IL-4 receptor targeting as an effective immunotherapy against triple-negative breast cancer

Abstract

In many solid tumors including triple-negative breast cancer (TNBC), IL-4 receptor (IL-4R) upregulation has been shown to promote cancer cell proliferation, apoptotic resistance, metastatic potential and a Th2 response in the tumor microenvironment (TME). Immunosuppressive cells in the TME including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) also express the IL4-R. We hypothesized that selective depletion of IL4-R bearing cells in TNBC may have dual cytotoxic and immunotherapeutic benefit. To selectively target IL-4R+ cells, we genetically constructed, expressed and purified DABIL-4, a fusion protein toxin consisting of the catalytic and translocation domains of diphtheria toxin fused to murine IL-4. We found that DABIL-4 has potent and specific cytotoxic activity against TNBC cells in vitro. In murine TNBC models, DABIL-4 significantly reduced tumor growth, splenomegaly and lung metastases, and this was associated with reductions in MDSC, TAM and regulatory T-cells (Tregs) populations with a concomitant increase in the proportion of IFNγ+ CD8 T-cells. The anti-tumor activity of DABIL-4 was absent in IL-4R KO mice directly implicating IL-4R directed killing as the mechanism of anti-tumor activity. Moreover, NanoString analysis of DABIL-4 treated TNBC tumors revealed marked decline in mRNA transcripts that promote tumorigenesis and metastasis. Our findings demonstrate that DABIL-4 is a potent targeted antitumor agent which depletes both IL-4R bearing tumor cells as well as immunosuppressive cell populations in the TME.STATEMENT OF SIGNIFICANCEIn solid tumors like breast cancer, Interleukin-4 receptor (IL-4R) expression in the tumor microenvironment aids tumor growth and metastasis. IL-4R expression upon host immune cells further dampens antitumor immunity. In this study, we have genetically constructed a fusion protein toxin, DABIL-4, composed of the catalytic and translocation domains of diphtheria toxin and murine IL-4. DABIL-4 showed specific cytotoxicity against triple-negative breast cancer (TNBC) cells in vitro. DABIL-4 also markedly inhibited TNBC tumor growth and metastasis in vivo. The primary activity of DABIL-4 was found to be depletion of IL-4R+ immune cells in combination with direct elimination of tumor cells. In conclusion, DABIL-4 targeting of both tumor and immunosuppressive host cells is a versatile and effective treatment strategy for TNBC.

Effective host-directed therapy for tuberculosis by targeted depletion of myeloid-derived suppressor cells using a diphtheria toxin-based fusion protein

“…We first scored the effect upon myeloid cells and noted a significant increase in the total myeloid cell population (CD11b + of all CD45 + cells) in both lungs and spleens (Fig S1A and S1B). Interestingly, despite an overall increase in myeloid cell abundance in the lungs, the population of IL-4R + myeloid cells in lungs was reduced by 27% (p < 0.05) (Fig 3A), the observation is consistent with our breast cancer study [21]. We then characterized the impact of DABIL-4 administration specifically on MDSCs, a cell population known to drive immunosuppression in the granuloma.…”

“…DABIL-4 is a diphtheria toxin-IL4 fusion protein related to the FDA-approved drug denileukin diftitox [31]. DABIL-4 kills IL-4Rbearing cells with an IC50 of 2 nM [21]. Related diphtheria toxin fusion proteins with similarly low IC50 values have been evaluated in humans and animals and are known to have short half-lives of ~90 min [32].…”

“…In light of that work suggesting that DABIL-4 was able to effectively modulate the immune responses, we constructed a gene fusion between the catalytic and translocation domains of diphtheria toxin and murine IL-4. In order to achieve expression and secretion of the fusion protein into the culture medium the structural gene also included the native diphtheria tox promoter, a mutant form of the tox operator, and the native tox signal sequence [19,21]. Following expression and purification of DABIL-4, we confirmed and extended the earlier observations of Lakkis et al by showing that the in vivo administration of the fusion protein toxin was well tolerated and resulted in the depletion of IL-4R+ positive myeloid cells in the spleen [21].…”

“…In order to achieve expression and secretion of the fusion protein into the culture medium the structural gene also included the native diphtheria tox promoter, a mutant form of the tox operator, and the native tox signal sequence [19,21]. Following expression and purification of DABIL-4, we confirmed and extended the earlier observations of Lakkis et al by showing that the in vivo administration of the fusion protein toxin was well tolerated and resulted in the depletion of IL-4R+ positive myeloid cells in the spleen [21]. The results reported here are consistent with earlier results using non-specific MDSC depletion strategies in the mouse TB model.…”

“…We demonstrated that DABIL-4 targets and depletes IL-4R + murine breast cancer cells, as well as tumor-associated MDSCs in a murine 4T1 adenocarcinoma model resulting in reductions of tumor growth and metastases [21]. Given its potency and immunotherapeutic role in cancer, we sought to determine whether DABIL-4-mediated MDSC depletion may play a beneficial role during tuberculosis.…”