CSPG4 CAR-redirected Cytokine Induced Killer lymphocytes (CIK) as effective cellular immunotherapy for HLA class I defective melanoma.

BACKGROUND: Even acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40-60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed. METHODS: In this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules. RESULTS: We successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice. CONCLUSIONS: In this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors.

Full chimaeric CAR.CIK from patients engrafted after allogeneic haematopoietic cell transplant: Feasibility, anti-leukaemic potential and alloreactivity across major human leukocyte antigen barriers.

Cytokine-induced killer lymphocytes (CIK) are a promising alternative to conventional donor lymphocyte infusion (DLI), following allogeneic haematopoietic cell transplantation (HCT), due to their intrinsic anti-tumour activity and reduced risk of graft-versus-host disease (GVHD). We explored the feasibility, anti-leukaemic activity and alloreactive risk of CIK generated from full-donor chimaeric (fc) patients and genetically redirected by a chimeric antigen receptor (CAR) (fcCAR.CIK) against the leukaemic target CD44v6. fcCAR.CIK were successfully ex-vivo expanded from leukaemic patients in complete remission after HCT confirming their intense preclinical anti-leukaemic activity without enhancing the alloreactivity across human leukocyte antigen (HLA) barriers. Our study provides translational bases to support clinical studies with fcCAR.CIK, a sort of biological bridge between the autologous and allogeneic sources, as alternative DLI following HCT.

Integrated Antitumor Activities of Cellular Immunotherapy with CIK Lymphocytes and Interferons against KIT/PDGFRA Wild Type GIST.

Gastrointestinal stromal tumors (GISTs) are rare, mesenchymal tumors of the gastrointestinal tract, characterized by either KIT or PDGFRA mutation in about 85% of cases. KIT/PDGFRA wild type gastrointestinal stromal tumors (wtGIST) account for the remaining 15% of GIST and represent an unmet medical need: their prevalence and potential medical vulnerabilities are not completely defined, and effective therapeutic strategies are still lacking. In this study we set a patient-derived preclinical model of wtGIST to investigate their phenotypic features, along with their susceptibility to cellular immunotherapy with cytokine-induced killer lymphocytes (CIK) and interferons (IFN). We generated 11 wtGIST primary cell lines (wtGISTc). The main CIK ligands (MIC A/B; ULBPs), along with PD-L1/2, were expressed by wtGISTc and the expression of HLA-I molecules was preserved. Patient-derived CIK were capable of intense killing in vitro against wtGISTc resistant to both imatinib and sunitinib. We found that CIK produce a high level of granzyme B, IFNα and IFNγ. CIK-conditioned supernatant was responsible for part of the observed tumoricidal effect, along with positive bystander modulatory activities enhancing the expression of PD-L1/2 and HLA-I molecules. IFNα, but not In, had direct antitumor effects on 50% (4/8) of TKI-resistant wtGISTc, positively correlated with the tumor expression of IFN receptors. wtGIST cells that survived IFNα were still sensitive to CIK immunotherapy. Our data support the exploration of CIK immunotherapy in clinical studies for TKI-resistant wtGIST, proposing reevaluation for IFNα within this challenging setting.

Recruitment, Infiltration, and Cytotoxicity of HLA-Independent Killer Lymphocytes in Three-Dimensional Melanoma Models.

Cancer adoptive cell therapy (ACT) with HLA-independent tumor killer lymphocytes is a promising approach, with intrinsic features potentially addressing crucial tumor-escape mechanisms of checkpoint inhibitors. Cytokine-induced Killer (CIK) and Natural Killer (NK) lymphocytes share similar tumor-killing mechanisms, with preclinical evidence of intense activity against multiple solid tumors and currently testing in clinical studies. To improve the effective clinical translation of such ACT approaches, several fundamental questions still need to be addressed within appropriate preclinical contexts, capable of overcoming limitations imposed by most traditional two-dimensional assays. Here, we developed a novel experimental approach to explore, dissect, and visualize the interactions of CIK and NK lymphocytes with melanoma tumors in vitro in 3D. Primary melanoma cells were assembled into small tumors that were dispersed in a 3D matrix and challenged with patient-derived CIK or the NK-92 cell line. By means of imaging-based methods, we reported, visualized, and quantitatively measured the recruitment of CIK and NK on the 3D targets, their infiltration, and cytotoxic activity. Our results support the effective tumor recruitment and tumor infiltration by CIK and NK. Such features appeared dependent on the specific geometric aspects of the environment but can be explained in terms of directional migration toward the tumor, without invoking major feedback components. Overall, our 3D platform allows us to monitor the processes of tumor recruitment, infiltration, and killing by means of live measurements, revealing important kinetic aspects of ACT with CIK and NK against melanoma.

CSPG4-Specific CAR.CIK Lymphocytes as a Novel Therapy for the Treatment of Multiple Soft-Tissue Sarcoma Histotypes.

PURPOSE: No effective therapy is available for unresectable soft-tissue sarcomas (STS). This unmet clinical need prompted us to test whether chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CAR.CIK) are effective in eliminating tumor cells derived from multiple STS histotypes in vitro and in immunodeficient mice. EXPERIMENTAL DESIGN: The experimental platform included patient-derived CAR.CIK and cell lines established from multiple STS histotypes. CAR.CIK were transduced with a retroviral vector encoding second-generation CSPG4-specific CAR (CSPG4-CAR) with 4-1BB costimulation. The functional activity of CSPG4-CAR.CIK was explored in vitro, in two- and three-dimensional STS cultures, and in three in vivo STS xenograft models. RESULTS: CSPG4-CAR.CIK were efficiently generated from patients with STS. CSPG4 was highly expressed in multiple STS histotypes by in silico analysis and on all 16 STS cell lines tested by flow cytometry. CSPG4-CAR.CIK displayed superior in vitro cytolytic activity against multiple STS histotypes as compared with paired unmodified control CIK. CSPG4-CAR.CIK also showed strong antitumor activity against STS spheroids; this effect was associated with tumor recruitment, infiltration, and matrix penetration. CSPG4-CAR.CIK significantly delayed or reversed tumor growth in vivo in three STS xenograft models (leiomyosarcoma, undifferentiated pleomorphic sarcoma, and fibrosarcoma). Tumor growth inhibition persisted for up to 2 weeks following the last administration of CSPG4-CAR.CIK. CONCLUSIONS: This study has shown that CSPG4-CAR.CIK effectively targets multiple STS histotypes in vitro and in immunodeficient mice. These results provide a strong rationale to translate the novel strategy we have developed into a clinical setting.

Cytokine Induced Killer cells are effective against sarcoma cancer stem cells spared by chemotherapy and target therapy.

Metastatic bone and soft tissue sarcomas often relapse after chemotherapy (CHT) and molecular targeted therapy (mTT), maintaining a severe prognosis. A subset of sarcoma cancer stem cells (sCSC) is hypothesized to resist conventional drugs and sustain disease relapses. We investigated the immunotherapy activity of cytokine induced killer cells (CIK) against autologous sCSC that survived CHT and mTT. The experimental platform included two aggressive bone and soft tissue sarcoma models: osteosarcoma (OS) and undifferentiated-pleomorphic sarcoma (UPS). To visualize putative sCSC we engineered patient-derived sarcoma cultures (2 OS and 3 UPS) with a lentiviral sCSC-detector wherein the promoter of stem-gene Oct4 controls the expression of eGFP. We visualized a fraction of sCSC (mean 24.2 ± 5.2%) and confirmed their tumorigenicity in vivo. sCSC resulted relatively resistant to both CHT and mTT in vitro. Therapeutic doses of doxorubicin significantly enriched viable eGFP+sCSC in both OS (2.6 fold, n = 16) and UPS (2.3 fold, n = 29) compared to untreated controls. Treatment with sorafenib (for OS) and pazopanib (for UPS) also determined enrichment (1.3 fold) of viable eGFP+sCSC, even if less intense than what observed after CHT. Sarcoma cells surviving CHT and mTT were efficiently killed in vitro by autologous CIK even at minimal effector/target ratios (40:1 = 82%, 1:4 = 29%, n = 13). CIK immunotherapy did not spare sCSC that were killed as efficiently as whole sarcoma cell population. The relative chemo-resistance of sCSC and sensitivity to CIK immunotherapy was confirmed in vivo. Our findings support CIK as an innovative, clinically explorable, approach to eradicate chemo-resistant sCSC implicated in tumor relapse.

BRAF and MEK Inhibitors Increase PD-1-Positive Melanoma Cells Leading to a Potential Lymphocyte-Independent Synergism with Anti-PD-1 Antibody.

Purpose: BRAF and MEK inhibitors (BRAF/MEKi) favor melanoma-infiltrating lymphocytes, providing the rationale for current combinatorial trials with anti-PD-1 antibody. A portion of melanoma cells may express PD-1, and anti-PD-1 antibody could have a direct antitumor effect. Here, we explore whether BRAF/MEKi modulate rates of PD-1+ melanoma cells, supporting an additional-lymphocyte-independent-basis for their therapeutic combination with anti-PD-1 antibody.Experimental Design: With data mining and flow cytometry, we assessed PD-1, PD-L1/2 expression on melanoma cell lines (CCLE, N = 61; validation cell lines, N = 7) and melanoma tumors (TCGA, N = 214). We explored in vitro how BRAF/MEKi affect rates of PD-1+, PD-L1/2+ melanoma cells, and characterized the proliferative and putative stemness features of PD-1+ melanoma cells. We tested the functional lymphocyte-independent effect of anti-PD-1 antibody alone and in combination with BRAF/MEKi in vitro and in an in vivo immunodeficient murine model.Results: PD-1 is consistently expressed on a small subset of melanoma cells, but PD-1+ cells increase to relevant rates during BRAF/MEKi treatment [7.3% (5.6-14.2) vs. 1.5% (0.7-3.2), P = 0.0156; N = 7], together with PD-L2+ melanoma cells [8.5% (0.0-63.0) vs. 1.5% (0.2-43.3), P = 0.0312; N = 7]. PD-1+ cells proliferate less than PD-1- cells (avg. 65% less; t = 7 days) and are preferentially endowed with stemness features. In vivo, the direct anti-melanoma activity of PD-1 blockage as monotherapy was negligible, but its association with BRAF/MEKi significantly delayed the development of drug resistance and tumor relapse.Conclusions: BRAF/MEKi increase the rates of PD-1+ melanoma cells that may sustain tumor relapse, providing a lymphocyte-independent rationale to explore combinatory strategies with anti-PD-1 antibody. Clin Cancer Res; 24(14); 3377-85. ©2018 AACR.

Post-Transplant Cyclophosphamide and Tacrolimus-Mycophenolate Mofetil Combination Prevents Graft-versus-Host Disease in Allogeneic Peripheral Blood Hematopoietic Cell Transplantation from HLA-Matched Donors.

Allogeneic hematopoietic cell transplant (HCT) remains the only curative therapy for many hematologic malignancies but it is limited by high nonrelapse mortality (NRM), primarily from unpredictable control of graft-versus-host disease (GVHD). Recently, post-transplant cyclophosphamide demonstrated improved GVHD control in allogeneic bone marrow HCT. Here we explore cyclophosphamide in allogeneic peripheral blood stem cell transplantation (alloPBSCT). Patients with high-risk hematologic malignancies received alloPBSCT from HLA-matched unrelated/related donors. GVHD prophylaxis included combination post-HCT cyclophosphamide 50 mg/kg (days +3 and +4) and tacrolimus/mofetil mycophenolate (T/MMF) (day +5 forward). The primary objective was the cumulative incidence of acute and chronic GVHD. Between March 2011 and May 2015, 35 consecutive patients received the proposed regimen. MMF was stopped in all patients at day +28; the median discontinuation of tacrolimus was day +113. Acute and chronic GVHD cumulative incidences were 17% and 7%, respectively, with no grade IV GVHD events, only 2 patients requiring chronic GVHD immunosuppression control, and no deaths from GVHD. Two-year NRM, overall survival, event-free survival, and chronic GVHD event-free survival rates were 3%, 77%, 54%, and 49%, respectively. The graft-versus-tumor effect was maintained as 5 of 15 patients (33%) who received HCT with evidence of disease experienced further disease response. A post-transplant cyclophosphamide + T/MMF combination strategy effectively prevented acute and chronic GVHD after alloPBSCT from HLA-matched donors and achieved an unprecedented low NRM without losing efficacy in disease control or impaired development of the graft-versus-tumor effect. This trial is registered at clinicaltrials.gov as NCT02300571.

Cytokine-Induced Killer Cells Kill Chemo-surviving Melanoma Cancer Stem Cells.

Purpose: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and relapses.Experimental Design: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter oct4 Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP+ mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed in vivo in two distinct immunodeficient murine models.Results: We visualized eGFP+ mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP+ MCs (1/42) compared with the eGFP- counterpart (1/4,870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (n = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP+ mCSCs (P = 0.001). Sequential CHT-immunotherapy treatment retained antitumor activity (n = 12, P = 0.001) reducing mCSC rates (P = 0.01).Conclusions: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided. Clin Cancer Res; 23(9); 2277-88. ©2016 AACR.

Cytokine-induced killer cells as immunotherapy for solid tumors: current evidence and perspectives.

Cytokine-induced killer (CIK) cells are ex vivo expanded T lymphocytes endowed with potent MHC-independent antitumor activity. CIK cells are emerging as promising therapeutic approach in the field of cancer adoptive immunotherapy, with biologic features favoring their transferability into clinical applications. Aim of this review is to present the biologic characteristic of CIK cells, discussing the main preclinical findings and initial clinical applications in the field of solid tumors.

Adoptive immunotherapy against sarcomas.

INTRODUCTION: Conventional treatments reached an unsatisfactory therapeutic plateau in the treatment of advanced unresectable bone and soft tissue sarcomas that remain an unsolved medical need. Several evidences support the concept that adoptive immunotherapy may effectively integrate within the complex and multidisciplinary treatment of sarcomas. AREAS COVERED: In this work we reviewed adoptive immunotherapy strategies that have been explored in sarcoma settings, with specific focus on issues related to their clinic transferability. We schematically divided approaches based on T lymphocytes specific for MHC-restricted tumor-associated antigens or relying on MHC-independent immune effectors such as natural killer (NK), cytokine-induced killer (CIK) or γδ T cells. EXPERT OPINION: Preclinical findings and initial clinical reports showed the potentialities and drawbacks of different adoptive immunotherapy strategies. The expansion of tumor infiltrating lymphocytes is difficult to be reproduced outside melanoma. Genetically redirected T cells appear to be a promising option and initial reports are encouraging against patients with sarcomas. Adoptive immunotherapy with MHC-unrestricted effectors such as NK, CIK or γδ T cells has recently shown great preclinical potential in sarcoma setting and biologic features that may favor clinical transferability. Combination of different immunotherapy approaches and integration with conventional treatments appear to be key issues for successful designing of next clinical trials.

Immunotherapy of cancer stem cells in solid tumors: initial findings and future prospective.

INTRODUCTION: Conventional chemotherapies seemed to have reached a therapeutic plateau in the treatment of solid tumors and many metastatic diseases are still incurable. Events of chemo-resistance and relapses appear to be sustained by a subset of putative cancer stem cells (CSCs). New anticancer strategies need to face this new challenge exploring their efficacy against CSCs. Immunotherapy has raised enthusiasms in cancer therapy and its potential against CSCs is an intriguing field of research. AREAS COVERED: In this work we reviewed the immunotherapy approaches directed against CSCs in solid tumors. We schematically divided adaptive immunotherapy strategies, mainly based on dendritic cell-vaccination, and strategies exploiting MHC-unrestricted effectors like natural killer cells, γδ T lymphocytes and cytokine-induced killer cells. Findings, strength and limitations of these models are discussed and compared highlighting their potential clinical relevance. EXPERT OPINION: The important biologic role and clinical relevance of CSCs introduced a 'noble target' for immunotherapy and cancer treatments in general. Initial evidences suggest that CSCs may be susceptible to various types of immunotherapy attacks, overcoming their chemo-resistance. Investigation of important safety issues, based on shared features with 'normal' stem cells, along with intriguing synergisms with modulatory agents are open challenges for the next future and effective clinical translation.

Cytokine-induced killer cells eradicate bone and soft-tissue sarcomas.

Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem-like cells (sCSC) that are likely responsible for relapses and drug resistance. In this study, we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS, including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs, autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4, a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients, we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas, including putative sCSCs, supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease.

Ex vivo-activated MHC-unrestricted immune effectors for cancer adoptive immunotherapy.

Adoptive immunotherapy is considered a promising strategy for the treatment of metastatic tumors and current research efforts are directed to define the optimal approach and facilitate the transferability from preclinical to clinical settings. Among several approaches it is possible to schematically distinguish strategies based on either MHC-restricted or MHC-unrestricted immune effectors. The first are mainly based on the infusion of tumor-specific T lymphocytes capable of recognizing determined MHC-restricted tumor associated antigens (TAA) through their T cell receptor. MHC-unrestricted approaches do not target specific tumor associated antigens and are mainly mediated by effectors of the innate immune system, like natural killer (NK) cells or NKT cells, first barrier against pathogens and tumorigenesis processes, or by ex vivo activated lymphocytes like cytokine-induced killer (CIK) cells. MHC-unrestricted effectors are usually more abundant than TAA-specific precursors and easier to expand. Furthermore their activity is not restricted to precise HLA-haplotypes, not limited to a single tumor histotype and could overcome downregulation of MHC molecules operated by tumor cells as immune escape mechanism. In this review we will discuss the main cancer immunotherapy strategies based on MHC-unrestricted immune effectors. The topic will be approached from the angle of ex vivo expansion protocols in clinical prospective, as well as potential approaches to favorably modulate their functions.

Effective activity of cytokine-induced killer cells against autologous metastatic melanoma including cells with stemness features.

PURPOSE: We investigate the unknown tumor-killing activity of cytokine-induced killer (CIK) cells against autologous metastatic melanoma and the elusive subset of putative cancer stem cells (mCSC). EXPERIMENTAL DESIGN: We developed a preclinical autologous model using same patient-generated CIK cells and tumor targets to consider the unique biology of each patient/tumor pairing. In primary tumor cell cultures, we visualized and immunophenotypically defined a putative mCSC subset using a novel gene transfer strategy that exploited their exclusive ability to activate the promoter of stemness gene Oct4. RESULTS: The CIK cells from 10 patients with metastatic melanoma were successfully expanded (median, 23-fold; range, 11-117). Primary tumor cell cultures established and characterized from the same patients were used as autologous targets. Patient-derived CIK cells efficiently killed autologous metastatic melanoma [up to 71% specific killing (n = 26)]. CIK cells were active in vivo against autologous melanoma, resulting in delayed tumor growth, increased necrotic areas, and lymphocyte infiltration at tumor sites. The metastatic melanoma cultures presented an average of 11.5% ± 2.5% putative mCSCs, which was assessed by Oct4 promoter activity and stemness marker expression (Oct4, ABCG2, ALDH, MITF). Expression was confirmed on mCSC target molecules recognized by CIK cells (MIC A/B; ULBPs). CIK tumor killing activity against mCSCs was intense (up to 71%, n = 4) and comparable with results reported against differentiated metastatic melanoma cells (P = 0.8). CONCLUSIONS: For the first time, the intense killing activity of CIK cells against autologous metastatic melanoma, including mCSCs, has been shown. These findings move clinical investigation of a new immunotherapy for metastatic melanoma, including mCSCs, closer.