María Betina Pampena

Phenotypic and Functional Dysregulated Blood NK Cells in Colorectal Cancer Patients Can Be Activated by Cetuximab Plus IL-2 or IL-15

The clinical outcome of colorectal cancer (CRC) is associated with the immune response; thus, these tumors could be responsive to different immune therapy approaches. Natural killer (NK) cells are key antitumor primary effectors that can eliminate CRC cells without prior immunization. We previously determined that NK cells from the local tumor environment of CRC tumors display a profoundly altered phenotype compared with circulating NK cells from healthy donors (HD). In this study, we evaluated peripheral blood NK cells from untreated patients and their possible role in metastasis progression. We observed profound deregulation in receptor expression even in early stages of disease compared with HD. CRC-NK cells displayed underexpression of CD16, NKG2D, DNAM-1, CD161, NKp46, and NKp30 activating receptors, while inhibitory receptors CD85j and NKG2A were overexpressed. This inhibited phenotype affected cytotoxic functionality against CRC cells and interferon-γ production. We also determined that NKp30 and NKp46 are the key receptors involved in detriment of CRC-NK cells’ antitumor activity. Moreover, NKp46 expression correlated with relapse-free survival of CRC patients with a maximum follow-up of 71 months. CRC-NK cells also exhibited altered antibody-dependent cellular cytotoxicity function responding poorly to cetuximab. IL-2 and IL-15 in combination with cetuximab stimulated NK cell, improving cytotoxicity. These results show potential strategies to enhance CRC-NK cell activity.

Phase II Study of Adjuvant Immunotherapy with the CSF-470 Vaccine Plus Bacillus Calmette–Guerin Plus Recombinant Human Granulocyte Macrophage-Colony Stimulating Factor vs Medium-Dose Interferon Alpha 2B in Stages IIB, IIC, and III Cutaneous Melanoma Patients: A Single Institution, Randomized Study

The irradiated, allogeneic, cellular CSF-470 vaccine plus Bacillus Calmette–Guerin (BCG) and recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) is being tested against medium-dose IFN-α2b in stages IIB–III cutaneous melanoma (CM) patients (pts) after surgery in an open, randomized, Phase II/III study. We present the results of the Phase II part of the ongoing CASVAC-0401 study (ClinicalTrials.gov: NCT01729663). Thirty-one pts were randomized to the CSF-470 vaccine (n = 20) or to the IFN-α2b arm (n = 11). During the 2-year treatment, immunized pts should receive 13 vaccinations. On day 1 of each visit, 1.6 × 107 irradiated CSF-470 cells plus 106 colony-forming units BCG plus 100 µg rhGM-CSF were administered intradermally, followed on days 2–4 by 100 µg rhGM-CSF. IFN-α2b pts should receive 10 million units (MU)/day/5 days a week for 4 weeks; then 5 MU thrice weekly for 23 months. Toxicity and quality of life (QOL) were evaluated at each visit. With a mean and a maximum follow-up of 39.4 and 83 months, respectively, a significant benefit in the distant metastasis-free survival (DMFS) for CSF-470 was observed (p = 0.022). Immune monitoring showed an increase in antitumoral cellular and humoral response in vaccinated pts. CSF-470 was well tolerated; 20/20 pts presented grades 1–2 dermic reactions at the vaccination site; 3/20 pts presented grade 3 allergic reactions. Other adverse events (AEs) were grade 1. Pts in the IFN-α2b arm presented grades 2–3 hematological (7/11), hepatic (2/11), and cardiac (1/11) toxicity; AEs in 9/11 pts forced treatment interruptions. QOL was significantly superior in the vaccine arm (p < 0.0001). Our results suggest that CSF-470 vaccine plus BCG plus GM-CSF can significantly prolong, with lower toxicity, the DMFS of high-risk CM pts with respect to medium-dose IFN-α2b. The continuation of a Phase III part of the CASVAC-0401 study is encouraged.

Abstract A37: Immunization of cutaneous melanoma patients with the allogeneic cell vaccine CSF-470 enhances immune infiltration of metastatic lesions and would favor subsequent response to Vemurafenib

Therapeutic cancer vaccines are aimed at promoting tumor-specific immunity with long-term memory. We have developed the CSF-470 therapeutic vaccine, a mini-allograft of four irradiated allogeneic cutaneous melanoma cell lines, combined with BCG and rhGM-CSF as adjuvants; that is currently being assayed in adjuvancy against medium-dose IFN-alfa2b in the CASVAC-0401 phase II/III clinical trial (ClinicalTrials.gov identifier: NCT01729663). An update to September 2014 reveals that after inclusion of 31 patients, a significant benefit in the distant metastasis-free survival for the CSF-470 arm is obtained (p=0.034). Of the 20 patients assigned to the CSF-470 arm, with a mean follow-up of 26 months, six patients developed distant metastases, of whom two died, and fourteen patients are distant metastases-free (70%). Five patients of the vaccine arm were treated with Vemurafenib after progression; two of them achieved complete remission and two of them partial remission (>50%); only one developed severe dermatitis. Before Vemurafenib treatment, metastases biopsies from only three patients could be obtained; two of them later achieved complete responses. In this work, we present the analysis of the peripheral blood and in situ immune populations during CSF-470 immunization in the two patients that achieved complete responses with Vemurafenib treatment. To attain this task, histopathological characterization of tumor biopsies, along with immune populations determined by immunohistochemistry, was performed by microscopy and image analysis. Peripheral blood mononucleated cells were obtained from patients at different times during CSF-470 immunization and immune populations were assessed by flow cytometry. Patient #006 entered the CASVAC-0401 study after a primary and lymph node tumor resection. By the end of CSF-470 immunization, she developed simultaneously subcutaneous and lung metastases. Following a 3-month cycle of Vemurafenib 960mg bid, patient #006 achieved complete remission of lung tumor nodules. Analysis of immune response evolution during protocol revealed a marked increase in in situ immune infiltration at the cutaneous metastasis relative to the primary tumor, especially in CD8+, CD4+ and CD20+ cells. Several dying tumor cells could be distinguished in the cutaneous metastasis, with attached and surrounding CD8+, CD4+ and CD45Ro+ T lymphocytes and CD68+ cells; and with Granzyme B vesicles and DNA strand breaks. Patient #005 developed sequential in transit metastases; CD8+, CD4+ and CD20+ lymphocyte infiltration increased in situ in biopsies following CSF-470 immunization. Vemurafenib treatment allowed achieving a complete response characterized by brisk infiltration of CD68+ macrophages, CD11c+ dendritic cells, CD8+ and CD4+ lymphocytes. In both patients, analysis of peripheral immune populations revealed an increment of NK cells detected after 6-months treatment, followed by an increase in CD4+ and CD8+ cells by the end of the protocol (2 years). Also, serum reactivity to CSF-470 cutaneous melanoma cells increased during vaccination. We suggest that previous immunization of melanoma patients with CSF-470 vaccine increases tumor immune infiltration and subsequent response to Vemurafenib. These encouraging results may open new options for combined therapies in cutaneous melanoma. Citation Format: Mariana Aris, Maria Betina Pampena, Estrella M. Levy, Alicia I. Bravo, Florencia P. Madorsky-Rowdo, Ana Mordoh, Julio Kaplan, Antonela Baron, Mariela Urrutia, Paula A. Blanco, Maria Marcela Barrio, Jose Mordoh. Immunization of cutaneous melanoma patients with the allogeneic cell vaccine CSF-470 enhances immune infiltration of metastatic lesions and would favor subsequent response to Vemurafenib. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A37.

Abstract CT118: A randomized phase II study of the CSF-470 therapeutic vaccine plus BCG plus rhGM-CSF versus IFN-α2b in cutaneous melanoma patients stages IIB, IIC and III

Adjuvant treatment of high-risk cutaneous melanoma (CM) patients (pts) is still an unsolved issue, since the cost-benefit ratio of high-dose IFN-α2b is under discussion. The CSF-470 therapeutic vaccine, a mini-allograft of four lethally-irradiated allogeneic CM cell lines, with BCG and rhGM-CSF as adjuvants, is currently being tested in post-surgical adjuvancy vs medium-dose IFN-2b in stage IIB-III CM pts (phase II/III trial CASVAC-0401, NCT01729663).We present here the results of the phase II part of the study. A total of 31 pts (stage IIC = 2; stage III: 29) were enrolled: 20 pts were randomized to receive CSF-470 vaccine and 11 pts to IFN-α2b. Pts assigned to the vaccine arm received i.d. 1.6×10 7 CSF-470 irradiated cells plus 10 6 cfu BCG and 100μg rhGM-CSF (first day); 100 μg rhGM-CSF/day/3days were injected consecutively i.d. at the vaccination site. During the two-year treatment, pts in the vaccine arm received a total of 13 vaccinations. Pts assigned to the IFN-α2b arm received 10 MU/day/5 days a week for 4 weeks; then 5 MU 3/week for 23 months. Imaging studies were performed to follow the clinical evolution of the disease. Analysis of blood chemistry and differential white blood cell counts were performed to monitor systemic toxicity. Immune monitoring was performed at baseline and at 6, 12 and 24 months from protocol start. Also, pts were evaluated by Quality of Life Questionnaires (QOL) along the study. After including 20 pts who received a total of 176 vaccinations, we conclude that: CSF-470 vaccine was well tolerated; the main toxicity was a grade 2 reaction at the injection site; 3/20 pts presented grade 3 allergic reactions that were easily handled with anti-histamines and corticosteroids. Pts in the IFN-α2b arm presented grade 2-3 hematologic toxicity; 9 pts developed adverse events that forced treatment discontinuation provisionally or permanently. With a mean follow-up of 28 months and a maximum follow-up of 67 months, a significant benefit in the distant metastasis-free survival (DMFS) for CSF-470 was observed: 14/20 pts (70%) immunized with CSF-470 vaccine and only 4/11 pts (36.4%) in the IFN-α2b arm remain without distant metastases (p = 0.032). No significant differences in OS were yet observed. QOL was significantly superior for CSF-470 vaccine as compared to IFN-α2b treatment (p th and 12 th vaccine as compared to progressing pts. These results demonstrate a clear superiority of CSF-470 vaccine plus BCG plus GM-CSF vs IFN-α2b in the adjuvant setting in pts with high-risk CM. Citation Format: Jose Mordoh, Maria Betina Pampena, Mariana Aris, Paula Blanco, Alicia I. Bravo, Juan Manuel O9Connor, Julio Kaplan, Franco Ramello, Estrella M. Levy, Maria M. Barrio. A randomized phase II study of the CSF-470 therapeutic vaccine plus BCG plus rhGM-CSF versus IFN-α2b in cutaneous melanoma patients stages IIB, IIC and III. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT118. doi:10.1158/1538-7445.AM2015-CT118

High neutrophil to lymphocyte ratio and decreased CD69 + NK cells represent a phenotype of high risk in early-stage breast cancer patients

Purpose Breast cancer (BC) is a highly heterogeneous disease presenting a broad range of clinical and molecular characteristics. In the past years, a growing body of evidence demonstrated that immune response plays a significant role in cancer outcome. However, immune prognostic markers are not completely validated in clinical practice in BC patients. Materials and methods With the aim to characterize immune features, several parameters were analyzed in peripheral blood at diagnosis of 85 nonmetastatic BC patients between April 2011 and July 2014. Results With a median follow-up of 38.6 months, peripheral blood analysis of BC patients (stages I, II, and III) showed that total lymphocyte and T lymphocyte counts were augmented in nonrelapsed patients. Also, a higher neutrophil-to-lymphocytes ratio was associated with prolonged disease-free survival. Natural killer cell receptor analysis revealed that early activation receptor CD69 was associated with a better outcome. Conclusion This preliminary evidence is in accordance with the concept of immune surveillance. We suggest an “immune phenotype” that provides relevant prognostic information in early-stage BC patients and which could be useful in the decision-making process.

Avelumab, an IgG1 anti-PD-L1 Immune Checkpoint Inhibitor, Triggers NK Cell-Mediated Cytotoxicity and Cytokine Production Against Triple Negative Breast Cancer Cells

The standard treatment for Triple Negative Breast Cancer (TNBC) patients is cytotoxic chemotherapy, but it is restricted since the duration of response is usually short. Blocking the PD-1/PD-L1 pathway through monoclonal antibodies (mAbs) appears to be a promising therapeutic strategy for TNBC patients. Avelumab is a human IgG1 anti-PD-L1 mAb being tested in clinical trials that may also trigger antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells as an additional antitumor activity. In the present work, we studied in vitro Avelumab-mediated ADCC against a panel of TNBC cells with different PD-L1 expression using peripheral blood mononuclear cells (PBMC) or purified NK cells from healthy donors. We determined that Avelumab significantly enhanced NK-cell mediated cytotoxicity against TNBC cells and that tumor cells expressing higher levels of PD-L1 were more sensitive to Avelumab-mediated ADCC. IFN-γ treatment upregulated PD-L1 expression in tumor cells but had a variable impact on Avelumab-mediated ADCC, which could be related to the simultaneous effect of IFN-γ on the expression of NK cell ligands. Moreover, IL-2 and IL-15 stimulation of NK cells enhanced Avelumab-triggered cytokine production and degranulation along with increased lytic activity against tumor cells. Improving the treatment of TNBC remains still a considerable challenge. This in vitro study suggests that Avelumab-mediated ADCC, independently of the blockade of the PD-1/PD-L1 pathway, could be a valuable mechanism for tumor cell elimination in TNBC. Avelumab combination with immunomodulators such as IL-15 or IL-2 could be taken into consideration to increase the therapeutic efficacy of Avelumab in TNBC.

Changes in the TCRβ Repertoire and Tumor Immune Signature From a Cutaneous Melanoma Patient Immunized With the CSF-470 Vaccine: A Case Report.

The allogeneic therapeutic vaccine CSF-470 has demonstrated a significant benefit over medium-dose IFNα2b in the distant metastasis-free survival for stages IIB–IIC–III cutaneous melanoma patients in a randomized phase II/III clinical trial (CASVAC-0401, NCT01729663). At the end of the 2-year CSF-470 immunization protocol, patient #006 developed several lung and one subcutaneous melanoma metastases; this later was excised. In this report, we analyzed the changes throughout vaccination of immune populations in blood and in the tumor tissue, with special focus on the T-cell repertoire. Immunohistochemistry revealed a marked increase in CD8+, CD4+, and CD20+ lymphocytes infiltrating the metastasis relative to the primary tumor. Lymphocytes were firmly attached to dying-tumor cells containing Granzyme-B granules. Whole-exon sequencing assessment indicated a moderate-to-high tumor mutational burden, with BRAFV600E as the main oncogenic driver. Mutational signature presented large numbers of mutations at dipyrimidines, typical of melanoma. Relevant tumor and immune-related genes from the subcutaneous metastasis were addressed by RNA-Seq analysis, revealing expression of typical melanoma antigens and proliferative tumor-related genes. Stimulatory and inhibitory immune transcripts were detected as well as evidence of active T-cell effector function. Peripheral blood monitoring revealed an increase in CD4+ and CD8+ cells by the end of the immunization protocol. By CDR3-T-cell receptor β (TCRβ) sequencing, generation of new clones and an increase in oligoclonality was observed in the peripheral T-cells immune repertoire throughout immunization. A shift, with the expansion of selected preexisting and newly arising clones with reduction of others, was detected in blood. In tumor-infiltrating lymphocytes, prevalent clones (50%) were both new and preexisting that were expanded in blood following CSF-470 immunization. These clones persisted in time, since 2 years after completing the immunization, 51% of the clones present in the metastasis were still detected in blood. This is the first report of the modulation of the TCRβ repertoire from a melanoma patient immunized with the CSF-470 vaccine. After immunization, the changes observed in peripheral immune populations as well as in the tumor compartment suggest that the vaccine can induce an antitumor adaptive immune repertoire that can reach tumor lesions and persists in blood for at least 2 years.

Early Events of the Reaction Elicited by CSF-470 Melanoma Vaccine Plus Adjuvants: An In Vitro Analysis of Immune Recruitment and Cytokine Release

In a previous work, we showed that CSF-470 vaccine plus bacillus Calmette–Guerin (BCG) and granulocyte macrophage colony-stimulating factor (GM-CSF) as adjuvants resulted in a significant benefit in the distant metastasis-free survival when comparing vaccinated vs. IFN-α2b-treated high-risk cutaneous melanoma patients in a Phase II study. Immune monitoring demonstrated an increase in anti-tumor innate and adaptive immunities of vaccinated patients, with a striking increase in IFN-γ secreting lymphocytes specific for melanoma antigens (Ags). In an effort to dissect the first steps of the immune response elicited by CSF-470 vaccine plus adjuvants, we evaluated, in an in vitro model, leukocyte migration, cytokine production, and monocyte phagocytosis of vaccine cells. Our results demonstrate that leukocytes recruitment, mostly from the innate immune system, is an early event after CSF-470 vaccine plus BCG plus GM-CSF interaction with immune cells, possibly explained by the high expression of CCL2/MCP-1 and other chemokines by vaccine cells. Early release of TNF-α and IL-1β pro-inflammatory cytokines and efficient tumor Ags phagocytosis by monocytes take place and would probably create a favorable context for Ag processing and presentation. Although the presence of the vaccine cells hampered cytokines production stimulated by BCG in a mechanism partially mediated by TGF-β and IL-10, still significant levels of TNF-α and IL-1β could be detected. Thus, BCG was required to induce local inflammation in the presence of CSF-470 vaccine cells.

Abstract B80: Natural killer and dendritic cells collaborate in the immune response induced by Cetuximab and IL-15 against triple negative breast cancer

Background: While the majority of Triple Negative Breast Cancer (TNBC) overexpress epidermal growth factor receptor (EGFR), EGFR inhibition as a monotherapy with Cetuximab is not effective. The study of its mechanism of action is important to find out potentially synergistic combinations. Previously, we demonstrated that Cetuximab can trigger antibody-dependent NK cell-mediated cytotoxicity against TNBC cells. The aim of this study was to investigate the effect of NK cell activation by Cetuximab-coated TNBC cells on Dendritic Cell (DC) function, and the result of its combination with IL-15. Methods: Human monocyte-derived DCs were co-cultured with autologous isolated NK cells, TNBC cells, Cetuximab and/or IL-15 for 24hs. Costimulatory molecule and activation marker expression in DC and NK cells were analyzed by flow cytometry, and cytokine concentration in culture supernatants was assessed by ELISA. DCs obtained after co-cultures were incubated for 24hs with CD40 ligand transfected fibroblasts to mimic the interaction with CD40L expressing Th cells, and IL-12 was analyzed in culture supernatants by ELISA. Results: Cetuximab treatment of TNBC cells enhanced DC maturation only when NK cells were present in the co-culture (%CD83 + : 6.6±0.7 vs 61.0±17, p Conclusions: Our results suggest that besides its anti-proliferative mechanism of action, Cetuximab has immunological effects in an in vitro model of TNBC, promoting NK cell activation and DC maturation, which could be potentiated by the combination with IL-15. This could ultimately result in the priming of T cell-based immunity, which will be evaluated in further studies. Citation Format: Estefania P. Julia, Maria Betina Pampena, Yamila S. Rocca, Jose Mordoh, Estrella M. Levy. Natural killer and dendritic cells collaborate in the immune response induced by Cetuximab and IL-15 against triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B80.

Abstract 3677: Early effects of CSF-470 melanoma vaccine plus adjuvants in immune cell stimulation

Background: In a Phase II/III clinical trial (CASVAC-0401, NCT01729663), cutaneous melanoma (CM) patients (pts) stages II/III were randomized to receive CSF-470 vaccine or IFN-α (2:1). Vaccine CSF-470 is composed of four lethally-irradiated allogeneic CM cell lines; BCG (Bacillus Calmette-Guerin) and rhGM-CSF were used as adjuvants. At the end of the Phase II part of the study (30 pts), a higher distant-metastases-free-survival was observed in vaccinated pts. All vaccinated pts developed inflammation at the vaccination site. Therefore, in an attempt to understand these first steps of immune reaction, leukocytes migration, cytokines synthesis and monocyte phagocytosis promoted by the vaccine components were studied in vitro. Methods: Cell migration of healthy donors’ leukocytes or mononuclear cells (MN) towards vaccine components was assayed in 0,5 μm transwells. Cytokines were measured by ELISA in supernatants of CSF-470 vaccine and MN (1:1, 0,5x106 each) cocultures. For phagocytosis assays, CM cell lines were labeled with PKH67 lipophilic dye, irradiated, and cocultured with purified monocytes. After 24 hs, cultures were stained with CD14 and double staining (CD14+PKH67+) was analyzed by flow cytometry. Results: In migration assays, CSF-470 vaccine induced higher attraction of leukocytes as compared to culture medium (p=0.003, unpaired-T-test). GM-CSF exerted little attraction, although combined with BCG it increased the migration triggered by CSF-470 (vaccine vs vaccine plus adjuvants, p=0.034, unpaired-T-test). When using MN, CSF-470 alone or plus adjuvants, highly attracted monocytes, which represented 10%-65% of the migrating CD45+ population (before migration 6-15%) (p Conclusions: Our in vitro results suggest that recruitment of leukocytes, mostly from the innate immune system, is an early event after CSF-470 vaccine + BCG + GM-CSF interaction with immune cells. Also, early release of TNFα and IFNγ proinflammatory cytokines and efficient vaccine phagocytosis by monocytes would favor subsequent antigen processing and presentation. Further experiments will be performed to address the initial steps triggered after CSF-470 vaccination. Citation Format: Maria B. Pampena, Estefania P. Julia, Yamila S. Rocca, Paula A. Blanco, Maria M. Barrio, Jose Mordoh, Estrella M. Levy. Early effects of CSF-470 melanoma vaccine plus adjuvants in immune cell stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3677. doi:10.1158/1538-7445.AM2017-3677