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Despite tremendous clinical success of chimeric antigen receptor (CAR) expressing T cell (CAR-T) therapies, targeting B-phenotypic antigens in ALL, CLL, NHL or multiple myeloma, there are still major limitations for broader clinical application. CAR-Ts are capable to generate a specific immune response against defined surface-expressed antigens leading to sustained depletion of target antigen expressing tissues e.g. B cells. While B cell function can be substituted by repetitive IgG infusions, prolonged depletion of vitally essential tissues is not compatible with life. In AML for instance, most promising target antigens are expressed along myeloid lineage differentiation, limiting the therapeutic applicability. Therefore, CAR-Ts targeting essential shared antigens must allow tight regulation of CAR-T function and/or be able to differentiate between cancerous and healthy tissue. To address these issues, we have developed the adapter CAR-T cell (aCAR-T) system. By splitting antigen recognition and CAR-T activation, introducing adapter molecules (AMs), the system allows precise quantitative (on-/off-switch) as well as qualitative (change and combine target antigens) regulation of CAR-T function. aCAR-Ts are based on the unique properties of a novel scFv targeting a "neo"-epitope-like structure consisting of the endogenous vitamin biotin in the context of a specific linker, referred to as linker-label-epitope (LLE). LLEs can be easily conjugated to novel or preexisting AM formats like monoclonal antibodies (mAbs) or mAb fragments in a GMP-compliant manner. We were able to demonstrate that aCAR-Ts allow simultaneous targeting of various antigens ("OR"-gate) , preventing antigen evasion by selection of antigen or epitope-loss variants. In the present study we intended to investigate whether aCAR-Ts are capable to identify and differentiate target cells due to versatile antigen expression profiles ("AND"-gate). In theory, AMs against different target antigens can be assembled on the surface of a target cell , leading to aCAR-T activation independent of the targeted antigens (surface painting) , by binding to the presented LLE-tags. Therefore, combinatorial AMs treatment might allow to translate complex and multiple antigen-dependent target cell identification into an aCAR-T activation. To test this hypothesis, we have generated LLE-AMs against ALL/NHL - (CD10, CD19, CD20, CD22, CD37, CD138, ROR1) and AML - (CD32, CD33, CD38, CD123, CD135, CD305, CLL1) associated antigens. Individual threshold concentrations for aCAR-T activation by different AMs, targeti ng the model cell lines Nalm 6 (ALL), JeKo1 (NHL), HL-60 , U973 and Molm13 ( all AML), have been analyzed. Cut offs were found to be between 10 and 100 pg/ml, dependent on target expression and target cell line. Importantly, combinations of 2, 3 or 5 AMs, targeting different antigens expressed on the same target cell, cause target- cell lysis at concentrations below the activation threshold for single AMs (exemplified for HL-60 in Figure A) . Our results clearly demonstrate an additive effect in combining different AMs to hurdle the activation threshold. Moreover, in a JeKo 1 CD19 and/or CD20 knock out (KO) antigen-loss model, combinations of AMs targeting CD19, CD20 and ROR1 can differentiate between wild type and antigen -1 (CD19 or CD20 KO) or antigen -2 (CD19 and CD20 KO) variants in medi ating target- cell lysis, even though at least one target antigen is expressed. Finally, we found that combinations of CD10, CD19, CD22 and CD138 sufficiently eliminate Nalm-6 BCP-ALL cells, while sparing healthy B cells in co - culture experiments. Similar results were obtained in co - culture experiments of HL-60 AML cells with monocytes, neutrophils as well as CD34- enriched hematopoietic progenitor cells, applying combinations of CD32, CD33, CD38, CD123, CD305 and CLL1. Co - culturing experiments using autologous blasts, monocytes, neutrophils and aCAR-Ts are ongoing. Together, our results indicate that aCAR-Ts in combination with selected AM combinations might have the ability to identify and specifically eliminate cancer cells based on complex antigen expression profiles. This would have major implications for clinical translation, enabling combinatorial therapy, essential to avoid antigen evasion, and the possibility to spare vitally essential tissue from elimination. Figure. Figure. Disclosures Seitz: Miltenyi Biotec: Patents & Royalties, Research Funding. Mittelstaet:Miltenyi Biotec: Employment, Patents & Royalties. Lock:Miltenyi Biotec: Employment. Kaiser:Miltenyi Biotec: Employment, Patents & Royalties. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding. Lang:Miltenyi Biotec: Patents & Royalties, Research Funding. Schlegel:Miltenyi Biotec: Patents & Royalties, Research Funding.

Chimeric antigen receptor (CAR) expressing T cells (CAR-Ts) have demonstrated tremendous clinical success, especially when targeted against the B-phenotypic antigens CD19 and CD22 in ALL, CLL as well as NHL. Despite the recent success, production of CAR-Ts still requires an extensive and time consuming manufacturing process. Moreover, CAR-Ts have to be prepared individually for each patient. Especially in heavily pretreated and rapidly progressing patients, which often lack sufficient numbers of healthy T cells for CAR-T production, alternatives are a significant clinical need. NK cells might represent a promising alternative effector cell source. The continuously expandable and well established NK cell line NK-92 can provide a safe and consistent way to produce NK effector cells in a GMP-compliant and cost-effective way. Irradiated NK-92 CARs as an "off-the-shelf on-demand" cell therapeutic are currently tested in pre-clinical and early-phase clinical trials. Furthermore, NK-92 can be redirected by CARs to mediate direct antigen specific lysis. We have recently developed a universal adapter CAR (aCAR) system. By splitting antigen recognition and CAR-immune cell activation, introducing adapter molecules (AMs), the system allows precise quantitative (on-/off-switch) as well as qualitative (change and combination of target antigens) regulation of immune cell function. aCARs are based on the unique properties of a novel scFv targeting a "neo"-epitope-like structure consisting of the endogenous vitamin biotin in the context of a specific linker, referred to as linker-label-epitope (LLE). LLEs can be easily conjugated on novel or preexisting AM formats like monoclonal antibodies (mAbs) or mAb fragments in a GMP-compliant manner. In the present study, we intended to combine the universal and flexible targeting as well as controllability of the aCAR with the "off-the-shelf" properties of NK-92 cells. NK-92 was obtained from ATCC and transduced with aCARs containing either CD28 or 4-1BB co-stimulatory plus CD3-ζ signaling domains. Importantly, only CD28 containing aCARs sufficiently mediated specific target cell lysis in the presence of biotinylated antibodies (LLE-AMs). Using single cell sorting, aCAR NK-92 clones with the highest CAR expression were selected and demonstrated significantly improved target cell lysis, in a LLE-AMs dependent manner. Both, LLE-AMs against CD19 and CD20, were capable of inducing significant NK-92-mediated lysis against the NHL cell lines Raji, Daudi and JeKo-1. Cytotoxicity experiments using aCAR NK-92 cells and primary lymphoma cells are ongoing. Specificity of the LLE-AM directed effector cell elimination was further proven using a JeKo-1 CD19 and/or CD20 knock out (KO) antigen-loss model. aCAR NK-92 mediated antigen specific lysis only in the presence of the target antigen and the specific LLE-AM. Moreover, combinations of anti-CD19 and anti-CD20 LLE-AMs are capable of avoiding antigen evasion. To test the universal applicability of aCAR NK-92, specific target cell lysis against a variety of different tumor entities was demonstrated using LLE-conjugated therapeutic antibodies. Importantly, irradiation of effector cells, as required in all active clinical trials using NK-92, prior to testing had no observable effect on target cell lysis. Finally, the investigation of potential on-target off-tumor reactivity against healthy B cells showed no cytotoxic effects of aCAR NK-92 cells in combination with LLE-AMs against CD19 or CD20. In conclusion, we have generated an NK cell line, aCAR NK-92, whose effector function can be tightly regulated and redirected against one or multiple antigens allowing tunable and universal targeting. Moreover, aCAR NK-92 cells can be manufactured as an "off-the-shelf on-demand" standardized product improving the practicality of NK CAR therapy combined with the possibility of tailoring a specific LLE-AM platform for patient-individualized treatment. Disclosures Seitz: Miltenyi Biotec: Patents & Royalties, Research Funding. Mittelstaet:Miltenyi Biotec: Employment, Patents & Royalties. Kaiser:Miltenyi Biotec: Employment, Patents & Royalties. Schlegel:Miltenyi Biotec: Patents & Royalties, Research Funding. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding.

CD4+ T cells can differentiate into multiple effector subsets, but the potential roles of these subsets in anti-tumor immunity have not been fully explored. Seeking to study the impact of CD4+ T cell polarization on tumor rejection in a model mimicking human disease, we generated a new MHC class II-restricted, T-cell receptor (TCR) transgenic mouse model in which CD4+ T cells recognize a novel epitope in tyrosinase-related protein 1 (TRP-1), an antigen expressed by normal melanocytes and B16 murine melanoma. Cells could be robustly polarized into Th0, Th1, and Th17 subtypes in vitro, as evidenced by cytokine, chemokine, and adhesion molecule profiles and by surface markers, suggesting the potential for differential effector function in vivo. Contrary to the current view that Th1 cells are most important in tumor rejection, we found that Th17-polarized cells better mediated destruction of advanced B16 melanoma. Their therapeutic effect was critically dependent on interferon-γ (IFN-γ) production, whereas depletion of interleukin (IL)–17A and IL-23 had little impact. Taken together, these data indicate that the appropriate in vitro polarization of effector CD4+ T cells is decisive for successful tumor eradication. This principle should be considered in designing clinical trials involving adoptive transfer–based immunotherapy of human malignancies.

Relapsed and refractory B-precursor acute lymphoblastic leukemia (B-ALL) remains a major therapeutic problem. Chimeric antigen receptor (CAR) modified T cells targeting CD19 are promising treatment options for these patients with the potential to induce hematological remission in adult and pediatric patients with refractory B-ALL. Despite the promising data, some patients do not respond to T-cell treatment. Until now it is not possible to fully understand and predict critical factors for response or non-response, but proliferation and persistence of CAR T cells in vivo is an essential precondition for treatment efficacy. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to be the best candidates for a sustained in vivo expansion after T-cell therapy with small cell doses. Therefore, we set up a protocol for the generation of anti-CD19 CAR T cells in a closed system that is compliant to current GMP regulations. Starting samples were mononuclear cells from pediatric ALL patients at diagnosis and under chemotherapy using up to 100cc peripheral blood. After separation for CD4+/CD8+ cells, T cells were activated with anti-CD3/CD28 beads. The lentiviral vector encoded the anti-CD19 single-chain variable fragment, 4-1BB (CD137) co-stimulation and T cell receptor (TCR) zeta chain. The whole process including separation of T cells, activation, transduction and cultivation was performed in a closed and fully automated system. Despite a broad variety in cellular composition including high blast counts, low cell numbers and a rather exhausted phenotype in the starting fraction, a robust T-cell composition was achieved at day five after activation with a mean of 63% CD4+ and 37% CD8+ T cells and a transduction rate of up to 38 %. The vast majority of CAR T cells were of a Tcm (47%) and Tscm (44%) phenotype leading to a strong proliferative potential of more than 100-fold expansion. In addition, a reduced sensitivity to inhibitory signals was documented (programmed cell death protein 1 (PD-1) expression ≤10%). CAR T cells showed effective cytotoxic functionality when co-cultured with CD19+ target cells with only little background of the un-transduced control. At an effector to target ratio of 5:1 up to 80 % of the CD19+ target cells were killed. In addition, a significant release of Interferon gamma (IFN-ɣ), Tumor necrosis factor (TNF-α) and Interleukin-2 (IL-2) was detected upon recognition of the target cell lines, confirming a strong and target-specific Th1 response. In conclusion, generation of CAR T cells from small pediatric blood samples was feasible in a closed GMP-compatible fully automated system. Despite variety of cell numbers, cellular composition and T cell phenotype in the starting sample, a uniform T cell product of Tcm and Tscm could be produced with a balanced CD4 / CD8 ratio leading to high expansion potential and functionality of the T cell graft. Disclosures Blaeschke: Miltenyi Biotec GmbH: Other: Miltenyi Biotec GmbH provided reagents free of charge.. Kaiser:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment.

BACKGROUND Genetically modified T cells have enormous potential for the treatment of relapsed and refractory haematopoietic malignancies. CD19-positive B-cell malignancies including acute lymphoblastic leukaemia (ALL), chronic lymphocytic leukaemia (CLL) or B cell non-Hodgkin lymphomas (NHL) have been shown to be an excellent target for adoptive immunotherapy with T cells expressing CD19-specific chimeric antigen receptors (CARs). The increasing need for genetically modified T cells is hampered by the limited number of centres with the required infrastructure and expertise to produce this complex therapeutic product. Ex vivo modification of T cells requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, Miltenyi Biotec has developed the CliniMACS Prodigy platform and is automating complex cell manufacturing processes. These have now been adapted for lentiviral transduction of T cells and we show the feasibility and effectiveness of the device for adoptive immunotherapy using chimeric antigen receptors. METHODS A self-inactivating third generation lentiviral vector encoding a CAR specific for CD19 (CAR19) was used for automated T-cell transductions (TCT). Using closed single-use tubing sets (TS520), fresh or cryopreserved peripheral blood mononuclear cells from non-mobilised leukapheresis collected from healthy donors were loaded onto the CliniMACS Prodigy, washed and activated in TexMACS media with TransAct, a polymeric nanomatrix activation reagent agonist for CD3 and CD28. Cells were transduced 24-48h after activation and expanded in the CentriCult-Unit of the tubing set, allowing for stable culture conditions as well as automated feeding and media exchange. Small and large scale comparison transductions were run in parallel to assess the efficiency of the automated T-cell modification. Finally, cells were harvested and cryopreserved to assess the functional capabilities of CAR19 T cells. RESULTS Three automated TCT runs were performed and continuously monitored to assess cell expansion, transduction efficiency and the phenotype of the final cell product. On average, expansion during automated cultivation was 11.7x (range: 5.4 - 22.8x) which was comparable to the expansion achieved in small scale controls (12.3x ± 1.2x). The average yield from the automated process was 11.8x108 total lymphocytes/run (ranging between 4 - 23.2x108 lymphocytes/run). Notably, this was comparable to existing CAR19 T cell manufacturing processes using a WAVE-Bioreactor. In all three runs in the Prodigy, successful transduction was observed with an average transduction efficiency of 32% CAR19-positive cells (range: 22- 45%). Again, this was similar to transduction efficiencies (32% CAR19-positive; range: 27-40%) in previous WAVE-production campaigns using X-Vivo15 media and magnetic beads conjugated with anti-CD3/CD28 antibodies for T-cell activation (Dynabeads). Flow cytometry analysis of the final cell product showed a high purity of CD45+/CD3+ cells (90%) as well as a relatively high frequency of CD8-positive cytotoxic T cells (56%). Immunophenotyping revealed high expression of CD45RA, CD62L, CD27 and CD95 with moderate expression of CCR7. Importantly, no significant difference in PD-1 expression was observed between automatically and manually processed cells. Finally, functional analysis showed cytotoxic activity as well as IFN-γ/TNF-α production upon co-cultivation with CD19-expressing target cells. CONCLUSION In summary, we have demonstrated the feasibility of the CliniMACS Prodigy for the generation of CAR+ T cells for adoptive immunotherapy. Automated activation, transduction and expansion resulted in clinically relevant doses of CAR19 T cells with very little 'hands-on' operator time. Given the closed-system nature of the device, and automated features, the CliniMACS Prodigy should widen applicability of T-cell engineering beyond centres with highly specialised infrastructures. Disclosures Mock*: Miltenyi Biotec GmbH: Research Funding. Nickolay*:Miltenyi Biotec GmbH: Research Funding. Peggs:Cellectis: Research Funding; Autolus: Consultancy, Equity Ownership. Johnston:Miltenyi Biotec GmbH: Employment. Kaiser:Miltenyi Biotec GmbH: Employment. Pule:CELLECTIS: Research Funding; AUTOLUS: Employment, Equity Ownership, Research Funding; AMGEN: Honoraria; UCLB: Patents & Royalties. Thrasher:Miltenyi Biotec GmbH: Research Funding; Autolus Ltd: Consultancy, Equity Ownership, Research Funding. Qasim:Cellectis: Research Funding; Miltenyi Biotec GmbH: Research Funding; Autolus Ltd: Consultancy, Equity Ownership, Research Funding; Cell Medica: Research Funding.