Ponatinib Averts αCD40 Antibody Mediated Toxicity by Lowering MAPK38 Expression and Shows Proimmunogenic Effects in a Murine Tumor Model

Abstract

The use of the agonist CD40 antibody (αCD40) is associated with several disadvantages including the cytokine release syndrome (CRS), hepatotoxicity, and induced PD-L1 expression. Previously, we have demonstrated that ponatinib, a tyrosine kinase inhibitor, could inhibit induced programmed death ligand 1 (PD-L1) expression. In this study, we showed that combinatorial treatment of αCD40 and ponatinib delayed the tumor growth and overall survival in mice bearing B16-F10 melanoma and 4T1 orthotopic tumors. The combination treatment increased the CD45⁺CD8⁺ T cell population in the tumor; induced CD86 expression; and lowered the expression of PD-L1, FOXP3, and Arginase-1 in both the tumor and spleen. Interestingly, ponatinib averted both immuno- and hepatotoxicity of αCD40 monotherapy by lowering alanine aminotransferase (ALT), aspartate aminotransferase (AST), IL-6, IL-10, and IL-1β levels through downregulating MAPK38 and ERK1/2 expression. Our results suggest that this combination can be further explored in clinics to improve the in vivo antitumor efficacy of αCD40 while reducing the associated toxicities.

Introduction

αCD40 has shown promising antitumor efficacy in both preclinical and clinical studies as it can induce potent antitumor responses either directly by killing the cancer cells or indirectly by activating the cells of adaptive and innate immunity, sensitizing them to other therapies. − Similarly, the ligation of αCD40 to the CD40 receptor has been shown to induce significant changes in the tumor microenvironment (TME) by promoting the activation of antigen presenting cells (APCs), enhancing Th1 cytokine release and macrophage repolarization from M2 (protumor) to M1 (antitumor) phenotype, and improving tumor-specific CD8⁺ T cell infiltration resulting in increased antitumor response. ,

Despite achieving favorable antitumor efficacy, αCD40 use is often linked to several potential disadvantages that mainly include CRS, organ-specific toxicities, autoimmune reactions, thromboembolic syndromes, etc. , Although some studies reported that the toxicity associated with αCD40 is transient, dose-limiting toxicities associated with αCD40 still remain as one of the bottlenecks for further clinical development. , Recently, various strategies have been developed to improve the efficacy of αCD40 by combining different therapeutics to improve the overall antitumor efficacy and survival. − In addition to the combination therapy of αCD40 with anti-PD-1 or gemcitabine, new CD40 agonists have been developed with improved tolerability profiles. − Moreover, αCD40 administration has been observed to increase the expression of PD-L1, PD-1, and CTLA-4 in TME, leading to the dysfunction and apoptosis of tumor killing immune cells, ultimately causing immune resistance and supporting tumor progression. The increase in the expression of PD-L1 is mainly due to the surge in IFN-γ that is released by the activated APCs following αCD40 treatment. The surge in IFN-γ creates a negative feedback loop that supports tumor growth by increasing the expression of PD-L1. The increase in PD-L1 expression eventually hampers the αCD40-induced tumor-specific T cell response and impairs the ability of αCD40 to produce long-lasting antitumor immunity, resulting in the limited anticancer efficacy of αCD40 monotherapy. In our previous studies, we reported that ponatinib, an FDA-approved tyrosine kinase (BCR-ABL) inhibitor used for the treatment of chronic myeloid leukemia patients, can lower IFN-γ-induced PD-L1 expression and improve in vivo antitumor efficacy in solid tumors. − Although prior reports have shown that ponatinib has exhibited some vascular and cardiotoxicity in humans, the preclinical usage in mice is well tolerable. Recently, a few reports have shown that ponatinib has the ability to reduce CRS in SARS-CoV2 and influenza infection via inhibiting some key cytokine-releasing pathways like MAP3K8, ERK1/2, EPHA3, and JAK1. ,

In this study, we have used a novel combination of αCD40 with ponatinib to improve the antitumor efficacy of αCD40 and to address the drawbacks associated with αCD40 treatment. The combination treatment significantly delayed the tumor growth and improved the overall survival of mice bearing B16-F10 melanoma and 4T1 orthotopic breast tumor. Moreover, combination treatment showed a significant increase in effector CD8⁺ T cell population in tumors and lowered PD-L1 and FOXP3 expression, which correlates with decreased tumor burden. Also, ponatinib enhanced the M2 to M1 repolarization efficacy of αCD40 by significantly increasing the expression of M1 markers like CD86 while reducing Arginase-1 (M2 marker) expression in both the tumor and spleen. Similarly, combination-treated mice showed a significant increase in pro inflammatory cytokine (IL-2, IFN-γ, and TNF-α) levels in the spleen. In addition, the combination treatment showed an increase in both CD4⁺ and CD8⁺ T cell population in the spleen and in cytotoxic T-lymphocyte (CTL) activity and a decrease in PD-L1, FOXP3, and Arginase-1 expression. Most interestingly, ponatinib treatment lowered the in vivo toxicities associated with αCD40 monotherapy by significantly lowering the levels of ALT, AST, ALP, IL-6, IL-10, and IL-1α in serum through MAPK38 inhibition. Taken together, these data suggest that ponatinib treatment could be a feasible strategy to reduce αCD40-induced toxicities and PD-L1 expression while improving the in vivo antitumor efficacy and warrant the use of this novel combinatorial therapy in clinical settings.

Materials and Methods

Mice

Female 6–8 week old C57BL/6 and BALB/c mice were housed in a pathogen-free environment at the animal facility at the Regional Center for Biotechnology, Haryana, and Central Drug Research Institute (CSIR), Lucknow, respectively. All animal experiments in this study have ethical approval from the RCB (ethical approval RCB/IAEC/2020/081) and CDRI, Lucknow (IAEC/2022/1/Renew-0), Institutional Animal Ethics Committee, respectively.

Cell Lines

B16-F10 and RAW 264.7 cells were purchased from the National Centre for Cell Science (NCCS), India. The 4T1 cell line was a kind gift from Dr. Smrati Bhaduaria from the Central Drug Research Institute (CSIR), Lucknow, India. The cells were cultured and maintained in DMEM (cat. #A007S, HIMEDIA) supplemented with 10% FBS and antibiotics (penicillin and streptomycin) in an incubator at 37 °C with 5% CO₂. Post thawing, the cells were cultured for a duration spanning between 3 and 10 passages, but the culture never exceeded 1 month. All cell lines used in this study were tested for the absence of Mycoplasma contamination by PCR.

Cytokines and Growth Factors

LPS (cat. #L2654) was purchased from Sigma-Aldrich. IFN-γ (cat. #575306) and IL-4 (cat. #431101) were purchased from Biolegend USA.

In Vitro Cytotoxicity Activity of Ponatinib

To estimate the cytotoxic activity of ponatinib on macrophages. RAW 264.7 cells at a density of 8 × 10³were seeded in a 96-well plate overnight. The next day, the cells were treated with different concentrations of ponatinib (cat. #A10080, Adooq Biosciences) ranging from 0.001 to 100 μM for 48 h at 37 °C with 5% CO₂. After 48 h of treatment, cell cytotoxicity was evaluated as described elsewhere. Briefly, MTT solution (5 mg/mL) in DMEM was added and incubated for 4 h. After 4 h, 100 μL of DMSO was added to each well, and absorbance was measured at 570 nm using a microplate spectrophotometer (Epoch, BioTek, USA). The experiment was performed in triplicate, and the IC₅₀ value was obtained.

In Vitro PD-L1 Expression

RAW 264.7 cells at a density of 2.5 × 10⁵ were seeded in a six-well plate in DMEM containing 10% FBS and 1% penicillin–streptomycin and incubated overnight at 37 °C with 5% CO₂. Before the treatment, αCD40 (FGK4.5, cat. #BE0016-2, BioXcell) was cross-linked using goat anti-rat IgG (Biolegend, cat. #405401) by incubation for 30 min at a 2:1 molar ratio (goat anti-rat IgG to FGK4.5) at room temperature. Then, the cells were differently treated with PBS, isotype control, 20 μg/mL αCD40 (cross-linked to IgG) alone, 0.25 μM ponatinib alone, and a combination of both and incubated for 24 h at 37 °C with 5% CO₂. After 24 h, the cells were harvested, 0.1 × 10⁶ cells were stained with PE labeled anti-PD-L1 (cat. #124308, Biolegend USA), and PD-L1 expression was evaluated by a flow cytometer.

In Vitro Repolarization Studies

RAW 264.7 cells were seeded at a density of 2.5 × 10⁵ and were treated with IL-4 at a concentration 20 ng/mL for 48 h to make M2 polarized RAW 264.7 cells. Before the treatment, αCD40 was cross-linked with goat anti-rat IgG as mentioned in the previous section. Then, the cells were differently treated with PBS, isotype control, 20 μg/mL αCD40 (cross-linked to IgG) alone, 0.25 μM ponatinib alone, and a combination of both and incubated for 24 h at 37 °C with 5% CO₂. Post treatment, the cells were then harvested, 0.1 × 10⁶ cells were stained with APC labeled anti-CD206 (cat. #141708, Biolegend USA) and APC labeled anti-CD86 (cat. #105012, Biolegend USA), and M1 and M2 population was evaluated by a flow cytometer. The gates were applied with respect to the unstained population. The levels of TGF-ß and IL-10 were also estimated from the culture media of the treated cells using ELISA kits according to the manufacturer’s instructions.

In Vivo Tumor Experiments

Tumor Regression and Survival Studies

A B16-F10 mouse melanoma model was established by inoculating 2.5 × 10⁵ cells mixed with Matrigel (cat. #E6909, Sigma) subcutaneously near the back of 6–8 week old female C57BL/6 mice on day 1. When the tumor was in the range of 80–110 mm³, the mice were randomized into four groups of five mice, namely, untreated control, αCD40, ponatinib, and combination. The mice received a dose of αCD40 (FGK4.5, 3 mg/kg) ip on days 17 and 22 in both the αCD40 alone and combination groups. However, mice in the ponatinib alone group received ponatinib (15 mg/kg) ip on days 17, 18, 22, and 23 and on days 19, 20, 24, and 25 in the combination group, as shown in Figure A. The tumor size and body weight were measured every alternate day. The tumor size was measured by calipers, and the volume was calculated by using (D*d*d)/2, with D and d being the longest and shortest tumor diameter, respectively, in millimeters.

2.

Ponatinib improved the in vivo antitumor efficacy of αCD40. (A, B) Schematic representation of αCD40 and ponatinib tumor inoculation and dosing regimen in mice bearing B16-F10 and 4T1 orthotopic tumors, respectively. (C–H) Tumor regression and survival studies in C57BL/6 mice bearing B16-F10 tumors. Briefly, 2 × 10⁵ B16-F10 tumor cells were implanted in the right flank of 6–8 week old C57/BL6 mice on day 1. When the tumors volume reached ∼80–110 mm³, mice were treated with αCD40 (3 mg kg^–1 BW), ponatinib (15 mg kg^–1 BW), and their combination. (C) Tumor volume over time of C57BL/6 mice bearing B16-F10 (n = 5). (D–G) Tumor volume over time of the individual mouse in the control (D), αCD40 (E), ponatinib (F), and combination (G) treatment group (n = 5). (H) Kaplan–Meier survival analysis of mice bearing B16-F10 tumors in each group (n = 5). (I) Body weight of mice bearing B16-F10 tumors over time up to day 27 (n = 5). (J–O) Tumor regression and survival studies in BALB/c mice bearing 4T1 tumors up to day 48. Briefly, 1 × 10⁶ 4T1 cells were subcutaneously injected into the mammary fat of 6–8 week old female BALB/c mice on day 1. When the tumor was in the range of 80–120 mm³, mice were treated with αCD40 (3 mg kg^–1 BW), ponatinib (15 mg kg^–1 BW), and their combination. (J) Tumor volume over time of BALB/c mice bearing 4T1 tumors (n = 5). (K–N) Tumor volume over time of the individual mouse in the control (K), αCD40 (L), ponatinib (M), and combination (N) treatment group (n = 5). (O) Kaplan–Meier survival analysis of mice bearing 4T1 tumors in each group (n = 5). (P) Body weight of mice bearing 4T1 tumors over time up to day 17 (n = 5). The values are mean ± SD (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05).

In the 4T1 tumor model studies, 1 × 10⁶ 4T1 cells were subcutaneously injected into the mammary fat of 6–8 week old female BALB/c mice. When the tumor was in the range of 80–120 mm³, the mice were randomized into four groups of five mice, namely, untreated control, αCD40, ponatinib, and combination. The treatment schema used for the treatment is shown in Figure B. The mice were sacrificed when the tumor volume reached >2500 and >1000 mm³ in the case of the B16-F10 melanoma and 4T1 orthotopic breast murine model, respectively. Cumulative survival curves were compared using Kaplan–Meier analysis.

Immunophenotyping, ELISA, Western Blotting, IHC, and Toxicity Analysis

The B16-F10 mouse melanoma model and 4T1 orthotopic breast model were established, and the dosing regimen mentioned in the previous section was followed. After 48 h of the completion of the dosing of the combination group, at days 27 and 17, mice bearing B16-F10 and 4T1 tumor were sacrificed, respectively. Post sacrifice, the tumor, spleen, liver, and kidney were excised and used for further study. Blood was also collected on the same day.

CTL Assay

Ex vivo cytotoxicity was evaluated by using the MTT assay as described previously. − Briefly, after the completion of the therapy, the spleen was isolated and processed into a single cell suspension, and splenocytes were incubated with B16-F10 cells at E/T ratios of 20:1 and 40:1. After 24 h of treatment, images were captured at 10× using a multimode cell imager (Cytation 3, Biotek, USA). To evaluate cytotoxicity, MTT solution (5 mg/mL) was added to the cells and incubated for 4 h at 37 °C with 5% CO₂. After 4 h, 100 μL of DMSO was added to each well, and absorbance was measured at 570 nm using a microplate spectrophotometer (Epoch, BioTek, USA). The experiment was performed in triplicate.

Western Blot Analysis of Various Markers of the Spleen and Tumor

The tumor and spleen lysate was prepared as described elsewhere. Briefly, the spleen and tumor tissue were homogenized with Mini Bead beater 16 in RIPA buffer and centrifuged at 12,000g at 4 °C for 30 min. The supernatant was collected, and the total protein content was estimated using a Micro BCA kit. Twenty micrograms of the tissue lysate was resolved by 12% SDS-PAGE. The gel was then transferred to the PVDF membrane for 45 min at 400 mA. After transfer, the PVDF membrane was blocked using 2% BSA in TBST for 2 h at RT. After blocking, the membrane was incubated with a primary antibody directed against the indicated antigens for 2 h at RT. The membrane was washed five times with TBST and incubated with antigen-specific secondary antibodies for 1 h at RT followed by five times washing with TBST and twice with PBS. After washing, the blot was developed with luminol and hydrogen peroxide solution, the image was captured using Syngene G:BOX, and bands were carefully observed.

Cytokine Analysis in the Tumor, Spleen, and Serum

Two days after treatment ended, the tumors and spleen were excised and mechanically dissociated into single-cell suspensions with the syringe plunger. The cell suspensions were passed through a 70 μm strainer. Cells were centrifuged at 350g for 10 min at 4 °C, and the supernatant was collected and stored at −80 °C. Different cytokine levels of IL-2, IFN-γ, IL-4, and TNF-α etc were estimated from the supernatant using murine ELISA kits according to the manufacturer’s instructions.

Flow Cytometry Analysis of the Tumor

As mentioned in the in vivo tumor experiments, mice were sacrificed 2 days after the completion of doses of the combination group on day 27 and 17 for the B16-F10 model and 4T1 model, respectively. B16-F10 and 4T1 tumors were excised out, and a single-cell suspension was prepared. Briefly, tumors were cut into small pieces and mechanically dissociated with the back of the syringe in the dissociation buffer containing 0.25 M EDTA with 2% FBS and 2% BSA. The cell suspension was allowed to settle for 5–10 min at RT, and the upper layer was passed through a 28G syringe in a fresh tube. Cells were washed multiple times with PBS to remove the debris. For staining, 1 × 10⁶ cells were stained with the APC-CD8 antibody (cat. #100712, Biolegend USA), APC-Cyc7 CD45 antibody (cat. #147718, Biolegend USA), FITC-CD4 antibody (cat. #100405, Biolegend USA), and Alexa Fluor 488 FOXP3 antibody (cat. #126406, Biolegend USA). The gates were applied with respect to the unstained population. Cell populations were analyzed on a BD Fortessa, and data were analyzed by the Flow JO software.

Flow Cytometry Analysis of the Spleen

Spleens were harvested at the end of the experiment. The single-cell suspension of the spleen was prepared as mentioned above. Red blood cells (RBCs) were lysed using the RBC lysis buffer. A total of 1 × 10⁶ cells were stained with the following antibodies: FITC-CD3 antibody (cat. #100204, Biolegend USA), APC-CD4 antibody (cat. #100412, Biolegend USA), APC-CD8 antibody (cat. #100712, Biolegend USA), Alexa Fluor 488 FOXP3 antibody (cat. #126406, Biolegend USA), and PE labeled anti-PD-L1 (cat. #124308, Biolegend USA). The gates were applied with respect to the unstained population. Cell populations were analyzed on the BD Fortessa, and data were analyzed by the Flow JO software.

Immunohistochemistry

Tumors were collected at the end of the experiment and fixed in 4% paraformaldehyde followed by paraffinization. For analysis, 0.2 μm microsections were prepared. The sections were blocked with fetal bovine serum for 1 h and then incubated with anti-CD40-PE (cat. #E-AB-F10280D, purchased from Elab Bioscience), PE labeled anti-PD-L1 (cat. #124308, Biolegend USA), anti-Ki-67 (cat. #Sc-15402, Santa Cruz Biotechnology, USA), and anti-Granzyme-B (cat. #EAB-60730, Elabscience, USA). Images were taken at 10× using a multimode cell imager (Cytation 3, Biotek, USA), and mean fluorescent intensity was quantified using the ImageJ software.

In Vivo Toxicity Studies

After 48 hr of completion of the therapy, blood was isolated from the mice by the retro-orbital bleed protocol. The liver, lungs, spleen, and kidney were also excised from the animals and were fixed with 4% paraformaldehyde. The sections were taken, and hematoxylin and eosin staining was performed. The sections were given random numbers and were handed over to an experienced pathologist expert in gastrointestinal and liver disorders. The cases were examined blindly. Spotty necrosis was identified per 10× objective field area (an Olympus BX43 bright field microscope was used). Spotty necrosis was defined as the loss of up to two hepatocytes by a lobular lymphocytic infiltrate. In addition, we also noted features such as evidence and the site of neutrophilic infiltrate and the presence of microgranulomas. All of these parameters were carefully compared among different treatment groups.

For the hepatic myeloid population, after the completion of the treatment, livers were harvested at the end of the experiment. The single-cell suspension of the liver was prepared as mentioned above. Red blood cells (RBCs) were lysed using the RBC lysis buffer. A total of 1 × 10⁶ cells were stained with the following antibodies: APC-CD86 antibody (cat. #105012, Biolegend USA), FITC-F4/80 antibody (cat. #123108, Biolegend USA), APC-Cyc7 CD45 antibody (cat. #147718, Biolegend USA), PE-CD40 antibody (cat. #124308, Biolegend USA), and CD11b (cat. #101208, Biolegend USA). Cell populations were analyzed on the BD Fortessa, and data were analyzed by the Flow JO software.

For blood parameter analysis, the serum was separated from the blood by centrifugation at 350g for 10 min at 4 °C, and levels of IL-6, IL-10, IL-1α, TNF-α ALT, and AST (ELISA kits, Elabscience) were measured by ELISA kits according to the manufacturer’s instructions. A Transasia/XL-200/B151110 instrument was used for blood biochemistry analysis. H and E staining was also performed from the fixed liver and kidney samples, and images were obtained using a compound light microscope (Nikon E600, Japan) fixed with a camera (Nikon DS Fi3, Japan). The expression of MAPK38 and ERK1/2 was determined in both the serum and tumor by Western blot analysis using anti-p38 MAPK Phospho (Thr180/Tyr182; cat. #690201, Biolegend USA) and Direct-Blot HRP anti-ERK1/2 Phospho (Thr202/Tyr204; cat. #675506), respectively.

Statistical Analysis

All of the experiments were performed in triplicate. The results show the mean of at least three experiments ± SD. Statistical analysis was carried out using GraphPad Prism 7, and P values were calculated by the unpaired t test and one-way ANOVA. The significance of the difference between the two treatments was analyzed by Tukey’s multiple comparison tests.

Results

Ponatinib Lowered αCD40-Induced PD-L1 Expression and Promoted the Repolarization of M2 to M1 In Vitro

To determine the effect of ponatinib on the repolarization of macrophages and PD-L1 expression, we first checked the in vitro cytotoxicity of ponatinib on RAW 264.7 macrophage cells and found an IC50 of 3.1 μM (Figure A). Next, RAW 264.7 cells were treated with αCD40, and the PD-L1 expression was determined by flow cytometry analysis. Figure B shows a 30% increase in the PD-L1 expression with αCD40 treatment in comparison to the PBS control. In contrast, when RAW 264.7 cells were treated with a combination of both αCD40 and ponatinib, a 50% reduction in the levels of PD-L1 was observed compared to those of αCD40 monotherapy (Figure B).

1.

Ponatinib lowered αCD40-induced PD-L1 expression in vitro and enhanced the repolarization of M2 to M1. (A) In vitro cytotoxicity of ponatinib on RAW 264.7 cells. (B) Flow cytometry analysis of PD-L1 expression in RAW 264.7 cells (n = 3). Gates were applied to the unstained population. (C) Flow cytometry analysis of CD206 expression in RAW 264.7 cells (n = 3). (D) Flow cytometry analysis of CD86 expression in RAW 264.7 cells (n = 3). (E) M1/M2 ratio. (F) TGF-β and (G) IL-10 secreted by the M2 macrophage (n = 3). The values are mean ± SD (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05).

Repolarization of the macrophage from M2 to M1 is a key function of any immunomodulatory drug that improves the tumor-specific activity of the macrophage. Hence, to evaluate the repolarization potential, we treated M2 cells with both αCD40 and ponatinib. Interestingly, the flow cytometry data revealed that a 50 and 45% decrease in the population of CD206⁺ cells in comparison to the control was observed when M2 cells were treated with the combination and αCD40 alone, respectively (Figure C). To further confirm the repolarization effect of the combination treatment, the population of CD86⁺ was also estimated by flow cytometry analysis. The flow data showed a 3- and 2-fold increase in the population of CD86⁺ with combination treatment compared to control and αCD40, respectively (Figure D). Furthermore, M1/M2 was also determined by calculating the ratio of CD86⁺ to CD206⁺ cells, and the M1/M2 ratio was found to be superior with combination treatment in comparison to other groups (Figure E). Moreover, the levels of TGF-β and IL-10 (M2 markers) were estimated, and the concentration of both TGF-β and IL-10 was significantly reduced in the combination group compared to PBS, αCD40, and ponatinib treatment (Figure F,G). Together, these results confirmed that ponatinib not only lowered the surged PD-L1 expression but also aided αCD40 in repolarizing M2 macrophages to the M1 phenotype in vitro.

Ponatinib Improved the In Vivo Antitumor Efficacy of αCD40

To investigate the in vivo antitumor efficacy, B16-F10 melanoma (Figure A) and 4T1 orthotopic mouse (Figure B) models were established, and dosing was given as shown in Figure A,B. The growth of B16-F10 tumors was significantly delayed when mice were treated with αCD40 + ponatinib compared to αCD40, ponatinib, and the control (Figure C). Figure D–G shows tumor growth curves of individual mice in each group. Furthermore, the mice that received the combination treatment had a median survival of 61 days versus 46, 45, and 36 days in the αCD40, ponatinib, and control group, respectively (Figure H). Furthermore, Ki-67 is well reported to play a critical role in tumor cell proliferation and is a key marker for assessing tumor aggressiveness. Hence, we estimated the expression of Ki-67 in B16-F10 tumors by IHC. Interestingly, Ki-67 expression was significantly reduced in the tumors of mice receiving the combination treatment compared to other groups (Figure S1A). No significant change in the body weight of the mice in any group was observed throughout the course of the treatment (Figure I).

To check the antitumor potential of this novel combination across tumor types, mice bearing 4T1 orthotopic tumors were treated with αCD40, ponatinib, and their combination (Figure B). Like the B16-F10 model, the combination treatment delayed the growth of the 4T1 tumor significantly better than αCD40, ponatinib, and the control (Figure J). Figure K–N shows tumor growth curves of individual mice in each group. Furthermore, the mice that received the combination treatment had a median survival of 47 days versus 32, 32, and 26 days in the αCD40, ponatinib, and control group, respectively (Figure O). No significant change in the body weight of the mice in any group was observed throughout the course of the treatment (Figure P). Therefore, these results showed that the combined therapy of αCD40 and ponatinib outperformed the monotherapy in multiple tumor models.

Combination Treatment Enhanced Cytotoxic CD8⁺ T Cell Infiltration and Reduced Treg Cell Activity

Infiltration of cytotoxic CD8⁺ T cells is key to any immunotherapy as cytotoxic CD8⁺ T cells are the prime effector cells that have the ability to neutralize the function of cancer cells and inhibit their growth and proliferation. Hence, we estimated CD8⁺ T cell infiltration in the tumor. Strikingly, the population of CD45⁺CD8⁺ was found to be significantly higher in the mice bearing B16-F10 tumors when treated with the combination of αCD40 and ponatinib in comparison to other treatment groups (Figure C). Similar results were observed in 4T1 tumors where the combination treatment showed the highest expression of CD8a compared with other groups (Figure D). Similarly, IHC data also revealed that the mice bearing either B16-F10 or 4T1 tumors showed significantly higher expression of CD8a with the combination treatment compared with other groups (Figure E). Moreover, Western blot analysis of the B16-F10 tumors that received combination treatment showed a 3-fold increase in CD8a expression compared to the control, whereas only a 1.5-fold increase was observed with αCD40 treatment (Figure F). Various studies have highlighted the tumoricidal role of Granzyme B (GzmB) that is secreted by activated cytotoxic T cells. Hence, we estimated the expression of GzmB in B16-F10 tumors by IHC. Interestingly, the combination treatment significantly increased the expression of GzmB in the tumors compared to other groups (Figure S1B).

3.

Combination treatment enhanced cytotoxic CD8⁺ T cell infiltration, reduced Treg cell activity, and polarized M2 TAMs to M1 TAMs. (A, B) Schematic representation of αCD40 and ponatinib tumor inoculation and dosing regimen in mice bearing B16-F10 and 4T1 orthotopic tumors, respectively (n = 3). After 48 h of the completion of doses of the combination group, on day 27 and 17 for the B16-F10 model and 4T1 model, respectively, all mice were sacrificed for immunophenotyping, ELISA, Western blot analysis, and IHC studies. (C) Flow cytometry data showing CD8⁺ T infiltration in B16-F10 tumors (n = 3). (D) Flow cytometry data showing CD8⁺ T infiltration in 4T1 tumors (n = 3). (E) IHC images of tumor sections of B16-F10 (top panel) and 4T1 (bottom panel) tumors showing the levels of CD8a expression (n = 3). (F) Western blot analysis of CD8a expression in B16-F10 tumors across treatments (n = 3). (G) Flow cytometry data showing the population of Treg cells in B16-F10 tumors across different groups (n = 3). (H) Western blot analysis of FOXP3 expression in B16-F10 tumors (n = 3). (I, J) Flow cytometry data showing M1 TAMs (F4/80⁺CD86⁺) (I) and M1 TAMs (F4/80⁺CD206⁺) (J) in B16-F10 tumors (n = 3). (K) Western blot analysis showing the expression of Arginase-1 (n = 3). The values are mean ± SD (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05).

T regulatory cells (Tregs) are known to suppress the tumor-specific immune attack and favor tumor development and metastasis. Hence, to determine Treg activity, we estimated the population of CD4⁺FOXP3⁺ cells by flow cytometry. Strikingly, the population of CD4⁺FOXP3⁺ cells was significantly reduced in the mice bearing B16-F10 tumors when treated with the combination of αCD40 and ponatinib in comparison to other groups (Figure G). Additionally, we also checked the expression of FOXP3, a transcription factor of Tregs, in the tumor tissue by Western blotting. Interestingly, Western blot data of mice bearing B16-F10 tumors revealed that FOXP3 levels were reduced by 75 and 50% in the combination-treated tumor compared to control and αCD40 monotherapy, respectively (Figure H). Taken together, the combination treatment not only enhanced the infiltration of cytotoxic T cells in tumors but also reduced Treg activity, leading to a potent antitumor response.

Ponatinib Improved the TAM Repolarization Potential of αCD40

To evaluate the repolarization of M2-type tumor associated macrophages (M2 TAMs) toward M1-type tumor associated macrophages (M1 TAMs) through combination therapy, we examined the population of macrophages in B16-F10 tumors by flow cytometry and Western blotting analysis. M1 macrophages in tumors have the potential to directly kill the cancer cells by their intrinsic phagocytic ability and through the release of proinflammatory cytokines. , Therefore, we sought to estimate the population of F4/80⁺CD86⁺ cells (M1 TAMs) by flow cytometry and found a significantly higher population of M1 TAMs in the B16-F10 tumors that received the combination treatment in comparison to other groups (Figure I). Also, many studies have reported that M2 macrophages support tumor growth by releasing various anti-inflammatory cytokines like IL-10 and IL-4 and help the tumor to escape the immune surveillance. Hence, we estimated the population of F4/80⁺CD206⁺ (M2 TAMs) macrophages in the B16-F10 tumors. Interestingly, the population of F4/80⁺CD206⁺ macrophages was significantly reduced with the combination treatment compared to the control, αCD40, and ponatinib monotherapy (Figure J). Further, we also checked the level of Arginase-1 in the tumors by Western blot analysis and found that the combination treatment lowered the expression of Arginase-1 by 3- and 4-fold compared to αCD40 alone and the control, respectively (Figure K). Various reports suggest that the ratio of M1 to M2 TAMs is very crucial to achieve a robust antitumor response. Hence, we also sought to check the ratio of M1 to M2 TAMs in B16-F10 tumors by calculating the ratio of F4/80⁺CD86⁺ to F4/80⁺CD206⁺ cells, and we found that the M1/M2 ratio in the tumors that received combination treatment was superior in comparison to other groups (Figure S2A). Therefore, these results clearly demonstrated that the combined therapy of αCD40 and ponatinib facilitated the repolarization of TAMs from M2 toward the M1 phenotype to produce a robust antitumor response.

Ponatinib Boosted the Antitumor Splenic Immunity of αCD40 Therapy

To achieve a robust antitumor response, cross talk of TME with the spleen is crucial. Thus, we checked the ability of αCD40 and ponatinib combination in boosting splenic antitumor immunity. First, we sought to analyze the population of CD8⁺ T cells by flow cytometry and found that the combination therapy significantly increased the splenic CD3⁺CD8⁺ T cell population when compared to control and αCD40 monotherapy in mice bearing B16-F10 tumors (Figure A,B). Similar results were also observed in the 4T1 tumor model where the combination treatment showed a significantly higher population of CD8⁺ T cells in comparison to the control, αCD40, and Ponatinib monotherapy (Figure C,D). CTLs are the specific effector T cells that have the potential to neutralize the function of tumor cells. Therefore, we checked the CTL activity of splenocytes (E) isolated from mice against B16-F10 cells (T). The CTL activity of splenocytes isolated from the mice that received both αCD40 and ponatinib at E/T ratios of 20:1 and 40:1 showed better cytotoxicity toward B16-F10 cells among all groups (Figure E,F). Together, these results along with the CD8⁺ T cell population were related to the improved antitumor T cell activity in the spleen.

4.

Ponatinib boosted the antitumor splenic immunity of αCD40 therapy. Mice bearing B16-F10 and 4T1 tumors were treated as mentioned in Figure A. At days 27 and 17, mice bearing B16-F10 and 4T1 tumor were sacrificed, respectively; spleens were excised; and various immunomodulatory markers were evaluated by flow cytometry, Western blotting, and ELISA. (A–D) CD8⁺ T cell population in the spleen in mice. (A, B) Flow cytometry analysis of the CD3⁺CD8⁺ T cell population in the spleen of mice bearing B16-F10 tumors (n = 3). (C, D) Flow cytometry analysis of CD3⁺CD8⁺ T cell population in the spleen of mice bearing 4T1 tumors (n = 3). (E) CTL assay in the B16-F10 model. After the completion of the treatment, splenocytes from mice bearing B16-F10 tumors were isolated, and a CTL assay was performed at 20:1 and 40:1 ratio to determine the CTL activity of splenocytes (n = 3). (F) Representative microscopic images after 24 h of treatment (n = 3). (G) Flow cytometry analysis of CD4⁺FOXP3⁺ Treg cell population in the spleen of mice bearing B16-F10 tumors (n = 3). (H–K) Western blot analysis showing the expression of FOXP3 (I), Arginase-1 (J), and CD86 (K) (n = 3). (L–O) Estimation of different proinflammatory and anti-inflammatory cytokines: IFN-γ (L), TNF-α (M), IL-2 (N), and IL-4 (O), respectively (n = 3). The values are mean ± SD (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05).

Modulation of the spleen microenvironment from protumor to antitumor is very important for effective antitumor efficacy. FOXP3⁺ regulatory T cells have the ability to effectively suppress the differentiation of naïve T cells into myelopoietic cytokine-producing CD4⁺ T cells and dampen antitumor response. Therefore, we estimated the population of CD4⁺ FOXP3⁺ Tregs in the spleen and found a significant decrease in the Treg population in the mice that received the combination treatment in comparison to that of the αCD40 monotherapy (Figure G). Similarly, we estimated the expression of FOXP3 by Western blot analysis and found that when ponatinib was used in combination with αCD40, the expression of FOXP3 was lowered by 2-fold in comparison to αCD40 treatment (Figure H,I). To further check the status of M1 vs M2 macrophages with combination treatment, we estimated Arginase-1 and CD86 levels in the spleen of mice bearing the B16-F10 tumor by Western blot analysis. Interestingly, the Arginase-1 level was found to be the least in mice that received the combination treatment compared to αCD40 monotherapy and control (Figure H,J). Furthermore, mice treated with the combined therapy showed a 3- and 2-fold higher expression of CD86 in comparison to the control and αCD40, respectively (Figure H,K). Together, the combination treatment of αCD40 and ponatinib showed excellent M2 to M1 repolarization activity in the spleen and thus helped in boosting the antitumor efficacy.

To establish continuous antitumor splenic immunity, a balanced Th1/Th2 ratio is crucial. Hence, we evaluated the levels of various Th1 (IFN-γ, IL-2, and TNF-α) and Th2 (IL-4) cytokines. IFN-γ is known to exhibit immunomodulatory effects in the spleen that promote the generation of cancer-specific myeloid cells. We found that the IFN-γ level in the spleen was significantly higher in the mice treated with αCD40 and ponatinib in comparison to the control, αCD40, and ponatinib treatment (Figure L). Similarly, TNF-α has a key role in the development of tumor-specific splenic immunity, and we found that the TNF-α level was significantly higher in the mice that received combination treatment compared to control, αCD40, and ponatinib (Figure M). Additionally, the levels of IL-2, which is one of the key Th1 cytokine that helps to eradicate cancer, were increased in all treated groups where IL-2 levels surged 2-fold in the combination group compared to the control (Figure N). Moreover, we also checked the levels of IL-6 in the spleen and found a significant increase in the IL-6 level with the combination treatment in comparison to the control (Figure S4A). In the spleen, IL-4 is known to promote the apoptosis of cancer-specific B cells, and it was slightly lowered in the αCD40 treatment group (Figure O). Interestingly, IL-4 levels were significantly reduced in the group that received combination treatment with respect to the control (Figure O). Furthermore, Th1/Th2 was also determined by calculating the ratio of IFN-γ to IL-4, and we found that the Th1/Th2 ratio in the mice that received combination treatment was 1.523 ± 0.1015 in comparison to 0.553 ± 0.1295 and 0.5777 ± 0.1572 for αCD40 and ponatinib, respectively, indicating a dominant Th1 immunity than Th2 in combination-treated mice over αCD40 and ponatinib monotherapy (Figure S3A). Taken together, these data suggest that Ponatinib boosted the antitumor splenic immunity of αCD40 therapy.

Ponatinib Reduced PD-L1 Expression in Both the Tumor and Spleen

A high PD-L1 expression is often linked with the immune escape and progression of cancer cell proliferation and metastasis. Therefore, we sought to check the expression of PD-L1 in the tumor and spleen. The IHC data of both tumor models revealed that the combination treatment lowered the PD-L1 expression in comparison to the control and αCD40 monotherapy (Figure A–C). Moreover, we sought to estimate the PD-L1 expression in immune cells and found a significant decrease in the population of CD45⁺PD-L1⁺ cells in the B16-F10 tumors that received the combination treatment in comparison to control and αCD40 monotherapy (Figure D). Similarly, Western blot analysis of the mice bearing B16-F10 tumors showed that ponatinib treatment lowered the PD-L1 expression in the tumor by more than 50% compared to the control (Figure E,F). Interestingly, when ponatinib was used in combination with αCD40, we found a 50 and 32% decrease in the PD-L1 expression in tumor compared to the control and αCD40 alone, respectively (Figure E,F). Next, we wanted to check the PD-L1 expression in the spleen as high PD-L1 expression in the spleen dampens antitumor splenic immunity and is related to poor prognosis. Interestingly, in our study, flow cytometry analysis of the spleen showed a significant decrease in the population of CD45⁺PD-L1⁺ cells with the combination treatment in comparison to the αCD40 monotherapy (Figure G). Moreover, the Western blot analysis of the spleen of mice bearing B16-F10 tumors showed a 50% surge in the PD-L1 expression with the αCD40 treatment in comparison to the control (Figure H,I). Surprisingly, when ponatinib was used in combination with αCD40, the PD-L1 expression was significantly lowered compared to the αCD40 treatment (Figure H,I). Therefore, these results showed that ponatinib can lower PD-L1 expression in both the tumor and spleen, which correlates with better antitumor efficacy.

5.

Ponatinib reduced PD-L1 expression in both the tumor and spleen. Mice bearing B16-F10 and 4T1 tumors were treated as mentioned in Figure A,B. At days 27 and 17, mice bearing B16-F10 and 4T1 tumors were sacrificed, respectively; the tumor and spleen were excised; and levels of PD-L1 were determined. (A) IHC images of mice bearing B16-F10 and 4T1 tumors showing the levels of PD-L1 expression (n = 3). (B, C) MFI of PD-L1 expression in B16-F10 (B) and 4T1 (C) tumors (n = 3). (D) Flow cytometry analysis of the CD45⁺PD-L1⁺ population in the B16-F10 tumors across various treatment groups (n = 3). (E, F) Western blot analysis of PD-L1 expression in B16-F10 tumors (n = 3). (G) Flow cytometry analysis of the CD45⁺PD-L1⁺ population in the spleen of the mice bearing B16-F10 tumors across various treatment groups (n = 3). (H, I) Western blot analysis of PD-L1 expression in the spleen from B16-F10 mice (n = 3). The values are mean ± SD (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05).

Ponatinib Lowered the In Vivo Toxicity Associated with αCD40 Therapy

αCD40 treatment is known to cause hyperstimulation of CD40 expression, which ultimately leads to hyperactivation of immune response and results in cytokine release syndrome (CRS) and grade 2 and 3 hemato- and hepatotoxicity in vivo. , In our study, similar results were observed in mice bearing B16-F10 tumors where the expression of CD40 was significantly increased with αCD40 treatment, which is correlated with the hyperstimulation of CD40 (Figure A,B). In contrast, when treated with the combination, the expression of CD40 in the tumor was significantly reduced, confirming the decrease in the hyperstimulated expression of CD40 (Figure A,B). Similar results were obtained from the flow cytometry analysis where the B16-F10 tumors that received the combination treatment showed a significant decrease in the population of CD45⁺CD40⁺ cells in comparison to αCD40 monotherapy (Figure C). Previously, it has been shown that αCD40 treatment targets hepatic myeloid cells like CD11b⁺Gr-1⁺ and M1 macrophages (CD86⁺F4/80⁺) to cause liver damage. − Hence, we sought to estimate these myeloid cell populations in our study. Interestingly, the mice bearing B16-F10 tumors that received combination treatment showed a significant decrease in the hepatic myeloid population of both CD11b⁺Gr-1⁺ and CD86⁺F4/80⁺ cells in comparison to the αCD40 monotherapy (Figure D,E). As shown in previous studies, mice bearing 4T1 tumors when treated with αCD40 showed an enlarged spleen in comparison to the untreated control and the ponatinib monotherapy (Figure S4B). However, when treated with the combination, the spleen weight was significantly reduced in comparison to the αCD40 monotherapy and was found to be equivalent to the weight of the spleen in control mice (Figure S4B).

6.

Ponatinib mitigated the overall toxicity induced by αCD40 monotherapy. (A) Mice bearing B16-F10 tumors were treated as mentioned in Figure A. At day 27, mice bearing B16-F10 tumors were sacrificed, tumors were excised, and the expression of CD40 was determined by IHC (n = 3). (B) MFI of CD40 expression in B16-F10 tumors (n = 3). (C) Flow cytometry analysis of the CD45⁺CD40⁺ population in the B16-F10 tumors (n = 3). Mice bearing B16-F10 and 4T1 tumors were treated as mentioned in Figure A. At days 27 and 17, mice bearing B16-F10 and 4T1 tumors were sacrificed, respectively; the liver was excised out; and various myeloid cell populations were checked by flow cytometry. (D) Flow cytometry analysis of the CD11b⁺Gr-1⁺ population (n = 3). (E) Flow cytometry analysis of the CD86⁺F4/80⁺ population (n = 3). At days 27 and 17, mice bearing B16-F10 and 4T1 tumors were sacrificed, respectively, and blood was collected for serum analysis. (F) Serum IL-6 concentration in the mice bearing 4T1 tumors (n = 3). (G) Serum IL-10 concentration in the mice bearing 4T1 tumors (n = 3). (H) Serum IL-1α concentration in the mice bearing 4T1 tumors (n = 3). (I, J) After the completion of the treatment at day 17, serum circulating levels of hepatic biomarkers ALT (I) and AST (J) were determined in mice bearing 4T1 tumors. (K) After the completion of the treatment on days 27 and 17, mice bearing B16-F10 and 4T1 tumors were sacrificed, respectively, to excise the liver, and H & E staining was performed to determine the degree of organ damage. Representative images of liver H & E (scale bar = 100 μm; 10× magnification) in the mice bearing B16-F10 and 4T1 orthotopic tumors where the black arrows indicate the spotty necrosis and blue arrows indicate the microgranulomas (n = 3). (L) Quantification of necrotic lesions observed per 10× field in the liver of mice bearing 4T1 tumors (n = 3) (M, N) Western blot analysis of MAKP38 expression in the serum of mice bearing B16-F10 tumors (n = 3). (O, P) Western blot analysis of MAKP38 expression in B16-F10 tumors (n = 3). (Q, R) Western blot analysis of ERK1/2 expression in the serum of mice bearing B16-F10 tumors (n = 3). The values are mean ± SD (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05).

To measure CRS, we checked the serum IL-6 and TNF-α levels in mice bearing 4T1 tumors and found a significant 3- and 2-fold increase in the IL-6 and TNF-α concentration, respectively, with αCD40 treatment when compared to the control (Figure F, Figure S4C). However, in the combination group, IL-6 and TNF-α levels were lowered by 40 and 30%, respectively, in comparison to αCD40 monotherapy (Figure F, Figure S4C). Similar results were also observed in the B16-F10 model where the serum IL-6 levels were significantly reduced by more than 80% with combination treatment compared to αCD40 monotherapy (Figure S4D). A similar trend was also observed with the circulating levels of IL-10 and IL-1α in the serum of mice bearing 4T1 tumors where the levels of both cytokines were significantly reduced with the combination treatment in comparison to the αCD40 monotherapy (Figure G,H). To measure the degree of hepatotoxicity, we estimated the levels of toxicity biomarkers like ALT and AST in the serum. − In both tumor models, when αCD40 was administered, a sudden spike in the levels of ALT and AST in serum was observed in comparison to the control, which correlates with severe liver damage (Figure S4E and Figure I,J). Strikingly, when ponatinib was administered along with αCD40, the levels of these enzymes showed a 3-fold decrease in comparison to the αCD40 alone (Figure S3E and Figure I,J).

To further confirm the in vivo toxicity, H & E staining of the liver was performed in both B16-F10 and 4T1 tumor models. H & E staining showed that the mice that received αCD40 showed signs of moderate to severe microgranulomas, portal inflammation, spotty necrosis, and evidence of periportal inflammation indicating severe liver damage (Figure K, Figure S5A5). Interestingly, when ponatinib was used in combination with αCD40, H & E staining of the liver showed mild to moderate microgranulomas (indicated by blue arrows) and significant decreases in the spotty necrosis in comparison to the αCD40 monotherapy (Figure K, Figure S5A). Furthermore, in both tumor models, using H & E staining, the number of lesions (indicated by black arrows) observed per 10× field was significantly decreased by 40% in mice that received combination treatment (Figure S4B and Figure L). However, no significant toxicity was observed in the kidney, lungs, and spleen among different treatment groups (Figure S5C–E). Ponatinib is known to reduce CRS through the inhibition of cytokine-releasing signaling pathways including MAPK38 and ERK1/2 expression. , Also, in our previous study, we have demonstrated that the ponatinib has significantly decreased the expression of MAPK38 and ERK1/2 expression in the mice bearing B16-F10 tumors. Hence, we estimated the levels of MAPK38 in both the serum and tumor and found that the combination-treated mice showed decreased expression of MAPK38 in comparison to that of αCD40 monotherapy in the mice bearing B16-F10 tumors (Figure M–P). Similarly, the expression of ERK1/2 was significantly reduced in the serum of mice bearing B16-F10 tumors when treated with the combination treatment in comparison with the αCD40 monotherapy (Figure Q,R). Together, these data suggest that the levels of cytokines and toxicity biomarkers were elevated with αCD40 treatment, whereas when ponatinib was used in combination with αCD40, the levels were not significantly altered in the serum and were similar to the control mice.

Discussion

In preclinical and clinical studies, αCD40 has shown promising anticancer efficacy across various tumor types. Ligation of αCD40 with CD40 has been shown to induce antitumor immune responses by increasing the effector CD8⁺ T cell infiltration and the activation and proliferation of tumor-specific DC and B cells via MAPK/p38/ERK1/2/PI3K and the downstream signaling. Also, the interaction of αCD40 with CD40 present in immune cells activates the JNK signaling cascade and stimulates the release of various proinflammatory cytokines including IL-2, TNFα, IL-6, NO, and IL-12 that enhance the antitumor responses. Together, these mechanisms help the modulation of immunosuppressive TME toward an immunogenic state. To further improve the tumor regression efficacy, many research groups have explored IL-15, VEGF inhibitor, IL-23, and TLR-9 in combination with αCD40 therapy. − In a study, when IL-15 was used in combination with αCD40, the antitumor efficacy and survival of the mice bearing CT26 and MC38 tumors were significantly improved in comparison to either monotherapy. Moreover, the αCD40/VEGF inhibitor combination has significantly improved CD8⁺ T cell infiltration and overall survival in the mice bearing MC38 tumors.

However, despite having favorable antitumor efficacy, αCD40 administration has been observed to increase the expression of immune checkpoint proteins like PD-L1, PD-1, and CTLA-4 in TME, leading to the dysfunction and apoptosis of tumor-specific immune cells, ultimately causing immune resistance and supporting tumor progression. Previous studies showed that αCD40 treatment activates APCs that release IFN-γ, resulting in the increase in the PD-L1 expression.

Ponatinib, has shown promising antitumor efficacy in chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) patients. Interestingly, in our previous study, we have shown that ponatinib decreased IFN-γ-induced PD-L1 expression both in vitro and in vivo and exhibited potent antitumor activity across different murine tumor models. ,

Notably, in this study, we have investigated the antitumor efficacy of αCD40 in combination with ponatinib and found that ponatinib has significantly improved the in vivo antitumor efficacy of αCD40 in mice bearing B16-F10 and 4T1 tumors. The dosage of ponatinib used in this study was found to be well tolerable as previously reported. , Various studies have shown that the antitumor efficacy of any immune modulatory drug depends on the infiltration of effector CD8⁺ T cells in the tumor. In this study, we found that ponatinib in combination with αCD40 has significantly increased the CD8⁺ T cell population in tumors in comparison to other treated groups across both tumor models. It has been observed that an increase in the expression of FOXP3 in TME increased Treg activity, which suppresses antitumor immunity and favors tumor development and progression. Surprisingly, when ponatinib was administered in combination with αCD40, the population of Treg cells and expression of FOXP3 in tumors were significantly decreased, indicating a reduced Treg cell activity.

TAMs have been known to create an immunosuppressive tumor microenvironment by repolarizing the antitumor state (M1) to the protumor state (M2) and by releasing various immune suppressive cytokines including IL-4 and IL-10, which ultimately favor tumor progression and induce angiogenesis and metastasis. αCD40 is known to repolarize macrophages from the M2 to M1 phenotype. Strikingly, in our study, the combination of αCD40 and ponatinib exerted better M2 to M1 repolarization potential in comparison to either monotherapy by significantly increasing the population of M1 TAMs (F4/80⁺CD206⁺) and reducing the number of M2 TAMs (F4/80⁺CD206⁺). Taken together, these results showed that the combination treatment exerts better M2 to M1 polarization, which favors the overall antitumor efficacy.

To generate a robust antitumor response, continuous support from systemic immunity to TME is very crucial. To get an insight into systemic immunity, we checked the CD8⁺ T cell population; expression of immunomodulatory markers like FOXP3, CD86, and Arginase-1; and levels of cytokines like IFN-γ, IL-2, TNF-α and IL-4 in the spleen. The CD8⁺ T cell population in the spleen is known to produce tumor lytic function, which hampers tumor growth and metastasis. In this study, we found a significant increase in the effector CD8⁺ T cell population with the combination treatment in comparison to that in all other treated groups. To determine tumor-specific killing of any immunomodulatory drug, evaluation of CTL activity against cancer cells is very important. In addition to the increase in the CD8⁺ T cell population, the combination treatment showed significantly superior CTL activity against cancer cells in comparison to the other treated groups, suggesting improved systemic immunity. Interestingly, we have found that Treg activity was significantly reduced by the combination treatment as it reduces the population of Treg cells and expression of FOXP3 significantly in comparison to the control and αCD40 monotherapy. Further, ponatinib boosts the macrophage repolarization activity of αCD40 by significantly increasing the expression of CD86 while reducing the expression of Arginase-1, which corresponds to the improved M1/M2 ratio in the combination-treated group. Furthermore, the cytokine profile indicated the superior Th1/Th2 ratio toward a dominant Th1 in the spleen of mice that received combination treatment over αCD40 and ponatinib monotherapy. Taken together, these results showed that the combination treatment improved splenic immunity that correlates with the decrease in the tumor burden and improved overall survival.

Various reports have shown that αCD40 treatment activates APCs, which further release IFN-γ. The elevated IFN-γ levels often create a negative feedback loop that supports tumor growth and hampers tumor-specific T cell response by elevating the PD-L1 expression. , Remarkably, ponatinib, when used in combination with αCD40, significantly reduced the population of immune cells expressing PD-L1. Moreover, the combination treatment significantly reduced the expression of PD-L1 in both the tumor and spleen, contributing to better antitumor activity that correlates with delayed tumor growth in the combination group.

A key challenge in the broad implementation of αCD40 immunotherapy in clinics is linked with the αCD40 treatment associated adverse effects that mainly include CRS, organ-specific toxicities, autoimmune reactions, and thromboembolic syndromes. The CRS induced by αCD40 treatment is generally observed within a few minutes of administration, leading to persisting hepatotoxicity and resulting in liver dysfunction. Although prior reports suggested that the toxicity associated with αCD40 is transient and could be manageable with the use of improved novel CD40 agonists, some reports also reveal that due to the dose-limiting toxicities, αCD40 may never reach an optimal therapeutic dose, resulting in limited antitumor activity. However, various combination strategies of αCD40 with the anti-PD-1 antibody or chemotherapy have been developed and found to be tolerable in patients. , Despite showing promising antitumor activity in preclinical studies, αCD40 agonists have demonstrated only moderate efficacy and dose-limiting toxicity in clinical settings. To enhance antitumor effects while minimizing associated toxicities, various research groups have explored different strategies. For example, a fully human anti-CD40 agonist antibody (2141-V11) was developed with optimized Fc binding to FcγRIIB. This modification significantly improved CD40 agonist activity and reduced the toxicity when administered intratumorally. However, intratumoral administration is primarily limited to the treatment of topical tumors. Additionally, it restricts the activation of tumor-specific immune cells in secondary lymphoid organs. In another strategy, researchers fused CD40 ligand (CD40L) directly to agonistic anti-CD40 antibodies, creating a “superagonist” with enhanced immune cell activation and reduced required dosing. However, this study did not evaluate dose-limiting toxicities or in vivo tumor regression studies. A further approach involved developing a bispecific antibody, ABBV-428, which combines a CD40-targeting arm with a mesothelin-tumor-associated antigen (TAA). Preclinical data suggested that ABBV-428 could provide comparable antitumor immunity to a monoclonal CD40 antibody with reduced systemic toxicity. However, in phase 1 clinical trials, ABBV-428 showed limited antitumor efficacy, with no significant responses observed in patients. There are only a few studies that have explored the use of combination therapies or engineered αCD40 to enhance antitumor efficacy while minimizing associated toxicities.

It has been known that CD40 signaling induces the expression of JAK3, MAPK, ERK1/2, PI3K, etc. In particular, the MAPK38 and ERK1/2 signaling cascade is linked to the release of key cytokines like IL-1β, IL-6, IL-8, IL-10, CCL2, and TNF-α that are involved in CRS. Like other TKIs including imatinib, dasatinib, and nilotinib, the clinical usage of ponatinib in hematologic malignancy patients has shown signs of cardiovascular toxicity and hypertension. However, the causes of these events are unclear. Moreover, unlike in humans, ponatinib did not show any adverse toxicity in mice. Interestingly, it has been shown in several reports that ponatinib has the potential to reduce CRS via inhibiting various cytokine-releasing signaling pathways like MAP3K8, ERK1/2, EPHA3, and JAK1. , In addition, in our previous study, we have also demonstrated that ponatinib significantly reduced the expression of MAPK38 and ERK1/2 in mice bearing B16-F10 tumors. Surprisingly, when ponatinib was administered in combination with αCD40, the levels of ALT, AST, ALP, IL-6, IL-10, and IL-1α in the serum were significantly lowered, resulting in lowered in vivo toxicity. Further to check the mechanistic effect of ponatinib on averting αCD40 toxicity, we estimated the expression of MAPK38 and ERK1/2 in serum and found a significant reduction in its expression in comparison to αCD40 monotherapy. In addition, the combination of ponatinib and αCD40 significantly reduced the population of liver myeloid cells particularly CD11b⁺GR-1⁺cells that are responsible for the liver damage. Moreover, the combination therapy has shown a great potential in reducing the magnitude of spotty necrosis, microgranulomas, etc., which usually surges in the case of αCD40 monotherapy.

Notably, the combination of ponatinib and αCD40 has demonstrated improved antitumor effects across multiple tumor models, along with a reduction in αCD40-related toxicities. Therefore, this therapeutic approach warrants further investigation in higher animal models, and its antitumor efficacy and dose-limiting toxicity profiles should be carefully evaluated in human studies prior to clinical application.

Glossary

Abbreviations:

ALT

alanine transaminase

AST

aspartate transaminase

ALPU

alkaline phosphatase

CRS

cytokine release syndrome

IAA

immune agonist antibodies

PD-L1

programmed death-ligand 1

TGF-β

transforming growth factor beta

TNF-α

tumor necrosis factor

TME

tumor microenvironment

MFI

mean fluorescent intensity

All the materials used to produce the data in this study are available from the corresponding authors upon reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.5c00227.

• Additional data including tumor immunohistochemistry (IHC) studies for Ki-67 and Granzyme B; flow cytometric analysis of macrophage phenotypes within the tumor; various cytokine ratios in the spleen; spleen weight analysis; and assessments of organ-specific toxicities in the liver, lungs, spleen, etc. (PDF)

V.G. and J.B. designed the study. V.G. performed all the experiments. A.B., W.T., U.M.A., J.M., S.B., P.Y., S.D., and A.B. helped V.G. in establishing the 4T1 orthotopic breast tumor and B16-F10 melanoma murine models, respectively. K.S. and A.N. helped in IHC experiment. P.D. helped in taking the H & E images of the liver, lungs, and spleen and in the quantification of liver toxicity. V.G. and J.B. performed the data analysis and wrote the manuscript. The manuscript was reviewed and approved by all authors.

This research was supported by a grant from the Indian Council of Medical Research (grant IRIS 2021-9962, IIRP-2023-2184) sanctioned to J.B.

The authors declare no competing financial interest.