Nanobody-mediated targeting of immunoregulatory cells for cancer therapy

Cancer is one of the most challenging diseases facing the human race. Although several treatment options exist, it remains the second leading cause of mortality worldwide. This highlights the urgent need to develop new therapeutic modalities that can enhance the prognosis of the disease. The expanding knowledge on tumor development and composition is fueling research with new promising targets. For instance, immune checkpoint blockade (ICB) is one of the recent additions to the treatment of solid cancers and it has revolutionized the therapy of several cancer types. However, the majority of cancer patients remain refractory to ICB. Moreover, a fraction of patients can experience disease hyper-progression in which the initiation of ICB causes a dramatic acceleration of tumor growth and metastasis. The exact molecular and cellular components causing those two phenomena are not yet completely deciphered. Nevertheless, several pieces of evidence point towards the involvement of immune cells, specifically tumor-infiltrating regulatory T cells (tiTregs) and tumorassociated macrophages (TAMs), as both cell types are known for their pro-tumoral activities. Hence, targeting those cells can have a positive impact on cancer therapy. However, Tregs and macrophages are essential for maintaining homeostasis of the human body, highlighting the need to specifically target their tumor-infiltrating counterparts.
Our transcriptomic analysis has shown that interleukin-1 receptor 2 (Il1r2) is one of the most highly upregulated genes in tiTregs. In this work, we verified that IL1R2 protein is absent from peripheral Tregs and from anti-tumoral immune cells infiltrating mouse tumor models. We also showed that IL1R2 marks a highly activated and suppressive population of tiTregs. However, a deficiency of this receptor, either in the whole body or specifically within Treg, did not affect tumor characteristics nor tiTreg infiltration or phenotype, suggesting that this receptor is not functionally important on these cells in a cancer setting. Next, we generated nanobodies (Nbs), the smallest naturally occurring antigen-binding fragments, against IL1R2 and functionalized them with the Fc region of mouse IgG2a, known for its capacity to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). This construct could indeed successfully deplete a proportion of the tiTregs which, in combination with ICB, caused a significant delay in mammary tumor growth. Furthermore, we enhanced the efficacy of the Nb construct by introducing point mutations in the Fc region known to potentiate the ADCC activity. We showed that the combination of ICB with this potentiated construct was superior to the combination with the original construct, causing full regression of 60% of the mammary carcinoma tumors with maintenance of immunological memory against a later challenge with the same cancer cells.
TAMs are another cell type implicated in the negative outcome of ICB. Indeed, it is well documented that the high infiltration of TAMs is linked to a worse prognosis in the majority of human cancers. However, TAMs are highly plastic cells that retain the capacity to shift their phenotype to an antitumoral state in response to certain stimuli, such as toll-like receptor (TLR) 7/8 agonists. Therefore, the delivery of a TLR7/8 agonist specifically to TAMs could re-educate them to fight cancer. Evidence from our lab has identified the macrophage mannose receptor (MMR) as a marker of pro-tumoral TAMs. We have also shown in previous work that our own anti-MMR Nb is capable of penetrating solid tumors and delivering a therapeutic payload. Here, we conjugated anti-MMR Nb to imidazoquinoline, a TLR7/8 agonist, and showed that the conjugation did not interfere with the targeting capacity of the Nb. Then we showed in vivo that the conjugate was able to delay tumor growth of a murine lung carcinoma model while reprogramming TAMs to a more anti-tumoral M1like state and remodeling the immune infiltrate.
Altogether, My PhD work contributed to the characterization of IL1R2 as a specific marker for tiTregs and showed the therapeutic benefit of targeting pro-tumoral cell types, like tiTregs and TAMs, in the tumor microenvironment, possibly paving the way for further clinical evaluation.