Neoantigens

Question 1

1. What are the ways to classify tumour antigens, shared and unique?

Answer 1

Universal, oncoefetal, differential, mutated, viral, overexpressed, altered glycoprotein.
They can be classic tumour antigen or neoantigens.
TAA: present in all tissues, central tolerance can trigger autoimmunity.
Neoantigens: unique and specific, can be stem like, not found in genome mutated self peptides, increased immunogenicity, governed by peripheral tolerance

Question 2

2. What are the technologies used for tumour antigen and epitope identification and what are their limitations?

Answer 2

• Expression cloning: Take a tumour, CDNA library get the whole sequence, infect it in a t-cell from the patient, expand the cell population, get CDNA and identify epitope.
Limitations: Maybe t-cells don’t respond, long process and difficult, limited throughput.
• Epitope prediction: CDNA sequence, identify gene, identify peptide, rank epitope based on affinity
Limitations: High false positives because : t-cells deleted from thymic otogeny, no TCR, inaccurate algorithms, some epitopes not produced by proteolytic cleavage, or peripheral tolerance. Have to have extensive knowledge of peptide interaction with HLA, therefore the ones we know about most are the ones most studied. Doesn’t account for epitope generation.
• Peptide elution: Take tumour cells, purify HLA molecule, elute peptide from it and mass spec this peptide to identify it. Carry out the same procedure with normal cells and subtract the background.
Limitations:
• Need a bunch of tumour cells, poor efficiency of recovering peptide, need to do t-cell assay to confirm repertoire, time consuming and expensive.
• Neoeptiope discovery: Immunoaffinity of HLA complexes from tumour tissue, recover HLA peptides, identify high mutation sequence and canditates of class 1 and class 2 MHC using mass spec , shared and unique mutations, validation of identification and immunogenicity, create vaccine and do immunotherapy
Limitations:
Time consuming,expensive, immune escape, selecting the best epitope, different immunogenicity, might not work for mutations with low mutation rates, how to deliver the epitope.

Question 3

3. Briefly explain how the neoantigens are used in immunotherapy? What are the diversities found of neoepitopes found across aall tumour types? How are they being employed?

Answer 3

• They are selected from tumour samples and identified using mass spec along with HLA class found in the complex. Once the RNA and sequence is identified, they use this peptide to create a vaccine, they can use the RNA or the DNA, after validating the effectiveness they are injected into the patient and monitored, the theory is that it should induce an immune response against a certain tumour type bearing this antigen, it does this by priming the peripheral immune system using this vaccine.
• Neoepitope effectiveness depends on the cancer tissue type and the patient themselves. There can be immunediting of the neoepitope, they can be driver epitopes or non drivers.