gamida cell - cell and immune therapy technologies - … · 2019. 12. 12. · cell cycle mmps...

Figure 3. Upregulation and downregulation of families of transcription factors in CD34+ cells expanded with NAM

Figure 4. Enrichment analysis showing NAM upregulates transcription factors responsible for stem cell renewal and DNA repair while down-regulating transcription factors that activate cell differentiation, inflammation, and apoptosis

• HPC differentiation (mTOR, JNK, cMYC, NF-kB, cJUN)

• Inflammatory signaling (IFNγ,TNFα, TLR2/4, CCL2/7)

• ROS and RNS production (CYBB, NCF1/2/4, NOS2)

• Apoptotic signal (IL1β, BCL2L4/8, IRF1, NRP1, PARP)

• MMP production (MMP2/7/9/12/19, TIMP2, LRP1, A2M)

p-value: 3.0x10-8

p-value: 1.6x10-6

p-value: 6.6x10-8

p-value: 1.6x10-6

p-value: 2.4x10-3

•Self-Renewal (SP1, cJUN)

•Cell Cycle regulation (E4F1, SP4)

•Stem Cell development (ETS1)

•DNA repair (UNG, NDN)

•Chromatin remodeling (SMC2/4, ESCO2)

p-value: 0.6x10-3

p-value: 2.4x10-3

p-value: 0.4x10-3

p-value: 0.2x10-3

p-value: 1.7x10-2

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 Gene Therapy, Jerusalem, Israel

Background

Nicotinamide (NAM)

Results

Figure 1. A master regulator of NAD-related signaling pathways

• Historic efforts at expansion of umbilical cord blood, (UBC) derivedCD34+ hematopoietic stem cells, (HSCs) ex-vivo with cytokines yieldedlarge numbers of progenitors for transplantation but impaired theirengraftment.

• Ex-vivo HSC expansion resulted in accelerated cell proliferation,elevated levels of reactive oxygen and nitrogen species (ROS andRNS), and upregulation of inflammatory signaling leading to celldifferentiation and loss of in-vivo functionality.1

• We used nicotinamide (NAM), an allosteric inhibitor of NAD-dependentenzymes, to create omidubicel, an investigational cell therapy designedto improve the expansion of CD34+ HSCs for bone marrow transplant.2

• A Phase 1/2 trial of omidubicel in patients with high-risk hematologicmalignancies showed rapid neutrophil engraftment and a favorableimmune reconstitution profile in patients compared to historicalcontrols. We hypothesized that NAM treatment maintains the stemnessand engraftment potential of omidubicel, which is associated withclinical benefit.3

Cluster analysis of differentially expressed genes in CD34+ cells expanded ±NAM for 3-weeks compared to non-cultured CD133+ cells (Affymetrics).

Figure 2. Similarity between CD34+ cells cultured with NAM and non cultured CD34+ cells

Study Design

Exp.1

Exp.2

Exp.3

Exp.1

Exp.2

Exp.3

Exp.1

Exp.2

Exp.3

CD34+ cells cultured W/O NAM

CD34+ cells cultured with NAM

Non-culturedCD34+ cells

DATA SET “A”. Day 21

299 genes downregulated

3

NAM effect

83 genes upregulated

Transcription Factor enrichment

PU.1 0.009SRF 0.022FOS/b 0.039JUN/b/d 0.019SP1 0.033E4F1 0.039PLAGL1 0.032

DATA SET ”B”. Day 4

650 genes downregulated

664 genes upregulated1

2

1

SP1 0.000YY1 0.000E2F1 0.000SP4 0.000RAR/RXR 0.001CREB1 0.003PPAR/a/b 0.014HOXB7 0.041

ELF1 0.000PU.1 0.000CEBPD 0.007JUN/b/d 0.019FOS/b 0.011JUN/b/d 0.011

Fresh NAM

ObjectiveIdentify pathways leading to the preservation of engraftment after ex-vivo expansion of omidubicel with NAM compared to CD34+

cells grown in the absence of NAM.

Pathway Inhibition

P-value

Day 2, 4 and 21: NAM VS Control

Culture Seeding

RNA sampling

d2 d21

Culture Harvest

d4

- NAM+ NAM

DATA SET “A”

Day 4: ± fresh NAM

+ NAM

DATA SET “B”

Culture Seeding

+ NAM +6hd4

RNA sampling Biological / Functional enrichment

Conclusions

Nicotinamide (NAM) Modulates Transcriptional Signature of Ex Vivo Cultured UCB CD34+ cells (Omidubicel) and Preserves Their Stemness and Engraftment Potential

• Next Generation Sequencing (Cel-SEQ, NGS) was performed on HiSeq 2500, rapidv2, Illumina.

• Gene annotation was done based on Human genome GRCh38.p12 versionEnsembl/BioMart.

• Total 3,816 Differentially Expressed (DE) genes was defined based on absolutelinear fold change of 1.3 and unadjusted p-value <0.01.

• DE-Genes were clustered to “CliCK”-Heatmap. 17 and 5 Clusters were defined inDataset “A” and “B” respectively.

Figure 6. Bone marrow niche preserves HSC pool

Quiescent environmentHigh ROS scavengingVery low Oxygen (1%)Dense Extra Cellular Matrix

ROS

Differentiation

Cell Cycle

MMPs activity

1. Reactive Oxygen Species Regulate Hematopoietic Stem Cell Self-Renewal, Migration and Development, As Well As Their Bone Marrow Microenvironment. Aya Ludin, Shiri Gur-Cohen, Karin Golan, Kerstin B. Kaufmann, Tomer Itkin, Chiara Medaglia, Xin-Jiang Lu, Guy Ledergor, Orit Kollet, Tsvee Lapidot. ANTIOXIDANTS & REDOX SIGNALING. Volume 21, Number 11, 2014. DOI: 10.1089/ars.2014.5941.

2. Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment. Tony Peled , Hadas Shoham, Dorit Aschengrau , Dima Yackoubov , Gabi Frei , Noga Rosenheimer G , Batya Lerrer , Haim Y. Cohen , Arnon Nagler , Eitan Fibach , and Amnon Peled. Experimental Hematology 2012;40:342–355.

3. Phase I/II Study of Stem-Cell Transplantation Using a Single Cord Blood Unit Expanded Ex Vivo With Nicotinamide. Horwitz M.E., et. al., J Clin Oncol. 2019 Feb 10;37(5):367-374.

NAM NAD+ precursor

NAD+

enzymesCellular

stress,

ROS

- Histone acetylation- Gene expressionBalancing NAD+

catabolism- Mitochondrial mass

- Energy metabolism

• Transcription Factor (TF) enrichment analysis was done using FunRich software (http://funrich.org/).

• GO - Biological Process Enrichment was performed (http://geneontology.org/).• INGENUITY (IPA) software was used for Pathway Analysis (Qiagen).

Bone Marrow NicheNAM platform is unique in mimicking the bone marrow niche during ex vivo expansion of HSCs.

NAM attenuates genes responsible for:• Reactive oxygen and nitrogen species production.• Inflammatory and apoptotic signaling.• HSC differentiation and maturation.• Secretion of matrix metalloproteinases (MMP’s).

Our gene expression data: • Reinforce the mechanism of action underlying our NAM

technology platform.• Provide additional insights into the pathways leading to clinically

significant engraftment in patients.

The NAM platform has also been used for ex vivo expansion of otherimmune cells (GDA-201 ASH 2019 Abstract #777). Poster #3718.

References

Induction of iNOS IFNg induced

differentiation

Generation of Nitric OxideAgingInflammation Differentiation

Generation of Super Oxide

InflammationDifferentiation

Introduction of iNOS by cytokines

ROS secretion

Figure 5. NAM Inhibits Pathways Responsible for ROS / RNS and Inflammation

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0

Normalized Enrichment Score