# Presentation: 472073

##  Cellular and Tissue Therapies Branch (CTTB) Site Visit November 3, 2006

- Steven R. Bauer, PhD, Chief
- Deborah Hursh, PhD, Senior Staff Fellow
- Gerald Marti, MD, PhD Senior Investigator
- Brenton McCright, PhD, Senior Staff Fellow
- Malcolm Moos, PhD, MD Senior Investigator
- John Terrig Thomas, PhD Visiting Scientist

## The Problems

- Clinical benefit is highly variable, often hard to demonstrate
- In many cases, most cells administered die immediately
- Products may ‘misdifferentiate’
- Inadequate supply

## Cell Therapy Challenges

- Poor understanding of how cells  interact with their microenvironment
- Inadequate markers predictive of cell state and cell fate
- Poor survival of cells post transplantation

## CTTB Approaches

- Complementary Systems
- Frogs, Flies, Mouse, and Man
- Gene, Protein, Cell, Tissue Interactions
- Normal Development and Tumorigenicity
- Knowledge and manipulation of growth factor pathways
- Knowledge and utilization of transformation pathways

## Microenvironment in cell therapy: manufacturing, patient - Bauer

_**In vitro: reduced stromal dlk**_
**No differentiation or apoptosis upon removal of IL-7**
**No changes in typical pre-B markers**
** **_**In vivo effects: Dlk1-/- mouse**_
**Alters B-cell development and function**

**Pre-B**

**Abnormal stroma=> **
**Abnormal B cells**

- Remove IL-7

## Systematic approach for identification and qualification of tumorigenicity biomarkers for cell therapy products- Bauer

- Mechanisms of transformation
- Identify biomarkers of transformation

**Systematic approach for identification and qualification of tumorigenicity biomarkers for cell therapy products- Bauer**

## Impact for Cell Therapy

- Stroma can alter cell product in a way that is not revealed in lot release tests
- Efficacy may be affected by
- Microenvironment during cell product manufacturing
- Microenvironment in patient
- Improved tumorigenicity assessments

## Approach: Genetically modify the mouse to the study function of proteins required for mammalian organ development in vivo.

- Mammalian organogenesis as a model for cellular and tissue engineered therapies – McCright

- Genetic modification of the mouse by blastocyst injection

- _Mouse models created:_
- Mice that allow us to either inactivate or over express Notch2 in a tissue specific manner
- Mice that allow us to isolate stem cells based on their expression of Notch2
- Mice to study a putative anti-oncogene, B56gamma

## Notch2 expression and heart specific inactivation demonstrates a cell autonomous requirement for Notch2 during murine heart development

- Notch2-

- *

- pa

- Notch2+

- *

- pa

- Notch2 is highly expressed in the embryonic heart

- Notch2 inactivation results in developmental defects in the outflow tract and the right ventricle

- Hearts from newborn mice, 2/3 of the mice with Notch2 heart specific inactivation die perinatally

- Notch2-gal expressing cells
- stain blue with Xgal in the E17 heart

## Impact for Cell Therapy

- Identification and analysis of molecules required for mammalian organogenesis
- Notch2 as a biomarker for evaluating the developmental potential of cells used in cardiac repair.
- Notch1 or Notch2 activation may have similar effects on cell products.
- Exogenous Notch activation and the functional requirements for Notch2 can be studied in most tissues

## A Genetic Model of Growth Factor Action to Develop Markers of Safety and Efficacy of Cell-Based Products-Hursh

- A system to study cell communication in intact tissues
- Ability to alter gene expression within a microenvironment
- High throughput screens identify critical control points
- Markers predictive of pathway activity
- Analyze cell stress and viability
- Markers predictive of survival

## Genetic interaction screens identify and place genes in a functional pathway

- Asks the organism to identify critical control points.
- Avoid bias of abundance, immunogenicity, modifications.
- Model organisms allow screens of sufficient size.
- Knowledge of control points for cellular products is critical.
- identified > 20 genes that interact with BMP signaling.

## Loss of BMP causes cell death, induces JNK pathway: a model of cell competition

**WT****,****,****caspase**

**Mut,**** caspase**
**,****JNK activity**

**TGF-ß/BMP, insulin, EGF serve **
**as cell survival factors. **
**Loss of growth factor causes **
**inability of cells to “compete” **
**with normal neighbors, can cause **
**apoptosis, and  overgrowth**

## Impact for Cell Therapy/Tissue Engineering

- Improve our ability to predict the survival of transplanted cells in their new location
- Cell interactions in tissue development

## Protein: Protein Interactions in Joint Development- Moos

- Proprotein Convertases and GDF5 colocalize
-  to establish normal joint structure

## A novel BMP antagonist copurified and colocalizes with CDMP-1/GDF-5

**Joint interzones**

**A novel BMP antagonist**** ****copurified**** ****and**** ****colocalizes**** ****with**** ****CDMP-1/GDF-5**

## Feedback and Crosstalk in Cell and Tissue Specification

- Colocalization of several signals is necessary to instruct formation of articular cartilage
- Developmental signals in characterization of cell and tissue engineering products

## Chronic Lymphocytic Leukemia in Mouse and Man- Marti

- Molecular Lesion in CLL
- Precursor states:  Monoclonal B Cell Lymphocytosis (MBL)
- Familial Chronic Lymphocytic Leukemia (CLL)
- NZB mouse model of CLL
- Shared micro RNA lesion in human and mouse CLL
- Biomarker:  ZAP70
- Quantitative Flow Cytometry (QFCM)
- Cell therapy product characterization
- Flow cytometry-based cell separation
- Standards Development:
- Fluorescence reference materials
- CLSI documents

## Mir16-1 mutations in  Mouse and Man

- MicroRNA lesions in Human CLL
- and Mouse model of CLL

- _NZB model of CLL reveals similar mutation_
- - Identification of critical lesion in CLL
- Animal model for elucidation of mechanisms of transformation
-  Improved diagnosis/treatment of CLL

## Applicability

- Early Detection:
- Molecular lesions
- Leukemogenesis
- Intervention
- Product Characterization
-  In-process
- Lot Release
- Cellular and Gene Therapy Products
- Clinical Use of Flow Sorted Cells
- Quantitative Flow Cytometry (QFCM) Standards
- Instrument/Assay Performance
- Linearity of detectors
- Compensation  Controls

## CTTB Research: Addressing Cell Therapy Challenges 

- Complementary approaches
- Cell-Cell interactions
- Genetic interaction screens
- Protein-Protein interactions
- Organogenesis
- Tumorigenesis

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