A First-in-Human Phase I Trial of NKG2D Chimeric Antigen Receptor-T Cells in AML/MDS and Multiple Myeloma

Sarah Nikiforow1,2, Joana Murad3, Heather Daley1, Helene Negre1, Jake Reder3, Charles L. Sentman4, Frédéric Lehmann5, Sarah Snykers5, Rachel Allen5, Ilene Galinsky1, Nikhil Munshi1,2, Richard Stone1,2, Robert Soiffer1,2, Jerome Ritz1,2, Susanne Baumeister1,2,6

Abstract

Basic CM-CS1 CAR Construct and Pre-clinical Efficacy Phase 1 Dose-Escalation Trial– Single Infusion Background: Canonical CAR- constructs encode a single chain antibody variable #patients Control T cells #patients fragment, costimulatory domain, and signaling domain of CD3 zeta. This restricts • Chimeric NKG2D receptor generated by NKG2D CAR T cells Murine Cohort 1 6 1x10 CM-CS1 T cells 3-6 recognition to 1 tumor antigen and a limited set of . This study employs a novel fusion between native full-length Myeloma MTD 6 CAR fusing full-length human Natural Killer Group 2D (NKG2D) gene with the human CD3 extracellular domain (EC) and Model Cohort 2 AML/MDS-RAEB zeta signaling domain. NKG2D CAR receives costimulation via naturally-expressed DAP10 transmembrane domains (TM) of human 3x106 CM-CS1 T cells 3-6

(5T33MM)7 and activates T cells directly to kill and secrete upon recognition of MIC-A, MIC- Natural Killer Group 2D (NKG2D) with MTD 6 Cohort 3 B, and UL-16 binding . These NKG2D-ligands are up-regulated in many solid cytoplasmic signaling domain of human 1x107 CM-CS1 T cells 3-6 Multiple Myeloma tumors and hematologic malignancies but absent or poorly expressed on healthy CD3 zeta.5 Cytoxan Control T cells tissues. In multiple murine models, NKG2D CAR-T cells induced complete Cohort 4

•SFG vector, Moloney Murine Leukemia Control T NKG2D T cells +Cy 3x107 CM-CS1 T cells 3-6 Tumor assessment with Follow-up/Research studies escalation

NKG2D T NKG2D T cells expansion - remissions, T-cell memory, and altered tumor microenvironment via cytokines. We - virus-based vector, used for retroviral BM aspirate/biopsy demonstrated manufacture of autologous NKG2D CAR-T cells in a GMP-environment from Cohort 5 3x/week until day 21

transduction. Transcription of NKG2D CAR 1x108 CM-CS1 T cells Baseline Dose healthy adults and patients with AML and myeloma was feasible. 3-6 Dose gene regulated by the 5’ and 3’ long Day 28 Monthly x 5 months 6 At 3 months Following isolation of mononuclear cells, T cells are activated with anti-CD3 mAb and IL- terminal repeats of Mo-MuLV. Cohort 6 8 3x10 CM-CS1 T cells 3-6 At 6 months 2, undergo 2 transductions with SFG retroviral vector containing NKG2D CAR construct, T cell infusion T cell infusion and expand in media containing IL-2. Validation studies yielded consistent viability, At 9 months Cohort 7 robust cell expansion, high vector-driven NKG2D expression on T cells, potent IFN-g GMP Manufacturing Scheme – 8 days 1x109 CM-CS1 T cells 3-6 At 12 months At 12 months At 15 months production during tumor cell co-culture, viral copy number/cell < 5, and no replication- NKG2D CAR Cohort 8 competent retrovirus in NKG2D CAR-T (CM-CS1) cells. 3x109 CM-CS1 cells 3-6 At 24 months IV Dap 10 Methods: A phase 1 dose-escalation study to establish safety and feasibility of a single administration of infusion of CM-CS1 T cells without lymphodepleting conditioning is currently enrolling autologous CM- CD3ζ Trial Design and Experience to Date subjects with AML/MDS-RAEB without standard therapy options or relapsed/refractory CS1 T cells progressive multiple myeloma (ClinicalTrials.gov NCT02203825). Dose-escalation will g-retroviral g-retroviral proceed in 8 cohorts [1x106 to 3x109 CM-CS1 T cells] according to a 3+3 design followed Inclusion Criteria Key Design Aspects transduction transduction by expansion cohorts at the MTDs in AML/MDS and myeloma. Native NKG2D • Histologically confirmed AML, MDS-RAEB not in • Single dose IV administration of CM-CS1 T cells #1 #2 MIC-A Release remission and without reasonable standard treatment • No prior lymphodepleting chemotherapy As of May, 2016, 9 subjects have been treated. Three cohorts have been completed Grex testing options • 3+3 escalation - classical Fibonacci sequence without DLTs. Enrollment to Cohort 4 is proceeding as planned. Future studies may culture on CD8+ • Relapsed or relapsed/refractory multiple myeloma • At least 1 patient with each disease per cohort include lymphodepletion, multiple infusions, and cytoreductive chemotherapy in both Activation with IL-2 T-cell T cells with progressive disease • Escalation design allows determination of separate hematologic and solid tumor malignancies. with IL-2 and Mock Mock MTDs for individual diseases collection soluble AML/MDS/MM Tumor cell Exclusion Criteria processing processing Native NKG2D • Multiple safety evaluation periods • Chemotherapy or radiotherapy within 3 weeks prior to Patient antiCD3-Ab #1 #2 infusion Correlative Immunobiology Objectives Dap 10 Background • Active , autoimmune disease or CNS • Persistence, trafficking and bioactivity of CM-CS1 cells involvement • Impact on endogenous milieu and secondary anti- • Pre-clinical and clinical observations emphasize the role of NKG2D-expressing cells in • Prior T-cell, stem-cell or gene-modified cellular tumor responses immune surveillance against tumor growth.1,2 therapy • Levels of soluble NKG2D-ligands and antibodies against • NKG2D is an activating receptor expressed on natural killer and other cells as a GMP Validations Objectives Release Criteria • Primary - Safety and feasibility • Tumor ligand expression to assess antigen-escape homodimer in association with the natural adaptive DAP10, which provides NKG2D CM-CS1 CD8+ Cells NKG2D CM-CS1 CD4+ Cells 6000 (median: n=5) • Secondary – Progression-free survival and clinical control over surface expression and provides a co-stimulatory signal analogous to that Experience to Date of CD28.3 5000 Initial Sterility Negative anti-tumor effect • 9 patients treated in first 3 cohorts • Killing activity by NK and CD8+ T cells through the 6-protein receptor complex is 4000 Viable T-cell # (from < 60cc PB) 1.5x10^8 • Up to 15 years of gene-therapy safety monitoring • Feasibility and safety – no DLTs to date dependent on NKG2D ligand expression and independent of inhibitory signaling. 3000 Trypan Blue Viability 93.6%

• NKG2D ligands consist of MHC Class I-related proteins MIC-A, MIC-B and various 2000 Endotoxin <5 EU/kg/hr UL16 binding proteins (ULBPs 1-6), also called RAET1 proteins. Multiple Potential Mechanisms of Action Affiliations/Disclosures/References Vector-specific NKG2D expression 1000 53.6% •NKG2D ligands are found on lymphomas, leukemias, myelomas, melanomas, ovarian on CD8+ T cells 1. Dana-Farber Cancer Institute; 2. Harvard Medical School; 3. Celdara Medical, LLC; 4. Geisel School of Medicine at Mock-Processed CD8+ Cells Mock-Processed CD4+ Cells (pg/ml)

g production g 0

cancers and many other malignancies but protein expression is absent or minor in

- Dartmouth; 5. Celyad, SA; 6. Boston Children’s Hospital healthy tissues. A given tumor cell may express multiple NKG2D ligands. NKG2D CAR Vector-copy Number 2.3 copies/cell -1000 • In murine models of leukemia, myeloma, colorectal cancer, melanoma, and ovarian IFN Mock NKG2D Replication-competent Retrovirus No evidence JM is Principal Scientist at Celdara Medical, LLC Transduced CM-CS1 T JReder is CEO of Celdara Medical, LLC CM-CS1 cancer, adoptive therapy with T cells expressing an NKG2D CAR construct led to T Cells Cells CLS is Scientific Founder of Celdara Medical, LLC;US patents 4 Mycoplasma DNA None Detected complete remissions. Cells for NKG2D CAR technology CAR Cells Only 0.0 3.9 SS is R&D Manager at Celyad, SA Murine Line - •NKG2D CAR-T cells target and eliminate immunosuppressive regulatory T cells and 0.0 0.1 Final Product Gram Stain Negative RA is Clinical Scientist at Celyad, SA Ligand Neg myeloid-derived suppressor cells and activate within the tumor Murine Line - Informational Testing 1. Guerra, Immunity 2008; 28:571-580 microenvironment. 0.0 723.9 huMICA Pos 2. Rebmann, Clin Immunol 2007; 123: 114-120. 3. Spear. Cancer Immunity 2013; 13: 8-22 •These cells can also target tumor vasculature based on NKG2D ligand expression of huMyeloma % CD8 among T cells 79.4% NKG2D Expression 17.2 4512.2 4. Sentman. Cancer J 2013;20(2): 156-159 Line tumor-associated endothelial cells. Vector-specific NKG2D expression 5. Barber. Cancer Res 2007;67(10):72-78 huPancreatic 90.3% 6. Riviere. PNAS 1995; 92(15):6733-6737 • NKG2D CAR-T cell therapy, in pre-clinical models, induces durable CD4+ and CD8+ 0.0 5596.2 on CD4+ T cells Tumor 7. Barber. Gene Tx 2011; 18:509-516 IFNg secretion T-cell memory against tumor antigens capable of protecting against tumor re- * Graphs shown are representative examples from 5 GMP validation runs 2400 pg/mL (against Myeloma line) This work was supported by NIH NHLBI 2R44HL099217, challenge, despite relatively short-term circulation of CAR-T cells themselves. Celdara Medical, LLC, and Celyad, SA