Targeted α-Therapy Using ²²⁵Ac Radiolabeled Single-Domain Antibodies Induces Antigen-Specific Immune Responses and Instills Immunomodulation Both Systemically and at the Tumor Microenvironment

DISCUSSION

In this study, we showed that α-TRT with ²²⁵Ac-9079 in the B16-huCD20 melanoma mouse model has therapeutic potential as it delays tumor growth. We further showed stimulation of cytokine release, tumor-specific CD8^pos T-cell responses, and a shift from a tumor-promoting TME to a TME characterized by an immune contexture that enables tumor cell rejection, including activated (PD-1^pos) T-cells, classically activated macrophages, and DCs. Finally, we showed that ICB with anti-PD-L1 mAbs combined with α-TRT improves tumor control compared with either therapy alone, albeit coinciding with adverse events (i.e., weight loss).

We evaluated the potential of ²²⁵Ac-9079 as an α-TRT agent and showed specific accumulation in B16-huCD20 tumors of immunocompetent mice, sparing muCD20^pos cells. This observation is in line with previous work performed with the same sdAb, albeit conjugated to another linker and radionuclide (11). For therapeutic efficacy, a dose approximating 5 Gy was desired as literature described little therapeutic effect given solid tumors’ resilience to radiation (14). Hence 180 and 300 kBq of ²²⁵Ac-9079 were administered, corresponding to respective delivery of 4 and 6 Gy in tumor. The observed delay in tumor growth with ²²⁵Ac-9079 is in line with published studies that highlight the potential of ²²⁵Ac as a therapeutic radionuclide (9).

Serum cytokine levels were quantified as markers for systemic immune activation, and various cytokines were enhanced. Increased levels of MCP-1 and GM-CSF suggest monocytes or macrophage migration or infiltration and DC maturation, respectively (15,16). Furthermore, elevation of antitumoral cytokines such as IFN-γ and CCL5 advocates activation of CD8^pos T-cells and serves as predictor of response to ICB (17). A recent study investigating adoptive cell transfer and α-TRT described the secretion CCL5 and IFN-γ from tumor on treatment (18). These results prompted us to restimulate isolated spleens with cancer-associated epitopes. Intriguingly, IFN-γ secretion revealed that splenocytes recognized and reacted to ADPGK and ovalbumin but not toward melanoma differentiation antigens. Evidence of high-avidity antitumoral CD8^pos T-cells responses on α-TRT has not yet been published.

After analyzing systemic responses, we evaluated α-TRT–mediated alteration of the TME, which is emphasized by an increase in professional antigen presenting cells at the expense of immunosuppressive subsets of myeloid cells. More specifically, alternatively activated macrophages (M2) are significantly decreased in numbers. These cells are involved in wound healing under physiologic conditions and attenuating immune responses to prevent collateral tissue damage (19). In malignancy, M2 macrophages are enthralled by tumor cells to overexert immunosuppressive functions, quench uprising antitumoral responses, and facilitate tumor progression and metastasis (19). Unlike, M2 macrophages, we documented an increase in M1 macrophages and DCs, professional antigen presenting cells involved in the education of T-cell responses (20). This observation is consistent with the cytokine profile of MCP-1 and GM-CSF, hinting at macrophage infiltration and DC maturation. DCs can acquire antigens, migrate, and present these to T-cells within lymph structures to elicit a clonal T-cell response, which ties in with the identification of antitumoral T-cells in the spleen (21). Regarding the expression pattern of immune checkpoint receptors in the TME, we noted that α-TRT upregulated expression of PD-1 on CD4^pos T-cells as well as its ligand, PD-L1, on various cell types in the TME. In recent studies involving α-TRT, a similar observation was made (18). Ligation of PD-1 to PD-L1 impairs T-cell receptor signaling, in turn interfering with the effector function of antitumoral T-cells (20). In short, we observed that ²²⁵Ac-9079 altered the tumor immune compartment, favoring myeloid cells that likely facilitate antigen processing, presentation, and education of T-cell responses, were it not for the presence of inhibitory markers such as PD-1 and PD-L1. To overcome this hurdle we performed ICB of the PD-1–PD-L1 axis to enhance the therapeutic effect of sdAb-mediated α-TRT.

Synergy between α-TRT and ICB occurred only in combination with PD-L1 blockade in these experiments, despite literature stating PD-1 synergizes with α-TRT as well (22). Intriguingly, PD-L1 ICB on its own did not amount to a therapeutic effect, compared with the IC, whereas PD-1 had therapeutic benefit on its own. We surmise that PD-L1 ICB after α-TRT is this effective because of the immune alterations induced on α-TRT, setting the stage to unleash all this immunologic potential. Besides the effect of anti-PD-L1 mAbs as a blocking agent, antibody-dependent cellular cytotoxicity (ADCC) also comes into play. Unlike the PD-1 mAb, the PD-L1 mAb used in this study is able to induce ADCC (23). Hence, we surmised that PD-L1^pos tumor and stromal cells in the TME are clad with mAbs on PD-L1 ICB, flagging them for ADCC by FcγR^pos effector cells, such as B cells, NK cells, and M1 macrophages, which highly infiltrate tumors subjected to α-TRT (24). Unfortunately, mice that received a combination of α-TRT and PD-L1 ICB experienced extensive weight loss, resulting in premature attainment of a humane endpoint. We presume that this phenomenon is the result of the combination therapy’s ensuing cytokine storm as mice receiving a similar amount of radioactivity or ICB, as monotherapy, did not lose weight during the course of the experiment. Curiously, α-TRT with PD-1 ICB did not result in weight loss, suggesting the onset of ADCC by the PD-L1 mAb may have played a crucial role in this process.

To investigate late-stage systemic radiation burden, we administered similar therapeutic doses to healthy mice, which allowed long-term follow up. We noted that 4 mo after administration of ²²⁵Ac-9079, mice started losing weight. The spleen and kidneys of these mice were severely reduced in size compared with unirradiated mice, suggesting that α-TRT inflicted severe damage to these organs. Damage to tubular epithelial cells and loss of renal function on α-TRT was described by Scheinberg et al. (25). Unlike the kidneys, spleen biodistribution data does not advocate for a substantial accumulation of ²²⁵Ac-9079. Hence, we surmise that the loss of spleen weight is not directly related to the radiation aspect and is more likely a systemic effect due to the extensive weight loss. Cheal et al. demonstrated that injection of 296 kBq of ²²⁵Ac is tolerated by mice; however, the study relied on a pretargeted approach using haptens, resulting in minimal kidney retention (26). With about 10 times the size of a hapten, a 15 kDa sdAb is excreted via the kidneys’ glomerulus with moderate retention, resulting in kidneys being the dose-limiting organ of sdAb-mediated TRT. Hence, the pretargeting approach is worthwhile investigating to circumvent the limitations imposed by the kidneys for sdAb-mediated TRT (27). Other possible avenues are modulation of sdAb protein sequence, cleavable linkers, and novel coupling methods to avoid a high radiation burden on the kidneys (28). In this regard, a limitation of this study is the lack of histologic evaluation of kidneys, which would have yielded more insight into the extent of renal damage inflicted upon α-TRT. Furthermore, immune analyses were performed at a single time point. Though this provided valuable insights, it might not reflect the dynamic nature of the immune system and its contribution to immune-related adverse events. Finally, the antihuCD20 sdAb used in this study does not bind mouse CD20, thus introducing a bias in the healthy organ dosimetry. However, antihuCD20 TRT using mAbs was previously performed in patients and displayed an acceptable safety profile (5).

Future perspectives for ameliorating the therapeutic index of α-TRT consists of combination strategies to reduce the radiation burden on dose-limiting organs such as kidneys as well as titration of ICB. Further tweaking of the therapy regimen should circumvent kidney damage as well as immune-related adverse events.