeostatic proliferation (HP) in a lymphopenic environment acquire a central memory-like phenotype (CD44(+) CD62L(+) Ly6c(+)). Such cells are readily functional in vitro, with a strong capacity to secrete IFNγ and IL-2 and to lyse target cells upon antigen recognition. In vivo, these memory-like T cells display potent anti-tumor reactivity. When addressing whether these remarkable properties were "acquired" or dependent on sustained HP, we observed, for the first time, that memory-like T cells retained full anti-tumor functions even when removed from their lymphopenic environment and retransferred into non-lymphopenic P14/Rag2(-/-) recipients (where HP is prevented). Moreover, memory-like T cells were superior to in vitro expanded effector T cells. We next sought to determine the conditions required to reproduce such a potent phenotype in vitro, in order to obtain optimal cells for adoptive cell transfer therapy. Assessing ex vivo lymph node cultures, dendritic cells, fibroblastic reticular cells, and HP-associated cytokines, we found that stimulation of naïve T cells with anti-CD3/CD28 beads and IL-15 (IL-7 was dispensable) led to the generation of memory-like T cell with a similar phenotype. Both in vitro and in vivo memory-like T cells retained the capacity to efficiently control tumor growth in non-lymphopenic hosts upon adoptive cell transfer. A similar phenotype could be imparted to human peripheral blood leukocytes with comparable culture conditions. Our data reinforce the idea that in vitro-generated memory-like T cells could benefit adoptive cell transfer therapies.

Reduced tumor-antigen density leads to PD-1/PD-L1-mediated impairment of partially exhausted CD8⁺ T cells.

Clinical progression of cancer patients is often observed despite the presence of tumor-reactive T cells. Co-inhibitory ligands of the B7 superfamily have been postulated to play a part in this tumor-immune escape. One of these molecules, PD-L1 (B7-H1, CD274), is widely expressed on tumor cells and has been shown to mediate T-cell inhibition. However, attempts to correlate PD-L1 tumor expression with negative prognosis have been conflicting. To better understand when PD-1/PD-L1-mediated inhibition contributes to the functional impairment of tumor-specific CD8(+) T cells, we varied the levels of antigen density and/or PD-L1 expression at the surface of tumor cells and exposed them to CD8(+) T cells at different levels of functional exhaustion. We found that the gradual reduction of cognate antigen expression by PD-L1-expressing tumor cells increased the susceptibility of partially exhausted T cells to PD-1/PD-L1-mediated inhibition in vitro as well as in vivo. In conclusion, chronically stimulated CD8(+) T cells become sensitive to PD-1/PD-L1-mediated functional inhibition upon low antigen detection; a setting which is likely involved during tumor-immune escape.

Overall survival and PD-L1 expression in metastasized malignant melanoma.

BACKGROUND: Cancers are known to elude the immune system, for example, by MHC loss, FAS up-regulation, or increased secretion of TGF-beta. Recently, ligands of coinhibitory receptors like programmed cell death ligand-1 (PD-L1, B7-H1) have come to attention for their role in tumor immune escape. Various tumors have been tested for PD-L1 expression, and conflicting results were obtained regarding its correlative impact on patient survival. This study aimed to determine the prognostic relevance of PD-L1 expression for the survival of melanoma patients. METHODS: Paraffin-embedded nevi, primary melanoma, and in-transit, lymph node, and distant organ metastases from a set of 63 stages III-IV melanoma patients referred to the Netherlands Cancer Institute between 2000 and 2004 for a sentinel-node procedure or systemic therapy were studied. A large effort was invested in validating specific PD-L1 staining. In addition to immunological factors such as T-cell infiltration (CD8, CD4, and regulatory T cells), TGF-beta and MHC-I expression were assessed. RESULTS: Longitudinal analysis revealed no relevant PD-L1 expression on primary melanoma compared with metastatic disease. No significant correlations with prognosis were found regarding immunological factors, whereas known prognostic markers such as Breslow thickness and sex could be confirmed. Analyses of the overall survival of our patient cohort did not reveal a negative association with PD-L1 expression. CONCLUSIONS: Correlation of overall survival with PD-L1 expression by melanoma cells remains controversial, and future clinical studies should focus on antibody validation and time of analysis in respect to disease progression.

Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies.

T-cell receptor (TCR) gene therapy enables for the rapid creation of antigen-specific T cells from mice of any strain and represents a valuable tool for preclinical immunotherapy studies. Here, we describe the superiority of γ-retroviral vectors compared with lentiviral vectors for transduction of murine T cells and surprisingly illustrate robust gene-transfer into phenotypically naive/memory-stem cell like (TN/TSCM; CD62L(hi)/CD44(low)) and central memory (TCM; CD62L(hi)/CD44(hi)) CD8+ T cells using murine stem cell-based γ-retroviral vectors (MSGV1). We created MSGV1 vectors for a major histocompatibility complex-class I-restricted TCR specific for the melanocyte-differentiation antigen, glycoprotein 100 (MSGV1-pmel-1), and a major histocompatibility complex-class II-restricted TCR specific for tyrosinase-related protein-1 (MSGV1-TRP-1), and found that robust gene expression required codon optimization of TCR sequences for the pmel-1 TCR. To test for functionality, we adoptively transferred TCR-engineered T cells into mice bearing B16 melanomas and observed delayed growth of established tumors with pmel-1 TCR engineered CD8+ T cells and significant tumor regression with TRP-1 TCR transduced CD4 T cells. We simultaneously created lentiviral vectors encoding the pmel-1 TCR, but found that these vectors mediated low TCR expression in murine T cells, but robust gene expression in other murine and human cell lines. These results indicate that preclinical murine models of adoptive immunotherapies are more practical using γ-retroviral rather than lentiviral vectors.

Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.

The transfer of T cell receptor (TCR) genes can be used to induce immune reactivity toward defined antigens to which endogenous T cells are insufficiently reactive. This approach, which is called TCR gene therapy, is being developed to target tumors and pathogens, and its clinical testing has commenced in patients with cancer. In this study we show that lethal cytokine-driven autoimmune pathology can occur in mouse models of TCR gene therapy under conditions that closely mimic the clinical setting. We show that the pairing of introduced and endogenous TCR chains in TCR gene-modified T cells leads to the formation of self-reactive TCRs that are responsible for the observed autoimmunity. Furthermore, we demonstrate that adjustments in the design of gene therapy vectors and target T cell populations can be used to reduce the risk of TCR gene therapy-induced autoimmune pathology.