Simultaneous targeting of PD-1 and IL-2Rβγ with radiation therapy inhibits pancreatic cancer growth and metastasis

Abstract

In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to radiation therapy (RT) is characterized by increased IL-2Rβ and IL-2Rγ along with decreased IL-2Rα expression. The bispecific PD1-IL2v is a PD-1-targeted IL-2 variant (IL-2v) immunocytokine with engineered IL-2 cis targeted to PD-1 and abolished IL-2Rα binding, which enhances tumor-antigen-specific T cell activation while reducing regulatory T cell (Treg) suppression. Using PD1-IL2v in orthotopic PDAC KPC-driven tumor models, we show marked improvement in local and metastatic survival, along with a profound increase in tumor-infiltrating CD8⁺ T cell subsets with a transcriptionally and metabolically active phenotype and preferential activation of antigen-specific CD8⁺ T cells. In combination with single-dose RT, PD1-IL2v treatment results in a robust, durable expansion of polyfunctional CD8⁺ T cells, T cell stemness, tumor-specific memory immune response, natural killer (NK) cell activation, and decreased Tregs. These data show that PD1-IL2v leads to profound local and distant response in PDAC.

Keywords: NK cells; T cell stemness; Tregs; antigen specificity; cancer immunotherapy; cytotoxic T lymphocyte; immune memory; metabolomics; metastasis; tumor immunology.

Figure 1:. PD1-IL2v treatment enhances survival of pre-clinical murine PDAC models, which is further improved by radiation therapy

(A) 1.) Conventional IL-2 and 2.) aPD-1 therapy mechanisms. 3.) Model of PD1-IL2v antibody complex mechanism of targeted delivery of IL-2v through cis-binding of PD-1 4.) PD1-IL2v has been optimized for IL-2Rβγ binding without binding in the presence of IL-2Rα. Created with BioRender.com (B) Kaplan-Meier survival analysis of PK5L1940 orthotopically implanted pancreatic tumor-bearing mice treated with RT, aPD-1, DP47-IL-2v, PD1-IL2v, or a combination therein. (C) Kaplan-Meier survival analysis of PK5L1940 orthotopically implanted pancreatic tumor-bearing mice untreated and treated with PD1-IL2v with and without RT. (D) Kaplan-Meier survival analysis of PK5L1940 orthotopically implanted pancreatic tumor-bearing mice untreated and treated with aPD-1 with and without RT. (E) Kaplan-Meier survival analysis of PK5L1940 orthotopically implanted pancreatic tumor-bearing mice untreated and treated with DP47-IL-2v with and without RT. (F) Kaplan-Meier survival analysis of PK5L1940 orthotopically implanted pancreatic tumor-bearing mice untreated and treated with aPD-1 + DP47-IL-2v with and without RT. (G) Kaplan-Meier survival analysis of PK5L1940 orthotopically implanted pancreatic tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. (H) Kaplan-Meier survival analysis of FC1242 orthotopically implanted pancreatic tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. RT administered 7 days post-implantation. PD1-IL2v, DP47-IL-2v, and aCD25 dosed once per week beginning day 7 post-implantation. aPD-1 dosed twice per week beginning day 7 post-implantation. n ≥7 per group. See also Figure S1.

Figure 2:. PD1-IL2v increases infiltration, proliferation, and activation of polyfunctional, intratumoral CD8⁺ T cells

(A) Quantification of tumor-infiltrating immune subpopulations in untreated, RT, aCD25, PD1-IL2v, or RT + PD1-IL2v treated mice. Quantification determined by gating analysis using FlowJo software. Gating is performed on live CD45⁺ cells, n ≥6 per group. CD4⁺ T cell group does not include Tregs. Significance shown for differences in CD8⁺ T cell population frequency. (B) Representative histogram of CD8 MFI (left) and representative gating plots (right) of intratumoral leukocytes (CD45⁺ subset) in pancreatic tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. Gating is performed on live CD45⁺ cells. (C) Flow cytometric analysis of frequency of tumor infiltrating Tregs (top) and IL-10 MFI of intratumoral CD4⁺ T cells (bottom). MFI is calculated on live CD45⁺ CD4⁺ population, n ≥5 per group. (D) Ratio of CD8⁺ T cells to Tregs in the TME of pancreatic tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. n ≥5 per group. (E) Ki67 expression across CD8⁺, CD4⁺, and Treg subpopulations in pancreatic tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. Gating is performed on live CD45⁺ Ki67⁺ cells, n ≥5 per group. (F) Flow cytometric analysis of the expression of functional and activation markers IFNγ (left), IL-2 (center), PD-1 and TIM3 (right) in intratumoral CD8⁺ T cells. Gating is performed on live CD45⁺ CD8⁺ cells, n ≥5 per group. (G) Heat map showing the expression profile of FlowSOM-generated intratumoral CD8⁺ T cell clusters collected from tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v (L) Differences in cluster frequencies by treatment (R) Clustering is performed on live CD45⁺CD8⁺ cells. (H) Flow cytometric analysis of IFNγ expression of intratumoral CD8⁺ T cells in untreated, RT, aCD25, and PD1-IL2v treated pancreatic tumor bearing mice. Gating is performed on live CD45⁺ CD8⁺ cells, n ≥6 per group Mice were orthotopically implanted with PK5L1940 cells. RT administered 7 days post-implantation. PD1-IL2v and aCD25 dosed once per week beginning day 7 post-implantation. Serum collected at time of sacrifice. Tumors harvested 21 days post-implantation. Data represented as mean ± SEM. P-values calculated with Students T-test, *indicates p<0.05, **<0.01, ***<0.001, ****<0.0001. See also Figures S1, S2, and S3.

Figure 3:. RT+ PD1-IL2v simultaneously increases polyfunctional CD8⁺ T cells and reduces Treg immune suppression within the tumor draining lymph node

(A) viSNE dimensionality reduction analysis of immune subpopulations in the draining lymph node of tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. Clustering is performed on live CD45⁺ cells. (B) Quantification of immune subpopulations the draining lymph node of tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. Gating performed on live CD45⁺ cells, n ≥5 per group. (C) Flow cytometric quantification of polyfunctional (IFNγ⁺ TNFα⁺ GnzmB⁺) CD8⁺ T cells in the draining lymph node of tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v (L) or untreated, RT, aCD25, PD1-IL2v, and RT with PD1-IL2v (R) Gating is performed on live CD45⁺ CD8⁺ cells, n ≥5 per group. (D) Flow cytometric quantification of exhausted (PD-1⁺TIM3⁺) CD8⁺ T cells in the draining lymph node of tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v (E) viSNE dimensionality reduction analysis showing expression of IFNγ, CD44, TNFα, GnzmB, and PD-1 on CD8⁺ T cells in the draining lymph node of tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. Clustering is performed on live CD45⁺ CD8⁺ cells. (F) Flow cytometric analysis of the frequency of Tregs as a proportion of CD45⁺ cells (top), and FoxP3 (center) and IL-10 (bottom) MFI of CD4⁺ T cells in the draining lymph node of pancreatic tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. MFI is calculated on live CD45⁺ CD4⁺ population, n ≥5 per group. (G) Heat map showing the expression profile of FlowSOM-generated nodal CD8⁺ T cell clusters collected from tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. Clustering is performed on live CD45⁺ CD8⁺ cells. Mice were orthotopically implanted with PK5L1940 cells. RT administered 7 days post-implantation. PD1-IL2v and aCD25 dosed once per week beginning day 7 post-implantation. Lymph nodes harvested 21 days post-implantation. Data represented as mean ± SEM. P-values calculated with Students T-test, *indicates p<0.05, **<0.01, ***<0.001, ****<0.0001. See also Figure S4

Figure 4:. Anti-tumor response of PD1-IL2v with and without RT is antigen-specific

(A) Experimental timeline of OT-1 CD8⁺ T cell adoptive transfer experiment. Created with BioRender.com (B) Frequency of CD45.1⁺ CD8⁺ T cells as a proportion of live cells in OVA-expressing tumors of untreated, RT, PD1-IL2v, or RT+PD1-IL2v treated mice following CD45.1⁺ OT-1 adoptive transfer. (C) Frequency of CD45.1⁺ CD8⁺ T cells as a proportion of live cells in the blood (left) and draining lymph node (right) of OVA-expressing tumor-bearing mice following OT-I CD8⁺ T cell adoptive transfer and no treatment, RT, PD1-IL2v or RT + PD1-IL2v treatment. Gating is performed on live cells. (D) Flow cytometric analysis of activation and cytotoxicity marker MFI of CD45.1⁺ CD8⁺ T cells in the blood (top), draining lymph node (middle) and tumor (bottom) of OVA-expressing tumor-bearing mice following OT-I CD8⁺ T cell adoptive transfer and no treatment, RT, PD1-IL2v or RT + PD1-IL2v treatment. MFI is calculated on live CD45.1⁺ CD8⁺ population, I Flow cytometric analysis of IFNγ⁺ TNFα⁺ GnzmB⁺ CD8⁺ T cells in the blood (left) and draining lymph node (right) of OVA-expressing tumor bearing mice following OT-I CD8⁺ T cell adoptive transfer and no treatment, RT, PD1-IL2v or RT + PD1-IL2v treatment. Gating is performed on live CD3⁺ CD8⁺ cells. (F) Flow cytometric analysis of DNAM⁺ NKp46⁺ cells in the blood of OVA-expressing tumor bearing mice following OT-I CD8⁺ T cell adoptive transfer and no treatment, RT, PD1-IL2v or RT + PD1-IL2v treatment. Gating is performed on live cells. (G) Flow cytometric analysis of activation and cytotoxicity marker MFI of CD45.1⁻ and CD45.1⁺ CD8⁺ T cells in the blood (top) and draining lymph node (bottom) of OVA-expressing tumor-bearing mice following OT-I CD8⁺ T cell adoptive transfer and RT + PD1-IL2v treatment. MFI is calculated on live CD45.1⁻ CD8⁺ and CD45.1⁺ CD8⁺ (H) Pancreatic tumor incidence 5.5 weeks post-implantation in OVA-expressing tumor-bearing mice following OT-I CD8⁺ T cell adoptive transfer and no treatment, RT, PD1-IL2v or RT + PD1-IL2v treatment. Mice were orthotopically implanted with PK5L1940-OVA expressing cells. PD1-IL2v dosed once per week beginning day 7 post-implantation. All tissue collected 39 days post implantation, n ≥5 per group. Data represented as mean ± SEM. P-values calculated with Students T-test, *indicates p<0.05, **<0.01, ***<0.001, ****<0.0001. See also figure S5.

Figure 5:. PD1-IL2v in combination with radiotherapy increases peripheral activation of anti-tumor immune populations and reduces metastatic burden

(A) Flow cytometric analysis of the frequency of circulating tumor cells (CTCs) in the blood of pancreatic tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. CTCs defined as CD45⁻ EpCAM⁺ cells. Gating is performed on all live cells, n ≥5 per group. (B) viSNE dimensionality reduction analysis of circulating CD4⁺, CD8⁺, and NK cells in the blood of pancreatic tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. Clustering is performed on live CD45⁺ cells. (C) Quantification of the immune subpopulations in the blood of pancreatic tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. Gating performed on live CD45⁺ cells, n ≥5 per group. (D) Representative histogram of CD8 MFI (left) and representative gating plots (right) of circulating leukocytes (CD45⁺) in pancreatic tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. Gating is performed on live CD45⁺ cells. (E) Ki67 expression across peripheral CD8⁺, CD4⁺, and Treg subpopulations in pancreatic tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. Gating is performed on live CD45⁺Ki67⁺ cells, n ≥5 per group. (F) viSNE dimensionality reduction analysis performed on the circulating CD8⁺ T cell population showing relative expression of Ki67, TNFα, IFNγ, and CD44 in mice treated with RT, aCD25, and/or PD1-IL2v. Clustering is performed on live CD45⁺ CD8⁺ cells. (G) Flow cytometric analysis of the frequency of NK cells (left), and NK cell expression of functional markers DNAM1 (center) and GnzmB (right) in the blood of pancreatic tumor-bearing mice treated with RT, aCD25, and/or PD1-IL2v. n ≥5 per group. (H) Representative images of hepatic metastatic lesions following hemi-splenic implantation at the time of sacrifice in mice untreated or treated with RT, aCD25, and/or PD1-IL2v. (I) Kaplan-Meier survival analysis of mice implanted with PK5L1940 using a hemi-splenectomy model of metastatic pancreatic cancer. Mice were untreated or treated with RT, aCD25, and/or PD1-IL2v. n ≥7 per group. Mice were orthotopically implanted with PK5L1940 cells. RT administered 7 days post-implantation. PD1-IL2v and aCD25 dosed once per week beginning day 7 post-implantation. Blood collected 21 days post-implantation. Data represented as mean ± SEM. P-values calculated with Students T-test, *indicates p<0.05, **<0.01, ***<0.001, ****<0.0001. See also Figure S2.

Figure 6:. RT + PD1-IL2v with and without aCD25 induces an effector memory CD8⁺ T cell phenotype in tumor-eradicated mice

(A) Tumor growth curves of PK5L1940 flank tumors following tumor rechallenge in tumor-eradicated mice treated with RT + PD1-IL2v or RT + aCD25 + PD1-IL2v. (B) Flow cytometric analysis of the frequency of circulating CD8⁺ T cells early (21 days post-implant), prior to (4 days), and following (4 days) rechallenge in tumor-eradicated mice in (A) (C) Flow cytometric analysis of Ki67 MFI in circulating CD8⁺ T cells early (21 days post-implant), prior to (4 days), and following (4 days) rechallenge in tumor-eradicated mice in (A) CD69 (D), Eomes (E), and CD62L (F) MFI in circulating CD8⁺ T cells prior to (4 days) and following (4 days) rechallenge in tumor-eradicated mice in (A) (G) Kaplan-Meier survival analysis of mice implanted with FC1242 cells using a hemi-splenectomy model of metastatic pancreatic cancer. RT administered 7 days post-implantation. PD1-IL2v and aCD25 dosed once per week beginning day 7 post-implantation Treatment groups: RT alone (n ≥7), RT + aCD25 (n<7), RT + PD1-IL2v (n≥7), and RT + aCD25 + PD1-IL2v (n≥7) (H) Tumor growth curves of FC1242 flank tumors following tumor rechallenge in tumor-eradicated mice treated with RT + PD1-IL2v or RT + aCD25 + PD1-IL2v. Black circles denote ulcerated tumors. (I) Flow cytometric analysis of Treg functionality prior to (3 days) and following (4 days) rechallenge in tumor-rechallenge mice in (H) (J) Flow cytometric analysis of Treg (CD45⁺ CD4⁺ FoxP3⁺ CD25⁺) to CD45⁺ CD8⁺ cell ratio prior to (3 days) and following (4 days) rechallenge in tumor-rechallenge mice in (H) (K) CD62L MFI in circulating CD8⁺ T cells prior to (3 days) and following (4 days) rechallenge in tumor-rechallenge mice in (H). Gating performed on CD45⁺ CD8⁺ CD62L⁺ (L) Flow cytometric analysis of CD69⁺ circulating CD8⁺ T cells prior to (3 days) and following (4 days) rechallenge in tumor-rechallenge mice in (H) Data represented as mean ± SEM. P-values calculated with Students T-test, *indicates p<0.05, **<0.01, ***<0.001, ****<0.0001.

Figure 7:. Distinct proteome differences observed between mice treated with RT and/or aCD25 versus those treated with PD1-IL2v or combination

(A) Three-dimensional principal component analysis of proteome in NK and CD8⁺ T cells isolated from mice treated with RT, aCD25, and/or PD1-IL2v (left). Venn diagram showing significantly changed proteins based on treatment regimen and cell type as determined by ANOVA (right). (B) Hierarchical clustering analysis of the top 1500 proteins in RT, aCD25, and/or PD1-IL2v treated mice as determined by ANOVA. (C) Table of significantly altered metabolic pathways in CTLs harvested from RT + PD1-IL2v vs. RT treated mice. (D) Network view of the interactome of differentially expressed proteins in CTLs harvested from RT+ PD1-IL2v vs. RT treated mice. (E) Quantification of T cell receptor signaling pathway proteins in CD8⁺ T cells harvested from RT, aCD25, and/or PD1-IL2v treated mice. (F) Quantification of TCA, Glycolysis, and Fatty acid metabolism enzymes in CD8⁺ T cells harvested from RT, aCD25, and/or PD1-IL2v treated mice. Mice were orthotopically implanted with PK5L1940 cells. RT administered 7 days post-implantation. PD1-IL2v and aCD25 dosed once per week beginning day 7 post-implantation. CD8⁺ and NK cells isolated 17 days post-implantation. n=3 per group. See also Figure S6,7,8.

Figure 8:. Patient clinical response to RT is associated with significant changes in IL-2R signaling

(A) Normalized expression of IL-2Rα, IL-2Rβ, and IL-2Rγ in patient PDAC tumor tissue samples before (n=26) and after (n=29) RT as determined by RNA sequencing. (B) Normalized expression of PD-L1 and PD-1 in patient PDAC tumor tissue samples before (n=26) and after (n=29) RT as determined by RNA sequencing. (C) Normalized transcriptional levels of IL-2Rα, IL-2Rβ, and IL-2Rγ of patient PDAC tumor tissue samples collected before RT stratified by responders (n=11) and non-responders (n=4). (D) Normalized concentration of branched chain amino acids (BCAAs) leucine/isoleucine and valine in the serum of PDAC patients at baseline (n=12), during (n=12), and post (n=11) RT treatment. (E) Normalized concentration of the metabolite kynurenine in the serum of PDAC patients at baseline (n=12), during (n=12), and post (n=11) RT treatment. (F) Normalized enrichment score (NES) of the 20 most upregulated pathways in responders as compared to non-responders to RT treatment. Blue bars represent non-significant changes (p>0.05). Red bars represent significant changes (p<0.05). (G) Enrichment curve showing enrichment of the IL-2/STAT5 signaling pathway in responders compared to non-responders to RT. NES=1.42; p.adj=0.046. (H) Bar chart showing the genes most frequently appearing in the top 10 most upregulated pathways in responders relative to non-responders. (I) viSNE dimensionality reduction analysis of PBMCs collected from PDAC patients performed on live CD45⁺CD3⁺ cells showing expression of the immune cell markers CD8, CD4, and FoxP3, and the exhaustion marker PD-1 before, during, and after RT. Data represented as mean ± SEM. P-values calculated with Students T-test, *indicates p<0.05, **<0.01, ***<0.001, ****<0.0001. See also Figures S5 and S7.