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Typical targets of checkpoint inhibitors (usually humanized monoclonal antibodies) are molecules such as Programmed cell death protein 1 – Wikipedia (PD-1), PD-L1 – Wikipedia, CTLA-4 – Wikipedia. Applying checkpoint inhibitors (Cancer immunotherapy – Wikipedia) when such molecules are expressed by cells within a given tumor is akin to releasing the brakes of tumor antigen-specific Tumor-infiltrating lymphocytes – Wikipedia (TILs) present within them, empowering them to eliminate such tumors. This is the purported mechanism of action (MOA) of checkpoint inhibitors. However, their major problem is non-specificity, as in potential for off-target responses since they could release the brakes off of all T cells that express them, not just those that are tumor antigen-specific. To be able to effectively harness TILs to destroy any given tumor,

  1. Tumor should have TILs within it in the first place.
  2. Tumor should express ‘neoantigens’, i.e., tumor-specific antigens.
  3. TILs being used in immunotherapy (1) should be specific for peptides derived from antigens specifically expressed by tumor (2).

2 and 3 present formidable challenges because unless both conditions are met, cancer immunotherapy could end up attacking the body itself, with tragic results, since even death is possible. For example, while details haven’t been divulged, higher than anticipated clinical trial deaths have led leading cancer immunotherapy company, Juno Therapeutics – Wikipedia, to recently scrap its lead CAR-T therapy (1).

Thus, checkpoint inhibitors notwithstanding, to assess whether TILs might be induced to specifically target a given tumor, the experimental process entails

  1. Assessing whether the tumor has TILs and if yes, then isolating them.
  2. Assessing whether tumor expresses tumor-specific antigens by comparing tumor and matched healthy tissue.
  3. Confirming tumor cells themselves can indeed process and present these tumor-specific antigens within MHC class I (to tumor-specific cytotoxic CD8+ T cells) or MHC class II (to tumor-specific helper CD4+ T cells) molecules, an extremely technically challenging task made even more arduous by the following caveats,
    1. Even if a given tumor expresses several tumor-specific antigens, it might not present peptides derived from them since tumors are known to have aberrant antigen processing and presentation pathways (2).
    2. Differentiating immunologically relevant from irrelevant peptides is turning out to be more complex than envisaged (3).
    3. MHC class II-binding peptides are longer and bind more promiscuously compared to those that bind class I, making binding predictions much more technically challenging.
    4. Tumor cells rarely express MHC class II.
  4. Assessing whether isolated TILs are tumor-specific, i.e., specifically bind tumor-derived pMHC (peptides bound to Major histocompatibility complex – Wikipedia (MHC)).
  5. Assessing effect of checkpoint inhibitors on such tumor-specific TILs. If tumor-specific TILs don’t express the targets of such checkpoint inhibitors and yet also don’t attack and eliminate the tumor,
    1. They could be T regs (Regulatory T cell – Wikipedia) in which case it may be difficult to impossible to engineer effective anti-tumor immunity using them.
    2. If not Tregs, may need to assess whether such TILs express novel ‘brakes’, targets for novel checkpoint inhibitors.

Plenty of obstacles stand in the way of these experimental imperatives. Steps 3 and 4 the most fiendishly complicated, no one method exists to reliably assess both in a high-throughput fashion nor are existing In silico – Wikipedia approaches fool-proof (4). There are other hurdles as well.

  • Not all tumors express abundant tumor-specific antigens (4, 5, 6, 7).
  • Some tumors can mutate ferociously, i.e., cancer Immunoediting – Wikipedia (8, 9), which reduces the likelihood of finding tumor-specific TILs.
  • Rather than being tumor antigen-specific, some TILs may be specific for antigens that both tumor and normal tissue cells express (cross-reactivity).
    • T cells function by recognizing and binding pMHC through their T-cell receptor – Wikipedia (TCR). Central tolerance – Wikipedia, the developmental process of eliminating T cells reactive to cells of the body in which they develop, is incomplete.
    • The TCR is inherently cross-reactive, capable of binding >1 pMHC, i.e., can bind peptides derived from different antigens presented by the same MHC molecule (cross-reactivity) (10).
  • Thus, possible for checkpoint inhibitors to trigger autoimmunity.

However, promise of this approach is sustained by examples of tumor-specific TILs (11) having been found in

  • Human non-small cell lung cancer (12).
  • Human melanoma (13, 14, 15, 16, 17, 18, 19).
  • Human AML (Acute Myeloid Leukemia) (20).
  • Human CLL (Chronic Lymphocytic Leukemia) (21, 22).

Bibliography

1. http://www.xconomy.com/seattle/2…

2. Leone, Patrizia, et al. “MHC class I antigen processing and presenting machinery: organization, function, and defects in tumor cells.” Journal of the National Cancer Institute 105.16 (2013): 1172-1187. https://oup.silverchair-cdn.com/…

3. Gilchuk, Pavlo, et al. “Discovering protective CD8 T cell epitopes—no single immunologic property predicts it!.” Current opinion in immunology 34 (2015): 43-51. https://www.researchgate.net/pro…

4. Gfeller, David, et al. “Current tools for predicting cancer-specific T cell immunity.” OncoImmunology 5.7 (2016): e1177691. https://www.researchgate.net/pro…

5. Alexandrov, Ludmil B., et al. “Signatures of mutational processes in human cancer.” Nature 500.7463 (2013): 415-421. https://www.researchgate.net/pro…

6. Gubin, Matthew M., et al. “Tumor neoantigens: building a framework for personalized cancer immunotherapy.” The Journal of clinical investigation 125.9 (2015): 3413-3421. Tumor neoantigens: building a framework for personalized cancer immunotherapy

7. Schumacher, Ton N., and Robert D. Schreiber. “Neoantigens in cancer immunotherapy.” Science 348.6230 (2015): 69-74. http://pmpathway.wustl.edu/files…

8. Matsushita, Hirokazu, et al. “Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting.” Nature 482.7385 (2012): 400-404. https://www.researchgate.net/pro…

9. Rooney, Michael S., et al. “Molecular and genetic properties of tumors associated with local immune cytolytic activity.” Cell 160.1 (2015): 48-61. http://wulab.dfci.harvard.edu/si…

10. Tirumalai Kamala’s answer to How is it possible that a T Cell Receptor (TCR) recognises as few as 1-3 residues of the MHC-associated peptide?

11. McGranahan, Nicholas, et al. “Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade.” Science 351.6280 (2016): 1463-1469. https://www.ncbi.nlm.nih.gov/pmc…

12. Rizvi, Naiyer A., et al. “Mutational landscape determines sensitivity to PD-1 blockade in non–small cell lung cancer.” Science 348.6230 (2015): 124-128. https://www.ncbi.nlm.nih.gov/pmc…

13. Kvistborg, Pia, et al. “TIL therapy broadens the tumor-reactive CD8+ T cell compartment in melanoma patients.” Oncoimmunology 1.4 (2012): 409-418. http://www.tandfonline.com/doi/p…

14. Andersen, Rikke Sick, et al. “High frequency of T cells specific for cryptic epitopes in melanoma patients.” Oncoimmunology 2.7 (2013): e25374.; http://www.tandfonline.com/doi/p…

15. Robbins, Paul F., et al. “Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells.” Nature medicine 19.6 (2013): 747-752. http://ggdpathway.wustl.edu/file…

16. Lu, Yong-Chen, et al. “Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions.” Clinical Cancer Research 20.13 (2014): 3401-3410. http://clincancerres.aacrjournal…

17. Frøsig, Thomas Mørch, et al. “Broadening the repertoire of melanoma-associated T-cell epitopes.” Cancer Immunology, Immunotherapy 64.5 (2015): 609-620. https://www.researchgate.net/pro…

18. Linnemann, Carsten, et al. “High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma.” Nature medicine 21.1 (2015): 81-85.

19. Cohen, Cyrille J., et al. “Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes.” The Journal of clinical investigation 125.10 (2015): 3981-3991. Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes

20. Berlin, C., et al. “Mapping the HLA ligandome landscape of acute myeloid leukemia: a targeted approach toward peptide-based immunotherapy.” Leukemia 29.3 (2015): 647-659.

21. Rajasagi, Mohini, et al. “Systematic identification of personal tumor-specific neoantigens in chronic lymphocytic leukemia.” Blood 124.3 (2014): 453-462. ; http://www.bloodjournal.org/cont…

22. Kowalewski, Daniel J., et al. “HLA ligandome analysis identifies the underlying specificities of spontaneous antileukemia immune responses in chronic lymphocytic leukemia (CLL).” Proceedings of the National Academy of Sciences 112.2 (2015): E166-E175. http://www.pnas.org/content/112/…

https://www.quora.com/What-experiments-can-we-do-with-TILs-to-work-out-the-mechanism-of-action-of-checkpoint-inhibitors/answer/Tirumalai-Kamala

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