Antibody dependent cellular cytotoxicity-inducing anti-EGFR antibodies as effective therapeutic option for cutaneous melanoma resistant to BRAF inhibitors

Introduction: About 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots. Methods and results: By using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors' and patients' peripheral blood cells. Conclusion: Our data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors

A different immunologic profile characterizes patients with HER-2-overexpressing and HER-2-negative locally advanced breast cancer: implications for immune-based therapies

INTRODUCTION: The clinical efficacy of trastuzumab and taxanes is at least partly related to their ability to mediate or promote antitumor immune responses. On these grounds, a careful analysis of basal immune profile may be capital to dissect the heterogeneity of clinical responses to these drugs in patients with locally advanced breast cancer undergoing neoadjuvant chemotherapy. METHODS: Blood samples were collected from 61 locally advanced breast cancers (36 HER2- and 25 HER2+) at diagnosis and from 23 healthy women. Immunophenotypic profiling of circulating and intratumor immune cells, including regulatory T (Treg) cells, was assessed by flow cytometry and immunohistochemistry, respectively. Serum levels of 10 different cytokines were assessed by multiplex immunoassays. CD8+ T cell responses to multiple tumor-associated antigens (TAA) were evaluated by IFN-γ-enzyme-linked immunosorbent spot (ELISPOT). The Student's t test for two tailed distributions and the Wilcoxon two-sample test were used for the statistical analysis of the data. RESULTS: The proportion of circulating immune effectors was similar in HER2+ patients and healthy donors, whereas higher percentages of natural killer and Treg cells and a lower CD4+/CD8+ T cell ratio (with a prevalence of naïve and central memory CD8+ T cells) were observed in HER2- cases. Higher numbers of circulating CD8+ T cells specific for several HLA-A*0201-restricted TAA-derived peptides were observed in HER2+ cases, together with a higher prevalence of intratumor CD8+ T cells. Serum cytokine profile of HER2+ patients was similar to that of controls, whereas HER2- cases showed significantly lower cytokine amounts compared to healthy women (IL-2, IL-8, IL-6) and HER2+ cases (IL-2, IL-1β, IL-8, IL-6, IL-10). CONCLUSIONS: Compared to HER2- cases, patients with HER2-overexpressing locally advanced breast cancer show a more limited tumor-related immune suppression. This may account for the clinical benefit achieved in this subset of patients with the use of drugs acting through, but also promoting, immune-mediated effects

Multi-epitopic anti-tumor T cell responses and efficient Trastuzumab-mediated ADCC promote a favorable clinical outcome in HER2+ breast cancer patients after neoadjuvant chemotherapy (165.8)

Abstract The ability of drugs such as Trastuzumab and taxanes to act through immunomediated and/or immunomodulatory mechanisms suggest that anti-tumor immune responses may influence the clinical outcome of breast cancer patients. We have enumerated circulating CD8+ T cells specific for a broad spectrum of tumor-associated antigens (her2, muc-1, mammaglobin-A, trag-3, survivin, bcl-xL) in 13 patients with locally advanced breast cancer (6 HER2+ and 7 HER2-). At diagnosis, increased numbers of CD8+ T cells specific for all 13 HLA-A*0201 epitopes were observed in the whole series, with a markedly higher extent in HER2+ cases. This subset of patients retained an immunological profile similar to that of healthy women, whereas HER2- cases showed decreased circulating CD3+ T lymphocytes, higher numbers of regulatory T cells and alterations in serum cytokine levels (decreased IL-2, IL-6, and IL-8). Notably, patients with complete pathologic response (pCR), unlike those showing partial responses, retained high numbers of epitope-specific CD8+ T cells throughout the neoadjuvant chemotherapy (NC) treatment, particularly versus survivin and mammaglobin-A epitopes. Moreover, a higher efficacy of Trastuzumab-mediated ADCC in vitro correlated with the induction of pCR in HER2+ cases. These findings emphasize the crucial role of host anti-tumor T cell responses in promoting a favourable clinical outcome in the setting of neoadjuvant chemotherapy for breast cancer, particularly in HER2+ cases.</jats:p

Anthracycline-free neoadjuvant therapy induces pathological complete responses by exploiting immune proficiency in HER2+ breast cancer patients

Background: Neoadjuvant Chemotherapy (NC) including trastuzumab induces a high rate of pathological Complete Responses (pCR) in patients with locally advanced HER2-overexpressing Breast Cancer (BC), but is penalized by a severe cardiotoxicity when combined with anthracyclines. A phase II study was designed to assess whether an anthracycline-free NC regimen based on the early addition of trastuzumab to paclitaxel may increase the pCR rate without inducing severe cardiotoxicity in patients with locally advanced HER2-overexpressing BC. Immunomonitoring was performed to assess the contribution of patients' immunological background to the induction of clinical responses. Methods: Stage II-III HER2-positive BC patients received 24 weeks paclitaxel and trastuzumab NC, followed by 1 year adjuvant trastuzumab ± hormonal and/or radio-therapy. Assessment of pCR rate was the primary endpoint. A group of HER2-negative BC patients treated with neoadjuvant taxanes and anthracyclines was included. Serum levels of 10 cytokines and the efficiency of trastuzumab-mediated antibody-dependent cell cytotoxicity (ADCC) were monitored in vitro every 3 months. Results: From July 2006 to February 2013, we enrolled 109 patients including 46 evaluable HER2-positive cases. A pCR rate of 50% was reached and no severe cardiotoxicity occurred. Serum cytokine profiling revealed only an IL-10 decrease (P = 0.02) in patients achieving a partial response, while HER2-negative patients disclosed marked cytokines changes. Compared to the unfavourable F/F genotype, patients carrying the V allele in the FcγRIIIa-158 polymorphism showed a higher efficacy of trastuzumab-ADCC throughout treatment (P ≤0.05). Conclusions: In the absence of anthracyclines, trastuzumab and paclitaxel induced a high rate of pCR, exploiting the synergy between the immunomodulating properties of these drugs and the retained immunological proficiency of patients with HER2-overexpressing BC

Broadening specificity and enhancing cytotoxicity of adoptive T cells for nasopharyngeal carcinoma immunotherapy

Although promising, clinical responses to adoptive immunotherapy for nasopharyngeal carcinoma (NPC) are still limited by the restricted number of Epstein-Barr virus (EBV) antigens that can be targeted and their poor immunogenicity. Our previous work indicated that the immunogenic features of the NPC-associated viral antigen BARF1 may be exploited for immunotherapeutic purposes. Nevertheless, T-cell lines obtained with current protocols include only negligible numbers of BARF1-specific cytotoxic T lymphocytes, pointing to the need to enrich these effectors in BARF1 specificities. Considering that in B lymphocytes BARF1 is mainly a lytic EBV antigen, we tested different EBV lytic-cycle inducers (TPA/butyric acid, doxorubicin, and cisplatin) used at suboptimal concentrations for their ability to upregulate BARF1 expression in lymphoblastoid B-cell lines (LCL), the commonly used antigen-presenting cells, without compromising their survival. The LCLs treated with doxorubicin (DX-LCL) can reproducibly and efficiently generate EBV-specific effectors enriched in BARF1 specificities from both healthy donors and NPC patients. These DX-LCLs also had more pronounced immunogenic properties, including HLA class I upregulation and expression of immunogenic cell death markers, such as enhanced calreticulin exposure and HMGB1 release. In particular, doxorubicin triggers an HMGB1 autocrine/paracrine loop with its receptor, TLR4, which is also upregulated in DX-LCLs and is responsible for NF-\u3baB activation and a delayed apoptosis that allows a prolonged stimulation of EBV-specific T-cell precursors. This protocol may thus constitute a valid alternative to the use of engineered LCLs to generate EBV-specific T-cell lines for adoptive immunotherapy, being relatively simple, easily upgradable to Good Manufacturing Practice standards, and therefore more broadly applicable

Broadening specificity and enhancing cytotoxicity of adoptive T Cells for nasopharyngeal carcinoma immunotherapy

Although promising, clinical responses to adoptive immunotherapy for nasopharyngeal carcinoma (NPC) are still limited by the restricted number of Epstein-Barr virus (EBV) antigens that can be targeted and their poor immunogenicity. Our previous work indicated that the immunogenic features of the NPC-associated viral antigen BARF1 may be exploited for immunotherapeutic purposes. Nevertheless, T-cell lines obtained with current protocols include only negligible numbers of BARF1-specific cytotoxic T lymphocytes, pointing to the need to enrich these effectors in BARF1 specificities. Considering that in B lymphocytes BARF1 is mainly a lytic EBV antigen, we tested different EBV lytic-cycle inducers (TPA/butyric acid, doxorubicin, and cisplatin) used at suboptimal concentrations for their ability to upregulate BARF1 expression in lymphoblastoid B-cell lines (LCL), the commonly used antigen-presenting cells, without compromising their survival. The LCLs treated with doxo-rubicin (DX-LCL) can reproducibly and efficiently generate EBV-specific effectors enriched in BARF1 specificities from both healthy donors and NPC patients. These DX-LCLs also had more pronounced immunogenic properties, including HLA class I upregulation and expression of immunogenic cell death markers, such as enhanced calreticulin exposure and HMGB1 release. In particular, doxorubicin triggers an HMGB1 autocrine/paracrine loop with its receptor, TLR4, which is also upregulated in DX-LCLs and is responsible for NF-kappa B activation and a delayed apoptosis that allows a prolonged stimulation of EBV-specific T-cell precursors. This protocol may thus constitute a valid alternative to the use of engineered LCLs to generate EBV-specific T-cell lines for adoptive immunotherapy, being relatively simple, easily upgradable to Good Manufacturing Practice standards, and therefore more broadly applicable

Abstract B06: Loss of SPRY1 expression in BRAF-mutant cutaneous melanoma inhibits cell proliferation and improves response to targeted therapy

Abstract Background: Targeted therapy has proven efficacy in BRAF-mutant metastatic cutaneous melanoma (CM), but patients almost invariantly develop an early resistance. Although the underlying mechanisms are various and heterogeneous, resistance usually associates with an altered signaling. Sprouty 1 (SPRY1) has been described as an upstream mediator of MAPK signaling pathway, and although it has been found to be implicated in different biologic processes, its specific role in BRAF-mutant CM has been only partially addressed. Material and Methods: SPRY1 expression was assessed by quantitative real-time RT-PCR (qRT-PCR) and Western blotting (WB) analyses in BRAF-mutant CM cell lines and tissues. SPRY1 gene was knocked out using CRISPR-strategy. WB analyses evaluated the modulation of MAPK signaling pathways in SPRY1 knockout (SPRY1KO) clones. RNA-seq identified mRNAs that were differentially expressed in SPRY1KO clones respect to parental cells, and their expression was then validated by qRT-PCR. To further compare SPRY1KO clones to parental cells, cell proliferation was measured using xCELLigence instrument, whereas cell cycle progression, ROS induction, and apoptosis were assessed by flow cytometry. In vivo effects of SPRY1 silencing were investigated in xenografted mice treated with or without BRAF inhibitor (BRAFi). Results: SPRY1 was found highly expressed in a panel of BRAF-mutant CM cell lines and tissues, both at mRNA and protein level. To investigate the biologic role of SPRY1 in BRAF-mutant CM, SPRY1KO clones were obtained from 3 CM cell lines. RNA-seq identified 38 mRNAs that were significantly commonly modulated in SPRY1KO clones respect to parental cells. Ingenuity Pathway Analysis indicated that the top of the neighbor coding gene function of differentially expressed mRNAs was involved in cell proliferation and cell death. In addition, WB assays showed SPRY1 silencing was associated with i) decreased activation of p38 MAPK and beta-catenin signaling, ii) reduced expression of metalloproteinase 2, and iii) increased levels of p53. Consistent with these data, SPRY1 silencing markedly induced cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and impaired tumor growth in vivo. In addition, SPRY1 knockdown associated with improved response to BRAFi both in vitro and in vivo, which could be partly contributed by the higher levels of ROS observed in SPRY1KO clones. Interestingly, BRAFi treatment reduced mRNA and protein SPRY1 levels in parental BRAF-mutant CM cell lines, indicating that SPRY1 expression is downstream of the MEK-ERK signaling. Of note, some of the effects induced by BRAFi in parental cells were similar to those observed in SPRY1KO clones, suggesting that BRAFi activity may at least in part rely on SPRY1 downregulation. Conclusions: All together, our findings reveal important insights into SPRY1 role in BRAF-mutant CM and suggest its potential involvement in targeted therapy response. Citation Format: Barbara Montico, Giorgio Giurato, Francesca Colizzi, Alessia Covre, Elisa Comaro, Eliana Pivetta, Dania Benedetti, Alessandro Weisz, Agostino Steffan, Michele Maio, Luca Sigalotti, Elisabetta Fratta. Loss of SPRY1 expression in BRAF-mutant cutaneous melanoma inhibits cell proliferation and improves response to targeted therapy [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B06.</jats:p

Local high-dose radiotherapy induces systemic immunomodulating effects of potential therapeutic relevance in oligometastatic breast cancer

Local irradiation of cancer through radiotherapy can induce spontaneous regression of non-directly irradiated lesions, suggesting the involvement of systemic antitumor immune responses. In oligometastatic breast cancer (BC) patients, the use of stereotactic body radiotherapy (SBRT) favors the local control of treated lesions and may contribute to break local tolerance and release tumor-associated antigens (TAAs), improving host antitumor immunity. We performed a detailed immunomonitoring of BC patients undergoing SBRT to verify its ability to "switch on" the anti-tumor immunity both systemically, in peripheral blood, and locally, employing in vitro BC models. Twenty-one BC patients wit