DEVELOPING ADOPTIVE CELL TRANSFER (ACT) IMMUNOTHERAPY TO TARGET SPERMASSOCIATED ANTIGEN 9 (SPAG9), A NOVEL CANCER TESTIS ANTIGEN INVOLVED IN THE MAPK
PATHWAY. Luke D. Rothermel, MD, MPH1,2 Daniel J. Stephens, MD.2 Smita S. Chandran, PhD.2 Abhishek K.
Srivastava, PhD.2, Biman C. Paria, PhD.2 and Udai S. Kammula, MD2. 1University Hospitals Case Medical Center
& 2Surgery Branch, National Cancer Institute/National Institutes of Health
Background: In clinical trials, cancer testis antigens have been effectively targeted using ACT (i.e. NY-ESO-1 for
metastatic melanoma and synovial sarcoma). Recently, SPAG9, a novel cancer testis antigen, has been described to
be widely expressed in a variety of solid and hematologic cancers, contribute to the malignant phenotype of cells
through its impact on the MAPK pathway, and to elicit humoral immune responses. We aimed to validate these
claims and to isolate SPAG9-specific T cells for use in ACT for patients with a variety of malignancies.
Methods: Evaluation of SPAG9 mRNA and protein expression in cancer cell lines, fresh tumor, and normal tissue
samples was performed using real-time PCR, intracellular fluorescence-activated cell sorting (FACS), and
immunohistochemistry (IHC). Humoral reactivity against SPAG9 was characterized by ELISA using samples of
normal donor serum and the serum of patients with various malignancies. Forward and reverse immunologic
approaches were employed to identify T cells specific for SPAG9. T cell reactivity was assessed by the production
of interferon-g mRNA in response to target antigen.
Results: SPAG9 mRNA expression was identified at varying levels in all tumor cell lines tested, but was not found
in a panel of normal tissues except for testis. FACS analysis demonstrated SPAG9 protein expression in cell lines
that correlated with the mRNA data. IHC studies are ongoing to determine SPAG9 expression in freshly resected
tumor and normal samples. Antibodies against SPAG9 were found in high levels in patients with gastrointestinal
(GI) and lung cancers but not in patients with other malignancies and healthy volunteers. Forward immunologic
approaches are currently being pursued using tumor infiltrating lymphocytes selected from patients with high
humoral responses against SPAG9. The reverse immunologic approach has identified a patient with strong reactivity
against putative peptide epitopes. Testing is underway to determine if these T cells can also recognize SPAG9
expressing tumor cell lines and transfectants.
Conclusion: SPAG9 represents a novel cancer testis antigen highly expressed in GI and lung cancers. Identification
of SPAG9 specific T cells is underway and may extend ACT to the treatment of common malignancies.
1
Fig.1 (a) SPAG9 protein expression in representative cell lines
Fig.2 ELISA SPAG9 showing high frequency of
by FACS analysis; (b) correlation of SPAG9 mean fluorescence
SPAG9 antibodies in the serum of patients with GI
index (MFI) and SPAG9 mRNA copy number
and lung carcinoma vs. other malignancies and
normal donor serum
Arbitrary units
a.
H1299
HepG2
SNU387
1500
****
Anti- SPAG9 serum antibodies
(mU/ml)
T cells
SPAG9 (fluorescence)
b.
SPAG9 mRNA copies
per b-actin (x104)
1000000
--H1299
100000
10000
HepG2-SNU387--
1000
100
R² = 0.8938
1000
NS
NS
500
366
NS
10
25
--T cells
1
0
28
17
10
0
1000
2000
3000
SPAG9 (MFI)
n=
Normal
Donor
Soft Tissue
Sarcoma
Renal
Cancer
34
23
6
Cutaneous Carcinoma
Melanoma (Lung and GI)
46
27
Fig 3. Microculture reactivity against SPAG9 peptides after 14 day in-vitro stimulation
of peripheral blood lymphocytes (numbers on the x-axis correspond to the specific
IFN mRNA Stimulation Index
peptide used in testing; IFN=interferon gamma)
100
10
1
19
49
56
343
381
447
521
837
964
1016
Sensitizing SPAG9 peptides
(n = 48 microcultures/peptide)
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