Targeting 4-1BB and PD-L1 induces potent and durable antitumor immunity in B-cell lymphoma

Abstract

Introduction: Although PD-1/L1 mAb has demonstrated clinical benefits in certain cancer types, low response rate and resistance remain the main challenges for the application of these immune checkpoint inhibitors (ICIs). 4-1BB is a co-stimulator molecule expressed in T cells, which could enhance T cell proliferation and activation. Herein, the synergetic antitumor effect and underlying mechanism of 4-1BB agonist combined with PD-1/PD-L1 blockade were determined in B-cell lymphoma (BCL).

Methods: Subcutaneous transplantation BCL tumor models and metastasis models were established to evaluate the therapeutic effect of PD-L1 antibody and/or 4-1BB agonist in vivo. For the mechanistic study, RNA-seq was applied to analyze the tumor microenvironment and immune-related signal pathway after combination treatment. The level of IFN-γ, perforin, and granzyme B were determined by ELISA and Real-time PCR assays, while tumor-infiltrating T cells were measured by flow cytometry and immunohistochemical analysis. CD4/CD8 specific antibodies were employed to deplete the related T cells to investigate the role CD4+ and CD8+ T cells played in combination treatment.

Results: Our results showed that combining anti-PD-L1 ICI and 4-1BB agonists elicited regression of BCL and significantly extended the survival of mice compared to either monotherapy. Co-targeting PD-L1 and 4-1BB preferentially promoted intratumoral cytotoxic lymphocyte infiltration and remodeled their function. RNA-sequence analysis uncovered a series of up-regulated genes related to the activation and proliferation of cytotoxic T lymphocytes, further characterized by increased cytokines including IFN-γ, granzyme B, and perforin. Furthermore, depleting CD8+ T cells not CD4+ T cells totally abrogated the antitumor efficacy, indicating the crucial function of the CD8+ T cell subset in the combination therapy.

Discussion: In summary, our findings demonstrated that 4-1BB agonistic antibody intensified the antitumor immunity of anti-PD-1/PD-L1 ICI via promoting CD8+ T cell infiltration and activation, providing a novel therapeutic strategy to BCL.

Keywords: 4-1BB agonist; B-cell lymphoma; CD8+ T cells; anti-PD-L1 ICI; combination therapy.

Figure 1

4-1BB agonism and PD-L1 blockade elicited synergistic antitumor effect in B-cell lymphoma. (A, B) The subcutaneous A20 tumor model (n = 6) or WEHI-231 tumor model (n = 5) were well-established in BALB/c mice. Tumor volume was measured twice a week. Treatment was initialized when the tumor volume reached 100 mm³. (C, D) Tumor weight was measured at the end of the treatment. (E) The metastatic A20 model was established to evaluate the antitumor effect on survival (n = 10). * means p-value < 0.05, and ** means p-value < 0.01.

Figure 2

Mechanisms underlying the synergistic antitumor efficacy of the combination therapy. (A) Heatmap showed the differentially expressed genes between the anti-PD-L1 group and the combined therapy group. (B) Enrichment of KEGG pathway of differentially expressed genes. (C) GSEA showed an enrichment of the gene set related to CD8⁺ T cell activation. (D) GSEA showed enrichment of activation of IFN-γ response gene sets.

Figure 3

The combined therapy increased cytotoxic T-cell infiltration and expression of PD-L1. (A–C) The proportion of CD4/CD8 positive cells in the tumor tissue in each group (n = 3) after one week of treatment. (D) The infiltration of CD8⁺ T cell in tumor tissue detected by Immunohistochemistry (IHC) (Magnification × 20 (up), Magnification × 90 (below)). * means p-value < 0.05, and ** means p-value < 0.01.

Figure 4

Functions of CD8⁺ T cells in the combination therapy. (A, B) Combinational therapy significantly upregulated genes related to CD8⁺ and CD4⁺ T cells. (C) Genes were classified and grouped based on their molecular function. (D) Perforin, IFN-γ, and granzyme B in tumors were measured by RT-PCR or ELISA after combinational treatment (n = 3, mean ± SD). * means p-value < 0.05, ** means p-value < 0.01, and *** means p-value < 0.001.

Figure 5

T cell subsets in the antitumor efficacy of anti-PD-L1 mAb combined with 4-1BB agonist. The antitumor effect of the combined therapy was abolished by CD8⁺ T cell depletion. (A, B) Tumor volume was measured twice a week. (C) Tumor weight was measured at the end of the treatment. (n = 4, mean ± SD). ** means p-value < 0.01. ns means no significance.

Figure 6

A graphical description of how PD-L1 blockade and 4-1BB agonism elicited enhanced antitumor effect in BCL. The combination of 4-1BB agonist and anti-PD-L1 antibody can activate T cell function and increase the expression of cytokines such as IFN-γ. On the other hand, it can release the inhibition of T cells by tumor cells by blocking the PD-1-PD-L1 signaling axis. Thus, eliciting a more significant anti-tumor effect.