56. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Prof. Dr.

Vorsitz

Professor

Engineered T cells selectively depleting alloreactive B cells and resisting immunosuppressive treatment as tool to combat AMR following solid organ transplantation

Background One major complication after solid organ transplantation (SOT) is antibody-mediated rejection (AMR) by anti-donor HLA antibodies. However, the B-cell alloimmune response is only indirectly addressed by modern immunosuppression. Unselective B-cell depletion protocols are inefficient in preventing AMR and associated with an increased infection risk, emphasizing the need for a more precise targeting of alloreactive B cells.Methods B cells with anti-donor HLA specificity are uniquely characterized by expression of the corresponding B-cell receptors (BCRs). Using BCRs against a distinct HLA molecule as target, we redirected T cells towards alloreactive B cells by introducing a novel chimeric receptor comprising the respective HLA molecule fused to intracellular 4-1BB/CD3ξ signaling domains to generate T cells overcoming rejection by antibodies (CORA-Ts). As a proof-of-concept, CORA-Ts based on an HLA-A*02 molecule were generated and further modified to abrogate T-cell sensitization and confer resistance to immunosuppression. Their ability to recognize and selectively eliminate anti-HLA-A*02 B cells to limit antibody release was tested in vitro as well as in vivo.Results Upon co-cultivation with B-cell lines expressing and releasing anti-HLA-A*02 antibodies, CORA-Ts were specifically activated (expression of CD25, CD69, CD137), released pro-inflammatory molecules (e.g. IFN-γ, granzyme B), and exhibited strong cytotoxicity resulting in an effective reduction of the anti-HLA-A*02 antibody release. In a mouse model, CORA-Ts significantly reduced growth of an anti-HLA-A*02 hybridoma B-cell line. Modification of the HLA-A*02 α3-domain abrogated T-cell sensitization against the CORA receptor by prevention of CD8 binding. Additionally, CRISPR/Cas9-mediated knockouts of selected binding proteins endowed CORA-Ts with the ability to resist immunosuppressive treatment.Conclusion Our results demonstrate that CORA-Ts are able to specifically recognize and eliminate alloreactive B cells, and thus selectively prevent formation of anti-HLA antibodies even under immunosuppressive conditions. This suggests CORA-Ts as a potent novel approach to specifically combat AMR and to improve long-term graft survival in SOT patients while preserving their overall B-cell immunity.Offenlegung Interessenkonflikt: The authors declare no conflict of interest, except that authors A.C.D., C.F., R.B. and B. E.-V. submitted a patent application on CORA-Ts.

Immunotherapy

Freier Vortrag (Reinhold-Eckstein-Investigator-Award)

Zebrafish as a novel in vivo model to assess CAR-NK cell efficacy against metastatic breast cancer

Background NK cells are attractive effectors for adoptive immunotherapy of cancer. Results from first-in-human studies with chimeric antigen receptor (CAR)-engineered primary NK cells and NK-92 cells are encouraging in terms of efficacy and safety.Methods To further improve treatment strategies and to test the efficacy of CAR-NK cells in a personalized manner, high-throughput preclinical screening assays using patient-derived tumor samples are needed. Here, we established a flexible Danio rerio (zebrafish) larvae in vivo xenograft model and tested the efficacy of PD-L1-targeting CAR NK-92 cells (PD-L1.CAR NK-92) against the PD-L1-expressing breast cancer cell line MDA-MB-231. In addition, we used transgenic zebrafish models with labeled blood vessels to study CAR-NK migration in the vasculature.Results We have shown that MDA-MB-231 GFP cells injected into zebrafish larvae at 2 days post fertilization (dpf) are viable and migrate to peripheral parts of the zebrafish body, including the tail region. PD-L1.CAR NK-92 cells injected 2.5 hours later could also migrate to the zebrafish periphery and eliminate cancer cells throughout the body as early as 24 hours, in contrast to parental NK-92 or uninjected controls. Residual cancer cells were further eliminated at later time points, with 48 hours post-injection (hpi) being the best time point for analysis. Confocal live-cell imaging in vivo demonstrated intravascular migration and real-time interaction of PD-L1.CAR NK-92 with MDA-MB-231 cells, resulting in cytotoxicity.Conclusion Our data thus suggest that zebrafish larvae can be used for rapid assessment of CAR-NK cell potency in vivo to predict patient response to therapy.Offenlegung Interessenkonflikt: The authors declare that they have no competing interests.

Enhanced NK-cell cytotoxicity by combining genome editing and RIG-I activation

Background Natural killer (NK) cells can recognize and eliminate cellular targets without MHC-restriction, rendering them particularly amenable to off-the-shelf cellular immunotherapy. Nonetheless, potential target cells employ diverse molecular signals to limit NK-cell recruitment and evade cytotoxicity. Both innate immune activation and checkpoint inhibition on NK cells are promising approaches to improving the infiltration and cytotoxicity of NK-cell immunotherapy.Methods We examined the effect of the modulation of the immune checkpoint axis NKG2A/HLA-E on NK-cell tumor toxicity both alone and in combination with targeted activation of the innate immune receptor Retinoic-acid Inducible Gene I (RIG-I). Using CRISPR/Cas genome editing, we ablated the NKG2A-ligand HLA-E in diverse human tumor cell lines. Moreover, we developed a robust protocol for the targeted genome editing of primary and immortalized NK cells, allowing us to reduce NKG2A levels in effector cells. We then analyzed the activity and cytotoxic potential of these NKG2A-/- NK cells in vitro both in the presence and absence of HLA-E expression on tumor cells as well as alone and in combination with stimulation with a specific RIG-I agonist (3pRNA).Results All tumor cell lines tested demonstrated enhanced susceptibility to NK-cell killing after ablation of HLA-E on their targets. In line with this, NKG2A-/- NK cells also demonstrated greater degranulation, cytotoxicity towards target cells and cytokine induction, all of which occurred in an HLA-E dependent manner. Activation of RIG-I in tumor cells also enhanced NK cell activity overall but concurrently induced the expression of HLA-E in target cells. However, combining RIG-I activation with NKG2A-/- counteracted the effect of HLA-E induction and thus resulted in the highest cytotoxicity against target cells.Conclusion Altogether, our data demonstrate that combining NKG2A-/- NK cells with RIG-I activation is an effective approach to boosting allogeneic antitumor responses in vitro. Moreover, by establishing an efficient system for genome editing of primary NK cells, we provide an approach to targeting other promising candidate genes during NK-cell based therapies. Further studies will be needed to examine further NK checkpoints in this context and to investigate NKG2A-/- /RIG-I combination therapy in vivo.Offenlegung Interessenkonflikt: G.H. is an inventor on a patent covering synthetic RIG-I ligand and was a co-founder of Rigontec GmbH. All of others have no conflict of interest.Invited talks abstract/summary

Freier Vortrag

Next generation LMP2A-targeting TCR-recombinant T-Cells with inducible expression of Il-18 as promising tool to treat various EBV-associated malignancies

Background Epstein-Barr virus (EBV) infects &gt;90% of the population and remains in B-cell compartments life-long, passing through several latency stages. In latency stages II and III, latent membrane protein 2A (LMP2A) is expressed and therefore associated with post-transplant lymphoproliferative disorder (PTLD) as well as with various lymphomas and carcinomas. Recently, a clinically protective TCR recognizing an LMP2A-derived peptide (CLG) in context of HLA-A*02 (A*02/CLG) was identified (LMP2A_TCR).Methods We developed TCR-engineered T cells based on the LMP2A_TCR (LMP2A_TCR-Ts) and further equipped these with an inducible cassette for locally restricted IL-18 release (LMP2A_iIL18_TCR-Ts) to enhance T-cell functionality. Engineered TCR-Ts were generated by lentiviral transduction and their memory phenotype, replicative capacity, activation and exhaustion state during generation and after target cell encounter were analyzed. Their cytotoxicity towards HLA-A*02+ SPI-801 cells loaded with the CLG peptide and HLA-A*02+ EBV-transformed B-lymphoblastoid cell lines (B-LCLs) were analyzed using flow cytometry-, microscopy- and impedance-based assays. Furthermore, cell avidity (z-MOVI) as well intracellular Ca2+ signaling were evaluated.Results LMP2A_TCR-Ts and LMP2A_iIL18_TCR-Ts recognized HLA-A*02+ SPI-801 cells loaded with different concentrations of CLG in a dose-dependent manner. No signs of HLA cross-reactivity or recognition of an irrelevant HLA-A*02-restricted peptide were observed. Specificity and activation capacity of LMP2A_iIL18_TCR-Ts was confirmed by Ca2+ signaling analysis. Release of IL-18, which was shown to convert T cells into pro-inflammatory effector cells and reshape the immunosuppressive tumor milieu, resulted in significantly increased cytotoxicity when compared to LMP2A_TCR-Ts. Of note, LMP2A_iIL18_TCR-Ts but not LMP2A_TCR-Ts recognized and efficiently eliminated HLA-A*02+ B-LCLs serving as PTLD model and described to produce an anti-inflammatory milieu.Conclusion Our data indicate that LMP2A_iIL18_TCR-Ts specifically recognize A*02/CLG, leading to effective elimination of EBV+ HLA-A*02+ target cells. The release of IL-18 improved the functionality of the engineered T cells. In conclusion, ex vivo isolated, protective TCRs could be redirected into T cells from third-party donors with the potential to attract innate immune cells and alter the tumor environment, thereby widening the applicability of T-cell therapy to refractory viral infections.Offenlegung Interessenkonflikt: The authors declare no conflict of interest.

4th generation CAR-T cells with inducible cytokine expression targeting CD176 to overcome the immunosuppressive tumor milieu in lung cancer

Background In the development of effective CAR-Ts against solid tumors, both choosing an appropriate tumor-associated target and overcoming the immunosuppressive tumor microenvironment (TME) remain major challenges. The oncofetal carbohydrate CD176 (Thomsen-Friedenreich antigen), hidden in adult benign tissues by sialylation or prolongation with carbohydrates, is unmasked in 90% of carcinomas, predicting low "on-target/off-tumor"-toxicity.Methods To target CD176 and overcome the immunosuppressive TME, we developed 4th-generation CD176-specific CAR-Ts, also known as T cells redirected for universal cytokine-mediated killing (TRUCKs). In addition to the constitutive CD176-CAR expression, these cells were engineered with an inducible cassette expressing either IL-12 (iIL12_CD176_TRUCKs), IL-18 (iIL18_CD176_TRUCKs), or EGFP as a control (iEGFP_CD176_TRUCKs). Functionality of CD176_TRUCKs was tested by co-cultivation with CD176+ cell lines and human lung cancer tissue. To characterize activation and cytotoxicity of CD176-TRUCKs following target recognition, multicolor flow cytometry, confocal microscopy, multiplex assays and bulk-RNA transcriptomics were performed.Results Following co-culture with different CD176+ lung carcinoma cell lines, all CD176_TRUCKs increased NF-κB activity, became activated, released effector molecules (e.g. IFN-γ), and mediated effective cytotoxicity. They did not react towards CD176- control cells. The inducible cytokines IL-12 and IL-18 were released by respective TRUCKs in a target-specific manner and clearly improved particular effector functions in comparison with iEGFP_CD176_TRUCKs. Precision-cut lung sections (PCLS) were generated from explanted human lung adenocarcinoma tissue and shown to express CD176. Using PCLS as ex vivo model, specific cytotoxicity of CD176_TRUCKs against tumor tissue but not against healthy tissue of the same lung was demonstrated.Conclusion CD176_TRUCKs equipped with inducible cytokines were shown to be highly functional, suggesting them as promising strategy to overcome the TME. Based on the tumor-selective expression of CD176, CD176_TRUCKs have a high potential to effectively control lung carcinoma while minimizing "on-target/off-tumor"-toxicity.Offenlegung Interessenkonflikt: The authors declare no conflict of interest

Prof.

PD Dr.

Efficient CAR T cell generation starting with leukoreduction system chambers of thrombocyte apheresis sets

Background During plateletpheresis, the leukoreduction system chamber (LRSC) reduces the leukocyte amount within the subsequent platelet concentrate through saturated, fluidized, particle bed filtration technology. Normally, the LRSC is discarded after apheresis is completed. To explore if those retained leukocytes are attractive for research purposes, we isolated CD3+ T cells from the LRSCs via density gradient centrifugation in order to manufacture CD19-targeted chimeric antigen receptor (CAR) T cells.Methods Immunophenotyping of the LRSC derived mononuclear cells (MNCs) was performed via flow cytometry. T lymphocytes were isolated using the Pan T Cell Isolation Kit and stimulated with IL-2 and paramagnetic beads. Upon lentiviral transduction with concentrated self-inactivating viral vector supernatants, CD19 CAR T cells were further enriched through anti-CD19 CAR MACS, co-incubation with irradiated CD19+ Daudi cells, or through a vector containing an EGFP-T2A-puromycin resistance gene and a one-shot 2 day puromycin treatment. CAR T cell cytotoxicity was assessed using Luciferase labeled cell lines. In vitro CAR T cell expansion to clinically relevant numbers was achieved.Results Compared to peripheral blood, LRSC yields a 10-fold MNC concentration. Immunophenotypic characterization revealed viable and normal CD4+ and CD8+ T cell populations, with low CD19+ B cell counts. T cells showed low expression of exhaustion markers and a normal memory subpopulation distribution. Robust CD19 CAR cell surface expression on transduced T cells was confirmed by flow cytometry. Anti-CD19 CAR MACS resulted in 80% CAR+ cell populations, whereas puromycin selection achieved &gt;90% CAR T cell populations. To prove functionality, CAR T cells were co-incubated with the human CD19+ B cell precursor leukemia cell lines Nalm6 and Daudi. Compared to unmodified T cells, CD19 CAR T cells effectively eradicated Nalm6 and Daudi cells.Conclusion Stable transduction rates of the isolated T cells could be achieved by lentiviral transduction. Puromycin selection or anti-CD19 CAR-MACS can be used for further purification. These CAR T cells can be successfully expanded and show superior cytotoxicity against target cells compared to unmodified T cells. Taken together, we can show that lymphocytes isolated from LRSCs of plateletpheresis sets can be efficiently used for the generation of functional CAR T cells for experimental purposes.Offenlegung Interessenkonflikt: Die Autoren erklären, dass keine Interessenkonflikte bestehen.

Immunotherapy 2 - Engineered Cells

Appointment

Vorsitz

Programme