Arginine and Cancer Immunotherapy Flashcards
what are CARs?
Chimeric Antigen Receptors (CARs)
Artificially designed protein surface receptors:
- That recognise specific antigens on cancer cells
- Activate immune cells (like T cells) to directly recognise and destroy tumour cells
how do CARs become activated?
Recognises TAA on surface, signal through scFv to intracellular T cell domains = antigen-specific T cell activation
why do CAR-T cells improve anti-tumour immunity?
Only 1% of endogenous T cells recognise tumour antigen
CAR-T functions to expand population of T cells specific for tumour antigen
how are CAR-T cells limited by antigen expression?
Low expression of antigen on tumour means CAR-T function is poor
- the antigen can be cleaved and turned over so CAR-T can’t recognise and bind to tumour
how are CAR-T cells made?
- collect blood from patients - autologous
- use magnetic beads or FACS to isolate T cells
- transduce T cells in vitro with viral gene transfer (lentivirus, adenovirus) to express CAR - generates CAR-T
Efficiency depends on donor cells and technique
why is isolating T cells from blood difficult?
if patients have leukopenia or myeloid leukaemia with many blast cells diffuse in the blood, the T cell level will be low
what is the CAR-T cell mechanism of action?
CAR-T cells recognise TAA e.g. GD2 expressed on 70% of NBM cells
- CAR-T cells bind and release perforin, granzyme B which induce pores and lyse the tumour cell - direct tumour killing
in vitro - 90% killing
in vivo - more difficult due to TME
where has CAR-T been successful?
acute lymphoblastic leukaemia - most successful CAR-T trial
- anti-CD19 CAR-T killed 99% of blasts of patients
- Over time after infusion, CAR-T cells persist for weeks to kill the tumour - patient is responding to treatment and likely have good outcome
- they continue to be detectable in peripheral blood by flow cytometry
- CAR-T leads to ALL clearance
- When CD3+ CAR-Ts expand by day 10, the CD34+CD19+ ALL blasts are killed in the blood at day 10 – direct correlation of increased CAR-T and killed cancer
where has CAR-T been less successful?
solid tumours
- harder to target tissue tumours compared to circulating blasts - CAR-T needs to migrate to tissue
- success has not been seen for neuroblastoma (NBM)
- Most NBM patients don’t respond to CAR-T
- Also true for mesothelioma of lung and anti-mesothelin CAR-T, and in AML and anti-CD33 CAR-T - CAR-T not successful in this blood cancer
what does CAR-T depend on to function?
normally, when CAR-T recognises TAA they proliferate and expand
- the more CAR-T cells, the more chance for tumour lysis
- the environment enables their expansion - depends on cytokines like IL-2 and nutrients like amino acids
- CAR-T needs to also proliferate and expand to maintain anti-tumour response
- This means they are still detectable and can kill tumour
- Nutrients are needed to support T cell growth and expansion
- If there is nutrien and cytokine deficiency, CAR-T can’t expand
what markers show that CAR-T is less successful in solid tumours?
CAR-T less successful in solid cancers – cant infiltrate and persist
- Cells undetectable in blood with flow cytometry
- Only detectable by DNA amplification with qPCR - numbers are too low
- Absence/low persistence of CAR-T cells correlated with faster time to progression
- No benefit for patients
why does CAR-T fail in solid tumours?
Several tumour are characterized by an complex environment and highly immunosuppressive to T cells - TME is altered and this affects T cell function/expansion
- Made up of several cells types recruited by the tumour to support its growth - e.g. Tregs, MDSCs, TAMs
- These cells compete for nutrients, amongst which is the amino acid arginine
- neuroblastoma consumes a lot of arginine; due to high intrinsic expression of the enzyme Arginase
what amino acid is essential for T cells in tumours?
One of the most important T cell nutrients is arginine amino acid
- semi-essential amino acid – if in nutrient-rich media, T cells don’t need arginine, but if in infection/cancer, absence of arginine means T cells can’t divide
Tumours express arginase and consumes lots of arginine
- Metabolises arginine faster and removes from environment
what happens to CAR-T cells in a low arginine environment?
If lack arginine in environment, CAR-T can’t proliferate - no persistence
what is neuroblastoma?
Neuroblastoma is a cancer that starts in very early forms of nerve cells
- It is the most common extracranial solid cancer in children
- Has one of the lowest survival rates of all childhood cancers.
- The 5-year survival rate in England is ~65%
how was NBM affects on T cell proliferation shown via transwell?
Transwell -
- Tumour cells at bottom
- Membrane above with T cells on top
- this prevents physical cross-talk and contact-dependence
- Can see how tumour environment affects T cells with no direct contact between T cell and tumour
- In transwell system with no contact, T cells are still inhibited
Something in the environment that tumour cells release or consume that affects T cell expansion
how does neuroblastoma affect T cell proliferation?
Co-culture of NBM with T cells:
- Absence of tumour =100% proliferation
- Increasing conc of NBM cell lines = drastic reduction in proliferation, close to 0% in LAN-1 cell line
- Dose-dependent reduction and T cells can’t recover even with less NBM cells present
- something in NBM environment stops T cell proliferation
how was arginine shown to be depleted by NBM in mice?
used mice with spontaneous NBM development (TH-Myc-N mice) and measured arginine in serum
- In plasma of mice with large tumour, arginine is low compared to control mice
- If tumour is smaller, arginine level is similar to control
- Cancer growth affects levels of plasma arginine
Took tumour to look at arginine in TME - digested tumour
- Took healthy adrenal gland from mice with no tumour as control – high arginine
- Tumour tissue had reduced arginine compared to gland
- NBM is depleting the arginine
is it something that is released by NBM or consumed by NBM that inhibits T cell proliferation?
Screened secreted factors like IL-10, TGFb, IL-6 but none of these were found in NBM
- Instead found a change in arginine pathway in NBM
- Arginine uptake must be important for NBM growth - must be depleting arginine
how was arginine shown to be depleted by NBM in patients?
Human tests – isolate blood and then plasma and run ELISA for arginine - measured conc of arginine in plasma
- In healthy children, high plasma arginine levels
- In NBM patients, drastic reduction in plasma arginine levels
In these patients, T cell infusion won’t work as they won’t be able to expand in the arginine-low TME
what is arginine?
a semi essential amino acid required for cell viability and proliferation
how is arginine taken up?
arginine is external, so tumour cells and T cells uptake it with transporters (CAT-1) and transport it intracellularly
an unknown receptor enables access of arginine into cell
how is arginine metabolised?
Arginase-1/2 (two isoforms) = M2
iNOS = M1
- These transform arginine from plasma into products
- arginine is at the centre of metabolic pathways
how is arginine metabolised by arg1/2?
Arg1/2 convert arginine to ornithine and releases urea as byproduct
- Arg1 is cytoplasmic
- Arg2 is mitochondrial - Arg-2 is transcribed in the nucleus and imported to the mitochondria via characteristic N-terminal mitochondrial targeting sequences (MTS)
- They have same function and similar sequence
ornithine is important for polyamine synthesis and cell proliferation
