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Clinical Trial
. 2012 Oct 1;189(7):3759-66.
doi: 10.4049/jimmunol.1201529. Epub 2012 Sep 5.

Diversity of antigen-specific responses induced in vivo with CTLA-4 blockade in prostate cancer patients

Serena S Kwek  1 , Vinh Dao, Ritu Roy, Yafei Hou, David Alajajian, Jeffrey P Simko, Eric J Small, Lawrence Fong
Affiliations
Clinical Trial

Diversity of antigen-specific responses induced in vivo with CTLA-4 blockade in prostate cancer patients

Serena S Kwek et al. J Immunol. .

Abstract

CTLA-4 is a surface receptor on activated T cells that delivers an inhibitory signal, serving as an immune checkpoint. Treatment with anti-CTLA-4 Abs can induce clinical responses to different malignancies, but the nature of the induced Ag-specific recognition is largely unknown. Using microarrays spotted with >8000 human proteins, we assessed the diversity of Ab responses modulated by treatment with CTLA-4 blockade and GM-CSF. We find that advanced prostate cancer patients who clinically respond to treatment also develop enhanced Ab responses to a higher number of Ags than nonresponders. These induced Ab responses targeted Ags to which preexisting Abs are more likely to be present in the clinical responders compared with nonresponders. The majority of Ab responses are patient-specific, but immune responses against Ags shared among clinical responders are also detected. One of these shared Ags is PAK6, which is expressed in prostate cancer and to which CD4(+) T cell responses were also induced. Moreover, immunization with PAK6 can be both immunogenic and protective in mouse tumor models. These results demonstrate that immune checkpoint blockade modulates Ag-specific responses to both individualized and shared Ags, some of which can mediate anti-tumor responses.

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Figures

FIGURE 1
FIGURE 1. Modulation of antigen-specific IgG responses with CTLA-4 blockade
(A) Serum PSA levels of prostate cancer patients treated with CTLA4 blockade normalized to the baseline level are plotted over weeks after the initiation of treatment. Subjects received ipilimumab at either 3 mg/kg (filled symbols) or 10 mg/kg (open symbols). PBMCs and sera from these patients were used for subsequent experiments. (B) Scatter plot analysis of median centered log2 transformed and normalized fluorescence intensities are plotted for pre-treatment (x-axis) and post-treatment (y-axis) sera from clinical responders (subjects 19, 20, 24, 33, and 36) and (C) non-responders (subjects 21, 22, 23, 34, and 35). Diagonal lines delineate 4 fold change of the difference between post-and pre-treatment intensities above and below the x=y axis. (D) Box plots of up-modulated (upper panel) and down-modulated (lower panel) antibodies with 4 fold change for non-responders and responders. The box bounds the middle 50% of the values and the median is denoted by the thick line. The whisker lines span 1.5 times the interquartile range. Data points beyond that are considered as outliers and are shown as circles. Two-sided Wilcoxon rank sum test p-value for the difference in the number of up-modulated antibodies between non-responders and responders is 0.0043 and in the number of down modulated antibodies is 0.429. (*) denotes a significant p-value < 0.05.
FIGURE 2
FIGURE 2. Profiling of antibody responses in cancer patients to CTLA-4 blockade and GM-CSF with protein microarrays
(A) Unsupervised clustering of median centered log2 transformed and normalized fluorescence intensities in pre-treatment and post-treatment sera of 11 evaluable patients binding to proteins spotted on the arrays. Responders are highlighted in red and non-responders in black. (B) Venn diagram showing the number of up-modulated antibodies that are shared (shown by number in overlaps) or are unique in the 11 patients. Responders are represented by pink circles and non-responders in blue. Sizes of circles approximate the number of antigens.
FIGURE 3
FIGURE 3. Association between up-modulated antibodies with preexisting or non-preexisting antibody responses
Normal mixture modeling with estimation-maximization (EM) was used to define the boundary for determining the presence or absence of preexisting antibodies. (A) The left panel shows the number of two fold up-modulated antibodies in the post-treatment serum to antigens where there are no preexisting antibodies (light grey) and to antigens where there are preexisting antibodies (dark grey) in the pre-treatment serum for each patient. The right pane shows log odds ratios comparing two fold up modulation for preexisting versus non-preexisting antibody groups for each patient. Significant log odds ratio values are shown as solid circles (significance determined as Bonferroni adjusted p-value < 0.05 from performing 2-sided Fisher’s exact test for each patient). (B) Interaction plot using multiplicative poisson regression model with the number of antibodies up-modulated, clinical response status and pre-treatment preexisting or not status as dependent variables. Response main effect p-value: 2.6e-08; pre-treatment preexisting main effect p-value: 1.6e-05; and interaction p-value: 1.8e-06.
FIGURE 4
FIGURE 4. Detection of antibody and T cell responses to the candidate antigen Pak6
(A) Images of protein arrays showing levels of human IgG binding to Pak6 protein in the pre-treatment and post-treatment sera of clinical responders (patients 19 and 20). Control proteins and antibodies are also shown. Numbers shown below the Pak6 and influenza A antigen are the median-centered log2 transformed and normalized fluorescence values. (B) Flow cytometry of pre-treatment and post-treatment PBMCs from clinical responders (patients 19 and 20) and flow cytometry of post-treatment PBMCs from a clinical non-responder (patient 23) that had been incubated with media alone, Pak6 protein, CAMK protein, or PMA plus ionomycin for 48 h and then stained for CD4 and intracellular IFN-γ and IL-17. CD4+ T cells were gated upon and analyzed for cytokine production. Axes are log10 fluorescence of IFN-γ (y axis) and IL-17 (x axis) staining. The number in each quadrant indicates the percentage of cells in that quadrant.
FIGURE 5
FIGURE 5. Expression and localization of Pak6 in prostate cancer
(A) Western blots of prostate cancer cell lines (LNCAP, PC3, DU145 and CWR22) and human prostate epithelial cell lines (PWR-1E and RWPE) carried out with anti-Pak6 and anti-β-actin antibody. (B) Immunohistochemistry of responder patient 20’s prostate tumor biopsy and a representative prostate tumor with anti-Pak6 antibodies and with control rabbit IgG are shown. Scale bar represents 50 μm.
FIGURE 6
FIGURE 6. Immunogenicity and anti-tumor activity of Pak6 immunization
(A) Western blots carried out with sera from C57BL/6 and FVB mice immunized twice with PBS plus CFA or with human Pak6 plus CFA (3 mice/group. (B) Proliferation assays carried out with inguinal lymph nodes cells pooled from immunized C57BL/6 and FVB mice incubated with media alone or with the protein as shown. Concentrations are in μg/ml. Proliferation was measured as radioactive counts per min (CPM) of [3H]-thymidine incorporation. Error bars denote ± standard deviations of triplicate wells. (C) Kaplan-Meier survival curve of tumor challenge for C57BL/6 and FVB mice immunized twice with PBS plus CFA or human Pak6 plus CFA (5 mice/group) and challenged 14 days later with Tramp cells. Tumors were measured three times a week and mice were sacrificed when the tumor size reached 2 cm per institutional guidelines. p-values were calculated by using the log rank test (SPSS, IBM). (*) denotes a significant p-value < 0.05.
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References

    1. Greenwald RJ, Freeman GJ, Sharpe AH. The B7 family revisited. Annu Rev Immunol. 2005;23:515–548. - PubMed
    1. Brucher J, Domke I, Schroder CH, Kirchner H. Experimental infection of inbred mice with herpes simplex virus. VI. Effect of interferon on in vitro virus replication in macrophages. Arch Virol. 1984;82:83–93. - PubMed
    1. Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity by CTLA-4 blockade. Science. 1996;271:1734–1736. - PubMed
    1. Link KA, Balasubramaniam S, Sharma A, Comstock CE, Godoy-Tundidor S, Powers N, Cao KH, Haelens A, Claessens F, Revelo MP, Knudsen KE. Targeting the BAF57 SWI/SNF subunit in prostate cancer: a novel platform to control androgen receptor activity. Cancer Res. 2008;68:4551–4558. - PMC - PubMed
    1. Hernandez J, Ko A, Sherman LA. CTLA-4 blockade enhances the CTL responses to the p53 self-tumor antigen. J Immunol. 2001;166:3908–3914. - PubMed

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