In the Excel spreadsheet attached you will find a list of 2,000 x 9mer immunopeptides identified from an immunoprecipitation of MHC Class I peptides

In the Excel spreadsheet attached you will find a list of 2,000 x 9mer immunopeptides identified from an immunoprecipitation of MHC Class I peptides from a melanoma cell line. The list contains the gene name from which the peptides are derived and the peptide sequence. These peptides are TAAs (they are not sorted based on mutation of the DNA, which would make them TSAs). Remember, a major issue of using TSAs as a vaccine source is that some tumours have only 5-50 mutations in their DNA and it is highly unlikely that any of these 50 mutant proteins will produce mutant peptides that bind to a particular MHC Class I allele. As a result, we focus on TAAs, as there are a relatively large number of TAAs that can be found in any particular cancer, making them a more likely source of vaccine sequences.

Steps:

1. Using the tutorial for NetMHC4.0 as a guide, can you input this entire list of 2000 peptides into the box using peptide input NOT fasta input. You can then select the appropriate HLA subtypes to analyse against. Which HLA alleles should you choose? The patient from which this cell line was derived has the following alleles with differing HLA allele status and the indicated tabs can be used to chose each alleles:

HLA-A*02:01andHLA-A*01:01

HLA-B*44:03andHLA-B*57:01

HLA-C*06:02andHLA-C*16:02

2.The threshold for 'strong binders' can be chosen by using this tab:

"Threshold for strong binders"

This can be set at 0.1%. As a result, the software will list strong binders as 'SB' and weak binders (2%) as 'WB' and no annotation means very poor binders.

3.However, perhaps a key element is the actual affinity (Kd). Tick the box for Sort by predicted affinity and also save out put in XLS format. So that you will be able to count and copy the sequences that are of the highest affinity. In general, we can say that the best sequences are below 10nM affinity. You can save all the files in Excel spreadsheets.

Questions for the assessment:

Question 1. Out of the 2,000 peptides, howmanystrongbindingpeptides(below 10nM affinity, Kd) canyoufindineachof the six HLA classes?

Question2. Out of 'strong-binding peptides', are there any peptides in common betweenHLA-A*02:01and HLA-A*01:01?

Question3. DothestrongbindingpeptidesyoufindforHLA-A*01:01match themotifprediction for this allelefromtheSeqMotiftab?

Question 4. Do any of the 'weak-binding' peptides for HLA-A*01:01 share themotifprediction for this allelefromtheSeqMotiftab? What do you think makes them weak binding?

Question 5. These are not necessarily 'tumour specific antigens (TSAs) in our list of 2000 immunopeptides, although some of them could be. However, we should consider them at least TAAs because they all derive from the melanoma with the indicated alleles (i..e HLA-A*02:01,HLA-A*01:01, etc). Based on the results of the questions 1-4, are you optimistic that 'shared' TAAs can be found between patients with different HLA-A alleles? The probability of finding shared TAAs between patients has implications for the concept that vaccines that focus on TAAs might be shared between patients and is therefore more cost effective.

For example, you can take one of your strong binding peptides to HLA-A*01:01, such as this one: LTDRGVMSY with an affinity (Kd) of ~ 3 nM. Enter that single peptide into the PEPTIDE tab box, and choose 20 different HLA-A sub-alleles from the drop down tab (such as HLA-A*01:01 itself, and HLA-A*02:01, HLA-A*23:02, etc). When you do this, how many HLA-A alleles also bind this one peptide with a strong binding affinity to HLA-A*01:01? This will give you a feel for whether any single TAA can be shared between patients of a different HLA-A subclass.

Please write a 200-300 word summary of your findings from this analysis answering the questions and tasks above. Please note: For this assessment you will not be able to see the posts from other students until you post your answers. Once posted you will not be able to edit your submission so please make sure you are happy with your submission before posting. If you do have any problems please email us.