Peptide Characterization

Examination of the identified peptides reveals certain structural motifs. All peptides contain nine amino acid residues and display a conserved leucine residue at position 2 and limited (alanine, leucine, valine) variability in position 9. It is known that the peptide binding site of the MHC class one molecule contains six pockets (A-F). Of the six pockets, two are directly involved in peptide binding: the B and F pockets. The peptide’s N-terminal end is anchored in position 2, the B pocket, and the C-terminus is contained in position 9, the F pocket. The findings here support the prior observations that the B pocket anchor for the HLA-A*0201 are small aliphatic residues while the F pocket anchor are small,

aliphatic hydrophobic residues.6

Although the genes from which the peptides are derived are known (Table 5.2) 33 % of the peptides are from proteins that have only been detected on the mRNA level therefore the protein function in the cell is unclear. These peptides are in bold and are LLIPGLATA, ALSEKIVSV, FLISLVFLL, KLLVTVTAV, LLIAVNSPV, and ALTGWLPEV. Several other peptides could be good targets for therapeutic applications based upon such issues as their tissue distribution and immunogenicity. Immunotherapy trials directed at cancer associated

antigens have already been performed, and several of the peptides discovered in this study have a narrow tissue distribution for hematopoietic tissue, which could make them good targets for myeloid leukemia directed therapy.

Several peptides that were identified have interesting properties that may make them candidate anti-cancer immune targets. Ankyrin repeat domain 17

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(ANKRD17), the protein from which LLIERGASL is derived, is the source protein

from which the breast cancer antigen NY-BR-167 is found suggesting that this

protein is expressed in multiple cancer types and may be a good source of antigens for various HLA supertype. ALTPVVTL is from the cyclin dependent kinase 4 protein (CDK4), a member of the protein kinase superfamily. CDK4 is implicated in integrating oncogenic signaling and inactivation of the tumor

suppressor Rb.8 CDK4 small molecule inhibitors have been developed as

potential cancer therapeutics, and an immune therapy aimed at this regulatory

protein could be developed.9 Finally, LLLGGTALA is derived from the protein

neutrophil elastase (ELANE) which is the source protein of the leukemia

associated antigen PR1.10, 11

In addition, a deletion polymorphism has been reported for the

ALFPGVALL peptide, which would cause a frame shift mutation starting from the F pocket. If this polymorphism leads to a peptide that is unable to bind HLA- A*0201, the ALFPGVALL peptide could be a leukemia associated, minor

histocompatibility antigen. Minor histocompatibility antigens12 are important in

effecting cures for leukemia patients who undergo allogeneic stem cell

transplantation because the donor’s T lymphocytes have never been tolerized to

the allelic form of the peptide produced by the recipient/patient.13 In the case of

ALFPGVALL, if the donor is homozygous for the allele that yield the frame-shift mutation, the donor’s T lymphocytes which are transferred into the patient during the transplant procedure will have never “seen” the ALFPGVALL peptide and will regard it as foreign leading to immune-mediated attack of the cell presenting

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ALFPGVALL. Another peptide, FLLPTGAEA, is derived from cathepsin G and contains an A/G polymorphism in position 6, which could also make it a minor histocompatibility antigen like ALFPGVALL. Additionally, cathepsin G is a protein, like neutrophil elastase, that is produced in primary granules in myeloid hematopoietic tissue, and as such, is expressed primarily in the bone marrow potentially making it a good target for leukemia based immunotherapy.

5.4 Conclusions

Affinity chromatography and liquid chromatography coupled to mass spectrometry and database searching are effective in identifying HLA-A*201 presented peptides on leukemia. Eighteen peptides predicted to be tight binders have been identified. Peptides are nine amino acid residues in length and have leucine in position two and valine, leucine or alanine in postion nine. Several of the identified peptides have properties that may make them candidate anti- cancer immune targets.

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5.5 References

1. Delves, P. J.; Roitt, I. M., The immune system - First of two parts. New

England Journal of Medicine 2000, 343, (1), 37-49.

2. Delves, P. J.; Roitt, I. M., Advances in immunology: The immune system -

Second of two parts. New England Journal of Medicine 2000, 343, (2),

108-117.

3. Jensen, P. E., Recent advances in antigen processing and presentation.

Nature Immunology 2007, 8, (10), 1041-1048.

4. Cotran, R., Pathologic Basis of Disease. Saunders/Elsevier: Philadelphia,

PA, 2010.

5. Sette, A.; Sidney, J., Nine major HLA class I supertypes account for the

vast preponderance of HLA-A and -B polymorphism. Immunogenetics

1999, 50, (3-4), 201-212.

6. Sidney, J.; Peters, B.; Frahm, N.; Brander, C.; Sette, A., HLA class I

supertypes: a revised and updated classification. Bmc Immunology 2008,

9.

7. Hofmann, S.; Gluckmann, M.; Kausche, S.; Schmidt, A.; Corvey, C.;

Lichtenfels, R.; Huber, C.; Albrecht, C.; Karas, M.; Herr, W., Rapid and sensitive identification of major histocompatibility complex class I-

associated tumor peptides by nano-LC MALDI MS/MS. Molecular &

Cellular Proteomics 2005, 4, (12), 1888-1897.

8. Paternot, S.; Bockstaele, L.; Bisteau, X.; Kooken, H.; Coulonval, K.; Roger,

P. P., Rb inactivation in cell cycle and cancer The puzzle of highly

regulated activating phosphorylation of CDK4 versus constitutively active

CDK-activating kinase. Cell Cycle 9, (4), 689-699.

9. Shapiro, G. I., Cyclin-dependent kinase pathways as targets for cancer

treatment. Journal of Clinical Oncology 2006, 24, (11), 1770-1783.

10. Molldrem, J. J.; Clave, E.; Raptis, A.; Hensel, N.; Barrett, A. J., Cytotoxic T

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transfected B-cell line and inhibit growth of clonogenic chronic myeloid

leukemia (CML) cells. Blood 1997, 90, (10), 2242-2242.

11. Rezvani, K.; Yong, A. S. M.; Mielke, S.; Savani, B. N.; Musse, L.;

Superata, J.; Jafarpour, B.; Boss, C.; Barrett, A. J., Leukemia-associated antigen-specific T-cell responses following combined PR1 and WT1

peptide vaccination in patients with myeloid malignancies. Blood 2008,

111, (1), 236-242.

12. Mullally, A.; Ritz, J., Beyond HLA: the significance of genomic variation for

allogeneic hematopoietic stem cell transplantation. Blood 2007, 109, (4),

1355-1362.

13. Warren, E. H.; Fujii, N.; Akatsuka, Y.; Chaney, C. N.; Mito, J. K.; Loeb, K.

R.; Gooley, T. A.; Brown, M. L.; Koo, K. K. W.; Rosinski, K. V.; Ogawa, S.; Matsubara, A.; Appelbaum, F. R.; Riddell, S. R., Therapy of relapsed leukemia after allogeneic hematopoietic cell transplantation with T cells

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Chapter 6