rhIL-7-hyFc and hIL-2/TCB2c combination promotes an immune-stimulatory tumor microenvironment that improves antitumor efficacy of checkpoint inhibitors

Background: Recombinant human interleukin (rhIL)-7-hyFc (efineptakin alfa; NT-I7) is a potent T-cell amplifier, with two IL-7 molecules fused to IgD/IgG4 elements. rhIL-7-hyFc promotes extensive infiltration of CD8⁺ T cells into the tumor, concurrently increasing the numbers of intratumoral PD-1⁺CD8⁺ T cells. The hIL-2/TCB2 complex (SLC-3010) inhibits tumor growth by preferential activation of CD122 (IL-2Rβ)^high CD8⁺ T cells and natural killer cells, over regulatory T cells (Tregs). We investigated the underlying mechanisms of rhIL-7-hyFc and hIL-2/TCB2c antitumor activity and the potential synergistic efficacy, specifically focusing on tumor-specific CD8⁺ cells within the tumor and the tumor-draining lymph nodes (tdLN).

Methods: MC38 and CT26 tumor-bearing mice were administered with 10 mg/kg rhIL-7-hyFc intramuscularly and 0.9 mg/kg hIL-2/TCB2c intravenously. Anti-PD-1 monoclonal antibody was administered intraperitoneally three times at 3-day intervals at a dose of 5 mg/kg. Tumor volume was measured to assess efficacy. To compare the composition of immune cells between each monotherapy and the combination therapy, we analyzed tumors and tdLNs by flow cytometry.

Results: Our data demonstrate that the combination of rhIL-7-hyFc and hIL-2/TCB2c increases efficacy and generates an immune-stimulatory tumor microenvironment (TME). The TME is characterized by an increased infiltration of tumor-specific CD8⁺ T cells, and a decreased frequency of CD39^highTIM-3⁺ Treg cells. Most importantly, rhIL-7-hyFc increases infiltration of a CD62L⁺Ly108⁺ early progenitor population of exhausted CD8⁺ T cells (T_PEX), which may retain long-term proliferation capacity and replenish functional effector CD8⁺ T cells. hIL-2/TCB2c induces differentiation of CD62L⁺Ly108⁺ T_PEX rapidly into CD101⁺ terminally differentiated subsets (terminally exhausted T cell (T_{EX term})). Our study also demonstrates that rhIL-7-hyFc significantly enhances the proliferation rate of T_PEX in the tdLNs, positively correlating with their abundance within the tumor. Moreover, rhIL-7-hyFc and hIL-2/TCB2c can overcome the limited therapeutic effectiveness of PD-1 blockade, culminating in the complete regression of tumors.

Conclusions: rhIL-7-hyFc can expand and maintain the progenitor pool of exhausted CD8⁺ T cells, whereas hIL-2/TCB2c promotes their differentiation into T_{EX term}. Together, this induces an immune-stimulatory TME that improves the efficacy of checkpoint blockade.