Optimizing prostate cancer immunotherapy by ...

Optimizing prostate cancer immunotherapy by reducing vaccine induced PD-1 expression Christopher D Zahm and Douglas G McNeel Department of Medicine, University of Wisconsin Carbone Cancer Center

Abstract HLA-A2 MFI (Normalized)

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Immunization with high affinity peptides results in increased PD-1

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References 1.Smith, H. A., Cronk, R. J., Lang, J. M. & McNeel, D. G. Expression and immunotherapeutic targeting of the SSX family of cancer-testis antigens in prostate cancer. Cancer Res. 71, 6785– 95 (2011). 2.Smith, H. A. & McNeel, D. G. Vaccines targeting the cancer-testis antigen SSX-2 elicit HLAA2 epitope-specific cytolytic T cells. J. Immunother. 34, 569–80 (2011). 3.Smith, H. a., Rekoske, B. T. & McNeel, D. G. DNA vaccines encoding altered peptide ligands for SSX2 enhance epitope-specific CD8+ T-cell immune responses. Vaccine 32, 1707–1715 (2014).

Acknowledgments

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This work is supported by: Prostate cancer foundation 2014 Movember-PCF global treatment sciences challenge award

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Figure 6. OT-1 splenocytes were incubated with the various APLs and the phenotype of CD8 T-cells was assessed by flow cytometry every 24 hours for four days. A significant increase in CD3 and decrease in checkpoint expression was seen with lowered Kb affinity.

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T2 and RMA-S binding assays Altered peptide ligands were designed by modifying MHC class I anchor residues and determining predicted binding affinity at http://www.syfpeithi.de/. Peptide sequences with a range of affinities were chosen. TAP deficient T2 (human) or RMA-S (mouse) cells were pulsed with 2µg/mL peptide for 6 hours at 37⁰ C, stained with anti-HLA-A2 FITC (human, biolegend 343304) or anti-H-2Kb (mouse, eBioscience 17-5958-82) antibodies and analyzed by flow cytometry. SSX2 p103-111 Immunizations HHD mice were injected SQ with 100 µg of peptide in complete freund's adjuvant (N=3 per peptide). One week later the spleens were harvested, dissociated and pulsed with 2µg/mL native SSX2 p103-111 peptide for 24 hours. Golgi stop (BD) was added to the media for the last six hours after which surface markers and intracellular cytokines were analyzed by flow cytometry. In vitro simulations Splenocytes harvested from OT-1 mice were incubated with each of the five APLs from figure 3 and SSX2 p103-RF as a non-binding control (2µg/mL). At the time points indicated on the X axes the cells were stained, fixed, and frozen. Cells from all time points were thawed simultaneously and the levels of PD-1, 4-1BB, LAG3, and CTLA4 on CD3+ CD4- CD8+ cells were analyzed by flow cytometry.

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Figure 3. Altered peptide ligands (APLs) of the H2-Kb restricted SIINFEKL epitope were designed by altering Kb anchor residues (changes are designated by AA letter code on the X axis, LY – SIINFEKY; N.S. – Non specific). Peptides with varying affinity for the TCR but similar H2-Kb affinities were also selected (REFs). The ability of peptides to stabilize H2-Kb on RMA-S cells was assessed (2 µg/mL top; various, bottom left). The peptides used in further analysis are shown in blue (varying MHC-I affinity) and orange (varying TRC affinity). The functional avidity of the APLs was assessed by ex vivo stimulation of OT-1 splenocytes followed by intracellular cytokine staining (bottom right).

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Figure 7. OT-1 splenocytes were incubated with the various APLs and the phenotype of CD8 T-cells was assessed by flow cytometry every 24 hours for four days. A significant increase in CD3 and decrease in checkpoint receptor expression was seen with lowered TCR affinity.

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Figure 5. The mean contact time from 30 observed interactions (Figure 4) are shown, top right. The activation (4-1BB expression) and levels of immune checkpoint receptors was assess after T-cells and APC were incubated for three days. A significant decrease in PD1, LAG3 and TIM3 was observed with the lowered affinity FT APL, while no difference was in CTLA4 level was seen.

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Figure 2. Splenocytes from mice immunized with the APLs were harvested one week post immunization Above, percent SSX2 specific CD8 T-cells. Top right, Th1 cytokine and 4-1BB expression following ex vivo stimulation. Right, PD-1 expression.

TCR affinity alters T-cell phenotype

Contact time alters T-cell phenotype

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Figure 4. To determine if MHC-I affinity alters the length of time that T-cells spend with their cognate APCs, OT-1 T-cells (stained green) isolated from splenocytes were added to peptide loaded APCs (stained red). Fluorescent and brightfield (20x) images were taken every one min. for 60 min. Shown are cells undergoing the median (Figure 5, top left) contact time for the non-specific peptide (top row), FT (middle row) and OVA (bottom row). Images display the first observed contact and the following ten min at two min intervals. Color was removed from surrounding cells to highlight the intended interaction.

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Higher affinity results in greater T-cell response

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Epitope affinity alters the amount of time that T-cells spend with their APC; it is possible that changes in the immunological synapse occurring after a long duration are responsible for the changes in T-cell phenotype.

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Figure 1. Altered peptide ligands (APLs) of the HLA-A2 restricted SSX2 p103-111 epitope were designed by altering A2 anchor residues. The ability of each peptide to stabilize HLA-A2 on T2 cells is shown, five peptides were chose for immunization experiments (green).

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Similar findings were observed using OT-1 splenocytes and alterations of the SIINFEKL peptide, both in vitro and in vivo.

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Background Synovial sarcoma, X breakpoint 2 (SSX2) protein is a cancer-testis antigen expressed in metastatic prostate cancer1 Vaccines targeting SSX2 elicit HLA-A2 epitope-specific cytolytic T cells2 Vaccines encoding epitopes with Increased HLA-A2 binding elicits a greater frequency of CD8+ T cells specific for SSX2 when compared to the native vaccine3 These T cells are less effective against tumors due to higher expression of PD-1 (manuscript submitted)

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A single immunization with a high affinity SSX2 APL resulted in CD8+ T cells with increased multifunctional Th1 response but also higher PD-1 expression

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Determine the affect of increased MHC class I affinity on PD-1 expression Recreate this effect using a model system Determine the mechanism underlying increased PD-1 expression

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The MHC-I affinity of APLs alters T-cell/APC contact time

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Goals

Altering the MCH-I affinity of SSX2

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Through early phase clinical trials we have shown that a plasmid DNA vaccine can elicit antigenspecific CD8 T-cells that can persist for years. However, the persistence of antigen-expressing prostate tumors demonstrates that mechanisms of tumor escape are at play. To investigate these mechanisms we have focused on vaccine strategies using the synovial sarcoma, X breakpoint 2 (SSX2) protein as a model target antigen and utilized mice which express HLA-A2 but not murine MHC-I. We have shown that immunization with a DNA plasmid encoding SSX2 elicits robust CD8+ cells and that altering HLA-A2 affinity can augment this response. High affinity altered peptide ligand (APL)-encoding DNA vaccines elicited a greater frequency of specific T-cells; however, they fared worse in anti-tumor studies due to increased PD-1 expression on T-cells, an effect which could be abrogated with PD-1 blockade. Our current research has focused on why high-affinity epitopes elicit higher PD-1 expression, and whether this might be modulated at the time of antigen presentation. To that aim we have used the OT-1 mouse model, in which T-cells express a T-cell receptor that is specific for the dominant ovalbumin epitope SIINFEKL. We generated APLs of SIINFEKL and used them to stimulate OT-1 splenocytes ex vivo. The high affinity SIINFEKL epitope resulted in high, sustained expression of PD-1 following activation, while lower affinity APLs resulted in less, transient expression. However, lowered PD-1 expression can be indicative of T-cells that receive weaker signals via the T-cell receptor (TCR) and likewise display lowered effector function, therefore we also analyzed cytokine production and the in vivo effects of the APLs. In immunization experiments using B6 mice carrying adoptively transferred OT-1 T-cells the lower affinity peptide again resulted in transient PD-1 expression while the high affinity peptide led to sustained levels of PD-1 expression. One week after vaccination with the low affinity APL, PD-1 on tetramer+ CD8 T-cells had returned to baseline while cells from mice immunized with SIINFEKL maintained PD-1 expression. Furthermore, 38(±8)% of tetramer+ cells from the SIINFEKL immunized group were PD-1+ 4-1BB- indicating that PD-1 expression is maintained even after activation in vivo. We are currently assessing the ability of immunization with each peptide to prevent B16-OVA tumor growth in vivo. To assess the mechanism by which PD-1 is regulated after stimulation with each peptide we used a combination of live cell imaging and RNA sequencing to observe APC/T-cell interactions and T-cell gene expression respectively. This research indicates that while increasing antigen affinity may generate a more robust T-cell response it does not always result in better anti-tumor immunity and provides a model in which we can study the biology underlying PD-1 expression.

Summary

Results

Figure 8. To determine the effect of varying Kb affinity in vivo, OT-1 T-cells isolated from splenocytes were adoptively transferred into B6/N mice. Twenty-four hours later mice were immunized with 100µg peptide in CFA. Spleens were harvested two and seven days post immunization and activation and checkpoint receptor expression were assessed. CD44 was used as a marker of antigen experience (bottom right).

National Institutes of Health Molecular and Cellular Mechanisms of Tumor Development Training Grant T32 CA157322 University of Wisconsin Carbone Cancer Center Cancer Center Support Grant P30 CA014520