Antigen-spezifische T-Zellaktivierung durch rekombinante Immunrezeptoren: Evaluierung in einem immunkompetenten Maus-Modell

Ein erfolgsversprechender Ansatz der adoptiven Immuntherapie zur Behandlung maligner Tumore beruht darauf, T-Zellen durch einen rekombinanten Rezeptor Spezifität für ein Tumor-assoziiertes-Antigen (TAA) zu verleihen. Derartige Rezeptoren bestehen aus einer extrazellulären Bindedomäne, die von einem Einzelketten-Antikörper (scFv) abgeleitet ist und einer intrazellulären Signalkette, die der T-Zell Aktivierung dient. Zahlreiche Ansätze zur Evaluierung der Immunrezeptor-Strategie basierten bisher auf der Verwendung immundefizienter Maus-Modelle, die nur unzureichend die physiologische Immunsituation wiedergeben. Das Ziel der vorliegenden Arbeit war es, die Immunrezeptor-Strategie am Beispiel des anti-CEA Rezeptors in einem immunkompetenten Mausmodell zu evaluieren. Hierfür wurden murine Rezeptoren mit Spezifität für CEA und mit der CD3ζ oder der kombinierten CD28-CD3ζ Signalkette generiert und durch retroviralen Gentransfer in humanen und in murinen T-Zellen exprimiert. Die Rezeptor-vermittelte T-Zell Aktivierung wurde anhand der IFN-γ Sekretion und zytolytischen Aktivität in vitro charakterisiert. Dabei wurde gezeigt, dass humane und murine T-Zellen mit den rekombinanten CD28-CD3ζ Rezeptoren stärker zur IFN-γ Sekretion induziert werden als mit den CD3ζ Rezeptoren, jedoch wird die spezifische lytische Aktivität gleich stark induziert. Durch die Ko-Injektion der CEA+ Tumorzellen mit CD8+ T-Zellen mit dem anti-CEA-CD3ζ Rezeptor (#895) und T-Zellen mit dem anti-CEA-CD28-CD3ζ Rezeptor (#907) wurden die CEA+ Tumore in der immunkompetenten Maus effizient eliminiert. Bei einer erneuten Injektion der CEA+ Tumorzellen blieben Tiere, die zuvor die Tumorzellen mit Hilfe der T-Zellen mit dem CD3ζ Rezeptor (#895) eliminiert hatten, tumorfrei, wohingegen die Hälfte der Tiere, die zuvor mit T-Zellen mit dem CD28-CD3ζ Rezeptor (#907) injiziert wurden, einen Tumor entwickelte. Diese und weitere Untersuchungen zeigen, dass T-Zellen mit dem anti-CEA-CD3ζ Rezeptor (#895) zur Generierung eines Antigen spezifischen "immunologischen Gedächtnisses" in C57-Bl/6 Mäusen führen. Unsere Untersuchungen mit Hilfe immunkompetenter Mausmodelle legen nahe, dass die Immunrezeptorstrategie möglicherweise für die Vorbeugung früher Rezidive durch Ausbildung eines Antigen spezifischen Gedächtnisses geeignet sein könnte. Dieses würde die Indikation der Immunrezeptorstrategie erheblich erweitern

Intra-tumoral production of IL18, but not IL12, by TCR-engineered T cells is non-toxic and counteracts immune evasion of solid tumors

Adoptive therapy with engineered T cells shows promising results in treating patients with malignant disease, but is challenged by incomplete responses and tumor recurrences. Here, we aimed to direct the tumor microenvironment in favor of a successful immune response by local secretion of interleukin (IL-) 12 and IL-18 by sadministered T cells. To this end, we engineered T cells with a melanoma-specific T cell receptor (TCR) and murine IL-12 and/or IL-18 under the control of a nuclear-factor of activated T-cell (NFAT)-sensitive promoter. These T cells produced IL-12 or IL-18, and consequently enhanced levels of IFNγ, following exposure to antigen-positive but not negative tumor cells. Adoptive transfer of T cells with a TCR and inducible (i)IL-12 to melanoma-bearing mice resulted in severe, edema-like toxicity that was accompanied by enhanced levels of IFNγ and TNFα in blood, and reduced numbers of peripheral TCR transgene-positive T cells. In contrast, transfer of T cells expressing a TCR and iIL-18 was without side effects, enhanced the presence of therapeutic CD8+ T cells within tumors, reduced tumor burden and prolonged survival. Notably, treatment with TCR+iIL-12 but not iIL-18 T cells resulted in enhanced intra-tumoral accumulation of macrophages, which was accompanied by a decreased frequency of therapeutic T cells, in particular of the CD8 subset. In addition, when administered to mice, iIL-18 but not iIL-12 demonstrated a favorable profile of T cell co-stimulatory and inhibitory receptors. In conclusion, we observed that treatment with T cells engineered with a TCR and iIL18 T cells is safe and able to skew the tumor microenvironment in favor of an improved anti-tumor T cell response

Chimeric Antigen Receptor-Redirected Regulatory T Cells Suppress Experimental Allergic Airway Inflammation, a Model of Asthma

Cellular therapy with chimeric antigen receptor (CAR)-redirected cytotoxic T cells has shown impressive efficacy in the treatment of hematologic malignancies. We explored a regulatory T cell (Treg)-based therapy in the treatment of allergic airway inflammation, a model for asthma, which is characterized by an airway hyper-reactivity (AHR) and a chronic, T helper-2 (Th2) cell-dominated immune response to allergen. To restore the immune balance in the lung, we redirected Tregs by a CAR toward lung epithelia in mice upon experimentally induced allergic asthma, closely mimicking the clinical situation. Adoptively transferred CAR Tregs accumulated in the lung and in tracheobronchial lymph nodes, reduced AHR and diminished eosinophilic airway inflammation, indicated by lower cell numbers in the bronchoalveolar lavage fluid and decreased cell infiltrates in the lung. CAR Treg cells furthermore prevented excessive pulmonary mucus production as well as increase in allergen-specific IgE and Th2 cytokine levels in exposed animals. CAR Tregs were more efficient in controlling asthma than non-modified Tregs, indicating the pivotal role of specific Treg cell activation in the affected organ. Data demonstrate that lung targeting CAR Treg cells ameliorate key features of experimental airway inflammation, paving the way for cell therapy of severe allergic asthma

Genome Editing With TALEN, CRISPR-Cas9 and CRISPR-Cas12a in Combination With AAV6 Homology Donor Restores T Cell Function for XLP

X-linked lymphoproliferative disease is a rare inherited immune disorder, caused by mutations or deletions in the SH2D1A gene that encodes an intracellular adapter protein SAP (Slam-associated protein). SAP is essential for mediating several key immune processes and the immune system - T cells in particular - are dysregulated in its absence. Patients present with a spectrum of clinical manifestations, including haemophagocytic lymphohistiocytosis (HLH), dysgammaglobulinemia, lymphoma and autoimmunity. Treatment options are limited, and patients rarely survive to adulthood without an allogeneic haematopoietic stem cell transplant (HSCT). However, this procedure can have poor outcomes in the mismatched donor setting or in the presence of active HLH, leaving an unmet clinical need. Autologous haematopoeitic stem cell or T cell therapy may offer alternative treatment options, removing the need to find a suitable donor for HSCT and any risk of alloreactivity. SAP has a tightly controlled expression profile that a conventional lentiviral gene delivery platform may not be able to fully replicate. A gene editing approach could preserve more of the endogenous regulatory elements that govern SAP expression, potentially providing a more optimum therapy. Here, we assessed the ability of TALEN, CRISPR-Cas9 and CRISPR-Cas12a nucleases to drive targeted insertion of SAP cDNA at the first exon of the SH2D1A locus using an adeno-associated virus serotype 6 (AAV6)-based vector containing the donor template. All nuclease platforms were capable of high efficiency gene editing, which was optimised using a serum-free AAV6 transduction protocol. We show that T cells from XLP patients corrected by gene editing tools have restored physiological levels of SAP gene expression and restore SAP-dependent immune functions, indicating a new therapeutic opportunity for XLP patients

Selective Bispecific T Cell Recruiting Antibody and Antitumor Activity of Adoptive T Cell Transfer

Background: One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy. Methods: SV40 T antigen-specific T cells from T cell receptor (TCR)-I-transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR-anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40+ and EpCAM+). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met+ human tumor cell lines. Differences between experimental conditions were analyzed using the Student's t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided. Results: The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR-transduced T cells to immobilized c-Met and recognition of tyrosinase+ melanoma cells by TCR-, as well as of CEA+ colon cancer cells by chimeric antigen receptor (CAR)-modified T cells. Conclusions: BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of AC

Selective bispecific T cell recruiting antibody and antitumor activity of adoptive T cell transfer

Background: One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy.Methods: SV40 T antigen–specific T cells from T cell receptor (TCR)-I–transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR–anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40+ and EpCAM+). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met+ human tumor cell lines. Differences between experimental conditions were analyzed using the Student’s t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided.Results: The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR–transduced T cells to immobilized c-Met and recognition of tyrosinase+ melanoma cells by TCR-, as well as of CEA+ colon cancer cells by chimeric antigen receptor (CAR)–modified T cells.Conclusions: BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of ACT

TRUCKs: the fourth generation of CARs

Introduction: Adoptive cell therapy of malignant diseases takes advantage of the cellular immune system to recognize and destroy cancer cells. This is impressively demonstrated by redirecting T cells with a chimeric antigen receptor (CAR) towards CD19, inducing complete and lasting remission of leukemia in more than two-thirds of patients in early phase trials. Areas covered: We outline how the CAR strategy is highly specific in redirecting T cells towards pre-defined target cells, however, reaches its limits when targeting solid tumors with a tremendous phenotypic heterogeneity. After initial tumor reduction by CART cells, antigen-negative cancer cells not recognized by CAR may give rise to tumor relapse. The situation may be overcome by CAR-mediated activation of T cells in the tumor, releasing inducible IL-12 which augments T-cell activation and attracts and activates innate immune cells to eliminate antigen-negative cancer cells in the targeted lesion. Expert opinion: CART cells with a transgenic 'payload', so-called TRUCK T cells or the 'fourth-generation' CART cells, are worthwhile to explore to shape the tumor environment by the inducible release of transgenic immune modifiers. Such TRUCK T cells are moreover envisioned to be applied in fields beyond cancer therapy including the therapy of virus infections, auto-immune diseases or metabolic disorders

TRUCKS, the fourth generation CAR T cells: current developments and clinical translation

The overriding goal of adoptive cell therapy with chimeric antigen receptor (CAR) redirected T cells in oncology is to eliminate cancer cells from infiltrated tissues. Clinical trials document that this form of immunotherapy can induce lasting remissions of hematologic malignancies; however, the successes could not yet be transferred to the treatment of solid tumors. In this situation, modulating the immune regulation within the solid tumor tissue is thought to be a key point. In order to induce a pro‐inflammatory milieu CAR T cells were additionally engineered to release a transgenic cytokine upon CAR signaling in the targeted tumor tissue. Such TRUCKs (“T cells redirected for antigen‐unrestricted cytokine‐initiated killing”), also called “4th generation” CAR T cells, combine the direct antitumor attack of the CAR T cell with the immune modulating capacities of the delivered cytokine. Through CAR‐induced release, the cytokine is ideally deposited in the targeted tissue alleviating systemic side effects. The TRUCK concept is currently explored using a panel of cytokines, including IL‐7, IL‐12, IL‐15, IL‐18, IL‐23, and combinations thereof, and is entering early phase trials. Future developments will expand the application to a broader panel of released proteins converting CAR T cells to “living factories” of therapeutically active, locally deposited products with the potential to eliminate some clinical deficits of the currently used CAR T cells in the field of solid tumors