Nicotinamide (NAM) Modulates Transcriptional Signature of Ex Vivo Cultured UCB CD34+ cells (Omidubicel) and Preserves Their Stemness and Engraftment Potential Dima Yackoubov1, Yair Steinhardt1, Dorit Ashengrau1, Alina Maliutina1, Adi Bitansky1, Sherry Cohen1, Boaz Buhandler2, Moshe Shahor2, Nathan Dinowitz2, Vered Chalifa-Caspi3, Amnon Peled4 Julian Adams1, Tracey Lodie1 and Tony Peled1 1Gamida-Cell, Jerusalem, Israel and Boston, MA. 2PomiCell, Israel. 3NIBN, National Institute for Biotechnology in Negev Ltd. 4Goldyne Savad Institute of Therapy, Jerusalem, Israel

Background Study Design Results

• Historic efforts at expansion of umbilical cord blood, (UBC) derived DATA SET “A” DATA SET “B” Figure 3. Upregulation and downregulation of families of transcription factors Figure 4. Enrichment analysis showing NAM upregulates transcription factors responsible for CD34+ hematopoietic stem cells, (HSCs) ex-vivo with cytokines yielded in CD34+ cells expanded with NAM stem cell renewal and DNA repair while down-regulating transcription factors that activate cell large numbers of progenitors for transplantation but impaired their differentiation, inflammation, and apoptosis Culture RNA sampling Culture Transcription Factor engraftment. DATA SET “A”. NAM effect Seeding Harvest Culture RNA sampling Day 21 enrichment Biological / Functional enrichment • Ex-vivo HSC expansion resulted in accelerated cell proliferation, Seeding elevated levels of reactive oxygen and nitrogen species (ROS and - NAM + NAM P-value 299 Self-Renewal (SP1, cJUN) • p-value: 0.4x10-3 RNS), and upregulation of inflammatory signaling leading to cell + NAM + NAM PU.1 0.009 1 d2 d4 d21 d4 +6h downregulated SRF 0.022 differentiation and loss of in-vivo functionality. 1 Cell Cycle regulation (E4F1, SP4) • -3 FOS/b 0.039 p-value: 0.2x10 Day 2, 4 and 21: NAM VS Control Day 4: ± fresh NAM • We used nicotinamide (NAM), an allosteric inhibitor of NAD-dependent JUN/b/d 0.019 Stem Cell development (ETS1) • p-value: 0.6x10-3 enzymes, to create omidubicel, an investigational cell therapy designed 83 genes + 2 SP1 0.033 to improve the expansion of CD34 HSCs for bone marrow transplant. 3 upregulated DNA repair (UNG, NDN) • -3 • Next Generation Sequencing (Cel-SEQ, NGS) was performed on HiSeq 2500, rapid E4F1 0.039 p-value: 2.4x10 • A Phase 1/2 trial of omidubicel in patients with high-risk hematologic v2, Illumina. PLAGL1 0.032 Chromatin remodeling (SMC2/4, ESCO2) • malignancies showed rapid neutrophil engraftment and a favorable p-value: 1.7x10-2 • Gene annotation was done based on GRCh38.p12 version ELF1 0.000 immune reconstitution profile in patients compared to historical PU.1 0.000 controls. We hypothesized that NAM treatment maintains the stemness Ensembl/BioMart. DATA SET ”B”. p-value: 1.6x10-6 • HPC differentiation (mTOR, JNK, cMYC, NF-kB, cJUN) • Total 3,816 Differentially Expressed (DE) genes was defined based on absolute CEBPD 0.007 and engraftment potential of omidubicel, which is associated with Day 4 Fresh NAM linear fold change of 1.3 and unadjusted p-value <0.01. JUN/b/d 0.019 p-value: 3.0x10-8 • Inflammatory signaling (IFNγ,TNFα, TLR2/4, CCL2/7) clinical benefit.3 FOS/b 0.011 • DE-Genes were clustered to “CliCK”-Heatmap. 17 and 5 Clusters were defined in 650 genes JUN/b/d 0.011 p-value: 6.6x10-8 • ROS and RNS production (CYBB, NCF1/2/4, NOS2) Dataset “A” and “B” respectively. 2 downregulated SP1 0.000 Nicotinamide (NAM) -3 • Apoptotic signal (IL1β, BCL2L4/8, IRF1, NRP1, PARP) YY1 0.000 p-value: 2.4x10 E2F1 0.000 • MMP production (MMP2/7/9/12/19, TIMP2, LRP1, A2M) Figure 1. A master regulator of NAD-related signaling pathways • Transcription Factor (TF) enrichment analysis was done using FunRich software -6 664 genes SP4 0.000 p-value: 1.6x10 (http://funrich.org/). upregulated RAR/RXR 0.001 - Histone acetylation 1 Normalized Enrichment Score • GO - Biological Process Enrichment was performed (http://geneontology.org/). CREB1 0.003 - NAM PPAR/a/b 0.014 NAD+ precursor • INGENUITY (IPA) software was used for Pathway Analysis (Qiagen). -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 HOXB7 0.041 Balancing NAD+ - Mitochondrial mass catabolism NAD+ - Energy metabolism enzymes Pathway Inhibition Bone Marrow Niche Conclusions Cellular stress, ROS Figure 5. NAM Inhibits Pathways Responsible for ROS / RNS and Inflammation Figure 6. Bone marrow niche preserves HSC pool NAM platform is unique in mimicking the bone marrow niche during ex vivo expansion of HSCs. Introduction of Induction of ROS secretion + IFNg induced Figure 2. Similarity between CD34 cells cultured with NAM and non iNOS by cytokines iNOS NAM attenuates genes responsible for: cultured CD34+ cells differentiation • Reactive oxygen and nitrogen species production. • Inflammatory and apoptotic signaling. Non-cultured Exp.1 Exp.2 • HSC differentiation and maturation. CD34+ cells Exp.3 • Secretion of matrix metalloproteinases (MMP’s). CD34+ cells Exp.1 Our gene expression data: cultured Exp.2 • Reinforce the mechanism of action underlying our NAM W/O NAM Exp.3 technology platform. CD34+ cells Exp.1 • Provide additional insights into the pathways leading to clinically cultured Exp.2 significant engraftment in patients. with NAM Exp.3 The NAM platform has also been used for ex vivo expansion of other Cluster analysis of differentially expressed genes in CD34+ cells immune cells (GDA-201 ASH 2019 Abstract #777). Poster #3718. expanded ±NAM for 3-weeks compared to non-cultured CD133+ cells (Affymetrics). Cell Cycle Quiescent environment Differentiation References Objective High ROS scavenging 1. Reactive Oxygen Species Regulate Hematopoietic Stem Cell Self-Renewal, Migration and Development, As Well As Their Bone Marrow ROS Microenvironment. Aya Ludin, Shiri Gur-Cohen, Karin Golan, Kerstin B. Kaufmann, Tomer Itkin, Chiara Medaglia, Xin-Jiang Lu, Guy Ledergor, Orit Identify pathways leading to the preservation of engraftment after Very low Oxygen (1%) Kollet, Tsvee Lapidot. ANTIOXIDANTS & REDOX SIGNALING. Volume 21, Number 11, 2014. DOI: 10.1089/ars.2014.5941. + Generation of Super Oxide 2. Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow ex-vivo expansion of omidubicel with NAM compared to CD34 Generation of Nitric Oxide Dense Extra Cellular Matrix MMPs activity homing and engraftment. Tony Peled , Hadas Shoham, Dorit Aschengrau , Dima Yackoubov , Gabi Frei , Noga Rosenheimer G , Batya Lerrer , Differentiation Haim Y. Cohen , Arnon Nagler , Eitan Fibach , and Amnon Peled. Experimental Hematology 2012;40:342–355. cells grown in the absence of NAM. 3. Phase I/II Study of Stem-Cell Transplantation Using a Single Cord Blood Unit Expanded Ex Vivo With Nicotinamide. 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