Blockade of the Immune Checkpoint CD47 By TTI-621 Potentiates the Response to Anti-PD-L1 in Cutaneous T Cell Lymphoma

Zhen Han^1,2, Xiwei Wu^3,4, Yate-Ching Yuan^5,6, Hanjun Qin^3,4, Chingyu Su^1,2, Jasmine Zain⁷, Oleg E Akilov⁸, Steven T. Rosen^2,7, Mingye Feng^2,9, Christiane Querfeld^1,2,7,10

¹Division of Dermatology, City of Hope, Duarte, CA; ²Beckman Research Institute, City of Hope, Duarte, CA; ³Department of Molecular and Cellular Biology, City of Hope, Duarte, CA; ⁴Integrative Genomics Core, City of Hope, Duarte, CA; ⁵Department of Computational Quantitative Medicine, City of Hope, Duarte, CA; ⁶Translational Bioinformatics, Center for informatics, City of Hope, Duarte, CA; ⁷Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, CA; ⁸Cutaneous Lymphoma Program, Department of Dermatology, University of Pittsburgh Medical Center, Pittsburgh, PA; ⁹Department of Immuno-Oncology, City of Hope, Duarte, CA; ¹⁰Department of Pathology, City of Hope, Duarte, CA.

Background: Blockade of PD1/PD-L1 (NCT03011814) and CD47/SIRPα (NCT02890368) have shown promising and durable therapeutic outcomes in cutaneous T-cell lymphoma (CTCL), but resistance and/or relapses are seen in a subset of patients. To improve outcomes of immunotherapy regimens for CTCL patients, we investigated the effects of dual CD47 and PD-L1 blockade as a strategy for targeting both the innate and adaptive immune system.

Methods: We performed RNA sequencing, gene expression deconvolution (CIBERSORT) and flow cytometry analysis to assess the immune cell composition, and CD47, SIPRα and PD-L1 expression in 45 CTCL patient samples and CTCL cell lines. CTCL specimens at baseline and during treatment with anti-CD47/SIRPα (TTI621) and anti-PD-L1 (durvalumab) were used to analyze immune cell gene expression. We performed functional assays utilizing our macrophage and CTCL cell line culture systems to assess macrophage polarization, phagocytic activity of macrophages against CTCL cells, T cell-mediated cytotoxicity using a chromium release assay, and CTCL cell line proliferation before and after CD47/SIRPα (TTI-621) and anti-PD-L1 antibody (durvalumab) treatment.

Results: Our data showed that CTCL cells and CTCL cell lines overexpress CD47 and PD-L1 compared with healthy control cells, and overexpression of CD47 and PD-L1 was associated with high MYC expression in CTCL cell lines. CIBERSORT analysis of CTCL samples compared to healthy control indicated that the fraction of M2 tumor-associated macrophages (TAMs) was increased, while M0/1 were significantly lower. Our NanoString data showed decreased M2 genes, immature DC-related genes, and the NK cell inhibitory receptor related genes, but up-regulation of M1 genes and the NK cell stimulatory receptors associated genes for CTCL patients after treatment with TTI-621 compared to baseline. In vitro, treatment with TTI-621 increased the phagocytic activity of macrophages against CTCL cells and enhanced CD8⁺ T-cell-mediated killing. Moreover, TTI-621 synergized with anti-PD-L1 (durvalumab) to reprogram M2-like tumor-associated macrophages to M1-like phenotypes by inhibiting MYC expression. Simultaneous exposure to TTI-621 and anti-PD-L1 inhibited growth of CTCL cell lines more potently than either antibody alone. RNA sequencing analysis indicated these effects were mediated by cell‒death-related pathways, such as apoptosis, autophagy, and necroptosis.

Conclusions: Collectively, our findings demonstrate that CD47 and PD-L1 are critical regulators of the immune microenvironment in CTCL and that dual targeting of CD47 and PD-L1 may potentiate anti-tumor responses in CTCL.