Achieving a robust and continuous anti-tumor immune response to prolong patient survival remains challenging. While DC vaccines exhibit favorable clinical prospects due to their safety, treating certain large tumors still poses limitations. Immunoadjuvants, such as TLR agonists, exosomes, and metal- and plant-derived immunoadjuvants, are widely used in anti-tumor therapy [32–33]. HMGN1 is an endogenous immune adjuvant typically combined with ICIs or TLR agonists [21, 34]. Similarly, R848 is a synthetic small molecule or its analogue that is commonly used in combination with other ICIs [35]. These treatments are extensively employed in anti-tumor therapy. We present a straightforward and efficacious therapy for combating tumors via intertumoral injection of HMGN1 + R848, and intraperitoneal infusion of a-TNFR2 antibody. Abbreviations will be explained upon first use. The effectiveness of HMGN1 + R848 arises from their ability to promote the anti-tumor immunity of DCs by enhancing the activation of these cells, improving the homing capacity of the draining lymph nodes, and supporting the production and expansion of long-term memory T cells[22]. Simultaneously, the α-TNFR2 has the capacity to impede the expression of TNFR2 molecules on the surface of MDSCs or tumor cells, consequently curtailing immunosuppression in the microenvironment of the tumor[27, 36]. Ultimately, the concomitant administration of HMGN1 + R848 + α-TNFR2 resulted in total elimination of the tumor and an elongated survival period (Fig. 1). In comparison to the intertumoral injection of HMGN1 (10µg/injection) as reported previously[21], this study utilized the most efficient therapeutic dosage of HMGN1 (0.5µg/injection) for combination therapy, and the outcomes surpassed our presumptions. The abatement of this tumor type renders it conceivable to conduct surgery in a clinical setting, and we aim to enhance the delivery method and drug amount in forthcoming research.
CD8+ T-cells infiltrate "hot" tumors, aiding in the reversal of immunosuppression and improvement of tumor antigen immunity. Our study indicates a significant increase in the number of CD8+ T cells in the triple therapy group on day four. Furthermore, combined therapy enhances the production of cytokines, TNF-α and IFN-γ, by CD4+ T and CD8+ T cells which is essential for effective tumor attack by T-cells. The research also discovered that triple therapy can modify the tumor microenvironment, enhance the invasion of cytotoxic T cells and support long-lasting anti-tumor defenses (Fig. 2).
The HMGN1 and R848 compounds selectively recruit and activate an array of immune cells, encompassing NK cells, B cells, neutrophils, monocytes, macrophages, and DCs[37–38]. DCs play a pivotal role in absorbing antigens from the tumor microenvironment and subsequently migrating to the tumor drainage lymph nodes for T cell presentation, thereby exerting a critical impact on anti-tumor immunity[28, 39]. Hence, DCs homing to tumor lymph nodes holds great significance. On the fourth day of treatment, it was found that HMGN1 + R848 can promote the maturation of migrating cDC1s, compared to the PBS treatment group. HMGN1 + R848 + α-TNFR2did not further enhance the maturation of cDC1s, however, it did increase the proportion of migrating cDC1s, as expected. Moreover, HMGN1 + R848 have been shown to synergistically enhance IL-12 production in in vitro experiments. This subsequently initiates the differentiation of helper T cells into Th1 cells, resulting in the propagation and maturation of Th1 cells (Fig. 3) [40].
The combination of HMGN1 + R848 + α-TNFR2 antibody therapy demonstrated an immune memory effect, resulting in partial systemic immunity(Fig. 4). After the final treatment, CT26 tumors were effectively eliminated, and the subjects remained tumor-free for up to day 80. One month later, CT26 tumor cells were reintroduced at the same site in tumor-free mice without any recurrence. Previous transcriptome analysis revealed that CD8+ T cells underwent a process that stimulated the release of TCR, costimulatory signals, and signaling factors via metabolic or cell cycle changes after HMGN1 treatment. This is comparable to the immune memory effect induced by memory T cells[34]. Zuo et al. showed that the DCTEX-HMGN1ND vaccine, which utilizes HMGN1 as an adjuvant, provides long-lasting anti-tumor immunity and inhibits the growth of large, poorly immunogenic orthotopic hepatocellular carcinoma (HCC). This vaccine enhances memory T cells and aids lymphoid tissue homing[37]. In addition, the combined treatment of HMGN1 + R848 + α-TNFR2 antibody proved to be more effective in clearing the tumor compared to other treatments assessed in the study. This phenomenon could be attributed to several factors. Firstly, HMGN1 + R848 exhibit synergistic effects in stimulating DCs activation and recruitment, promoting T cell-mediated tumor cell death and hence prompting a systemic immune response[24]. Also, α-TNFR2 antibodies can reduce the immunosuppressive capacity of the tumor microenvironment and downregulate the expression levels of MDSCs and Treg populations. Ultimately, this leads to potent tumor elimination capabilities[41–42].
HMGN1 and R848 are TLR agonists, capable of activating the MyD88 and NF-κB pathways to stimulate DCs and T cells. Consequently, TLR agonists have demonstrated some benefits in the realm of anti-tumor treatment, although there are worries about their acute toxicity. The mice's behavior and body weight were monitored two to three times per week during the entire study. The findings indicate a minor decrease in the weight of the mice during the primary phase, which later progressed to a gradual increase (Fig. 5). This implies that the given therapy can potentially aid in improving the current cachexia status that develops alongside tumor growth whilst also generating a host-protective effect. Earlier reports also demonstrated the efficacy of R848 in curbing the cachexia-associated symptoms of PDAC[43].
Currently, certain immunotherapy techniques demonstrate promising outcomes, including tumor vaccines, cytokines, monoclonal antibodies, and immunostimulatory small molecules. However, there are significant unknown factors that restrict their clinical use[4]. This article's drug delivery approach mainly utilizes immune preparations to reduce host damage while ultimately obtaining the same tumor elimination effect, rather than utilizing radiotherapy and chemotherapy. TIGIT, a recently discovered molecule in the CD28 family, acts as an immune checkpoint by binding to its ligands CD155 and CD112, which are expressed in APCs, T cells, and tumor cells[44]. Studies suggest that TIGIT is involved in the depletion of NK cells in colon cancer mice and patients. Blocking TIGIT enhances the restoration of NK cell depletion, promotes the immune function of T cells, and is beneficial for NK cell-dependent tumors. Moreover, this approach maintains the immune memory effect and has a stronger anti-tumor effect when used in conjunction with checkpoint blockers, including anti-PD-1[45]. The TLR7/8 agonist, 3M-052, is a newly designed drug that can effectively release slowly at the injection point. It exhibits a positive outcome when combined with other checkpoint blockers[46]. The combination of HMGN1 + 3M-052 + α-TIGIT noticeably eliminated the growth of colon cancer, indicating the comparable functions of 3M-052 and R848, and α-TIGIT and α-TNFR2 (Fig. 6). This implies that the insufficient effectiveness and drug resistance of monotherapy immunotherapy accelerate the advancement of multidirectional combined immunotherapy, thus leading to the creation of individualized treatments for patients [47].