Cellular and Tissue Therapies Branch (CTTB) Site Visit November 1, 2020 • Steven R. Bauer, PhD, Branch Chief • Brenton McCright, PhD, Senior Investigator – Ma Ge, MD, PhD, Staff Fellow • Deborah Hursh, PhD, Senior Investigator – Haritha Vallabhaneni, PhD, Staff Fellow • Malcolm Moos, MD, PhD, Senior Investigator • Kyung Sung, PhD, Senior Staff Fellow – Johnny Lam, PhD, Staff Fellow www.fda.gov 1 CTTB Product Review • Cell Therapies – Stem cells – Adult cells – Combination products • Cell-based tissue engineered products • Therapies • Devices – Diagnostic – Cell purification www.fda.gov 2 Cell Therapy Challenges

• Inadequate markers predictive of cell state and cell fate • Poor understanding of how cells interact with their microenvironment • Poor understanding of cell fate and survival post transplantation 3 CTTB Approaches

• Complementary Systems –Frogs, Flies, Mouse, and Man • Gene, , Cell, Tissue, Microenvironment Interactions –In vivo and In Vitro development • Knowledge and manipulation of growth factor pathways www.fda.gov 4 ASSURING SAFETY AND EFFICACY OF STEM-CELL BASED PRODUCTS • Steve Bauer –Development of Quantitative Approaches to Measure Biological Activity of Cell-based Products • Improved characterization of multipotent stromal cells (MSCs)

5 www.fda.gov CBER/FDA MSC Consortium: Identification and correlation of MSC attributes with in vivo and in vitro assays of safety and efficacy in vivo, in vitro Puri Lab: genomics McCright Lab: MSC models of wound repair Characterization

Bauer Lab: in CORRELATE vitro quantitative PRODUCT CANDIDATE differentiation CHARACTERISTICS ATTRIBUTES Moos Lab: gene WITH ASSAY expression, OUTCOMES qRT-PCR, Wei/Bauer Labs: in single cell PCR, NGS vitro, in vivo Alterman Lab: proteomics immunosuppression Hursh lab: Sung Lab: 3D in epigenetics, vitro tissue/organ karyotypes microfluidic models 6 Quantitative MSC Differentiation Assays

Adipogenesis

Chondrogenesis

Immunomodulation/ Anti-inflammatory/ Osteogenesis

www.fda.gov 7 Quantitative Measures to Assess MSC Characteristics

• Proliferation - Lo Surdo, JL, and Bauer, SR. 2012. Tissue • Cell Size Engineering: Part C 18: 877‐889. - Lo Surdo, et al. 2013. Cytotherapy 15: 1527- • Colony forming units (CFU-F) 40 • Adipogenic Activity • Osteogenic Activity Detect differences among – Marklein, et al. 2016. Stem Cells, 34:935–947 MSCs from different donors, cultured for different lengths • Chondrogenic Activity of time, and manufactured – Lam J, et al . 2018. Stem Cells Translational Medicine, 7: 664-675 under different conditions! • Immunosuppressive Activity – Klinker , and Marklein et al. 2017. PNAS. 114: 2598-2607 – Marklein, et al 2018. Cytotherapy: available online 11/28/2018 8 www.fda.gov Identification of Immunosuppressive MSC Morphological Sub-populations Morphological Data

SPγ14

Manual “gating”

Low correlation with High correlation with immunosuppression immunosuppression

SPγ2 SPγ9

www.fda.gov Marklein et al. Cytotherapy 2018 9 Developing Predictive Indicators of Genome Stability and Cell Maturation as Measures of Cell Therapy Product Safety and Efficacy • Deb Hursh –Haritha Vallabhaneni • Evaluating Methods And Culture Parameters To Assess Cell Quality And Genomic Stability Of Pluripotent Stem Cells (PSCs) www.fda.gov 10 Induced Pluripotent Stem Cells (iPSCs) Have Great Potential for Regenerative and Personalized Medicine. How do we assess the quality of products and their sources? Developing methods to assess cell quality and genome stability in pluripotent stem cells

• Mitochondria as Indicators of Cell Quality • Mitochondrial Dynamics Are Required for Pluripotency • Are mitochondria connected to stem cell quality? • Interrogate iPSCs cultured in atmospheric and low oxygen • Genome Stability • Assess if γH2AX, a marker of DNA breakage, could be used to assess DNA damage in human PSC lines • Analysis of DNA damage and karyotypic stability in primary MSCs grown under varying culture conditions • Interrogation of in vitro culture methods • Examine iPSC cultured in 2D and 3D culture systems • Develop methods to assess growth factor action: Identifying markers of TGF-β/BMP activity: • Genetic screens in a model organism to identify that interact with the TGF-β/BMP • Identify markers predictive of pathway activity and cell survival 11 www.fda.gov iPSCs Grown at 3% (Physiologic) Oxygen Have Greater Mitochondrial Mass

3 MitoTracker 11 3 -1 Tom 20 • Greater mitochondrial mass by

) 2.5 mitochondrial staining and by 2 g measurement of mitochondrial DNA

Lo 2 (

o i t Mitochondrial activity is not increased a 1.5 • R

I on a per cell basis F 1 M 2 O

0.5 • Expression of pluripotency markers is A / P 0 increased WB00031 DF19-9 EB1 Fb -0.5 • Ask if there is an effect on cell differentiation by embryoid body Physiologic/Atmospheric Ratio of MFI formation.

12 www.fda.gov PSCs Show Increased Differentiation Potential When Grown Under Physiological Oxygen

* two-tailed student’s t-test was done to determine significance: p-value ≤ 0.05. Mean normalized expression of 4 associated with each germ layer D’Antonio et al. Stem Cell Reports (2017) 8, 1101-1111 www.fda.gov 13 iPSCs Show High Basal Levels Of γH2AX That Associate With Replication

Enhanced γH2AX positive cells in iPSCs γH2AX positive cells associate with EdU positive cells

Further work showed this association correlated to replication rate, not cell type, as it was seen in fibroblasts whose replication was synchronized by serum starvation

14 www.fda.gov Purpose: Need for large amounts of cells for clinical applications requires new large-scale methods of cell culture

Schematic For Expansion And Characterization Of PSCs In Suspension

Microcarriers Aggregates horizontal vertical wheel impeller impeller

Characterization • Viability • Pluripotency • Differentiation potential 2D culture • Replication rate • γH2AX expression • Genetic and epigenetic changes DASbox® Mini Bioreactor System • Mitochondrial parameters

15 www.fda.gov Initial Studies Show Suspension Culture Provides Adequate Expansion And High Expression Of Pluripotency Markers In PSCs

100 90 80 70

%) (llsec eivitsop (llsec %) 60 50 Spinner 40 30 20 10 0 Spinner Vertical Day 7

Tra-1-60 Tra-1-81 SSEA4 OCT4 SSEA1 Pax6

Vertical Pluripotency markers expressed at high levels in cells grown in spinners and vertical bioreactors

16 www.fda.gov Developing measures of safety and efficacy for tissue- engineered products

• Brent McCright – Study how Protein Phosphatase 2A (PP2A) function and activity are controlled by regulatory subunits – Development of Magnetic Resonance Imaging (MRI) techniques to track transplanted cellular therapies and improve anatomical imaging

• Ge Ma – Identification of molecular and morphological indicators of Neural Stem Cell differentiation

17 www.fda.gov Protein Phosphatase 2A exists primarily as a heterotrimeric complex

PP2A has 3 functional components: – Catalytic subunit (C) – Structural subunit (A) – Regulatory subunit (B)

• The regulatory B subunits control PP2A intracellular localization, substrate specificity and kinetic activities

• Via their regulation of PP2A activity, B56 subunits have been shown to be regulators of developmental and cancer signaling pathways

• Our goal is to identify functional requirements for PP2A- B56 during the formation of tissues and in cells used for cellular therapies 18 www.fda.gov Loss of PP2A-B56γ in Mouse Embryonic Fibroblasts (MEFs) causes instability

(A-D) Immunohistochemical analysis with antibodies specific for Histone H3 pSER10 (pH3, green), DAPI (blue) and α-Tubulin (red), showing lagging or misaligned (shown with yellow arrows) in the B56γ- MEFs treated with nocodazole. E) Time lapse microscopy live images of nocodazole treated B56γ- MEFs. Of the 21 B56γ- MEFs, 13 displayed abnormal chromosomal segregation (shown with yellow arrows). Note wild type MEFs all arrest during mitosis when treated with nocodazole. Varadkar P. et al., , 2017 19 www.fda.gov Neural Stem Cells (NSCs) are cultured extensively to make cellular therapy products

Cell Source

The ability to Neural Stem Used as a product Cells measure NSC qualities is or important for ensuring Dopaminergic Used as a product or product quality and for manufacturing efficiency. Regulatory issue: What characteristics should be used to evaluate whether the cells retain Neural Stem Cell features?

• Depending on the final product, NSC characterization could be used as part of a lot release specification, or as method to evaluate the quality of an intermediate cell bank. 20 www.fda.gov iPSC-derived NSCs have a distinct morphology and express Notch and

Cells with NSC features (Right Gate) have a high forward scatter (FSC) and low side scatter (SSC) morphology. Cells from the right gate can form more new neurospheres and a higher percentage express Notch and Sox2.

Flow cytometry based cell morphology and molecular marker analyses were used to analyze human iPSCs-derived NSCs from early passage neurospheres (P1 or P2) and later passage neurospheres (P6 or P7).

Ma, G. et al., Cytotherapy 2018 21 www.fda.gov Inductive resonance can improve Magnetic Resonance (MR) imaging of transplanted 19F labeled NSCs A An inductive resonator was designed to improve MR images of transplanted cells labeled with a 19F contrast reagent (A). Compare the signals in the red boxes with the inductive resonator, to those without (B). Transplanted NSCs were viable for at least 40 days (C).

Fluorine (19F) is useful for labeling cells for MR B C imaging because there is no 19F background in tissues, and 19F can be directly quantified.

However, commercially available 19F detection equipment provide non- Park, Bu et al., MAGMA 2018 uniform images. 22 www.fda.gov Identifying Quality Attributes Using Developmental and Systems Biology

• Malcolm Moos –SMOC modulation of BMP/Wnt signaling –Single cell mRNA sequencing to identify and characterize cell product subpopulations

www.fda.gov 23 SMOC protein induces neural genes in hours

Animal cap RNAseq (single assay explants)

Genes up > 5 fold* Genes down > 5 fold** 25 Genes up > 5 44 Genes down > 5 fold c-src BMP response genes: fold -1 • BAMBI cone-rod • DLX5 Otx1, 2 BMP response genes: • FOXI1 HES5, 7 • ID2, 3 SIX3 BAMBI FOXD4L1 • VENT) • NUFIP2 Other: KLF 2,4, 17 cone-rod • DLX3 Otx2 FGFR1, 2 homeobox • FOXI1 HES7, X1 • GATA2 POU3F2 • ID4 • MSX1,2 • VENT Other: Sizzled (Wnt antagonist), Wnt- 7A * Selected of 25 ** Selected of 44 www.fda.gov Thomas JT, Furtak V, Moos M (2018) unpublished 24 Bone marrow and adipose tissue-derived stem cells have different subpopulation structure

Bone Marrow Adipose

Furtak V, Sridhara V, Moos M (2019) unpublished 25 www.fda.gov Large batch effects can be corrected computationally and B- cell lines from a single donor:

Uncorrected—Cells cluster by fastMNN corrected—Cells institution/technology cluster solely by cell type

Chen et al. (2019) Nature Biotechnology, submitted 26 www.fda.gov Summary: •Secreted Modular Calcium binding protein • Blocks BMP & Wnt signaling1 • extends their range1 • Induces neural program within hours •Adipose- and bone marrow-derived MSCs show different subpopulation structure •Single cell RNAseq batch effects between laboratories and technologies can be corrected computationally2

1 Thomas JT, Dollins ED, Andrykovich K, Furtak, V, Moos M (2020), Elife, submitted 2 Chen et al. (2020) Nature Biotechnology, submitted 27 www.fda.gov INVESTIGATING THE EFFECT OF CELL- MATERIALS INTERACTIONS ON THE SAFETY AND EFFECTIVENESS OF CELL- BASED PRODUCTS

• Kyung Sung – Johnny Lam – Development of an MPS as a platform to evaluate the trophic effect of MSC preparations on vasculogenesis

28 www.fda.gov INVESTIGATING THE EFFECT OF CELL-MATERIALS INTERACTIONS ON THE SAFETY AND EFFECTIVENESS OF CELL-BASED PRODUCTS

• For cell-based products, biomaterials are being tested for product manufacturing, assay development, product delivery strategy, and tissue-engineered products. • Mounting evidence suggests that cell-biomaterial interactions may play important roles in controlling cells’ functional behaviors in vitro as well as in vivo by providing specific microenvironmental cues to which cells respond. • With advances in biomaterials and cell biology, we are gaining deeper understanding regarding which biomaterials could be used or modified to obtain desired outcome of cell therapy products. • However, there needs to be continued effort to fine-tune a number of parameters of biomaterials as well as cell source to enhance the consistency and quality of manufactured cell based products.

29 www.fda.gov Goal

• The Sung lab research program aims to investigate cell- biomaterial interactions which outcome will contribute to enhancing cell therapy product manufacturing strategies and to developing new assays for critical quality attribute development.

• The primary goals of the laboratory research are 1) developing microscale 3D biomimetic systems to reliably measure the functional capacity of multipotent stromal cells (MSCs) and induced pluripotent stem cells (iPSCs)-derived cells in physiologically relevant conditions; and 2) understanding the functions and behaviors of cellular products in specific microenvironments to predict the safety and efficacy of manufactured cellular products. This will help us see how the cellular products need to be designed to provide the best method of treatment for patients.

30 www.fda.gov Chondrogenic capacity of MSCs

• MSCs are proposed as alternative cell source for treating cartilage defect due to their capacity for easy isolation and chondrogenic differentiation.

• We used 3D cell aggregates as a platform to measure chondrogenic capacity of MSCs because the model is thought to recapitulate aspects of developmental processes during mesenchymal condensation that precede cartilage formation.

31 www.fda.gov MSC aggregate size matters

Lam et al. Stem Cell Transl Med (2018) 32 www.fda.gov Role of cell-materials interactions in the immunosuppressive activity of MSCs

• Biomaterials have been used to manufacture MSCs to enhance the functions and survival of these cells. However, the functional immmunosuppressive capacity of MSCs on these biomaterials have not been well characterized. • In this project, we aim to study whether cell-cell interactions and cell-integrin engagement with biomaterials are critical to the immunosuppressive activity of MSCs. 33 www.fda.gov A microphysiological system to explore paracrine interactions between endothelial cells and MSCs

www.fda.gov 34 Vasculogenic metrics were different between MSC preparations and were passage-dependent

Collaboration with CurioChips,Inc 35 CTTB Research: Addressing Cell Therapy Challenges

• Complementary approaches –In vivo and in vitro systems • Cell-Cell interactions • Genetic interaction screens • Protein-Protein interactions • Organogenesis www.fda.gov 36 Significance for Cell Therapy

• Findings may reveal cell product quality attributes that lead to: – Improved characterization of cell-based therapies – Methods to monitor manufacturing differently – Ability to choose donors differently – Scientific basis for: .policy development .guidance for sponsors . www.fda.gov standards development 37 MSC Consortium

Brent McCright Michail Alterman • Mandy Bush • Samuel Mindaye • Kristin Shultz-Kuszak • Natalia Pripuzova Malcolm Moos • Sema Rosinbum • Jennifer Mateshaytis Steve Bauer • Elaine Thompson • Jessica Lo Surdo • Alexa Bianchi • Heba Degheidy • Ross Marklein • Mike Mendicino Raj Puri • Saniya Godil • Ian Bellayr • Matthew Klinker • Jennifer Catalano Deb Hursh • Patrick Lynch Cheng-Hong Wei • Yasmin Rovira-Gonzalez • Cristina Nazarov • Brian Stultz • Matthew Klinker • Katie Steers • CBER/OTAT/DCGT Kyung Sung • Johnny Lam • FDA Targeted Research Funds • BARDA 38 www.fda.gov Contact Information • Steven R. Bauer, Ph.D. [email protected] • Regulatory Questions: OTAT Main Line – 240 402 8190 Email: [email protected] and [email protected]

FDA Headquarters Federal Research Center at White Oak • OCTGT Learn Webinar Series: 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 http://www.fda.gov/BiologicsBloodVaccines/NewsEvents/ucm232821.htm

• CBER website: www.fda.gov/BiologicsBloodVaccines/default.htm • Phone: 1-800-835-4709 or 240-402-8010 • Consumer Affairs Branch: [email protected] • Manufacturers Assistance and Technical Training Branch: [email protected] • Follow us on Twitter: https://www.twitter.com/fdacber www.fda.gov 39