President Obama Bench Bedside

Release Criteria for Hematopoietic Stem Cell Therapies

Curt Civin, Nancy Hardy, Tami KingsburyUniversity of Maryland School of Medicine

CASSS Feb 9, 2017

Financial disclosure Drs. Civin and Kingsbury own

shares and serve on the board of 3DBioWorks Inc, a small privately-held biotechnology company which develops stem cell expansion and bioreactor technology.

The University of Maryland holds patents on bioreactor technologyand related inventions.

These arrangements are being managed by the University in accordance with its conflict of interest policies.

Dr. Hardy has nothing to disclose.

Release Criteria for Hematopoietic Stem Cell Therapies

Overview: HSCs and HSCT Curt Civin MD

Conceptual basis for standards and quality control

Genetic Engineering of HSCs for Blood Diseases Tami Kingsbury PhD

Considerations for which new standards and QC needed

Clinical Use of HSCT Nancy Hardy MD

Current standards and QC (i.e. “Release Criteria” for HSCT Grafts)

Overview: HSCs and HSCT

Curt I. Civin, MDAssociate Dean for Research, University of Maryland School of Medicine

Director, Center for Stem Cell Biology & Regenerative MedicineProfessor of Pediatrics and Physiology

Hematopoiesis is essential to human life

~200x109 cells produced per day

HSPCs are the cells most sensitive to radiation and most antineoplastic

chemotherapy

Homeostatic and reactive production of all cells of the

blood and immune system for over a lifetime

Functions of HSCT preparative radiochemotherapy regimens

Create hematopoietic space in the recipient Reduce numbers of endogenous HSCs that compete with

the donor graft Esp, delete endogenous HSCs occupying the hematopoietic

nicheImmunosuppress the recipient Prevent grant rejection (host vs graft)

Note: Radiochemotherapy may benefit the cancer patient, but does only harm to the patient w/ non-malignant disorder Reduced intensity (non-myeloblative) regimens

Important features of HSCT graftsVolume: with respect to recipient cardiac/renal functionCryoprotectant: limit DMSO cardiac/renal toxicitiesHematopoietic Capacity: engraft and support full donor-derived lympho-hematopoiesis in the recipient for lifetime

Leukocytes Colony-forming cells CD34+ cells CD34+ cell subsets

Immunologic potential: graft vs leukemia, graft vs host disease (biggest toxicity of HSCT)

T, B, NK lymphocyteso Graft vs tumor potentialo Alloreactive potential

Cancer potential Contaminating cancer cells in autologous HSCT for cancer Leukemogenicity of unintended genetic modifications in HSCT gene therapy

Purified CD34+ HSPCs for research and clinical transplantation

Civin, J Immunol 1984

CD34+ cell subsetsHSC

CD34+/CD38-/Lin-

Mature Blood & Immune Cells

CLP CMP EP

CD34+/[CD38/Lin]++

Human CD34+/CD38-/Lin- HSC cell subset:

contains most of the primitive in vivo-engrafting HSCs includes few of the less primitive hematopoietic single-lineage-committed progenitor cells

CD34+/[Lineage/CD38]++ subset: enriched in later hematopoietic progenitors depleted of HSCs

HPC

Hematopoiesis in the nicheSelf-renewal

ProliferationTrumpp 2010

Kaplan, The Biomedical Engineering Handbook 2010

HSPC transplant

The CD34 antibody is widely, but not always, used to purify HSPCs for HSCT

Civin, J Clin Oncol 1996

Stem cells

Multipotentprogenitors

Committedprogenitors

Mature cells

Regulators:

IL-1 IL-6 G-CSFIL-3 IL-11 GM-CSFG-CSF IL-4 M-CSFIL-2 IL-6 Epo IL-7

HSC self-renewal is limited, in vitroSCF TPO FL

The ultimate stem cells: Pluripotent embryonic stem cells can self-renew indefinitely and generate all types cells of a mouse in vitro and contribute to all of the tissues and organs of a mouse in vivo

We had thought and taught that differentiation could be only a one-way trip in mammals

Differentiation of human ES cells (or iPSCs) into blood cells

Day 2: Differentiating

human embryonicstem cell

aggregates called “embryoid bodies” 1-2 weeks later:

Many differentiating human embryoid

bodies

Zambidis, Blood 2005

2 weeks later: Coloniesof functional blood cells

But lots of work and ES cells generate only a few mature blood cells, and no HSCs.

Reprogramming procedures are being enhanced: more efficient and safer iPSCs

Leukemias can originate in HSPCsTheir longevity and (self-)renewal capacity make stem(-progenitor) cells susceptible to the acquisition of both initiating and secondary oncogenic mutations (in the same cell): Theory: Stem(-progenitor) cells persist long enough to accumulate

the multiple oncogenic hits necessary for cancer development Theory: Stem(-progenitor) cells are already programmed to

generate huge clones of identical or similar progeny cells Fact: Gammaretroviral vector-mediated IL2RG gene transfer into

autologous human CD34+ HSPCs and subsequent transplantation resulted in activation of LMO2 and other proto-oncogenes, and T-cell leukemias developed in 4 of the 9 patients

Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient

First case of tumor development in a human following (fetal = “adult”) stem cell therapy, though similar findings have been made when embryonic stem cells were injected into rats.

The immunologic deficits of the boy’s A-T may itself have allowed the allogeneic tumors to develop.

Amariglio et al, PLOS Med, Feb 2009

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are needed to see this picture.

Genetic Engineering of HSCs for Blood Diseases

Tami Kingsbury PhDAsst Professor of Physiology

Center for Stem Cell Biology & Regenerative MedicineUniversity of Maryland School of Medicine

Gene Editing

What determines off-target site binding and cleavage despite mismatches in sgRNA?What dictate INDEL biases for a given sgRNA? How can we improve HDR efficiency and increase conversion track length?

Nucleases generate DSBs at target site (and off-target sites)

Ran Nature Protocols, 2013

MSCRF project: Genetic Modification of Sickle Cell Disease in Hematopoietic Stem Cells

Disrupt the DNA encoding an erythroid-specific enhancer that regulates levels of BCL11A via CRISPR/Cas9 genome editing

technology of human HSCsCurt Civin, Wen-Chih Cheng, MinJung Kim, Tami Kingsbury

HbS(α2βs

2)http://www.nhlbi.nih.gov/health/health-topics/topics/sca

Gene Editing to Treat Disease

BCL11A enhancer SNPs reduce SCD phenotypes via increasing g-globin production in adult erythrocytes

BCL11A (transcriptional repressor)

γ-globin

fetal hemoglobin, HbF, α2γ2

Patients with HPFH have reduced severity of SCD because HbS polymerization is inhibited by HbF

HPFH patients have rare SNPs in the enhancer of BCL11A that specifically disrupt BCL11A expression in erythroidcells

Bauer, D.E. et al. Science 342, 253-257 (2013).

Individuals with hereditary persistence of fetal hemoglobin syndrome (HPFH) contain SNPs in their genomes that reduce

sickling and complications of Sickle Cell Disease SCD HPFH Modify BCL11A in SCD

Hardison & Blobel, Science 341:206, 2013

sgRNA sites near critical GATA binding site

sgRNA sites are limited by PAM sequences

Sequencing INDELS

- + - + - +

K562 HSPCs

C.Cas9

Cas9 +sgRNA

Cas9 +sgRNA

CRISPR/Cas9 editing of K562 leukemia cell line vsprimary CD34+ HSPCs

50-60% <10%

Gene editing requires an efficient method to deliver editing reagents to cells of interest

Low yield of edited cells may impact safety, although in some cases, selective advantage conferred by editing may provide benefit even if editing was not efficiente.g. SCID-X1 mouse models: 1% not enough, but 10% is sufficient

Optimize CRISPR/Cas9 delivery to HSCs using Maxcyte flow electroporation

HSCs are very difficult to transfect, ~1% efficiency with high toxicity Maxcyte flow electroporation instrument is cGMP compliant, and is

already being used in clinical trials

57% viable

97% GFP+

Collaboration with Maxcyte: Madhusudan Peshwa, PhD and Linhong Li, PhD

Gene editing reagent formatsReagent format impacts editing efficiency, cell toxicity and genotoxicity

Cas9 can be delivered using:DNAmRNARNPvirus based strategies, (IDLV, Adeno)

Modified sgRNAs direct more efficient gene editing

For HR, repair templates are also required

www.mirusbio.com

Clinical Gene EditingMaximize specificity of editing nuclease

Develop new toolsSplit Cas9Chimeric nucleases

Zetsche, Nature 2015

Corrigan-Curay, Mol Therapy 2015

Optimize protocol

Clinical Gene Editing cont.Unbiased and global identification of off-target sites

SequencingGuide-seqDefine “rules” for off-target binding and cleavage

Develop appropriate cell-type specific assays to detect cell transformation

Assess impact of gene editing risks in context of background levels of DSBs and chromosome translocations (or generate alternative strategies-hit & run)

Clinical HSCT

Nancy Hardy MDAssociate Professor of Medicine, UMSOM

Director, Cellular Therapy LaboratoriesDirector, Allogeneic Stem Cell Transplantation

Medical Director, Cancer Center Quality and SafetyMarlene and Stewart Greenebaum Comprehensive Cancer Center

CELL THERAPY & TRANSPLANTATION

PRODUCT RELEASE

Autologous or Related-Donor Hematopoietic Stem Cells (HSC)• Current Good Tissue Practice:

prevent the introduction or transmission of communicable diseases

Unrelated HSC, Umbilical Cord Blood, T Cell Therapy, Gene Therapy• Current Good Manufacturing

Practice: specific requirements for identity, strength, quality, and purity

CHALLENGES➳Patient-directed products➳Customization

Red Cells, Plasma, CD34 ➳Clinical realities require

Exceptional Release

REGULATION OF HUMAN CELLS, TISSUES, AND CELLULARAND TISSUE-BASED PRODUCTS (HCT/PS)

361 ProductsNot subject to premarket clearance or approval• Minimally manipulated• Homologous use• Not combined• No systemic effect, function is not

dependent upon the activity of living cells, or it is intended for autologous use or allogeneic use in close relatives …

Minimally Manipulated: Processing that does not alter the relevant biological characteristics of cells or tissues:Density-gradient separation, cell selection, centrifugation, cryopreservation

351: HCT/P that are not 361Adulterated drugs and devicesUnrelated allogeneic HSC, Lymphocytes• Lymphocyte Immune Therapy• Gene Therapy Products

STANDARD

• Communicable disease testing (allogeneic)• HLA typing (allogeneic)• ABO group and Rh typing on 2 separate samples• Microbial testing after processing• Post-processing nucleated cell count and viability• Assay of target cell population for products that have been

enriched or depleted

The Processing Facility Director shall define tests and procedures for measuring and assaying cellular therapy products to:

- assure their safety, viability and integrity - document that products meet predetermined release specifications

PRODUCT RELEASE

HSC Release Criteria

Identity

Purity

Potency

Donor Screening

HSC Potency: Engraftment, Chimerism & Immunity

Allogeneic: Donor Chimerism

Engraftment: cell dose, viability, “stemness”• Hematopoietic recovery

• Confounders• Conditioning intensity• Histocompatibility, rejection• Disease, e.g., infection, damaged

marrow stroma, hypersplenism

Immune Reconstitution: CD34, T cell dose; viability• Therapeutic: Graft-versus-Leukemia

• Functional: Immunity• Confounders:

• Histocompatibility, Graft-vs-Host Disease

• Donor, recipient age, gender

PLT

ANC

TRM

OS

CD34 & Engraftment, Outcomes:Autologous vs Allogeneic

Alloreactive Potency: Graft-vs-Leukemia

Monitor: Graft-vs-Host Disease

Horowitz, M. M. et al. Blood 75:555, 1990

Relapse after Hematopoietic Stem Cell Transplantation: Syngeneic vs. T-cell Depleted vs. T-cell Replete

Unrelated-DonorBone Marrow BMT for 90 Kg adult with Diamond-Blackfan anemiaABO: A-pos O-pos

CD34 Cell Dose 0.76 x 106/KgAfter Red Cell Depletion: 0.61 x 106/Kg viable CD34+ cells“Fresh” infusion (72 hours)Day 40: no engraftment

Mobilized, peripheral blood stem cell collectionBoost 5 x 106 viable CD34+ cells/KgNeutrophil recovery 15 days later

Autologous PBSC 60 year-old “super-mobilizer” with MMCD34 Cell Dose: 4.8 x 106/Kg viable CD34+ cells

CD34 Boost: 2.4 x 106/Kg on ~ Day 14

CD34 Boost: 2.4 x 106/Kg on ~ Day 21

Neutrophil Engraftment: Day 29Engraftment Monitoring on All Products• Failures trigger investigation

• Neutrophils• Platelets• Donor Cells

• Potency• Fresh viable CD34+ cell dose• Fresh CFU dose

Effect of Overnight Storage Prior to Cryopreservation on Hematopoietic Engraftment after Autologous HPCT

ANC PLT 20K PLT 50K ANC PLT 20K PLT 50K ANC PLT 20K PLT 50K

Potency Assays

STEMCELL Technologies Inc. WHITE PAPER, 09/2012: “Potency” Assays for Measuring the Engraftment Potential of Hematopoietic Stem and Progenitor Cells.

Potency Assays for Release• Enumeration of viable CD34 cell dose and CFU dose

prior to cryopreservation correlate with engraftment

• Late engraftment may reflect viable cell loss with freeze/thaw

• Post-thaw enumeration of viable progenitors is limited

• DMSO effects on membrane integrity• Cell loss with washing; clumping, filtering• Rapid cell loss GM-CFC stability testing of thawed PBSC

products @ RT. The mean GM-CFC yield from the fresh harvests tested on immediate thaw was 68 ± 27%. British Journal of Haematology, 175, 5, 17 OCT 2016.

Biology of Blood and Marrow Transplantation 2010; 16, 500-508.

Flow cytometric evaluation of thawed Cord units. Modified gating more accurately measures viability.

• Tolerant: Transplant across HLA barriers• Progenitor cells have high “stemness” but marginal numbers• Quality and outcomes vary: collection, processing and

storage: sites; practices, procedures• Leukemic Clones in cord blood units???

Umbilical cord blood, 4/6 HLA match; Adult, GATA-2 deficiency and MDS CD34 Cell Dose: 0.787x106/KgCD3 Cell Dose: 3.48 x 106/KgNeutrophil Engraftment Day 76Donor Cell Leukemia ~ 16M after transplant

Umbilical Cord Blood• Collected at L&D• Processing @ CBB• Cryoshipped• Transplant Center Thaw, Infusion

TNC Dose Neutrophils

Platelets

CFC Dose Cell dose and engraftment after cord blood transplantation

Migliaccio et al. Blood 2000;96

Survival

Page et al., Biology of Blood and Marrow Transplantation, 17:9, 2011.

Postthaw

Postthaw CFU content best correlates with engraftment after Unrelated Umbilical Cord Blood Transplantation

Neutrophils

Platelets

Pre-Cryo

Umbilical Cord BloodMinimally Manipulated, Unrelated

Intermediate Product Requiring cGMP

Page et al., Biology of Blood and Marrow Transplantation, 17:9, 2011.

Aldehyde dehydrogenase (ALDH) is highly expressed in hematopoietic stem and progenitor cellsALDHbr CBU cells coexpress CD34 and CD133 and are highly enriched for CFUs

ALDHbr–based cord blood potency assay

Thawed segments vs fresh or thawed CB Units

ALDHbr–Based Cord Blood Potency Assay

Thawed cord blood segments

ALDHbr Cells & Engraftment

Cord blood segments

Release Criteria for Hematopoietic Stem Cell TherapiesBlood and marrow transplantation is used to treat hematopoietic disorders, malignancies susceptible to the immunologic graft-versus-leukemia effect, and as autologous rescue after myeloablative therapy for cancer or auto-immune disease. Release criteria are employed by the collection center prior to transfer to the processing center, including verification of donor eligibility or documentation of exceptional release, product labeling, and integrity of product packaging. Release criteria are employed by the processing center prior to distribution for clinical use, which, in addition to the criteria employed by the collection center include verification of tissue typing and ABO/Rh compatibility, donor communicable disease testing, sterility testing, and potency assessment of viable nucleated cell and CD34+ hematopoietic stem-progenitor cell content. Additional testing for target cell content and viability are performed if cell deletion or enrichment processing is performed. Practical constraints limit product evaluation for standard, minimally manipulated hematopoietic stem-progenitor cell products; e.g., treatment timelines can preclude full characterization of products prior to clinical release, and exceptional releases are frequently employed. For example, allogeneic hematopoietic stem-progenitor cell products are often infused fresh, so exceptionally released prior to sterility testing and tissue culture results. Incorporation of genome-edited cells into transplants raises additional concerns over cell viability and ex vivo expansion, gene editing efficacy and safety, and hematopoietic stem-progenitor cell functional capacity. Ongoing efforts in Sickle Cell Disease genome editing will be presented as an example of challenges in bringing this technology to the clinic. Since transplant outcomes are influenced by a plethora of extrinsic factors, few potency release criteria effectively predict engraftment. For example, thawed hematopoietic stem-progenitor cells must be infused immediately, precluding assessment of potency. Cryopreserved hematopoietic stem-progenitor cell product potency is not routinely included in clinical release criteria; rather, QA/QC monitoring includes assessment of post-thaw potency and clinical engraftment data.