Abstract 900: Intermittent cyclophosphamide and vinorelbine reshape the immune cell environment, induce T cell clonal replacement and increase the efficacy of PD-1 inhibition in models of triple negative breast cancer
Authors: Falvo Paolo , Orecchioni Stefania , Hillje Roman , Raveane Alessandro , Mancuso Patrizia , Camisaschi Chiara , Luzi Lucilla , Bertolini Francesco 
Published: September 2020

Abstract
The clinical success of cyclophosphamide (Cy)-based haploidentical stem cell transplants in hematological malignancies indicates that this drug has the potential to reorchestrate the immune system against cancer cells. Along a similar way, Cy is administered before the infusion of CAR-T cells to improve their clinical efficacy. We have previously found that daily, low/medium-dose Cy (20mg/Kg), in association with vinorelbine (V) was able to improve the preclinical efficacy of checkpoint inhibitors (CIs) anti-PD-1 and anti-PD-L1 in preclinical models of breast cancer and lymphoma (Orecchioni et al, 2018). Randomized clinical trials have recently indicated that the addition of CIs to other chemotherapy drugs such as taxanes (T), doxorubicin (D), or platinum (P) might be beneficial in triple negative breast cancer (TNBC) patients. Thus, we designed the present study in two orthotopic, immunocompetent, local and metastatic models of TNBC (CI-resistant 4T1 and EMT6, moderately responsive to CIs) to investigate whether Cy and V, in different schedules and doses, were more effective than T, D, or P. In vivo studies in local and metastatic models using both 4T1 and EMT6 TNBC cells clearly indicated that intermittent (ie every 6 days), medium-dosage (140 mg/Kg) Cy, was more effective than any other combinatorial regimens including CIs plus daily low/medium-dose Cy, or T, D, or P at their regular dosages. The association of V further increased the preclinical efficacy of intermittent Cy plus CIs, and abrogated tumor growth in both 4T1 and EMT6 models. In vivo studies with neutralizing monoclonal antibodies targeting in separate experiments T, B, NK, and myeloid cells demonstrated that CD3+CD4+, CD3+CD8+ T cells as well as CD11b+ monocyte/macrophage dendritic cells were crucial to abrogate tumor growth in TNBC-bearing mice treated with intermittent Cy, V and CIs. Single-cell transcriptome analysis of more than 60,000 intratumoral immune cells and flow cytometry studies in circulating and intratumoral subsets of immune cells indicated that V promoted the generation and maturation of myeloid APC cells, and that intermittent Cy generated new clones of tumor-infiltrating CD3+CD4+ and CD3+CD8+ TCR alpha beta cells. After treatment with intermittent Cy, V and CIs, intratumoral immune cells showed a unique gene signature of 42 genes (CD3D, Ptprc, CD69, Lat, Lck, CD2, CD28, CD27, and CD3E among others) suggestive of a change in morphology and behavior resulting from exposure to a mitogen, cytokine, chemokine, cellular ligand, or an antigen for which it is specific, as well as APC-to-T cell adhesion. Taken together, our data support the hypothesis that APC priming and T cell clonal replacement can significantly improve CI efficacy in vivo in TNBC models. We are further investigating the Cy-related mechanisms inducing intratumoral T-cell clonal replacement and how these T cells cooperate with V-induced APCs.
Citation Format: Paolo Falvo, Stefania Orecchioni, Roman Hillje, Alessandro Raveane, Patrizia Mancuso, Chiara Camisaschi, Lucilla Luzi, Francesco Bertolini. Intermittent cyclophosphamide and vinorelbine reshape the immune cell environment, induce T cell clonal replacement and increase the efficacy of PD-1 inhibition in models of triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 900.