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Cytokine-Induced Killer Cells Engineered with Exogenous T Cell Receptors Directed Against Melanoma Antigens: Enhanced Efficacy of Effector Cells Endowed with a Double Mechanism of Tumor Recognition
Abstract and Figures
Cytokine-Induced Killer (CIK) cells consist of a heterogeneous population of polyclonal T lymphocytes displaying NK phenotype and HLA-unrestricted cytotoxic activity against a broad range of tumors. We sought to determine whether transduction of CIK cells with T-cell receptor (TCR) genes specific for tumor associated antigens could generate effector cells endowed with a double mechanism of tumor recognition. HLA-A2 restricted TCR transduced (TD) CIK directed against the melanoma antigens Mart1 and NY-ESO1 were generated by lentiviral transduction and successfully expanded over a 3-4 weeks period. TD-CIK cells were both CD3+/CD56- and CD3+/CD56+ (31±8% and 59±9%, respectively), indicating that both MHC-restricted T-cells and MHC-unrestricted CIK could be targeted by lentiviral transduction. At the end of the culture, the majority of both unmodified and TD-CIK displayed an effector memory phenotype, without considerable expression of replicative senescence and exhaustion markers. Functionally, TD-CIK specifically recognized tumor cells expressing the relevant antigen as well as maintained their MHC-unrestricted tumor activity. The cytotoxic activity of TD-CIK against HLA-A2+ melanoma cell lines was significantly higher than the untransduced counterparts at a low effector:target ratio (cytotoxic activity of TD-CIK was from 1.9 to 4.3 fold higher than untransduced counterparts). TD-CIK were highly proficient in releasing high amount of IFN-γ upon antigen specific stimulation and were able to recognize primary melanoma targets. In conclusion, we showed that: i) the reproducibility and simplicity of CIK transduction and expansion might solve the problem of obtaining adequate numbers of potent antitumor effector cells for adoptive immunotherapy; ii) the presence of both terminal effectors as well as of less differentiated progenitors might confer them long survival in vivo; iii) the addition of an MHC-restricted antigen recognition allows not only targeting tumor surface antigens but also a wider range of cytoplasmic or nuclear antigens, involved in tumor proliferation and survival. TD-CIK cells with a double mechanism of tumor recognition are an attractive and alternative tool for the development of efficient cell therapeutic strategies.
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... The PBMCs used in this study were HLA-A2 + and analyzed by staining with HLA-A2 mAb (FITC, Biolegend). The CATs were generated as described previously [28]. Briefly, 5 Â 10 6 cells/mL PBMCs were cultured in AIM-V medium (supplemented with 10% heat-inactivated FBS) and 1000 U/mL IFN-g (PeproTech Inc., Beijing, China) for 24 h. ...
... Tumor target cells were pre-incubated with 20 mg/mL (final concentration) high-affinity soluble 1G4 TCR (K D = 26 pmol/L) for 30 min at room temperature. For HLA class I blocking, the experiments were performed as described previously [28]. The plate was washed with culture medium (AIM-V + 10% FBS), and the culture medium was added as the blocking solution at room temperature for 2 h. ...
... In this study, we investigated the antitumor activity of CATs modified by TCR with various affinities for targeting HLA-A*02:01-restricted cancer/testis antigen NY-ESO-1 157-165 . CATs consist of a heterogeneous population of polyclonal CD3 + /CD56 -T lymphocytes and CD3 + / CD56 + NKT phenotype with high proliferation but with low cytotoxicity, which is able to mediate HLA-unrestricted cytotoxic activity against a broad range of tumors [14,28,31]. The easiness and safety of expanding CATs over a short period [32] are important factors to consider in clinical trials of cancer immunotherapy, as clearly demonstrated by our data showing that CATs expanded approximately 800-fold within 3 weeks of culture. ...
Cytokine-activated T cells (CATs) can be easily expanded and are widely applied to cancer immunotherapy. However, the good efficacy of CATs is rarely reported in clinical applications because CATs have no or very low antigen specificity. The low-efficacy problem can be resolved using T cell antigen receptor-engineered CAT (TCR-CAT). Herein, we demonstrate that NY-ESO-1157–165 HLA-A*02:01-specific high-affinity TCR (HAT)-transduced CATs can specifically kill cancer cells with good efficacy. With low micromolar range dissociation equilibrium constants, HAT-transduced CATs showed good specificity with no off-target killing. Furthermore, the high-affinity TCR-CATs delivered significantly better activation and cytotoxicity than the equivalent TCR-engineered T cells (TCR-Ts) in terms of interferon-γ and granzyme B production and in vitro cancer cell killing ability. TCR-CAT may be a very good alternative to the expensive TCR-T, which is considered an effective personalized cyto-immunotherapy.
... Among the various adoptive immunotherapy approaches, we focused on an MHC-independent strategy based on cytokineinduced killer (CIK) cells (28)(29)(30)(31)(32), which are ex vivo-expanded T-NK lymphocytes with MHC-independent antitumor activity (33)(34)(35)(36)(37)(38). The principal mechanism of tumor killing is recognition of stress-inducible tumor-restricted molecules (e.g., MIC A/B; ULBPs 1-6) by the NKG2D receptor (35,36,39). ...
... CIK cells were generate from 8 of our 11 patients (described above), as PBMCs were unavailable for three patients in the study cohort. Within 3 to 4 weeks, cells from fresh or cryopreserved PBMCs were successfully expanded ex vivo, per a standard protocol with timed additions of IFNg, Ab-anti-CD3, and IL2 (29)(30)(31)(32). The median expansion of CIK cells was 29-fold (range 16-to 125-fold). ...
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 lenti-viral 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 þ 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.
... BETA F2A 2H5ALFA were previously described. 20 Cells 54ζ17 hybridoma T cells were grown in DMEM supplemented with 10% fetal calf serum (FCS), 25 U/mL penicillin G, 25 μg/mL streptomycin, and 0.05 μM β-Mercaptoethanol. EBV-LCL cells, OVA transfected B16 cells, J76 T, J76 NFAT-GFP, 21 T2 cells, and mCherry T2 cells were grown in RPMI supplemented with 10% FCS, 25 U/mL penicillin G, and 25 μg/mL streptomycin. ...
... For this reason, we chose 6G9 and 2H5 TCRs, both specific for the Mart-1 (26)(27)(28)(29)(30)(31)(32)(33)(34)(35) peptide, which earlier data reported to have very low and intermediate pairing properties, respectively. 20 By using the pLenti expression system, we introduced the same mutations of the 1G4 TCR in 6G9 and 2H5 TCRs, and transduced the J76 NFAT-GFP cells in order to assess TCR surface expression and function (Supplemental Fig. 2, Supplemental Digital Content 2, http://links.lww.com/JIT/ A520 and Fig. 5). ...
Adoptive transfer of T lymphocytes (ACT) engineered with T-cell receptors (TCRs) of known antitumor specificity is an effective therapeutic strategy. However, a major constraint of ACT is the unpredictable interference of the endogenous TCR a and β chains in pairing of the transduced TCR. This effect reduces the efficacy of the genetically modified primary T cells and carries the risk of generating novel TCR reactivities with unintended functional consequences. Here, we show a powerful approach to overcome these limitations. We engineered TCR α and β chains with mutations encompassing a conserved motif (FXXXFXXS) required to stabilize the pairing of immunoglobulin heavy chain transmembrane domains. Molecular modeling supported the preferential pairing of mutated TCR and impaired pairing between mutated and wild-type TCRs. Expression of the mutated TCR was similar to wild type and conferred the expected specificity. Fluorescence resonance energy transfer analysis in mouse splenocytes transduced with mutated or wild-type TCRs showed a higher proximity of the former over the latter. Importantly, we show that mutated TCRs effectively outcompete endogenous TCRs and improve in vitro antitumor cytotoxicity when expressed in ex vivo isolated human T cells. This approach should contribute to improving current protocols of anticancer immunetherapy protocols.
... Adoptive cell therapy utilizes various immune cells including tumour infiltrating lymphocytes (TILs), lymphokine-activated killer cells and cytokine-induced killer (CIK) cells to induce effective immunity to clear cancer cells (2,37). CIK cells are derived from peripheral blood lymphocytes in the presence of CD3 monoclonal antibodies, IL-2 and IFN-g (38). The CIK cell population consists of CD3 + CD56 -T cell and CD3 + CD56 + T cell, with high anti-tumour activity and proliferation activity (39). ...
As one of the most common forms of solid tumours, gastric carcinoma has been revealed as the third leading cause of death worldwide. The symptom of gastric cancer is usually not obvious and thus difficult to detect at earlier stages. Therefore, gastric cancer is already in the advanced stage once detected in patients, which has a poor prognosis due to ineffective therapies and multiple resistance. Recent advance in understanding the microenvironment of cancer has significantly promoted the development of immunotherapy for advanced gastric cancer. Immunotherapy can induce immune responses in gastric cancer patients thus leads to the destruction of cancer cells. In comparison of traditional therapy, immunotherapy has demonstrated robust efficacy and tolerable toxicity. Therefore, this novel strategy for treatment of advanced gastric cancer has gain increasingly popularity. In this review, we summarize recent progress of immunotherapy in advanced gastric cancer, such as immune check point inhibitors, adoptive cell therapy, VEGF inhibitors, cancer vaccines and CAR-T cell therapy. We highlight immunotherapies involved in clinical applications and discuss the existing challenges of current immunotherapies and promising strategies to overcome these limitations.
... The expression of CD26 in these cell lines was analyzed before performing the experiments. CIK cells were generated from healthy donors as described previously [37]. All cells were cultured in RPMI1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% of penicillin/streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). ...
Simple Summary
Chronic myeloid leukemia stem cells (CML LSCs) are a rare and quiescent population that are resistant to tyrosine kinase inhibitors. CML LSCs have many features in common with hematopoietic stem cells (HSCs) and selectively targeting this population and sparing HSCs is of paramount importance. Targeted therapy by liposome via reducing side effects, controlled release, and versatile surface modifications is an effective way for the treatment of different cancers including leukemia. Here for the first time, we designed a liposome conjugated with Begelomab (anti-CD26) loaded with venetoclax to selectively target CD26+ CML LSCs/progenitor cells and to increase treatment outcome in CML patients. We proved that after antigen binding and drug release, the CD26+ LSCs/progenitor cells could be eliminated without any side effect on CD26− cells.
Abstract
CML is a hematopoietic stem-cell disorder emanating from breakpoint cluster region/Abelson murine leukemia 1 (BCR/ABL) translocation. Introduction of different TKIs revolutionized treatment outcome in CML patients, but CML LSCs seem insensitive to TKIs and are detectable in newly diagnosed and resistant CML patients and in patients who discontinued therapy. It has been reported that CML LSCs aberrantly express some CD markers such as CD26 that can be used for the diagnosis and for targeting. In this study, we confirmed the presence of CD26+ CML LSCs in newly diagnosed and resistant CML patients. To selectively target CML LSCs/progenitor cells that express CD26 and to spare normal HSCs/progenitor cells, we designed a venetoclax-loaded immunoliposome (IL-VX). Our results showed that by using this system we could selectively target CD26+ cells while sparing CD26− cells. The efficiency of venetoclax in targeting CML LSCs has been reported and our system demonstrated a higher potency in cell death induction in comparison to free venetoclax. Meanwhile, treatment of patient samples with IL-VX significantly reduced CD26+ cells in both stem cells and progenitor cells population. In conclusion, this approach showed that selective elimination of CD26+ CML LSCs/progenitor cells can be obtained in vitro, which might allow in vivo reduction of side effects and attainment of treatment-free, long-lasting remission in CML patients.
... Adoptive immunotherapy with CIK might greatly benefit from the new redirection opportunities offered by the developing strategies with engineered tumor-specific receptors. 6,7,21,22 In particular, CAR-based approaches showed impressive therapeutic potential in selected hematologic malignancies even if with relevant safety warnings. 23 CARs are constructed by fusing the single chain variable fragment (scFv) of a tumor surface antigen-specific monoclonal antibody with an intracellular TCR-derived signaling domain and costimulatory molecules. ...
Purpose of our study was to explore a new immunotherapy for high grade soft tissue sarcomas (STS) based on cytokine-induced killer cells (CIK) redirected with a chimeric antigen receptor (CAR) against the tumor-promoting antigen CD44v6. We aimed at generating bipotential killers, combining the CAR specificity with the intrinsic tumor-killing ability of CIK cells (CAR⁺.CIK). We set a patient-derived experimental platform. CAR⁺.CIK were generated by transduction of CIK precursors with a lentiviral vector encoding for anti-CD44v6-CAR. CAR⁺.CIK were characterized and assessed in vitro against multiple histotypes of patient-derived STS. The anti-sarcoma activity of CAR⁺.CIK was confirmed in a STS xenograft model. CD44v6 was expressed by 40% (11/27) of patient-derived STS. CAR⁺.CIK were efficiently expanded from patients (n = 12) and killed multiple histotypes of STS (including autologous targets, n = 4). The killing activity was significantly higher compared with unmodified CIK, especially at low effector/target (E/T) ratios: 98% vs 82% (E/T = 10:1) and 68% vs 26% (1:4), (p<0.0001). Specificity of tumor killing was confirmed by blocking with anti-CD44v6 antibody. CAR⁺.CIK produced higher amounts of IL6 and IFN-γ compared to control CIK. CAR⁺.CIK were highly active in mice bearing subcutaneous STS xenografts, with significant delay of tumor growth (p<0.0001) without toxicities.
We report first evidence of CAR⁺.CIK's activity against high grade STS and propose CD44v6 as an innovative target in this setting. CIK are a valuable platform for the translation of CAR-based strategies to challenging field of solid tumors. Our findings support the exploration of CAR⁺.CIK in clinical trials against high grade STS.
Recent development of methods to discover and engineer therapeutic T‐cell receptors (TCRs) or antibody mimics of TCRs, and to understand their immunology and pharmacology, lag two decades behind therapeutic antibodies. Yet we have every expectation that TCR‐based agents will be similarly important contributors to the treatment of a variety of medical conditions, especially cancers. TCR engineered cells, soluble TCRs and their derivatives, TCR‐mimic antibodies, and TCR‐based CAR T cells promise the possibility of highly specific drugs that can expand the scope of immunologic agents to recognize intracellular targets, including mutated proteins and undruggable transcription factors, not accessible by traditional antibodies. Hurdles exist regarding discovery, specificity, pharmacokinetics, and best modality of use that will need to be overcome before the full potential of TCR‐based agents is achieved. HLA restriction may limit each agent to patient subpopulations and off‐target reactivities remain important barriers to widespread development and use of these new agents. In this review we discuss the unique opportunities for these new classes of drugs, describe their unique antigenic targets, compare them to traditional antibody therapeutics and CAR T cells, and review the various obstacles that must be overcome before full application of these drugs can be realized.
Immunotherapy in the metastatic situation has changed the therapeutic landscape for a variety of cancers, including colorectal cancer. Immunotherapy has firmly established itself as a new pillar of cancer treatment in a variety of cancer types, from the metastatic stage to adjuvant and neoadjuvant settings. Immune checkpoint inhibitors have risen to prominence as a treatment option based on a better knowledge of the development of the tumour microenvironment immune cell-cancer cell regulation over time. Immunotherapy has lately appeared as the most potential field of cancer research by increasing effectiveness and reducing side effects, with FDA-approved therapies for more than 10 various tumours and thousands of new clinical studies.
Gastric cancer is considered as the third leading cause of deaths and the fifth most common cancers worldwide. Common treatment approaches include chemotherapy, radiation, gastric resection and targeted therapies. The emergence of gastric cancer immunotherapy has already shown some promising results and have altered the therapeutic procedures. Now, different combination therapies as well as novel immunotherapies targeting new molecules have been proposed. Despite ongoing investigations on the therapeutic options and significant advancements in this regard, the disease is poorly prognosed. In fact, limited therapeutic options and delayed diagnosis lead to the progression, dissemination and metastasis of the disease. Current immunotherapies are mostly based on cytotoxic immunocytes, monoclonal antibodies and gene transferred vaccines. The use of Immune checkpoint inhibitors (ICIs) have grown rapidly. In this review, we aimed to explore perspective and progression of different approaches of immunotherapy in the treatment of GC and the clinical outcomes reported so far. We also summarized the tumor immunosurveillance and tumor immunoescape.
Objective:
This study was undertaken to analyze disease response and immune response to assess treatment effectiveness and success in patients with advanced oral mucosal melanoma treated with cytokines injection, cryosurgery, and adoptive cell transfer therapy.
Study design:
Ten patients were enrolled in the study, and the relevant characteristics and immunologic differences were evaluated.
Results:
All patients achieved an objective clinical response according to the Response Evaluation Criteria in Solid Tumors, including 7 cases of continuing complete remission (55, 27, 87 + , 58+, 58 + , 45 + , and 37 + months) and 3 cases of partial remission (30, 12, and 9 months). Five responders are currently alive. After combination therapy, we observed that the proportion of CD3+ lymphocytes and the secretion of interferon-γ increased, whereas interleukin-10 decreased. In the assay of improved cytokine-induced killer cells, CD4+CD25+ regulatory T cells declined, and natural killer cells upregulated. Meanwhile, the proliferation rate of in vitro cultured improved cytokine-induced killer cells improved after courses of therapy.
Conclusions:
Combination therapy of cytokine injection, cryosurgery, and transfer of improved cytokine-induced killer cells may be a promising approach for patients with oral mucosal melanoma.
Tumors of the gastrointestinal system represent a significant share of solid tumors worldwide. Despite the advances in diagnosis and treatment, the prognosis of gastrointestinal tumors is still very poor and improved therapies are indispensable. Cytokine-induced killer (CIK) cells are feasible for an immunotherapeutic approach as they are easily available and have an advantageous biologic profile; they are rapidly proliferating and their high cytotoxicity is non-MHC-restricted. We summarize and discuss twenty recent clinical studies applying CIK cells for the treatment of gastric, pancreatic, hepatocellular, and colorectal cancer. Autologous CIK cells were transfused intravenously, intraperitoneally, or via the common hepatic artery. In all studies side effects and toxicity of CIK cell therapy were mild and easily controllable. The combination of CIK cell therapy with conventional adjuvant or palliative therapies was superior to the standard therapy alone, indicating the benefit of CIK cell therapy for cancer patients. Thus, CIK cells represent a promising immunotherapy for the treatment of gastrointestinal tumors. The optimal treatment schedule and ideal combination with conventional therapies should be evaluated in further clinical studies.
The profiling and monitoring of immune responses are key elements in the evaluation of the efficacy and development of new biotherapies, and a number of assays have been introduced for analyzing various immune parameters before, during, and after immunotherapy. The choice of immune assays for a given clinical trial depends on the known or suggested immunomodulating mechanisms associated with the tested therapeutic modality. Cell-mediated cytotoxicity represents a key mechanism in the immune response to various pathogens and tumors. Therefore, the selection of monitoring methods for the appropriate assessment of cell-mediated cytotoxicity is thought to be crucial. Assays that can detect both cytotoxic T lymphocytes (CTL) frequency and function, such as the IFN-γ enzyme-linked immunospot assay (ELISPOT) have gained increasing popularity for monitoring clinical trials and in basic research. Results from various clinical trials, including peptide and whole tumor cell vaccination and cytokine treatment, have shown the suitability of the IFN-γ ELISPOT assay for monitoring T cell responses. However, the Granzyme B ELISPOT assay and Perforin ELISPOT assay may represent a more direct analysis of cell-mediated cytotoxicity as compared to the IFN-γ ELISPOT, since Granzyme B and perforin are the key mediators of target cell death via the granule-mediated pathway. In this review we analyze our own data and the data reported by others with regard to the application of various modifications of ELISPOT assays for monitoring CTL activity in clinical vaccine trials.
Triple-negative breast cancer (TNBC) is a high risk form of this disease, even after surgery, due to the absence of targets for hormone treatment and anti-Her-2 therapy. Chemotherapy is the main therapeutic strategy for such breast cancer patients although the outcome is often unsatisfactory. Thus, the development of combination adjuvant therapies is essential for improved prognosis in TNBC patients. In this study, we investigated the efficacy of a sequential combination of cytokine-induced killer cell (CIK) infusion and chemotherapy for post-mastectomy TNBC patients.
From 2008 to 2012, 90 post-mastectomy TNBC patients were included in this retrospective study: 45 cases received chemotherapy alone or with sequential radiotherapy; a further 45 cases received chemotherapy with/without radiotherapy and sequential CIK infusion.
Survival analysis showed significantly higher disease-free survival (DFS) rate and overall survival (OS) rates in the CIK treatment group compared with the control group (p = 0.0382, p = 0.0046, respectively; log-rank test). Multivariate survival analysis showed that CIK adjuvant treatment was an independent prognostic factor for OS of TNBC patients. In subgroup analyses, CIK adjuvant treatment significantly increased the DFS rate of patients with pathological grade III, and significantly increased the OS rate of patients in N1, N2, N3-stage, IIB, III TNM stage and with pathological grade III.
These data indicate that adjuvant CIK treatment combined with chemotherapy is an effective therapeutic strategy to prevent disease recurrence and prolong survival of TNBC patients, particularly those with lymph node metastasis, advanced TNM-stage and poor pathological grade.
As significant numbers of acute myeloid leukemia (AML) patients are still refractory to conventional therapies or experience relapse, immunotherapy using T-cells expressing chimeric antigen receptors (CARs) might represent a valid treatment option. AML cells frequently overexpress the myeloid antigens CD33 and CD123, for which specific CARs can be generated. However, CD33 is also expressed on normal hematopoietic stem/progenitors cells (HSPCs), and its targeting could potentially impair normal hematopoiesis. In contrast, CD123 is widely expressed by AML, while low expression is detected on HSPCs, making it a much more attractive target. In this study we describe the in vivo efficacy and safety of using cytokine-induced-killer (CIK) cells genetically modified to express anti-CD33 or anti-CD123 CAR to target AML. We show that both these modified T-cells are very efficient in reducing leukemia burden in vivo, but only the anti-CD123 CAR has limited killing on normal HSPCs, thus making it a very attractive immunotherapeutic tool for AML treatment.Leukemia accepted article preview online, 7 February 2014; doi:10.1038/leu.2014.62.
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. Cancer Res; 74(1); 1-11. ©2013 AACR.
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Current therapeutic regimens for Acute Myeloid Leukemia (AML) are still associated with high rates of relapse. In the last years, great interest has been focused on the identification of surface molecules that are preferentially expressed by AML cells and leukemic stem cells (LSCs), in order to selectively target the tumor population, whilst sparing the normal counterpart of hematopoietic stem/progenitor cells (HSPC), and possibly impeding disease recurrence. Immunotherapy with T-cells genetically modified to express chimeric antigen receptors (CARs) represents a valid and innovative cell therapy approach for hematological malignancies. In this study we developed a new CAR molecule specific for the IL-3Rα (CD123) target antigen, which is overexpressed on AML blasts, CD34+ leukemic progenitors, and leukemic stem cells (AML-LSCs) compared to normal hematopoietic stem/progenitor cells (HSPCs), and whose overexpression is associated with poor prognosis. Cytokine Induced Killer (CIK) cells, ex-vivo expanded T cells with spontaneous antitumoral activity, were transduced with an SFG-retroviral vector encoding an anti-CD123.CAR and CAR functionality has been evaluated by short-term cytotoxicity assay. Transduced CIK cells strongly killed CD123+ THP-1 cell line (60%±5.4%, Effector:Target –E:T- ratio of 5:1, n=3), as well as primary AML blasts (59%±5.4%, E:T ratio of 3:1, n=4). With the aim to better characterize the ability of anti-CD123.CAR+CIK cells to kill leukaemia cells over time we performed long-term cytotoxicity assay, observing a leukemic cell recovery for THP-1 of 3.5%±1.5% (n=5) and for primary AML cells of 2.4%±1.4% (n=3) when co-cultured with CIK cells expressing anti-CD123.CAR, compared to an average target survival of up to 80%, when co-cultured with unmanipulated (NT) CIK cells. Interestingly, secondary colonies experiments after co-culture of healthy donor cord blood-derived HSPCs (Lin-) with anti-CD123.CAR+CIK cells demonstrated that this newly generated CAR molecule better preserved the normal haematopoietic reconstitution in contrast to a previously generated anti-CD33.CAR (total number of colonies of 146.8±6.6, 66.4±5.1, 117.6±4.6, for Lin- cells co-cultured with NT CIK cells, anti-CD33.CAR+CIK cells, anti-CD123.CAR+CIK cells respectively, n=4), while keeping identical cytotoxicity profile towards AML. Furthermore, a limited killing of normal CD123 expressing monocytes and CD123-low expressing endothelial cells was measured, accompanied by a lesser release of stimulatory cytokines such as IFN-gamma, TNF-alfa and TNF-beta when compared to the levels released after stimulation with CD123+ leukemic cells (THP-1 and AML), thus indicating a low toxicity profile of the anti-CD123.CAR. Taken together, our results indicate that CD123-specific CAR strongly enhances anti-leukemic CIK functions towards AML, while sparing HSPCs and normal CD123-expressing tissues, paving the way for the development of novel immunotherapy approaches for the treatment of resistant forms of AML, particularly for a precocious intervention in presence of minimal residual disease, in the context of early relapse after HSCT.
Disclosures
No relevant conflicts of interest to declare.
Renal cell carcinoma (RCC) is the most common malignancy of adult human kidney, which accounts for more than 2 % of all cancers. RCC generally does not respond well to conventional chemotherapy and radiotherapy. Cytokine-induced killer (CIK) cells are ex vivo activated lymphocytes with potent activity against various tumors and minimal side effects. Here, we summarize the data on preclinical and clinical efficacy of CIK cells for RCC treatment. Our preclinical data show that CIK cells have potent anti-tumor activity in vitro and in an in vivo nude mouse xenograft model. Clinical studies for the treatment of RCC patients indicate that CIK cell therapy can induce favorable responses with no serious side effects. These studies suggest that CIK cells may become a valuable strategy for the treatment of patients with RCC.
Genetically engineering of T lymphocytes to confer new antitumor specificities is a fascinating approach that may help the successful clinical translation of adoptive immunotherapy strategies. The recognition of tumor-specific antigens may be obtained inducing the membrane expression of transgene encoded antitumor T cell receptors (TCR) or chimeric antigen receptors (CAR). Few but very informative clinical trials with TCR or CAR redirected T lymphocytes have been attempted in the last years, reporting important clinical results along with disappointing failures and important warnings. In this work we will focus on TCR and CAR redirected T lymphocytes as adoptive immunotherapy for solid tumors. We will review the main topics of these strategies from the angle of clinical applications, discussing the main issues emerged from early clinical trials and their impact on next study designs.