Recombinant Costimulatory Fusion Proteins as Functional Immunomodulators Enhance Antitumor Activity in Murine B16F10 Melanoma

Blocking inhibitory signaling and engaging stimulatory signaling have emerged as important therapeutic modalities for cancer immunotherapy. This study aimed to investigate immunomodulatory features of three recombinant costimulatory ligand proteins in a mouse model, which are extracellular domains of OX40-ligand (OX40L), 4-1BB-ligand (4-1BBL), or two domains in tandem, fused with the transmembrane domain of diphtheria toxin (DTT), named DTT-COS1, DTT-COS2, and DTT-COS12, respectively. In vitro study showed that DTT-COS1 and DTT-COS12 had immunological activity increasing the ratio of CD8/CD4 T cells. Treatments with DTT-COS1 and DTT-COS12 dramatically generated immune protection against the B16F10 tumor challenge in both prophylactic and therapeutic efficacy. Furthermore, regarding tumor microenvironment (TME) immunomodulation, DTT-COS1 treatment increased the proportion of CD4+ effector T cells (Teff) and decreased the expression of a suppressive cytokine. Meanwhile, DTT-COS12 reduced regulatory T cells (Treg) and improved the level of stimulatory cytokines. In addition, endogenous antibodies against OX40L/4-1BBL were generated, which may help with antitumor responses. Unexpectedly, DTT-COS2 lacked antitumor effects in vitro and in vivo. Importantly, serum analysis of liver-function associated factors and pro-inflammatory cytokines demonstrated that treatments were safe formulations in mice without signs of systemic toxicity. Remarkably, DTT-COS1 and DTT-COS12 are functional immunomodulators for mouse B16F10 melanoma, creating practical preclinical value in cancer immunotherapy.


Introduction
T-cell activation is modulated by a number of factors that depend on costimulatory and inhibitory signals, in addition to T cell receptor (TCR) signaling that specifically recognizes complexes of peptide and major histocompatibility complex (MHC) [1][2][3]. Costimulatory agonists have been activating and supporting antitumor immune response by combination with immune checkpoint inhibition and other tumor-associated agents [3]. OX40L, tumor necrosis factor (ligand) superfamily, member 4, is a costimulatory ligand for OX40 and is expressed mainly on antigen-presenting cells (APCs), including dendritic cells, B cells, and macrophages [3]. Supporting studies suggested that OX40L-OX40 pathway promoted immune responses during T cell activation by establishing T cell memory as well as the expansion and survival of activated T cell subsets [4][5][6][7]. There are several OX40-specific 1 mM isopropyl-β-D-thiogalactoside (IPTG) when the E. coli culture reached OD 600 = 0.6. After culturing for an additional 20 h, the cells were collected by centrifugation, resuspended in PBS, lysed by sonication, and the debris removed by centrifugation. Purification of the supernatant was applied to His Trap HP column. The DTT-COS1, DTT-COS2, or DTT-COS12 proteins were further purified through Superdex G75 chromatography. The level of endotoxin was lower than 0.1 EU/mL by chromogenic Limulus Amebocyte Lysate assay (GenScript, Piscataway, NJ, USA).
For an immunized state, spleen cells were harvested from DTT-immunized C57BL/6 mice on the seventh day after the third injection and prepared into a single-cell suspension. An ammonium chloride-potassium (ACK) Lysis Buffer was used to remove the red blood cells. Splenocytes were cultured in total RPMI 1640 medium (RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin/streptomycin), stimulated with an amount (50 µg/10 6 cells) of DTT-COS1, DTT-COS2, DTT-COS12, or DTT control protein, and 150 U/mL IL-2 (Primegene). After 72 h stimulation, supernatants from cultured medium were collected and the cells were washed 3 times with PBS, stained with anti-CD3-PerCP (clone 17A2, eBioscience), anti-CD4-FITC (Clone GK1.5, BD Biosciences) and anti-CD8a-APC (Clone 53-6.7, BD Biosciences), and analyzed by flow cytometry. Ten mice were used for each experiment under different conditions in total with ex vivo stimulation.

Preventive and Therapeutic Tumor Models
For the preventive tumor models, C57BL/6 mice were injected s.c. with 7.5 × 10 4 B16F10 tumor cells, nine days after the third costimulatory fusion protein treatment. For the therapeutic tumor models, mice were s.c. challenged with 1 × 10 5 B16F10 tumor cells, subsequent three-time treatments of fusion proteins at weekly intervals. Tumor size was measured every 2 to 3 days with a caliper, and tumor volume calculated using the formula (width 2 × length × 0.5). The tumor size and survival were recorded until the tumor volume were reached 2000 mm 3 and mice were sacrificed for ethical reasons [24].
The amount of IFN-γ secretion in the supernatants diluted 2-fold from ex vivo stimulation above were measured by sandwich ELISA using the mouse IFN-γ DuoSet ELISA kits (R&D Systems) following the manufacturer's instruction. All standards and samples were assayed in duplicate and data were analyzed in Microplate Reader with a 6-parameter fit for the standard curve.

Serum Transfer Models
The serums were from C57BL/6 mice treated with DTT-COS1, DTT-COS2, DTT-COS12, or PBS three times and complement was inactivated by incubation for 30 min at 56 • C. Serum (300 µL) was administered by intraperitoneal (i.p.) injection 6 h before s.c. injection of 1 × 10 5 B16F10 cells [25]. Tumor size was measured every 2 to 3 days with a caliper, and tumor volume calculated using the formula (width 2 × length × 0.5). The tumor size and survival were recorded until the tumor volume were reached 2000 mm 3 and mice were sacrificed for ethical reasons.

Quantification of the Liver Injury
To assess liver injury, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations of serum from mice treated with DTT-COS1, DTT-COS2, DTT-COS12, or PBS (in the presence of aluminum hydroxide Gel adjuvant and CpG ODN 1826, as mentioned in 2.4.) were detected using the kit (Jiancheng Biologic Project Co., Nanjing, China) following the manufacturer's instructions. The absorbance at 510 nm was measured by EnSpire 2300 ELISA reader (PerkinElmer, Waltham, MA, USA).

Statistical Analysis
GraphPad Prism 6.0 and Image J was used to analyze data and assess the statistical significance of comparisons between groups by an ordinary one-way ANOVA test or unpaired two-tailed Student's test. p values < 0.05 were considered to be significant. Data were representative of two independent experiments. Comparison of Survival Curves were analyzed by the Kaplan-Meier method and p values were calculated using the Log-rank (Mantel-Cox) test.

Design and Biological Activities of Costimulatory Fusion Proteins
We designed three fusion proteins and expressed soluble forms in Escherichia coli, which was infrequent in previous studies. The extracellular domains of OX40L, 4-1BBL, and two domains in tandem (4-1BBL, aa 104-309; OX40L, aa  were fused with DTT, named as DTT-COS1, DTT-COS2 and DTT-COS12 respectively ( Figure 1A GraphPad Prism 6.0 and Image J was used to analyze data and assess the statistical significance of comparisons between groups by an ordinary one-way ANOVA test or unpaired two-tailed Student's test. p values < 0.05 were considered to be significant. Data were representative of two independent experiments. Comparison of Survival Curves were analyzed by the Kaplan-Meier method and p values were calculated using the Log-rank (Mantel-Cox) test.
These results show that costimulatory fusion proteins, DTT-COS1 and DTT-COS12, have some immunological activities to increase the percentage of CD8+ T cells and the secretion of IFN-γ in vitro. Results are presented as means ± SD. Ordinary one-way ANOVA test was used to analyze the data compared to DTT group, **** p <0.0001; *** p <0.001; ** p < 0.01; * p < 0.05.
These results show that costimulatory fusion proteins, DTT-COS1 and DTT-COS12, have some immunological activities to increase the percentage of CD8+ T cells and the secretion of IFN-γ in vitro.

DTT-COS1 and DTT-COS12 Protect Mice against Tumor Challenge in the Prophylactic Model and Therapeutic Tumor Models In Vivo
DTT-COS1 and DTT-COS12 significantly improved CD8+ T cells ratio and IFN-γ secretion in vitro, which indicated the proteins may elicit an antitumor immune response. To investigate the possibility, mice were pretreated three times with DTT-COS1, DTT-COS2, and DTT-COS12 respectively (50 µg/injection) 2 weeks apart, followed by B16F10 tumor challenge ( Figure 3A). Consistent with the data in vitro, pretreatment with DTT-COS12 generated a long-lasting tumor-protective effect, as all mice in this group remained tumor-free for up to 60 days ( Figure 3B). Similarly, pretreatment with DTT-COS1 also protected 80% of mice against the B16F10 tumor challenge ( Figure 3B). In contrast, DTT-COS2 treatment did not have any effect on survival compared to control groups, as all mice developed tumors ( Figure 3B).
Based on the dramatically protective effect, we next assessed the therapeutic efficacy of DTT-COS1 and DTT-COS12. From the second day after s.c. inoculation of B16F10 tumor cells, C57BL/6 mice were treated three times with DTT, DTT-COS1, and DTT-COS12, respectively (50 µg/injection) 7 days apart ( Figure 3C). Treatment with DTT-COS1 and DTT-COS12 resulted in potent inhibition of B16F10 tumor growth ( Figure 3D). The survival of mice challenged with B16F10 tumor cells was significantly prolonged in the DTT-COS1 and DTT-COS12 treated groups when compared to the DTT group ( Figure 3E). The median survival of DTT-COS1 and DTT-COS12 group were 32 and 29 days, respectively, while that of DTT control groups was 23 days ( Figure 3E). Delaying tumor challenge by 6 or 9 days resulted in distinct retardation in tumor growth.
The results suggest a striking long-lasting tumor-protective effect on DTT-COS1 and DTT-COS12 treatments in vivo.
These data indicated that DTT-COS1 could be associate with a reduction of Treg population and TGF-β expression in TME, as well as an increase of CD4+ Teff population in TILs. Meanwhile, DTT-COS12 seems to be more associated with an increase of IFN-γ and IL-2, as well as Treg reduction.

Therapeutic Tumor Models In Vivo
DTT-COS1 and DTT-COS12 significantly improved CD8+ T cells ratio and IFN-γ secretion in vitro, which indicated the proteins may elicit an antitumor immune response. To investigate the possibility, mice were pretreated three times with DTT-COS1, DTT-COS2, and DTT-COS12 respectively (50 μg/injection) 2 weeks apart, followed by B16F10 tumor challenge ( Figure 3A). Consistent with the data in vitro, pretreatment with DTT-COS12 generated a long-lasting tumorprotective effect, as all mice in this group remained tumor-free for up to 60 days ( Figure 3B). Similarly, pretreatment with DTT-COS1 also protected 80% of mice against the B16F10 tumor challenge ( Figure  3B). In contrast, DTT-COS2 treatment did not have any effect on survival compared to control groups, as all mice developed tumors ( Figure 3B).
Based on the dramatically protective effect, we next assessed the therapeutic efficacy of DTT-COS1 and DTT-COS12. From the second day after s.c. inoculation of B16F10 tumor cells, C57BL/6 mice were treated three times with DTT, DTT-COS1, and DTT-COS12, respectively (50 μg/injection) 7 days apart ( Figure 3C). Treatment with DTT-COS1 and DTT-COS12 resulted in potent inhibition of B16F10 tumor growth ( Figure 3D). The survival of mice challenged with B16F10 tumor cells was significantly prolonged in the DTT-COS1 and DTT-COS12 treated groups when compared to the DTT group ( Figure 3E). The median survival of DTT-COS1 and DTT-COS12 group were 32 and 29 days, respectively, while that of DTT control groups was 23 days ( Figure 3E). Delaying tumor challenge by 6 or 9 days resulted in distinct retardation in tumor growth.
The results suggest a striking long-lasting tumor-protective effect on DTT-COS1 and DTT-COS12 treatments in vivo.

DTT-COS1 and DTT-COS12 Can Generate Endogenous Antibodies Which May Contribute to Protective Effect against Tumors
Antibodies are known to have a variable and direct effect on tumors such as the killing of antibody-bound target cells via antibody-dependent cellular cytotoxicity (ADCC), antibodydependent phagocytosis (ADP) and though opsonization by antigen presentation and processing of APC [27]. We asked whether pretreatment with proteins can generate endogenous antibodies against The results are shown as means ± SD and the statistical significance was determined by Student's T test compared to DTT groups. ** p < 0.01; * p < 0.05.

DTT-COS1 and DTT-COS12 Can Generate Endogenous Antibodies Which May Contribute to Protective Effect against Tumors
Antibodies are known to have a variable and direct effect on tumors such as the killing of antibody-bound target cells via antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP) and though opsonization by antigen presentation and processing of APC [27].

DTT-COS1 and DTT-COS12 as Safe Formulations in Mice
For the safety evaluation of costimulatory fusion proteins, we measured the level of serum ALT and AST activity to reflect damage to hepatocytes [32]. All analysis of serum were from mice treated with DTT-COS1, DTT-COS2, DTT-COS12, or PBS in the presence of aluminum hydroxide Gel adjuvant (300 μg\200 μL; Invitrogen US) and CpG ODN 1826 (30 μg\200 μL; synthesis) on the Results are presented as means ± SD, and the statistical significance was determined by Log-rank test and Student's T test. ** p < 0.01; * p < 0.05. Different IgG subclass reveals different immune pathways [28]. In mice antibody subclass systems, Th1 cells are primarily involved with cellular immunity and associated with switching to IgG2a (IgG2c in C57BL/6) [29,30], while Th2 cells promote switching to IgG1 involved with humoral immunity [31]. Serum containing high antibody titers against anti-OX40L ( Figure 5B) was transferred i.p. into C57BL/6 mice (300 µL/mouse) 6 h before the B16F10 tumor challenge. These therapy-induced antibodies were functional, as serum transferred from DTT-COS1-treated and DTT-COS12-treated mice significantly prolonged untreated mice survival and slowed the growth of the tumor under B16F10 challenge ( Figure 5C,D). The result indicates that DTT-COS1 and DTT-COS12 generated endogenous antibodies in vivo which may contribute to the protective effect against the B16F10 challenge.

DTT-COS1 and DTT-COS12 as Safe Formulations in Mice
For the safety evaluation of costimulatory fusion proteins, we measured the level of serum ALT and AST activity to reflect damage to hepatocytes [32]. All analysis of serum were from mice treated with DTT-COS1, DTT-COS2, DTT-COS12, or PBS in the presence of aluminum hydroxide Gel adjuvant (300 µg\200 µL; Invitrogen US) and CpG ODN 1826 (30 µg\200 µL; synthesis) on the seventh day after the third treatment. DTT-COS1 and DTT-COS12 had minimal impact on serum ALT or AST levels ( Figure 6A), similar to the PBS group. DTT-COS2 significantly increased ALT and AST levels (~14.01 IU/L and~47.68 IU/L, respectively), compared to DTT-COS1 (~5.186 IU/L) and DTT-COS12 (~21.47 IU/L) ( Figure 6A). There were no significant differences between the DTT-COS2 and PBS groups in serum ALT and AST ( Figure 6A). The H&E staining showed that the liver, kidney, and lung tissue sections of the mice treated with costimulatory fusion proteins were almost the same as the PBS group ( Figure 6B). Serum concentrations of IL-6 and IL-8 were analyzed to show the inflammatory reactions by ELISA [33]. There were no obvious differences between the three proteins and the PBS group in the serum of the two pro-inflammatory cytokines IL-6 and IL-8 ( Figure 6C). Increasing the number of treatments, the body weights of mice treated with DTT-COS1, DTT-COS2, or DTT-COS12 did not show any differences with the PBS group ( Figure 6D). These results indicate that DTT-COS1 and DTT-COS12 were safe and less toxic to mice.
Vaccines 2020, 6, x FOR PEER REVIEW 11 of 17 seventh day after the third treatment. DTT-COS1 and DTT-COS12 had minimal impact on serum ALT or AST levels ( Figure 6A), similar to the PBS group. DTT-COS2 significantly increased ALT and AST levels (~14.01 IU/L and ~47.68 IU/L, respectively), compared to DTT-COS1 (~5.186 IU/L) and DTT-COS12 (~21.47 IU/L) ( Figure 6A). There were no significant differences between the DTT-COS2 and PBS groups in serum ALT and AST ( Figure 6A). The H&E staining showed that the liver, kidney, and lung tissue sections of the mice treated with costimulatory fusion proteins were almost the same as the PBS group ( Figure 6B). Serum concentrations of IL-6 and IL-8 were analyzed to show the inflammatory reactions by ELISA [33]. There were no obvious differences between the three proteins and the PBS group in the serum of the two pro-inflammatory cytokines IL-6 and IL-8 ( Figure 6C). Increasing the number of treatments, the body weights of mice treated with DTT-COS1, DTT-COS2, or DTT-COS12 did not show any differences with the PBS group ( Figure 6D). These results indicate that DTT-COS1 and DTT-COS12 were safe and less toxic to mice.

Discussion
In this study, we have demonstrated an effective strategy for cancer immunotherapy. Costimulatory fusion proteins, DTT-COS1 and DTT-COS12, protected mice from B16F10 tumor challenge in prophylactic and therapeutic models. Immunomodulation by DTT-COS1 in TME increased the proportion of CD4+ Teff cells and decreased the expression of TGF-β. Meanwhile, immunomodulation by DTT-COS12 in TME reduced the proportion of Treg cells and increased the expression of IL-2 and IFN-γ. The proteins elicited high-titer antibodies against OX40L/4-1BBL without systemic toxicity, which may affect tumor protection.

Discussion
In this study, we have demonstrated an effective strategy for cancer immunotherapy. Costimulatory fusion proteins, DTT-COS1 and DTT-COS12, protected mice from B16F10 tumor challenge in prophylactic and therapeutic models. Immunomodulation by DTT-COS1 in TME increased the proportion of CD4+ Teff cells and decreased the expression of TGF-β. Meanwhile, immunomodulation by DTT-COS12 in TME reduced the proportion of Treg cells and increased the expression of IL-2 and IFN-γ. The proteins elicited high-titer antibodies against OX40L/4-1BBL without systemic toxicity, which may affect tumor protection.
There have been few reports on the soluble expression of costimulatory ligands in E. coli expression systems among previous studies, and one demonstrated the recombinant human 4-1BBL inclusion body as the form of expression [34]. Our study is the first to achieve the soluble expression of OX40L, 4-1BBL, and their tandem forms in E. coli. Earlier reports indicated that native costimulatory protein ligands such as native 4-1BBL have no costimulatory activity [35,36], and some studies have fused OX40L or 4-1BBL with different carriers to promote T lymphocytes growth and exhibit potent biologic activity [37,38]. DTT, a transmembrane domain of diphtheria toxin with universal Th epitope, is an effective antigen carrier and scaffold to break immune self-tolerance [24,39,40], also used as a soluble membrane anchor [41][42][43][44]. Here, DTT was used as a carrier protein in our study, which fused with extracellular domains of costimulatory ligands, 4-1BBL, OX40L, and in combination. Our results showed that DTT-COS1 and DTT-COS12 had costimulatory activities on immune cells in vitro, regulating the proportion of CD3+CD4+ and CD3+CD8+ T cells, and DTT-COS12 notably increased the secretion of IFN-γ. DTT-COS12 have one more extracellular domain of 4-1BBL than DTT-COS1. Maybe the function of an additional costimulatory factor, 4-1BBL determined the difference of immune response between naïve and immunized state. As reported in previous reports, costimulatory signals, like 4-1BB/4-1BBL, have a pivotal role in the survival of activated effector and memory CD8+ T cells [45][46][47][48] and are important in establishment and maintenance of the CD8+ T cell recall response to antigens or viruses [48,49]. The stimulation of 4-1BB/4-1BBL preferentially induces Th1 responses by increasing IFN-γ, IL-2, and CD8+ T cell proliferation [16,45]. Therefore, DTT-COS12, a combination of two costimulatory factors, generated a higher immune response in the immunized state and enhanced the production of Th1 associated cytokines, like IFN-γ and IL-2.
Treatment with DTT-COS1 or DTT-COS12 had a long-term protective effect on mice inoculated with B16F10 cells, for up to 60 days, and more than 80% of mice had no tumor growth. This result is consistent with the recently reported preventive efficacy of the OX40 antibody and 4-1BBL fusion proteins [50,51]. Moreover, very few reports have demonstrated the role played by costimulatory proteins alone in the therapeutic model of tumors. To our surprise, after the B16F10 tumor challenge, there was a significantly prolonged survival and delay of tumor growth with DTT-COS1 or DTT-COS12 treatment. Disappointingly, we did not observe any antitumor effects on DTT-COS2 in vivo or in vitro, perhaps with other models. Recently, the combination of the OX40 antibody and CpG has a remarkable antitumor effect in situ [50], but the drawbacks of in situ immunization are the appropriate immune cells infiltration and adequate tumor injection site. A comparison in tumor-challenge trials using CT26 mice models was less effective compared to B16F10 models. Future studies should consider the optimization of doses, tumor models, and the mechanism of research.
Tumor-infiltrating Tregs play immunosuppressive roles in cancer and enhance suppression in the TME [52]. The antitumor mechanism has been reported in OX40/OX40L co-stimulation, preventing the induction of Foxp3+ Tregs from T effector cells [53]. Other studies have shown that OX40/OX40L engagement depleted intratumoral Tregs that correlated with tumor regression [54]. Consistent with previous researches, the function of OX40L showed a decrease of tumor-infiltrating regulatory T cells population in DTT-COS12 treatment. The population of tumor infiltrating CD4+ effector T cells increased distinctly in DTT-COS1 treatment. We also found IFN-γ and IL-2, involved in cellular killing immunity, were sharply increased by DTT-COS12 treatment, while the expression of TGF-β was significantly decreased by DTT-COS1 treatment. As one of the immunosuppressive cytokines, TGF-β inhibits the production and function of effector T cells as well as antigen-presenting dendritic cells (DC) [55,56]. The expression of IL-4, which is associated with humoral immunity [57,58], had no significant change in our experimental groups. Earlier studies have shown that OX40 was highly expressed by intratumoral Tregs [54]. Surprisingly, we have also found an increase in the expression of costimulatory receptor OX40 with DTT-COS1 treatment, and related receptor 4-1BB with DTT-COS12 treatment in TILs, which probably helped the infiltration of effect T cells and tumor resist in TME.
We can infer that costimulatory fusion proteins successfully decreased intratumoral Tregs to produce antitumor responses.
There are five antibody subclasses (IgA, IgD, IgE, IgG, and IgM) in mice, which are similar in structure and different in function [59]. We assessed the immune response and polarization of Th1 and Th2 through murine IgG (h+l), IgG1, IgG2b, IgG2c, IgG3, and IgM. Mouse IgG2a/2b (IgG2c in C57BL/6) is considered the same as human IgG1 [30,60], represent cellular immunity (Th1 polarization) [29][30][31]. Mouse IgG1 is considered the same as human IgG4, suggesting humoral immunity (Th2 polarization). IgM is the first immunoglobulin class to be synthesized by the neonate and plays a role in the pathogenesis of some autoimmune diseases [30]. It is reported that the IgM antibody can make a good biologic against cancer owing to its strong avidity, as well as complement fixation property [28]. Mouse IgG3 was demonstrated to be effective against several life-threatening bacterial infections and is the only IgG subclass able to agglutinate the cells when recognizing a surface antigen of red blood corpuscle [28,61]. Based on the results, DTT-COS1, DTT-COS2, and DTT-COS12 treatment produced higher titers of endogenous antibodies subtypes against OX40L and/or 4-1BBL. Serums with a high-titer antibody against OX40L could surprisingly contribute to a protective effect on B16F10 tumors. It has been shown that non-specific IgG substantially accumulates in solid tumors and endogenous IgG was used as a systemic drug delivery to solid tumors and enhance antitumor activity [62].

Conclusions
In conclusion, we demonstrate the unexpected immunomodulatory characteristics of different costimulatory ligand proteins with no combination of tumor-associated products, which showed potent immune responses against B16F10 tumor challenge in both preventive as well as therapeutic efficacy. This was a unique feature of the recombinant costimulatory molecule as an agonistic OX40/4-1BB antibody did not protect mice against tumor challenge. Moreover, treatments with DTT-COS1 significantly increased the percentage of effective TILs and decreased the expression of immunosuppressive cytokine TGF-β, meanwhile, DTT-COS12 decreased the percentage of regulatory TILs and enhanced the production of Th1-associated cytokines, IL-2, and IFN-γ. DTT-COS1 and DTT-COS12 generated endogenous antibodies that helped antitumor immunity, and without systemic toxicity. Thus, our design of recombinant costimulatory ligand proteins is a promising strategy for preclinical and clinical cancer immunotherapy.