A brief history of CAR T-cells

Question 1

What are CAR T-cells

Answer 1

Chimeric Antigen Receptor targeting T-cells

Question 2

What was the first development in CAR T-cells?

Answer 2

First development in CAR T-cells:
1909: Paul Erlich proposes that the immune system usually supresses tumour formation. This concept became known as the “immune surveillance” hypothesis. This was later confirmed.

Question 3

What is the difference in the way B-cells contribute to tumour cell death and the way T-cells contribute to tumour cell death?

Answer 3

B-cells secrete Abs that recognize tumour cell antigens. NK cells are guided by these Abs, recognize the tumour and kill it by secreting cytokines.
T-cells recognize peptides presented by the tumour cell. They secrete cytokines and granzymes to kill the tumour cell.
Difference: recognizing a surface antigen and recognizing a peptide presented by the tumour cell.

Question 4

What is adoptive T-cell immunotherapy? Name some examples.

Answer 4

Adoptive T-cell immunotherapy is the use of the T-lymphocytes to kill tumour cells.
E.g.:
•Haematopoeitic stem cell transplantation/bone marrow transplantation: Ablation of immune cells from patient and transplant of haematopoeitic stem cell of donor. Patient gets immune system donor.
•Tumor Infiltrating Lymphocytes (TIL) immunotherapy: TIL are enriched before the T-cell infusion. Showed impressive results.

Question 5

What is the CAR T-cell concept?

Answer 5

It unites the T-cell biology and the antibody biology. A CAR T-cell recognizes the surface antigen and has the strength of a T-cell

Question 6

Why was the first generation of CAR T-cells not so successful in clinical trials?

Answer 6

First generation CAR T-cells were not so succesfull in clinical trials. What was missing was the co-stimulation. Addition of CD28 or another costimulatory molecule gave not only a good response to the tumours, but also a sustained response with proliferation that remained for several activation rounds and in vivo activity. These are the second generation CAR T-cells.

Question 7

What are the differences between a CAR and a physiological T-cell receptor?

Answer 7

CAR:

• Extra-cellular antigens only
• No peptide processing required
• Not restricted by patient HLA
• Not limited by HLA down-regulation
• Designer signaling domain

Physiological T-cell receptor complex:

• Extra- and intracellular antigens
• Restricted by the patient’s HLA specificity
• No ligand on HLA-negative donor

Question 8

How do you make a CAR T-cell?

Answer 8

1. Make the DNA to have the sequence of all the elements
2. Clone it into a viral vector
3. Transfect a producer cell line (periferal blood T-cells) with the virus (e.g. lentivirus) with the viral vector. They start producing the CAR in the surface of the T-cell.

Tadaaa CAR T-cell

Question 9

Which types of cancer does a CD19 CAR T-cell target?

Answer 9

CD19 CAR T-cell targets the CD19 B-cell surface marker, so it’s on the surface of leukemia and lymphoma that have a B-cell origin. This lead to 2 products that are now on the market (but very expensive).

This sparked new research for MML and also solid tumours.

Question 10

What are the two types of challenges that development of CAR T-cells face

Answer 10

Biological challenges and manufacturing challenges

Question 11

What are biological challenges of developing CAR T-cells?

Answer 11

Biological challenges of developing CAR T-cells:

• Antigen escape
• On-target toxicity
• Off-target toxicity
• Intrinsic CAR T-cell dysfunction
• Suppressive tumour microenvironment

Question 12

What are manufacturing challenges of developing CAR T-cells?

Answer 12

• High cost
• Limited availability
• Manufacturing time delays
• Heterogeneity of personalized products

Question 13

What is a solution for antigen escape?

Answer 13

In antigen escape, the tumour downregulates the antigen

Solution: targeting two antigens at the same time, armoured CARs

Question 14

What is on-target toxicity?

Answer 14

o On-target toxicity: when the targeted antigen is not only expressed on the tumour, but also on healthy cells (maybe expressed lower). CAR T-cells recognize healthy tissue

Question 15

What is an example of on-target toxicity and how can this be solved?

Answer 15

You can infuse with Ig if B-cells are targeted and this gives problems.

When antigen is also expressed on mucosal tissue, colitis can occur.

Solved by: target selection, sometimes local infusion of the cells is used, transient expression of CAR to minimize toxicity, optimize affinity CAR.

Question 16

What is off-target toxicity?

Answer 16

Toxicity is not necessarily coming from the binding of the CAR T-cells.

Question 17

What are examples of off-target toxicity in CAR T-cells?

Answer 17

cytokine release syndrome, overactivation of the immune system

Question 18

What is CAR T-cell dysfunction?

Answer 18

CAR T cells get exhausted and disappear

Question 19

When is a suppressive tumour environment especially a big problem?

Answer 19

In solid tumours

Question 20

How expensive is CAR T-cell treatment per injection?

Answer 20

+- 250.000-300.00 euro/injection

Question 21

Why are manufacturing time delays a big problem for cancer patients?

Answer 21

Sometimes patients don’t have that much time left.

Question 22

How can CAR T-cells persist longer?

Answer 22

CAR T-cells that have 4-1 BB in their intracellular domain persist longer. The earlier in the differentation stage the T-cell is, the longer it can proliferate

Question 23

What is the advantage of CD28/CD3zèta CAR T-cells?

Answer 23

Advantage of CD28/CD3zèta CAR T-cells:

They have a higher cytokine production and better cytotoxic function

Question 24

What way of improving CAR T-cells has the most attention for in research at the moment?

Answer 24

Costimulation

Question 25

What are armoured CARs?

Answer 25

T-cells that co-express CAR with something else: cytokines, mAbs, chemokines to help CAR T cell with migration to destination, etc.

Question 26

How can you evaluate the efficacy of CAR T-cells?

Answer 26

You can evaluate the efficacy of CAR T-cells by:
•Flow cytometry: detection and immunophenotype of T-cells: restricted to looking at several surface molecules
•qPCR vector copy number in peripheral blood: CAR T-cell persistance: indirectly check if the CAR T-cells are there. DNA there? CAR T-cell also probably there.
•Genome sequencing for vector integration sites: clonality (follow the clones) and see the same clone that was transfected is the clone that is present.

Question 27

What are clonal kinetics?

Answer 27

Clonal kinetics is a new way of evaluating the efficacy of CAR T-cells in the patients:
Clonal kinetics (follow the clones) uses single-cell transcriptional profiling of CAR T-cells.
o Instead of using the bulk of the peripheral blood to go and see what is happening to every single cell, single cell RNA sequencing on blood which gives the opportunity to look at paired analysis of the T-cell receptor sequences. Every T-cell has their own T-cell receptor. Tracking the sequences of the T-cell receptors enables tracking the clones.
oThis can be combined with transcriptomics to make the transcriptional profile of this specific cell.
oUsing this method they found the majority of clones that proliferate and expand in the patient in vivo originate from cells that had a priori (from the beginning) higher levels of genes associated with cytotoxicity and proliferation in the original infusion product

Important for further development of CAR T-cells!