Summer Undergraduate Research Program - Fred Hutchinson ...

Identification of Candidate Minor Histocompatibility (H) Antigens for the Development
of T Cell Immunotherapy to Enhance the Graft Versus Leukemia (GVL) Effect
Amber Ortiz 1 , Julia Richardt, PhD 2 , Stanley Riddell, MD 2 and Marie Bleakley, MD/PhD 2
1 FHCRC – SURP, 2 Program in Immunology, Clinical Research Division, FHCRC, Seattle, WA
INTRODUCTION METHODS/RESULTS
METHODS/RESULTS CONTINUED
ATP8B4
A. Healthy
Cancer
Other Diseases B.
2. PCR Sequencing The reported population distribution of the allelic variants of SNPs is not always
accurate in the entrez SNP database. PCR sequencing was performed to confirm the population
ALL Prostate
Hematopoietic
distribution of the SNPS in order to validate the candidate minor H antigens and prioritize them for
AML
subsequent studies.
2011 Best Poster Presentation Award
Non-hematopoietic
Figure 5A. PCR Gel Primers were designed for each of the eight SNPs and PCR was conducted. The
above gel was run to show PCR products were formed in the reaction prior to sequencing.
7_ RS12601317_1F2R_2_2F
A/G
C.
AML
8_ RS12601317_1F2R_2_2F
G
9_ RS12601317_1F2R_2_2F
A
Acute Myeloid Leukemia (AML) is a life-threatening hematological cancer in
which the bone marrow makes an abundance of abnormal blasts or immature
blood cells. These malignant blasts divide rapidly and are unable to mature
preventing normal cells from being made and maturing into platelets, white,
or red blood cells. AML can often be cured by allogeneic stem cell
transplantation (SCT). The efficacy of SCT, where donor hematopoietic cells
including stem cells and lymphocytes are infused into the patient following
myeloablative chemotherapy and radiotherapy, is due in part to an immune
response, the GVL effect, where the donors T cells eliminate leukemia.
Unfortunately, AML relapse sometimes occurs after SCT and is generally
fatal. Another immune response that may occur is Graft versus Host Disease
(GVHD). GVHD occurs when transplanted T cells attack the recipient’s
normal organs like skin, gut, liver, and lungs. The major molecular targets of
GVL and GVHD are minor H antigens; polymorphic peptides that are
presented on the cell surface with MHC molecules and are derived from selfproteins
differing in sequence between donors and recipients due to genetic
polymorphisms, most commonly single nucleotide polymorphisms (SNPs). T
cells specific for minor H antigens predominantly expressed on
hematopoietic cells including leukemia can induce a selective GVL effect
without GVHD. Other T cells can recognize minor H antigens that are
expressed ubiquitously including epithelial tissues causing GVHD. T cell
therapy targeting hematopoietic-specific minor H antigens may prevent
relapse of AML following SCT.
Figure 5B. Sample Population Data This is an example of primer 1260 on three of our samples
showing a heterozygote (A/G), homozygote (G/G) and homozygote (A/A) in the SNP sequence. The
sequence data was used to obtain sample population information on the SNPs.
Figure 1. GeneSapiens Database This figure shows the expression pattern of the gene ATP8B4 in nonhematopoietic
tissue and AML. Figure A shows the overall expression of the gene in healthy tissue (left) and that
of certain cancers and other diseases (right). Figure B shows gene expression in normal tissue (nonhematopoietic
in red) Figure C shows gene expression in cancers and other diseases. (AML surrounded in red)
Previous minor H antigen discovery strategies have not been adequately
focused on identifying minor H antigens which are exclusively expressed on
hematopoietic cells. Our goal is to locate minor H antigens that can be used
to develop an immunotherapy to enhance the GVL effect after allogeneic
SCT and which will not induce GVHD. Specifically, we aim to identify minor H
antigens presented by HLA-A*0201, the most common Caucasian HLA
restricting allele in order to target a large population of AML donor/patient
pairs. We are evaluating a reverse immunology approach to the discovery of
hematopoietic-restricted minor H antigens for the development of
immunotherapy, first using bioinformatics to identify candidate minor H
antigens associated with genes expressed exclusively in hematopoietic tissue.
Why are Minor H Antigens Important?
oThey are molecular targets for GVL and GVHD
oThey can be used as targets for T cell immunotherapy to induce an antileukemic
effect
151
(GVL:
anti-leukemic anti leukemic effect)
Figure 5C. Sample Population Data Using PCR and Sequencing we have obtained the above population
data for phenotype distribution confirmation for the 30 samples on eight SNPs. We observed that
seven SNPs are adequately polymorphic to proceed for subsequent studies.
DISCUSSION
Figure 2. Entrez SNP/Gene Database. SNP data was obtained on 34 genes with expression restricted to
hematopoietic tissue. We aimed to identify non-synonymous SNPs with a heterozygosity of >.1 suggesting a
balanced population distribution. 26 SNPs with these characteristics were found within 15 of the 34 genes.
The Genesapiens database was used to identify 34 genes that are highly expressed in AML but not in
non-hematopoietic tissues. Using Entrez SNP we identified, in these genes, 26 non-synonymous SNPS
with a balanced population distribution which could be applicable to many patient/donor pairs. We
verified the population distribution using PCR-sequencing. Epitope prediction algorithms (IEDB) were
applied to amino acid sequences encompassing selected SNPs. IEDB identified 26 peptides predicted
to bind to HLA-A*0201 for eight genes. Using PCR sequencing we narrowed the list to seven peptides
for which there will be donor/patient pairs with the relevant directional disparity.
Peptide Data
Gene
AIMS
Peptide Data
Gene
1. To use a bioinformatic approach to identify candidate minor H antigens
that are A) encoded by hematopoietic restricted genes B) associated
with SNPs having a balanced population distribution C) predicted to bind
to HLA-A*0201.
2. To use PCR sequencing to verify the SNP population distribution in order
to prioritize candidate minor H antigens for further studies.
CONCLUSIONS and FUTURE DIRECTIONS
Figure 3. Entrez SNP Database This figure shows examples of SNP data available in the Entrez SNP database
for two candidate minor H antigens.
We identified seven candidate minor H antigens representing polymorphic peptide sequence
predicted to bind to HLA-A*0201 associated with SNPs confirmed (figure 4) to have a balanced
population distribution (figure 5c). Each candidate minor H antigen is encoded by a gene which is
highly expressed in AML and not hematopoietic tissue (figure 1).
METHODS/RESULTS
Future studies in the Riddell lab will be performed to demonstrate binding of the candidate minor H
antigens to HLA-A*201 in vitro. Studies will also be conducted to evaluate the immunogenic and
endogenous processing/presentation of the minor H antigens.
Ultimately, the candidate minor H antigens may be used as targets for immunogenicity to prevent and
treat relapse of AML after allogeneic SCT.
1. Bioinformatics
163 enes
Gene Sapiens Database
http://ist.genesapiens.org
http:// ist.genesapiens.org//
34 Genes with high expression levels in AML and low expression levels in
non-hematopoietic tissues
Entez Gene/SNP PubMed Database
http://www.ncbi.nlm.nih.gov
http:// www.ncbi.nlm.nih.gov/gene/ /gene/
http://www.ncbi.nlm.nih.gov/snp
http:// www.ncbi.nlm.nih.gov/snp
ACKNOWLEDGEMENTS
The Summer Undergraduate Research Program is supported in parts by the Cancer Center
Support Grant (CCSG) CURE Supplement: 5 P30 CA015704-37S1 and FHCRC Administrator
This work has been supported by the Damon Runyon Cancer Research Foundation via Dr. Marie
Bleakley’s Clinical Investigator Award
We would like to thank the MARC program supported by Award Number T34GM00851 from the
National Institute of General Medical Sciences
26 Non-synonymous SNPs with balance population distribution (15 Genes)
SYFPEITHI AND IEDB Database
http://www.syfpeithi.de
http:// www.syfpeithi.de/ /
http://www.immuneepitope.org
http:// www.immuneepitope.org//
Figure 4. IEDB Database Using the IEDB database for epitope predictions we identified 26 peptides from eight
SNPs predicted to bind to HLA-A*0201 with a high binding affinity (IC50