Optimizing In Situ Vaccination During Radiotherapy

Abstract

Effective in situ cancer vaccines require both a means of tumor cell death and a source of adjuvant to activate local dendritic cells. Studies have shown that the use of radiotherapy (RT) to induce tumor cell death and anti-CD40 to activate dendritic cells can result in in situ vaccination in animal models. Here, investigations are carried out on potential strategies to enhance such in situ vaccination. Strategies investigated include the use of smart immunogenic biomaterials (IBM) loaded with anti-CD40 in different tumor types including immunologically cold tumors like pancreatic and prostate tumors. The use of downstream checkpoint inhibitors to further boost such in situ vaccination is also examined. Results indicate that the use of IBM to deliver the anti-CD40 significantly enhances the effectiveness of in situ vaccination with anti-CD40 compared with direct injection in pancreatic and prostate cancers (p < 0.001 and p < 0.0001, respectively). This finding is consistent with significant increase in infiltration of antigen-presenting cells in the treated tumor, and significant increase in the infiltration of CD8⁺ cytotoxic T lymphocyte into distant untreated tumors. Moreover, in situ vaccination with IBM is consistently observed across different tumor types. Meanwhile, the addition of downstream immune checkpoint inhibitors further enhances overall survival when using the IBM approach. Overall, the findings highlight potential avenues for enhancing in situ vaccination when combining radiotherapy with anti-CD40.

Keywords: abscopal effect; cancer vaccine; dose-painting; immunogenic biomaterials; immunotherapy; pancreatic cancer; prostate cancer; radiotherapy.

Figure 1

Anti-CD40+RT generates effective in situ vaccination (abscopal effect) and can prevent distant lung metastasis. (A) Cartoon showing mouse treatment model with two pancreatic tumors in two flanks where only one tumor was treated and experimental schedule of the study observed for 6 weeks posttreatment of 5 Gy of IGRT or dAntiCD40 (20 µg), or the combination (RT+dAntiCD40), along with a control. (B) Showing ex vivo tumor weight of treated and untreated secondary/abscopal tumors, 6 weeks posttreatment. Inset, pictures of the representative tumors. Here, the developed palpable sized one of the subcutaneous tumors was given 5 Gy of IGRT (n = 8) or dAntiCD40 (n = 8), or RT+dAntiCD40 (n = 9). Antimouse CD40 antibody was injected intratumorally with of 20 µg in PBS for a single time. The control group (n = 8) was injected with same volume of PBS. (C). Macroscopic view (top) and hematoxylin and eosin-stained microscopic/histological view (bottom) of ex vivo lungs of the same group of mice, which were harvested 8 weeks postimplant (6 weeks posttreatment) and fixed in 10% formalin. Twenty-four-hour formalin-treated tumors were imaged for macroscopic pictures. For histological analysis, samples were imbedded in paraffin for tissue processing and generated 0.5-mm-thick slides. Scale bar represents 100 µm. A bar graph showing number of mice in each treatment group with microscopic lung metastases 8 weeks after tumor implantation. (D) Bar graph showing tumor weight of the abscopal response (untreated) tumors of the combination treated (RT+dAntiCD40) group corelating with presence of lung metastasis shown in histological analysis. (E) study design for the treatment timing showing early (2 weeks post implant) and late (3 weeks post implant) treatment onset with the combination treatment group (RT+dAntiCD40). (F). Kaplan Meier survival graph for early and late treatment onset of combination treatment with RT+dAntiCD40 in pancreatic adenocarcinoma mouse model (n = 5). ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001. Error bars are SD. ^*** p < 0.001.

Figure 2

Anti-CD40 agonist-loaded immune-biomaterial (IBM) may provide imaging contrast and induces higher immunogenicity in treated tumor microenvironment. (A) Schematic diagram of IBM made of immunogenic polymer with antibody core (red), which releases as it biodegrades. (B) CT image showing IBM in tumor (blue color) highlighted using Imalytics software (left) and gradually fading CT images of IBM from days 1 to 6, as polymer biodegrades (right). (C) Fluorescent images and representative bar graph of the average fluorescent intensity of immunofluorescence-stained prostate cancer tissue treated with mouse CD11b⁺ antibody at posttreatment day 7. Corresponding immunofluorescence (merged) pictures showing infiltration of CD11⁺ dendritic cells (red) in tumor tissue. Cancer cell nucleus (blue, DAPI) when IBM or dAntiCD40 was given intratumorally (n = 3). Scale bar is 2,000 µm. (D) Treatment design and (E) prostate tumors generated from TRAMP-C1-derived castration-resistant prostate cancer cells treated with IGRT (5 Gy) and/or IBM, representing CT images (Imalytics software analyzed) of animals from different treatment cohorts with treated tumors (blue) and untreated tumors (pink). Bar graph represents treated and untreated tumor volumes on day 10 posttreatment (n = 5). (F, G) The tumor volumes of pancreatic (n = 5, IGRT 5 Gy¹⁶) and cervical cancer (n = 5, IGRT 6 Gy²⁹), respectively 10 days after the same treatment regimen. Data represent the mean ± SD. *p < 0.5, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001.

Figure 3

IBM enhances higher abscopal effect when compared with direct intratumor injection of anti-CD40 agonist (dAntiCD40). (A) Study design. (B) Line graphs showing dynamics of tumor volume change of pancreatic cancer for both treated (solid line) and the other untreated cancer (dashed line, in situ vaccination effect) where IGRT of 5 Gy was given either as direct intratumoral injection of anti-CD40 (dAntiCD40) or IBM along with 5 Gy of radiation. (C) In prostate cancer model, line graphs showing dynamics of tumor volume change in prostate cancers of both treated (left) and the other untreated/metastatic cancer (right in situ vaccination effect) where image-guided radiotherapy (IGRT) at 5 Gy was administered either in combination with direct intratumoral injection of anti-CD40 (dAntiCD40) or IBM. (D) Corresponding Kaplan-Meier survival graph. (E) Representative images and bar graph showing IF staining of untreated pancreatic cancer tissue stained with CD4⁺ and CD8⁺ antimouse antibody, showing intratumoral infiltration of CD4⁺ (green) and CD8⁺ (red) T lymphocytes, comparing the abscopal effect of tumors treated with RT+dAntiCD40 (n = 3) and RT+IBM (n = 3). In all cases, nucleus is stained with DAPI (blue). Scale bar represents 200 µm. The corresponding bar graph showing average fluorescence intensity of infiltrating helper CD4⁺ and cytotoxic CD8⁺ T lymphocytes in untreated tumor tissue on the opposite flank (abscopal tumors) 14 days following the treatment. Data represent the mean ± SD. ^* p < 0.5, ^** p < 0.01, ^*** p < 0.001.

Figure 4

IBM further enhance the survival duration in combination with anti-PD1 and anti-CTLA4. (A) Research design to treat prostate cancers adding anti-PD1 and anti-CTLA4 antibody (IP) with the RT+IBM treatment. One treatment of IBM was given intratumorally (IT) in one out of two implanted tumors following 5 Gy IGRT in the same cancer on day 0. Anti-PD1and/or anti-CTLA4 was given intraperitoneally (IP) on days 0, 3, and 6. (B) Kaplan-Meier survival curve and (C) line graphs of dynamics of tumor volume change of both treated (left) and the other untreated/metastatic cancers (right) of this study with 5 Gy of IGRT+IT IBM followed by IP injection of anti-PDL-1 and/or CTLA4. (D, E) representing graph showing body score and body weight of the same cohort groups. All statistical significance was compared with controls. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001; NS, not significant.