IL-2 is one of the earliest immunotherapeutic agents to induce complete responses in patients with cancer, but its use has been limited by its toxicity and the need for frequent administration. In order to capitalize on the desirable effects of IL-2 while limiting its risks, Moynihan et al. set out to investigate ways to improve IL-2’s therapeutic profile. Their results were recently published in Cancer Discovery.

IL-2 is known to target a wide range of cell types expressing either IL-2Rβγ or IL-2Rαβγ. In previous studies, efforts have been made to target IL-2 only to cells not expressing IL-2Rα in order to reduce immunosuppression and toxicity associated with Tregs and innate lymphoid cells, respectively. However, the impact of this was limited, with treatments still simultaneously inducing antitumor immunity alongside immunosuppression and toxicity. A closer look into the mechanism in mice revealed that among affected cell types, not-α-mIL2 induced the strongest expansion and activation in NK cells, in line with their higher IL-2Rβ expression. However, NK cells were found to be largely responsible for mediating toxicity, while only CD8⁺ T cells were required for antitumor efficacy. Similarly, in human PBMCs, untargeted or not-α-targeted hIL-2 induced strong dose-dependent and antigen-agnostic IFNγ production by NK cells, but minimal IFNγ production by T cells in the absence of TCR stimulation.

Based on these observations, Moynihan et al. set out to develop an IL-2 molecule that would specifically target CD8⁺ T cells, but not NK cells or Tregs, via cis-targeting of CD8. To this end, they evaluated targeting CD8α and CD8β, and found that CDβ was more exclusive to CD8⁺ T cells. Next, they generated fusions between anti-CDβ and a set of IL-2 variants. Ultimately the selected design was an anti-CD8β (that bound to CD8β, but did not block co-receptor functioning) fused to an IL-2 variant with no binding to IL-2Rα and reduced binding to IL-2Rβ. This CD8-IL2β*γ molecule, termed AB248, demonstrated a strong preference for targeting and activating CD8⁺ T cells over other cell types in human PBMCs, avoiding both Treg activation and antigen-independent release of IFNγ and other proinflammatory cytokines by NK or other innate lymphoid cells. Further, only CD8⁺ T cells receiving TCR stimulation showed increased IFNγ and TNFα secretion.

To evaluate the antitumor efficacy of a CD8-targeted IL-2, the researchers generated a potency-matched murine surrogate, CD8-mIL2, that showed a strong preference for CD8⁺ T cells in both tissues and tumors in mice, while a control not-α-mIL-2 favored NK cells. In mice bearing MC38 tumors, treatment at day 8 with CD8-mIL2 demonstrated dose-dependent antitumor immunity, including a dose at which 7 of 10 mice achieved durable tumor rejection. Responses were accompanied by CD8⁺ T cell expansion in both the periphery and the tumor. Based on experiments with FTY720, which blocks lymph node egress, responses were dependent on both resident and newly infiltrating lymphocytes. Mice that responded to treatment also rejected rechallenge, indicating immune memory. Overall, CD8-mIL2 outperformed controls in terms of both increased antitumor immunity and reduced toxicity (as measured by weight loss). Further, even a single dose of the CD8-mIL2 outperformed daily dosing with standard IL-2.

To evaluate whether a CD8-targeted IL-2 could enhance the efficacy of other immunotherapies, A375 melanoma-bearing NSG mice were treated with antigen-specific human CAR T cells with or without CD8-hIL2. While the CAR T cells alone modestly delayed tumor growth, the addition of CD8-hIL2 resulted in complete tumor rejection in all mice.

To test whether CD8-targeted IL-2 could improve the efficacy of anti-PD-1 and overcome resistance, the researchers used T3 sarcoma-bearing mice. When mice were treated at day 12 – a time point at which they became resistant to anti-PD-1 monotherapy – treatment with CD8-mIL2 monotherapy induced transient tumor regression, while treatment with both anti-PD-1 and CD8-mIL2 induced complete and durable tumor elimination in all mice. Similar benefit of the combination of CD8-mL2 and anti-PD-1 was observed in anti-PD-1-resistant settings for B16F10, 1956, MCA205, and KP.mLama4 tumor models.

Further analyzing the antitumor responses in the T3 model, the researchers identified increased numbers of CD8⁺ T cells specific for the immunodominant antigen mLama4, as well as increased granzyme B after either CD8-mIL2 monotherapy or CD8-mIL2 plus anti-PD-1. Reduced exhaustion was also observed in the combination group. Following CD8-mIL2 monotherapy, TILs showed increased numbers of tumor antigen-specific CD8⁺ T cells, increased antigen-reactive T_CM, T_EFF, and T_EM cell numbers, and decreased expression of PD-1.

Single-cell RNA sequencing revealed clonal expansion, particularly in antigen-specific T cells in proliferating effector, effector/memory, and exhausted (precursor and terminal) CD8⁺ T cell populations. In animals treated with CD8-mIL2 monotherapy or CD8-mIL2 + anti-PD-1 combination therapy, antigen-specific T cells were mostly in the proliferating effector populations, while in animals treated with vehicle control or anti-PD-1, the majority of antigen-specific T cells fell into exhausted populations. T cells in CD8-mIL2-treated animals also showed enhanced proliferation scores, decreased exhaustion scores, and altered transcriptional profiles, even compared to those treated with not-α-IL-2, which induced similar T cell profiles, but with significant toxicity. Interestingly, in mice treated with CD8-mIL2, especially in combination with anti-PD-1, a new effector population emerged within tumor-reactive TILs around day 4. This population was characterized by genes for the expression of cytokines, effector molecules, and IL-7R, resembling a “better effector” T cell population. Similar results were observed in the MC38 tumor model, where researchers also noted a strong induction of the IFNγ response signature across myeloid clusters, consistent with increased IFNγ production by antigen-reactive CD8⁺ TILs.

Moving into cynomolgus monkeys, the researchers showed that in vitro, AB248 had similar CD8⁺ T cell binding and activation patterns in human and primate samples. Intravenous dosing of the monkeys resulted in induction of Ki67 and increased expansion of peripheral blood CD8⁺ T cells, with minimal changes to NK cell, CD4⁺ T cell, or Treg populations. Repeated administrations of AB248 could re-expand CD8⁺ T cells for at least 4 weekly doses. While some of the animals experienced transient diarrhea, treatment was generally well tolerated at dose levels comparable to those with potent antitumor efficacy in mice.

Overall, these findings from Moynihan et al. suggest that targeting IL-2 specifically to CD8⁺ T cells through cis-targeting of CD8 can enhance not only the safety profile of IL-2, but the antitumor efficacy as well. Further, CD8-IL2 molecules showed antitumor efficacy both alone and in combination with other immunotherapies, and could be used to enhance treatment and overcome resistance, supporting further research and clinical development. In an accompanying manuscript (see Spotlight below), the effects of AB248 were examined in detail in human patient-derived tumor fragments.

Write-up and image by Lauren Hitchings

Meet the researcher

This week, Kelly Moynihan answered our questions.

What was the most surprising finding of this study for you?
We knew that CD8⁺ T cells were necessary for IL-2-mediated therapeutic responses from the literature, but it was an open question whether signaling on CD8⁺ T cells alone would be sufficient for good therapeutic activity. Our cis-targeted molecule allowed us to address this question definitively: CD8⁺ T cells are both necessary and sufficient for significant IL-2-mediated antitumor activity in a variety of mouse models. Another finding that might be surprising is that CD8⁺ T cells could be safely expanded monkeys in excess of 20-fold. This may be explained by the observation that some IL-2-mediated effects are antigen-independent (proliferation), whereas others are antigen-dependent (enhancement of effector function and cytokine secretion).

What is the outlook?
It’s a very exciting time for AB248! We are now investigating AB248 in patients with solid tumors, both as monotherapy and in combination with pembrolizumab (NCT05653882) – stay tuned for updates!

What was the coolest thing you’ve learned (about) recently outside of work?
I had the incredible opportunity to witness the recent solar eclipse from the path of totality, and it was nothing short of fantastic and surreal. Leading up to the eclipse, I read several accounts of unusual animal behavior observed during previous total eclipses. I was particularly intrigued to discover that scientists at the Indianapolis Zoo, situated within the path of totality, had planned to meticulously study animal reactions during the event. I eagerly await their findings!