ALBERT

Description: Introduction: Chimeric Antigen Receptor (CAR) based cellular-immunotherapies have demonstrated significant clinical efficacy in haematological malignancies. However, the progress of cellular-immunotherapy for the treatment of Acute Myeloid Leukaemia (AML) has failed to gain momentum due to the lack of targetable tumour specific antigens. CD38 is a transmembrane glycoprotein expressed in lymphoid and myeloid cells with high expression in plasma B-cells, and is a well validated target for anti-CD38 therapy in Myeloma. A recent study has furthermore shown that a proportion of AML patients express CD38 on their leukemic blasts. TNF-related apoptosis-inducing ligand (TRAIL) receptor DR4 is another targetable antigen which has been shown to be expressed in 70% of AML patients. In this study, we investigate the therapeutic efficacy of "affinity-optimized" variant(s) of CD38 CAR and membrane bound TRAIL on NK-cell based platforms which can target AML blasts with high expression of CD38 (CD38high AML). The CAR variant is a CAR which binds with lower affinity to CD38 expressed on healthy immune cells such as CD38positive NK cells, while targeting CD38high AML. The membrane bound TRAIL variant (TRAIL4c9) is a mutant which binds with higher affinity to TRAIL-DR4 on AML cells, whilst avoiding binding to decoy receptors. We hypothesize that genetically modifying NK cells to express "affinity optimized" CD38 CARand/or TRAIL4c9 can effectively eliminate CD38high AML cells. Methods: AML cell lines THP-1, U937, and KG1a were immunophenotyped for CD38 and TRAIL-DR4 expression. Retrovirally transduced CD38 CAR-KHYG1 NK cells were used as immune effector cells and were co-cultured with AML cell lines in cytotoxicity assays. CD38low AML cell line KG1a was pre-treated with 10nM all-trans-retinoic acid (ATRA) to upregulate CD38 expression and were subsequently co-cultured with CD38 CAR-KHYG1 in cytotoxicity assays. CD38 CAR-KHYG1 was also co-cultured with n=4 patient derived AML cells in cytotoxicity assays. Using Maxcyte GT electroporation system primary donor derived IL-2 activated NK cells were either mock electroporated, or electroporated with TRAIL4c9 m-RNA orCD38 CAR m-RNA and subsequently co-cultured with THP-1 or ATRA pre-treated KG1a in a cytotoxicity assay. Expression of pro-apoptotic, anti-apoptotic and ligands for checkpoint inhibitory receptors was analysed by immunoblotting or flowcytometry. Results: Based on immunophenotyping, we classified AML cell lines as CD38high (THP-1), CD38moderate (U937) and CD38low (KG1a). CD38 CAR-KHYG1 was significantly more cytotoxic than MOCK KHYG1 against CD38high THP-1, at E:T ratios of 2.5:1, 5:1 and 10:1. CD38 CAR-KHYG1 were also more cytotoxic than MOCK KHYG1 against CD38moderate U937 at multiple E:T ratios; albeit the increase in cytotoxicity was at a much lower level in comparison to THP-1 (Fig 1a). Pre-treatment of CD38low KG1a cells with 10nM ATRA upregulated the cell surface expression of CD38, which were subsequently eliminated by CD38 CAR KHYG1 at E:T ratios of 2.5:1, 5:1 and 10:1. KG1a was intrinsically resistant to NK cells as compared to THP-1 and U937 (Fig 1b). This could partly be explained by the high intracellular expression of Bcl-xL, and higher cell surface expression of Nectin-1 and Sialic acid which are the ligands for checkpoint inhibitory receptors CD96 and Siglec-7/9 respectively on NK cell (Fig 1c). CD38 CAR-KHYG1 mounted a potent cytotoxic response against primary CD45intermediate AML blasts (n=4 patients) at multiple E:T ratios, and the extent of CAR induced cytotoxicity correlated with the cell surface CD38 expression on the primary AML blasts (R2=0.87) (Fig 1d,e). TRAIL4c9 or CD38 CAR m-RNA electroporated primary donor-derived NK cells were also potent in eliminating THP-1 and ATRA pre-treated KG1a at multiple E:T ratios (Fig 1f). This demonstrates the potential of therapeutically treating AML patients, with high CD38 expression, with a combination of NK cells expressing "affinity-optimized" CD38 CAR and membrane bound TRAIL variant. Conclusion: The study demonstrates the therapeutic potential of an "affinity-optimized" CD38 CAR NK cell-based therapy, which can potentially be combined with membrane bound TRAIL expressing NK cells to target CD38high AML. In patients with CD38low expressing AML blasts, patients could be pre-treated with ATRA followed by the combination therapy of CD38 CAR and TRAIL expressing NK cells. Disclosures Stikvoort: Onkimmune Ltd., Ireland: Research Funding. Kirkham-McCarthy:Onkimmune Ltd., Ireland: Research Funding. Van De Donk:Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Mutis:Celgene: Research Funding; Janssen Pharmaceuticals: Research Funding; Amgen: Research Funding; BMS: Research Funding; Novartis: Research Funding; Aduro: Research Funding; Onkimmune: Research Funding. Sarkar:Onkimmune: Research Funding. O'Dwyer:Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; GlycoMimetics Inc: Research Funding; AbbVie: Consultancy; BMS: Research Funding.

Description: Chimeric Antigen Receptors (CARs) are engineered transmembrane proteins consisting of an antibody-derived antigen recognition domain linked to intracellular cell signaling domains. CAR engineered autologous T cells have been successful in the treatment of a variety of hematologic malignancies. However, several major caveats, including lack of universal donors, long manufacturing times, and absence of a donor in immunologically frail patients, have limited the successful translation of CAR-T cell based therapy to a larger pool of patients. A universal, easy to manufacture, "off the shelf" CAR-based product could potentially address these limitations and result in a lower cost of goods. Towards developing an "off the shelf" CAR-based therapy for Multiple Myeloma (MM), we explored the feasibility and preclinical efficacy of expressing CD38 CARs in KHYG-1 cells, a natural killer (NK) cell line, first established by Yagita et al from a patient with aggressive NK leukemia (Leukemia, 2000). To this end, we effectively transduced KHYG-1 cells with high-affinity CD38 CARs as well as our recently reported affinity-optimized CD38 CARs, which can readily target MM cells with high CD38 expression, while ignoring non-malignant cells with intermediate, low or no CD38 expression when brought to expression on T cells (Drent et al, Molecular Therapy 2017). Moreover, we assessed performance of first and second generation CARs, with co-stimulatory domains CD28 and 4-1BB, and found the combination of CD28/CD3ζ to lead to the best results. After expanding the CAR transduced KHYG-1 cells, we analyzed their phenotype and efficacy in MM by analyzing their cytotoxic activity against CD38+ and CD38- MM and AML cell lines (UM9/THP-1 and U266/HL60, respectively), and against primary MM cells. The CD38-CAR transduced KHYG-1 cells showed no phenotypic alterations, and at effector to target ratios as low as 1:1, induced a high cytotoxicity towards CD38+ cell lines as compared to mock or non-transduced KHYG-1, demonstrating the important contribution of the CD38 CAR on the KHYG-1 NK cell surface. CD38- cell lines were unaffected by both CD38-CAR transduced KHYG-1 cells and mock or non-transduced KHYG-1 cells, indicating the specificity towards CD38 of the CAR and thus the potential safety of the CD38-CAR KHYG-1 cell. Similarly, ex vivo assays using primary MM cells revealed superior cytotoxic activity of CD38-CAR KHYG-1 cells as compared to mock or non-transduced KHYG-1 cells (median 86,5% vs 14% at 1:1 E:T ratio, n=2, Figure 1A). Confirming our previous results we identified an affinity-optimized CD38-CAR which mediated strong primary MM cell cytotoxicity with little or no "off tumor" effect. Normal immune cells (B, T, monocytes), which were either CD38 negative or only intermediate positive, were unaffected (Figure 1B-D), suggesting the potential safety of the CAR-NK cell therapy for clinical applications. As clinical administration would require irradiation of CD38-CAR KHYG-1 cells, we tested the effect of irradiation on their proliferative and cytotoxicity potential. Irradiation with 10Gy, while drastically inhibiting proliferative activity and viability (50% survival after 3 days), did not affect cytotoxicity, suggesting that repeated administrations of irradiated, CD38-CAR transduced KYHG-1 cells may exert effective in vivo anti-tumor activity, which is currently being evaluated in appropriate in vivo models, specifically the humanized bone scaffold in vivo model published by Groen et al (Blood, 2012). In conclusion, we demonstrate that the incorporation of CAR technology into the immortal NK cell line KHYG-1 has enormous potential to become a safe and effective "off the shelf" therapy for MM. Disclosures Stikvoort: Onkimmune: Research Funding. Sarkar:Onkimmune: Research Funding. van de Donk:Amgen: Research Funding; Janssen Pharmceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding. Zweegman:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corp.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. O'Dwyer:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Celgene: Research Funding; Glycomimetics: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees. Mutis:Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding; OnkImmune: Research Funding; Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (moAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells mediating tumour immunosurveillance in vivo. NK cells also play an important role during moAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their Fcγ RIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring high affinity variant of CD16 harbouring a single point polymorphism (F158V), and this variant has been linked to improved ADCC. However, the contribution of NK cells to the efficacy of Daratumumab remains debatable as clinical data clearly indicate rapid depletion of CD38high peripheral blood NK cells in patients upon Daratumumab administration. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach, on CD38low NK cells could significantly enhance therapeutic efficacy of Daratumumab in MM patients. In the present study, we investigate the optimal NK cell platform for generating CD38low CD16F158V NK cells which can be administered as an "off-the-shelf"cell therapy product to target both CD38high and CD38low expressing MM patients in combination with Daratumumab. Methods: MM cell lines (n=5) (MM.1S, RPMI-8226, JJN3, H929, and U266) and NK cells (n=3) (primary expanded, NK-92, and KHYG1) were immunophenotyped for CD38 expression. CD16F158V coding m-RNA transcripts were synthesized using in-vitro transcription (IVT). CD16F158V expression was determined by flow cytometry over a period of 120 hours (n=5). 24-hours post electroporation, CD16F158V expressing KHYG1 cells were co-cultured with MM cell lines (n=4; RPMI-8226, JJN3, H929, and U266) either alone or in combination with Daratumumab in a 14-hour assay. Daratumumab induced NK cell fratricide and cytokine production (IFN-γ and TNF-α) were investigated at an E:T ratio of 1:1 in a 14-hour assay (n=3). CD38+CD138+ primary MM cells from newly diagnosed or relapsed-refractory MM patients were isolated by positive selection (n=5), and co-cultured with mock electroporated or CD16F158V m-RNA electroporated KHYG1 cells. CD16F158V KHYG1 were also co-cultured with primary MM cells from Daratumumab relapsed-refractory (RR) patients. Results: MM cell lines were classified as CD38hi (RPMI-8226, H929), and CD38lo (JJN3, U266) based on immunophenotyping (n=4). KHYG1 NK cell line had significantly lower CD38 expression as compared to primary expanded NK cells and NK-92 cell line (Figure 1a). KHYG1 electroporated with CD16F158V m-RNA expressed CD16 over a period of 120-hours post-transfection (n=5) (Figure 1b). CD16F158V KHYG1 in-combination with Daratumumab were significantly more cytotoxic towards both CD38hi and CD38lo MM cell lines as compared to CD16F158V KHYG1 alone at multiple E:T ratios (n=4) (Figure 1c, 1d). More importantly, Daratumumab had no significant effect on the viability of CD38low CD16F158V KHYG1. Moreover, CD16F158V KHYG1 in combination with Daratumumab produced significantly higher levels of IFN-γ (p=0.01) upon co-culture with CD38hi H929 cell line as compared to co-culture with mock KHYG1 and Daratumumab. The combination of CD16F158V KHYG1 with Daratumumab was also significantly more cytotoxic to primary MM cell ex vivo as compared to mock KHYG1 with Daratumumab at E:T ratio of 0.5:1 (p=0.01), 1:1 (p=0.005), 2.5:1 (p=0.003) and 5:1 (p=0.004) (Figure 1e). Preliminary data (n=2) also suggests that CD16F158V expressing KHYG1 can eliminate 15-17% of primary MM cells from Daratumumab RR patients ex vivo. Analysis of more Daratumumab RR samples are currently ongoing. Conclusions: Our study provides the proof-of-concept for combination therapy of Daratumumab with "off-the-shelf" CD38low NK cells transiently expressing CD16F158V for treatment of MM. Notably, this approach was effective against MM cell lines even with low CD38 expression (JJN3) and primary MM cells cultured ex vivo. Moreover, the enhanced cytokine production by CD16F158V KHYG1 cells has the potential to improve immunosurveillance and stimulate adaptive immune responses in vivo. Disclosures Sarkar: Onkimmune: Research Funding. Chauhan:Onkimmune: Research Funding. Stikvoort:Onkimmune: Research Funding. Mutis:Genmab: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding. O'Dwyer:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Glycomimetics: Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.