CT Tech Process Details

Question 1

Nucleofector

Uses a specific combination of optimized pre-programmed electrical parameters and cell type-specific solutions. The different platforms provide different specifications for various applications.

Question 2

Nucleofector Kits:

Dedicated Solutions and Supplements

• Specified Nucleofection vessel
• Pipettes
• Fluorescent positive control vector pmaxGFP control vector

Question 3

Nucleofactor Features/Benefits:

• High transfection efficiencies of up to 90% for plasmid DNA and 99% for oligonucleotides, like siRNA
• Over 650 cell-type specific protocols that have led to direct transfection success
• Transfection of a wide range of substrates, including DNA, mRNA, miRNA, siRNA, peptides or proteins.
• Transfection of hard-to-transfect cells, including primary cells, stem cells, neurons and cell lines, as well as cells in adherence.

Question 4

CAR-T Treatment Relapse Factors

1. Antigen Loss/antigen escape (Target cells no longer expressing CD19)
   a. Antigen escape is tumor cells expressing alternative forms of the target antigen that lack the extracellular epitopes recognized by CAR T cells
   b. Antigen loss - cancer cells typically lose surface antigens following natural or therapy-induced selective pressure and these antigen-loss variants are often the population that causes therapy-resistant relapse
2. Lack of CAR T-cell persistence

Question 5

Co-stimulatory Domains

4-1BB domains:

• lower risk of toxicities
• higher T cell endurance
• lower peak level of T cell expansion

CD28 co-stimulatory domains:
- rapid Car-T cell activity onset and subsequent exhaustion

Question 6

APHERESIS:
Total Blood Volume (TBV)
Based on donor’s size, sponsor’s requested cell count, product volume targets
Processing centers use validated SOP’s
- Sponsor-required deviations may require SOP Revision
- Revision(s) may require validation of the new procedure
- Changes will require training of protocol-specific steps
T cell therapy starts with obtaining the patient’s WBCs by leukapheresis, an apheresis method that separates white blood cells from whole blood. The blood components are usually separated by density with continuous or intermittent centrifugation methods using density gradient media

Question 7

Maintenance of Cells in Culture

• Places selective pressure on the cells
• Cells in culture age - may accumulate genetic and epigenetic changes
• Cells may experience changes in differentiation behavior and function
• Understanding of genomic stability during cell culture are still evolving

The presence of cellular subsets at culture initiation can negatively impact T-cell activation and expansion

Question 8

CAR molecules consist of three parts

1. Extracellular domain - scFv receptor
2. Transmembrane domain
3. Intracellular domain

CAR T cells bind antigen independent of HLA’s, which are downregulated in cancer cells, a key mechanism for cell escape from immune surveillance

Question 9

CAR T cell processing

1. selection
2. pre-activation
3. activation
4. gene modification (transduction or transfection)
5. expansion ex vivo
6. freezing
7. re-infusion

Question 10

CAR T administration - patient pre-treatment

Lymphodepleting chemotherapy to enhance CAR T-cell EPP*
Typical treatment is with fludarabine and cyclophosphamide (2 to 14 days)

Question 11

Target cell purification

Enriching for T cells or specific T-cell subsets using antibody conjugated magnetic beads
Example: enrichment based on the expression of CD62L, CD4 and CD8

Question 12

Activation:

A T-cell activation step is required for adequate transduction and expansion

• Polyclonal stimulation using soluble anti-CD3 antibodies or immobilized CD3 and CD28 antibodies
• Immobilized anti-CD3 antibodies provide better cross-linking and activation of T cells
• CD28 antibodies activate costimulatory pathways in the target cells

Several methods that mimic the natural stimulation of T cells (TCRs) have been developed and implemented. A common approach is to add OKT3 (anti-CD3 monoclonal antibody (mAb)) and interleukin (IL) 2.

Question 13

Dynabeads

Paramagnetic beads
Bead removal
- Disrupting the T-cell/bead aggregates via agitation
- Passing the suspension through a strong magnetic field
- Beads are retained, cells are collected

Question 14

Autologous CAR T - Starting material

Challenges:

• Obtaining pure and viable starting material
• Significant batch-to-batch variability exists with autologous cells

Question 15

Allogeneic Protocols
Issues:
- Overcoming GvHD
- Gene editing methods - used to disrupt the expression of endogenous T-cell
receptors and/or MHC on allogeneic T cells
- CRISPR-Cas9
- Transcription activator-like effector nucleases (TALEN)
- Zinc finger nucleases (ZFN)

Question 16

T Cell Product Characterization

Most manufacturers characterize the final CAR T cell product by flow cytometry, with many characterization panels currently in use. Several ranges/measures exist to determine the total T-cell number via CD3 expression; to characterize the CD4+ and CD8+ subsets within this population; to detect transduced and not transduced cells; and to identify any contaminating cell subsets, such as monocytes/macrophages.

Question 17

CAR T - Issues

The early stage of the current clinical trials mean that target cell numbers have not been established. Therefore, the scale of the process is still not defined.

Transduction - oncogenic gene insertion

A relatively new method of CAR gene delivery is the use of transposon/transposase systems. A transposon is a sequence of DNA with the ability to change position within a genome via transposase excision and insertion

potency assays are one of the main challenges in CAR T cell product characterization during QC

Question 18

Activation

Anti-CD3/CD28 antibody coated magnetic beads as artificial antigen presenting particles

During expansion. the beads were used to continuously stimulate the cells. Cytokine production was 10-100 fold higher suggesting that activation is stronger using bead-activation compared to other methods such as activation with anti-CD3 antibodies and IL-2