Abstract

Introduction

Cancer is a major global public health problem and the second leading cause of death worldwide [1]. Around 10 million people die annually from cancer, and the number of cases is expected to roughly triple over the next two decades [2]. It has been demonstrated that cancer cells can be identified and eliminated prior to tumor formation by immune surveillance mechanisms [3] which includes communication between adaptive and innate immune cells. To escape the immune surveillance system and promote tumor growth, tumor cells evade immune system attacks through various mechanisms such as restricting antigen recognition, creating an immunosuppressive environment to suppress the immune system, and causing T cell exhaustion [4].

In 1891, William B. Coley showed the efficacy of immune stimulation as immunotherapy by injecting bacteria into a cancer patient in order to boost the immune system for tumor elimination.

Since then, the understanding and application of immunotherapy as a cancer treatment has advanced significantly. Numerous immunotherapeutic drugs have been investigated in preclinical models, and those that have demonstrated efficacy, such as immune checkpoint inhibitors, have been granted regulatory approval [3]. In recent years, the clinical usage of anti-PD1 (Nivolumab) and anti-CTLA-4 (Ipilimumab) checkpoint inhibitors has emerged with significant success in the treatment of malignancies [5]. In addition, immune co-stimulation to immune checkpoint inhibitors holds great potential to boost immune activation and destabilize the immunosuppressive tumor microenvironment.

CD137, also known as 4-1BB, is one of the potent immune costimulatory receptors that could be targeted for effective immunomodulation. 4-1BB, a member of the tumor necrosis factor (TNF) receptor family, is expressed on the surface of activated T, B, and NK lymphoid cells. Signaling via 4-1BB enhances the survival, proliferation, and differentiation of T cells into effector cells and the generation of long-term memory [6–8]. Activation of the 4-1BB receptor has been shown in preclinical studies to enhance the antitumor activity of T cells and increase the efficacy of cancer immunotherapy. With promising findings, this strategy has also been evaluated in clinical trials to assess the efficacy of agonistic 4-1BB antibodies alone or in combination with other immunotherapy and chemotherapy regimens. However, clinical trials with anti-4-1BB agonistic antibodies failed due to unacceptable systemic toxicity [9, 10].

Contrary to agonistic antibodies, 4-1BBL, the natural ligand for the 4-1BB receptor, does not induce toxicity upon binding to the 4-1BB receptor. 4-1BBL is a cell surface membrane protein that is expressed on dendritic cells, macrophages, B cells, and it is active only in its membrane-bound form. The trimeric (human) or dimeric (mouse) soluble form of 4-1BBL requires crosslinking to cluster the 4-1BB receptors for efficient signaling to immune cells [11–13].

Therefore, to construct a functional soluble natural ligand for the 4-1BB receptor, we fused the extracellular domain of murine 4-1BBL to a modified form of the streptavidin core (SA) to make a chimeric molecule (SA-4-1BBL). Due to the structural characteristics of SA, chimeric SA-41BBL formed tetramers and oligomers [14, 15]. Soluble SA-4-1BBL exhibited significant immunological activity in T effector cell activation, acquisition of effector functions, and long-term memory formation [16]. We previously reported that SA-4-1BBL prevented the conversion of T-conventional cells into T-regulatory cells, which was triggered by IFNγ production in T-conventional cells [17], and overcame Treg suppression through inducing the production of IL-2 by T-effector cells [14]. Furthermore, unlike 4-1BB antibodies, we did not observe systemic toxicity with repeated administration of SA-4-1BBL in mice [13]. Importantly, when used as an adjuvant in tumor-associated antigen-based subunit vaccines, SA-4-1BBL demonstrated therapeutic activity in a variety of preclinical tumor models [15, 16, 18–21]. We recently reported that mice treated with soluble SA-4-1BBL protein were protected against a challenge with transplantable tumors such as Lewis lung carcinoma (LLC), TC-1, and 3LL-huMUC1 [22]. Regardless of tumor type, this effect was long-lasting and required CD4 T cells, NK cells, and IFN-γ. In addition, treatment with SA-4-1BBL after surgical removal of tumors prevented recurrences [22].

Here, we demonstrated that the immunotherapeutic potential of SA-4-1BBL is not restricted to transplantable tumor models, but also extends to carcinogen-induced lung cancer in a rodent model with high susceptibility to carcinogen-induced tumors.

The data presented in this paper showed that utilizing an immune checkpoint stimulator as a single agent generated a protective immune response against lung cancer in the presence of carcinogen. A deeper understanding of the molecular basis of immune checkpoint stimulation may also offer opportunities to combine this therapy with potentially additive or synergistic biologics for enhanced immunological efficacy. Notably, this immunotherapeutic platform can be extended to a number of other cancer types, including breast and prostate cancers, for which improved diagnostics can detect disease at the preneoplastic stage.

Materials and methods

Results

Treatment with NNK-induced lung tumorigenesis

To evaluate the immunotherapeutic efficacy of SA-4-1BBL in genetically predisposed model [27], we used A/J mice because of their susceptibility to nicotine derivative-induced lung cancer, making them one of the best preclinical animal models for smoking-related lung cancer [28–31]. A/J mice are predisposed to spontaneous lung tumor development due to a mutation in the PAS1 gene, which is linked to the KRAS protooncogene [32]. The KRAS protooncogene regulates cell proliferation and division, and mutations in this gene are associated with the development of numerous cancers, including lung cancer. We used NNK (4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone), which is the most carcinogenic nitrosamine nicotine derivative in humans and animals, to accelerate tumor development [28–30]. Lung cancer development was accelerated in A/J mice by injecting NNK at a weekly dose of 25 mg/kg for eight weeks [27]. In the sixth- and eighth-week post-NNK injections, SA-4-1BBL protein was administered subcutaneously (Fig. 1A). Animals were assessed each week for 18 weeks to determine whether there were any differences in the animals' wellbeing after receiving NNK and SA-4-1BBL treatments. Body weight evaluation showed no significant differences across groups compared to untreated age-matched A/J mice (Fig. 1B). At the end of the study, necroscopy was performed to detect any NNK and/or SA-4-1BBL treatment-related adverse pathological changes in internal organs such as the kidney, liver, bladder, and stomach. No measurable differences were observed between the treatment and control groups (data not shown), which was consistent with the previously reported safety profile of SA-4-1BBL treatment [13, 22].

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Fig. 1

SA-4-1BBL treatment protected mice from developing lung tumors induced by NNK. A Experimental design, and treatment timeline. Female A/J mice were randomly divided into three groups: naïve (untreated), NNK control, and NNK+SA-4-1BBL-treated group. NNK control (NNK+vehicle) and NNK+SA-4-1BBL groups received 25 mg/kg of NNK (4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone) via i.p. injection weekly for the first eight weeks of the experiment. SA-4-1BBL treatment group (n=17) also received 100-µg SA-4-1BBL s.c. at weeks 6 and 8, two days post-NNK administration. B The animals body weight changes were assessed during weekly follow-up. Body weight changes were not significantly different across the groups compared to untreated age-matched A/J mice. C. Representative images of macroscopic tumor nodules in the lungs. In the 18th week, mice were euthanized, and lungs were perfused to evaluate macroscopic tumor nodules. Black dashed circles show macroscopic tumor nodules on the lungs. D. Macroscopic tumor numbers in NNK control and NNK+SA-4-1BBL treatment groups. Lungs were evaluated under the dissecting microscope, and macroscopic tumor nodules were counted and compared between the NNK control group and NNK+SA-4-1BBL treatment group. SA-4-1BBL-treated group showed significantly fewer macroscopic tumor nodules than the control group (p<0.01). E. Microscopic tumor nodule numbers in NNK control and NNK+SA-4-1BBL treatment groups. Lungs were sectioned and stained with H&E for immunohistochemistry analysis. NNK+SA-4-1BBL-treated group had significantly fewer microscopic tumor nodules than the NNK control group (p<0.0001). F. Representative images of sections containing tumor nodules stained with H&E and PCNA. Images in the first row (a and b) illustrate hyperplastic cell structures with increased nucleus-to-cytoplasm ratio in H&E-stained lung tissue (red dashed circle). Representative images were captured using EVOS M7000 Imaging System (ThermoFisher Scientific) with employing a 10X optical magnification. H&E-stained (a) and (b) images presented at 100X and 200X total magnification, with 500-µm and 100-µm scale bars, respectively. Lung sections were stained with proliferation marker PCNA antibody. PCNA staining (red) and SYTOX blue nuclear staining (blue) are depicted in the second row (c and d) captured with 20X objective in Leica confocal microscopy. Scale Bars: 100 µm and 25 µm for NNK+vehicle group (with zoom factor 1 and 1.35) and 100 µm and 75 µm for NNK+SA-4-1BBL (with zoom factor 1 and 1.56). The tumor area (yellow dashed circle) had a higher PCNA staining profile than the alveolar lung tissues surrounding the tumor area. The data presented in this figure were compiled from three to four independent experiments involving four to six mice per experiment and are shown as mean±standard deviation (SD). Body weight changes were analyzed using two-way ANOVA B, macroscopic D and microscopic E tumor nodule numbers were analyzed by unpaired students’ t test

SA-4-1BBL as monotherapy significantly decreased the number of tumor nodules in NNK-induced lung cancer

In our previous study, pretreatment with SA-4-1BBL, as an agonist of the 4-1BB costimulatory pathway, protected mice from subcutaneous tumor challenge without antigen priming. In this study, we investigated whether treatment with SA-4-1BBL protein inhibits tumor growth in a carcinogen-induced mouse model of lung cancer. A/J mice were injected with 100 µg of SA-4-1BBL at weeks 6 and 8 of NNK injections (Fig. 1A). At the end of the study (18 weeks), mice were euthanized, and two trained investigators counted macroscopic tumor nodules on the lung surfaces using dissecting microscope. Mice given SA-4-1BBL protein had significantly fewer macroscopic tumor nodules than the control group (NNK+vehicle) (Fig. 1C and D). The tumor prevention capacity of SA-4-1BBL was consistently seen in repeated experiments, further demonstrating the power of immune checkpoint stimulation. Visible tumor nodules were primarily observed in the periphery of the lungs (Fig. 1C, black circles), which is similar to smoking-related lung cancer in humans.

The macroscopic tumor nodules were further confirmed as cancerous lung nodules using histopathological analyses. Sectioned lung tissues were stained with H&E and tumor nodules were counted microscopically by two trained investigators in a blinded manner. PCNA immunofluorescence labeling was used to confirm the malignant phenotype with proliferative capacities of microscopic tumor foci defined by H&E staining (Fig. 1F). Mice treated with SA-4-1BBL had significantly fewer microscopic tumor foci than the control group (Fig. 1E). Moreover, one animal in the SA-4-1BBL treatment group showed no microscopic lung tumors. A strong significant reduction in the number of microscopic tumors may be attributable to the increased efficacy of SA-4-1BBL treatment on tumor nodules that have already formed prior to obtaining their first dose of SA-4-1BBL. Tumor nodule size was also measured by examining microscopic tumor areas to further analyze the proliferative capacity of the tumor cells associated with NNK exposure in A/J mice (Fig. 3A and C). Notably, both the tumor dimensions and the percentage of tumor area in lung sections in the SA-4-1BBL treatment group were significantly smaller than those in the control group (Fig. 3C and D).

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Fig. 3

T and NK cell depletion impaired the immunotherapeutic efficacy of SA-4-1BBL. As previously described, female A/J mice received weekly doses of NNK for eight weeks, followed by two doses of SA-4-1BBL treatment and depletion antibodies. In the experimental endpoint, lung tissues were sectioned, and H&E-stained slides were used to assess microscopic tumor size changes within different treatment groups. Microscopic tumor nodule margins were defined, and digital microscopy images were captured at 10X magnification. A. Representative microphotograph of H&E-stained lung tissue samples from naïve A/J mice, control group (NNK+vehicle), and NNK+SA-4-1BBL-treated group. The area marked by the black line shows microscopic tumor nodules in lung from the NNK control group. B. H&E-stained lung tissue samples from groups treated with SA-4-1BBL and then depleted of CD4⁺ T cells, CD8⁺ T cells, or NK cells. Microscopic tumor nodules marked with black lines. The EVOS M7000 Imaging System (ThermoFisher Scientific) was used to acquire representative microscopy images in A and B, at 100X, 200X, and 400X total magnification, with 500-µm scale bars, employing a 10X optical magnification. Variations in the dimensions of the embedded tissue resulted in non-uniform images when sections were magnified uniformly to compensate for ratio differences between sections. C. A representative image of tumor nodule dimension and total lung section dimension measurement in the H&E-stained section (left), and a comparison of microscopic tumor nodule areas between the groups (right). SA-4-1BBL-treated group microscopic tumor nodule dimensions were significantly smaller than the NNK control group and immune cell-depleted groups (n=15 tumor nodules/group, p<0.05 and p<0.01). D. The percent of the tumoral areas over the non-tumoral lung areas (n=15 tumor nodules/group, p<0.05). Tumor area dimension and the percent tumor areas to the lung section area in the SA-4-1BBL treatment group were significantly smaller than those in the control group. A p-value≤0.05 was considered significant. Microscopic tumor dimensions C and tumor area percentages D were compared using a one-way ANOVA. Data represented as mean±standard deviation (SD). Scale bars, 500 µm

SA-4-1BBL-mediated tumor prevention depended on CD4, CD8, and NK cells

In our reported study, the tumor preventive efficacy of SA-4-1BBL was shown to be dependent on cross-communication between CD4⁺CD44^high “memory-like” T cells and NK cells, with a requirement for IFN-γ [22]. A series of depletion experiments were conducted to determine the function of immune cell populations in mediating the immunotherapeutic efficacy of SA-4-1BBL in a NNK carcinogen-induced tumor model. Depleting antibodies against CD4⁺, CD8⁺ and NK cells were injected i.p. following SA-4-1BBL treatment during the sixth and eighth weeks of NNK administration (Fig. 2A). Immune cell depletion showed no adverse effects on the health of mice, including no differences in body weight between groups (Fig. 2B).

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Fig. 2 

In vivo depletion of CD4⁺, CD8⁺, and NK cells abolished SA-4-1BBL antitumor activity. A. Experimental design; NNK administration, SA-4-1BBL treatment, and in vivo depletion timeline. Female A/J mice were randomly divided into groups, and all groups received weekly 25 mg/kg NNK injections for eight weeks. In the sixth and eighth weeks of NNK injections, selected groups received SA-4-1BBL injection subcutaneously. Twenty-four hours after SA-4-1BBL treatment, antibodies against different immune cell populations were injected to deplete CD8⁺ cells (n=6), NK cells (n=6), or CD4⁺ cells (n=9). B. The animals body weight changes were assessed during weekly follow-up. Body weights were not significantly different in the SA-4-1BBL treatment and SA-4-1BBL+depletion groups compared to the control group. C. Representative images of macroscopic tumor nodules on the lung surface. Lungs were harvested, and macroscopic tumor numbers were assessed under a dissecting microscope at the experimental endpoint. Black dashed circles show macroscopic tumor nodules in the lungs of the mice from the NNK control group, CD4⁺ T cell, CD8⁺ T cell, and NK cell-depleted groups. D. Macroscopic tumor numbers between different treatment groups. Counted macroscopic tumor numbers were compared between the groups. The NNK+SA-4-1BBL-treated group (n=17) has significantly fewer macroscopic tumor nodules than the NNK control group (n=16) and CD4⁺ T cells depleted group (n=9, p<0.05). E. Lung tissue sections were stained with H&E, and microscopic tumor nodules were enumerated from each group. Microscopic tumor formation compared between NNK control (n=15), NNK+SA-4-1BBL treatment group (n=15), SA-4-1BBL+CD4 T cell-depleted (n=8), SA-4-1BBL+CD8⁺ T cell, and SA-4-1BBL+NK cells depleted groups (n=6). SA-4-1BBL-treated group had significantly fewer microscopic tumor nodules in the lungs (p<0.001) as compared to NNK control and depletion groups. The data presented in this figure were compiled from three to four independent experiments. A p-value≤0.05 was considered significant. Data are represented as mean±standard deviation (SD). Body weight changes were analyzed using a two-way ANOVA B, and a one-way ANOVA was used for analysis of macroscopic D and microscopic E tumor numbers

CD4⁺ T cell, CD8⁺ T cell and NK cell depletion abrogated the protective antitumor effect of SA-4-1BBL treatment. In mice depleted of CD4⁺ T cells (Supplementary Fig. S1A), there was a significant increase in both macroscopic and microscopic tumor nodules compared to mice that did not receive CD4⁺ T cell depleting antibody (Fig. 2C, D, E). In contrast, depletion of CD8⁺ T (Supplementary Fig. S1A) cells and NK cells resulted in a significant increase in microscopic tumor nodules, but no significant change was seen in macroscopic tumor nodule numbers (Fig. 2C, D, E). These results indicate that CD4⁺ T cell help is required for the generation of antitumor primary immune responses, which is consistent with our previous study [22].

The tumor burden was restricted to the lung, as detailed evaluation of organs using a dissecting microscope showed none of the mice in the study groups had tumor nodules in other organs. Notably, one mouse in CD4⁺ T cell-depleted group had a tumoral lesion on its chin. This lesion was also removed on the day of termination, and histopathologic analysis was performed. The analysis revealed that the tumor was metastatic, and it had the same histological phenotype as the tumors seen in the lung (Supplementary Fig. S2A-C). This metastatic nodule had 14-fold more PD1⁺CD4⁺ and PD1⁺CD8⁺ cells (PD-1, programmed cell death protein 1) than the lung tumors (Supplementary Fig. S2D and S2E). In contrast to previously published transplantable tumor models, depletion of CD8⁺ T cells had a significant effect on the antitumor activity of SA-4-1BBL in a NNK carcinogen-induced model [22]. These results suggest that CD8⁺ T cells play an essential role in the antitumor activity of SA-4-1BBL in the carcinogen-induced lung tumor (Fig. 2C and ​andE).E). As compared to SA-4-1BBL-treated controls, tumor areas were significantly increased in the lungs of SA-4-1BBL-treated mice depleted of either T or NK cells and were comparable to the NNK control (Fig. 3B, ​B,C,C, ​,D).D). These data further confirmed that the immunotherapeutic effectiveness of SA-4-1BBL treatment in inhibiting tumor growth was dependent on the presence of T and NK cells.

Overall, these findings provided important insights into the mechanisms underlying the immunotherapeutic effects of SA-4-1BBL treatment and highlighted the importance of T cells and NK cells in the immune response against cancer.

Treatment with SA-4-1BBL increased the number of naive and central memory CD4⁺ T cells and decreased the number of CD8⁺PD-1⁺ cells

We have previously shown that pretreatment with SA-4-1BBL elicited a long-lasting immune memory response, which was predominantly mediated by NK cells and CD4⁺ T cells expressing IFN-γ [22]. Here, we utilized deep immune phenotyping to assess the immune cell compositions in the lung and draining lymph nodes. SA-4-1BBL treatment resulted in a significant increase in the absolute cell number of CD4⁺ central memory T cells (CD4⁺CD44^hiCD62L^hi) in lung and lung draining lymph nodes compared to the NNK control group (p<0.05) (Fig. 4A, B). The increased number of central memory T cells in SA-4-1BBL-treated animals suggested that these cells were traveling between the lung and the lung-draining lymph nodes, where they had encountered antigen, been activated, and then migrated to lymphoid tissues where they survived for extended periods. The number of naive CD4⁺ T lymphocytes (CD4⁺CD44^loCD62L^hi) in the lung-draining lymph node of SA-4-1BBL-treated mice was also significantly higher, which may serve as a constant source of central memory cells and provide long-term protection against tumor growth (Fig. 4C). In addition, there was a significant decrease in the number of PD-1 positive CD8⁺ T cells in the draining lymph node of mice treated with SA-4-1BBL (Fig. 4D). PD-1 is an inhibitory receptor expressed on the T cell surface. The ligand of PD-1, named as Programmed Death-Ligand 1 (PD-L1), is found on the surface of many different cells, including cancer cells. When PD-L1 binds to PD-1, an inhibitory signal is transmitted that inhibits T-cell activation, thereby dampening the immune response. In preclinical models, it has been demonstrated that NNK-induced lung tumors express PD-L1 [28, 29]. The reduction in PD-1-positive CD8⁺ T cells could be an additional mechanism by which SA-4-1BBL exerts its antitumor growth effect. However, further research is needed to confirm this hypothesis and fully understand the role of SA-4-1BBL in modulating the immune responses against cancer in another induced or spontaneous tumor model.

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Fig. 4

SA-4-1BBL treatment increased the number of naive and central memory CD4⁺ T cells but decreased CD8⁺PD-1⁺ cells. Deep immune phenotyping was performed on lung tissue and lung draining lymph node at the experimental endpoint. A. SA-4-1BBL treated group (n=10) had significantly higher central memory CD4⁺ T cell numbers (CD4⁺CD44^hiCD62L^hi) in the lung, and B. lung-draining lymph nodes compared to NNK control group (n=10, p<0.05), and depletion groups (n=6–7, p<0.001). C. SA-4-1BBL treatment (n=10) increased naïve CD4⁺ T cell (CD4⁺CD44^loCD62L^hi) in the lung draining lymph nodes, compared to control groups (n=10, p<0.001), and immune cell-depleted groups (n=6–7, p<0.1). D. SA-4-1BBL treatment (n=10) significantly decreased the number of CD8⁺PD-1⁺ cells in lung-draining lymph nodes, compared to control group mice (n=10, p<0.05). The data presented in this figure were compiled from three to four independent experiments. A p-value≤0.05 was considered significant. Absolute cell numbers were analyzed using a one-way ANOVA A, B, C and D. Data represented as mean±standard deviation (SD)

Discussion

Immune checkpoint inhibitors such as anti-CTLA-4 and anti-PD-1/PD-L1 have been shown to restore T cell-mediated antitumor immunity and induce durable remissions in some patients with cancer. The FDA (Food and Drug Administration) has approved these treatments for the treatment of several types of cancer, including melanoma, lung cancer, bladder cancer, kidney cancer, head and neck cancer, and Hodgkin lymphoma [5, 30–34]. However, these treatments are not effective for all types of cancer, and not all patients respond equally well [35–38]. Therefore, there is a need for robust immune stimulation to obtain higher response rates and long-term remissions. Combining checkpoint inhibitors with robust immune co-stimulation thus becomes an attractive strategy.

One approach to achieving robust immune stimulation is combining two or more immunotherapeutic agents that can target different aspects of the immune system and work in synergy to potentially increase the antitumor immune response [39, 40]. Combining immune checkpoint inhibitors with other immunotherapeutic agents such as cancer vaccines, adoptive cell therapy, or immune stimulators has shown promising results in preclinical and clinical studies. In some cases, combination therapy has demonstrated improved response rates and longer survival than single-agent therapy [41, 42].

The 4-1BB/4-1BBL pathway has been shown to have a critical role in the regulation of immune responses[43]. Upon antigenic stimulation, such as exposure to a pathogen or tumor cells, the expression of 4-1BB receptor is upregulated on immune cells, including T cells and natural killer cells. The membrane-bound form of 4-1BBL binds to the 4-1BB receptor to provide a potent costimulatory signal that enhances immune cell activation, proliferation, and survival. Activation of this pathway has been shown to stimulate immune responses, particularly in the context of cancer immunotherapy [43, 44].

The soluble form of 4-1BBL has limited immunostimulatory activity because efficient signal transduction requires receptor crosslinking for multimerization. To generate a stable soluble form of 4-1BBL with potent co-stimulatory activity, we generated a chimeric molecule comprised of the extracellular portion of murine 4-1BBL and streptavidin (SA-4-1BBL). SA-4-1BBL, a novel form of 4-1BBL, forms tetramers and oligomers [14, 15] with high immunological potential to activate T effector cells. The soluble SA-4-1BBL has been shown to have antitumor effects in preclinical studies [13–15, 18, 20, 22, 45] with no detectable toxicities as compared to 4-1BB agonistic antibodies.

The aim of this study was to determine whether SA-4-1BBL could be used as an immune checkpoint stimulator in mice predisposed to spontaneous cancer development. For this purpose, SA-4-1BBL was administrated to A/J mice during ongoing exposure to tobacco carcinogens NNK, to determine whether treatment with SA-4-1BBL could halt or reverse the incidence of lung tumors compare to the group that did not receive SA-4-1BBL treatment. We adopted a previously described NNK-induced lung cancer model by administering intraperitoneal injections of 25 mg/kg NNK carcinogen weekly for eight weeks. We demonstrated that eight weekly injections of NNK were sufficient to induce smoking-related lung cancer by 18 weeks with 100 percent tumor incidence, compared to the intragastric gavage method, which requires 30 to 40 weeks to develop lung tumor nodules. The dose of SA-4-1BBL protein used in this study was based on our previous reported study, in which 100 µg was the optimal dose to protect mice from subsequent subcutaneous tumor challenges, and lower protein doses provided compromised protection compared to 100 µg [22]. Although we tested higher protein doses in A/J mice, the most efficacious regimen consisted of two 100-µg doses administered two weeks apart.

We herein report that the therapeutic doses of SA-4-1BBL administered to A/J mice did not result in detectable macroscopic toxicity, which was consistent with numerous preclinical cancer models reporting the same therapeutic doses without toxicity [13, 15, 18, 20, 22, 45].

Treatment of A/J mice, which are genetically predisposed to spontaneous cancer development, with the SA-4-1BBL protein during active carcinogen exposure prevented the development of lung tumor nodules. This prevention was significant compared to NNK carcinogen-exposed mice without SA-4-1BBL treatment which developed a higher number of macroscopic and microscopic tumor nodules (Fig. 1C, ​C,D,D, ​,E,E, ​,F).F). Furthermore, the depletion of CD4⁺, CD8⁺ T cells, and NK cells significantly impaired the antitumor activity of SA-4-1BBL, indicating that the therapeutic effect of SA-4-1BBL was mediated by these immune cells (Fig. 2). Mice depleted of CD4⁺ T cells had an increase in the number of both macroscopic and microscopic tumor nodules. In contrast, the depletion of CD8⁺ T and NK cells increased the number of microscopic tumor nodules significantly but had no effect on the number of macroscopic tumor nodules (Figs. 2 and ​and3).3). These findings suggested the antitumor activity of SA-4-1BBL immunotherapy is primarily mediated by CD4⁺ T cells, with active participation from CD8⁺ T and NK cells. These results also indicated a cross-communication between innate and adaptive immunity. It is well-known that CD4⁺ T cells play a crucial role in initiating an active immune response as they help activate other immune cells and boost antibody formation. CD8⁺ T and NK cells, on the other hand, are directly responsible for destroying infected or malignant cells. In the absence of these cells, the immune system's ability to target and eliminate malignant cells are compromised. Consistent with a previously published study [22], SA-4-1BBL treatment significantly increased CD4⁺ naive and central memory cell numbers in the lung and lung draining lymph node (Fig. 4A, B, C). In response to antigen exposure, naive CD4⁺ T cells become activated and differentiate into effector T cells, which can migrate to the site of an infection or tumor to eradicate the pathogen or tumor cells. Some of these effector T cells differentiate into memory T cells, including central memory T cells, which are able to persist in the target tissues and provide long-term protection against subsequent infections with the same pathogen or tumor. These cells might be the cells exposed to tumor antigens and further stimulated by SA-4-1BBL therapy, although their exact nature has not yet been fully determined. Treatment with SA-4-1BBL decreased the number of PD-1-expressing CD8⁺ T cells compared to NNK-exposed control mice that were not treated with SA-4-1BBL (Fig. 4D). PD-1 is expressed on the surface of activated T cells, including CD8⁺ T cells, and regulates the immune response. Chronic antigen exposure, such as during prolonged infections or in the presence of cancer cells, can induce the expression of PD-1, leading to T cell exhaustion, which limits T cells’ capacity to eliminate target cells. Therefore, a significant reduction in the number of CD8⁺ T cells expressing PD-1 suggests that SA-4-1BBL may enhance the immune response against NNK-induced cancers by reducing T cell exhaustion. It is important to note that without additional data, a complete interpretation of the results cannot be made.

In conclusion, our findings demonstrated the immunomodulatory effect of SA-4-1BBL during continuous carcinogen exposure in mice predisposed to cancer. These findings provide additional evidence that immunotherapy utilizing potent immunostimulatory molecules, such as SA-4-1BBL, has the potential to treat and prevent cancer by enhancing the immune system's ability to identify and eliminate cancer cells. Thus, co-stimulation-based immunotherapy can provide precise, targeted treatment that avoids many of the adverse side effects associated with chemotherapy and radiation in various cancer treatments.

Acknowledgements

Abbreviations

Author contributions

Conception and design were done by ESY and HS. AEG, RR, MT and VU performed the experiments. AEG, RR and MT analyzed data and prepared Figs. 1, ​,2,2, ​,33 and ​and4.4. ESY, HS and AEG wrote the manuscript. All authors reviewed the manuscript.

Funding

This study was supported by the Department of Defense (DoD) grant number LC190524.

Data availability

SA-4-1BBL protein is available through a material transfer agreement with the University of Missouri, Columbia, MO.

Declarations

Footnotes

Contributor Information

Haval Shirwan, Email: [email protected].

Esma S. Yolcu, Email: [email protected].

References