Cytokine-induced killer (CIK) cells, successes and challenges: report on the first international conference dedicated to the clinical translation of this unique adoptive cell immunotherapy.

On August 30, 2023, experts from Germany and abroad met to discuss the successes and challenges of cytokine-induced killer cell (CIK) therapy, that recently celebrated its 30th anniversary providing treatment for cancer. This first virtual conference was hosted by CIO Bonn, a certified Comprehensive Cancer Center (CCC) funded by German Cancer Aid (DKH). In addition to keynote speakers involved in CIK cell clinical trials or optimized preclinical models to improve this adoptive cell immunotherapy, more than 100 attendees from around the world also participated in this event. Initiatives to establish the International Society of CIK Cells (ISCC) and a stronger CIK cell network guiding preclinical research and future clinical trials were also announced.

Engineering tandem CD33xCD146 CAR CIK (cytokine-induced killer) cells to target the acute myeloid leukemia niche.

In acute myeloid leukemia (AML), malignant stem cells hijack the normal bone marrow niche where they are largely protected from the current therapeutic approaches. Thus, eradicating these progenitors is the ultimate challenge in the treatment of this disease. Specifically, the development of chimeric antigen receptors (CARs) against distinct mesenchymal stromal cell subpopulations involved in the maintenance of leukemic stem cells within the malignant bone marrow microenvironment could represent a new strategy to improve CAR T-cell therapy efficacy, which is still unsuccessful in AML. As a proof of concept, we generated a novel prototype of Tandem CAR, with one specificity directed against the leukemic cell marker CD33 and the other against the mesenchymal stromal cell marker CD146, demonstrating its capability of simultaneously targeting two different cell types in a 2D co-culture system. Interestingly, we could also observe an in vitro inhibition of CAR T cell functionality mediated by stromal cells, particularly in later effector functions, such as reduction of interferon-gamma and interleukin-2 release and impaired proliferation of the CAR+ effector Cytokine-Induced Killer (CIK) cells. Taken together, these data demonstrate the feasibility of a dual targeting model against two molecules, which are expressed on two different target cells, but also highlight the immunomodulatory effect on CAR CIK cells exerted by stromal cells, confirming that the niche could be an obstacle to the efficacy of CAR T cells. This aspect should be considered in the development of novel CAR T cell approaches directed against the AML bone marrow niche.

Selective homing of CAR-CIK cells to the bone marrow niche enhances control of the acute myeloid leukemia burden.

Acute myeloid leukemia (AML) is a hematological malignancy derived from neoplastic myeloid progenitor cells characterized by abnormal clonal proliferation and differentiation. Although novel therapeutic strategies have recently been introduced, the prognosis of AML is still unsatisfactory. So far, the efficacy of chimeric antigen receptor (CAR)-T-cell therapy in AML has been hampered by several factors, including the poor accumulation of the blood-injected cells in the leukemia bone marrow (BM) niche in which chemotherapy-resistant leukemic stem cells reside. Thus, we hypothesized that overexpression of CXCR4, whose ligand CXCL12 is highly expressed by BM stromal cells within this niche, could improve T-cell homing to the BM and consequently enhance their intimate contact with BM-resident AML cells, facilitating disease eradication. Specifically, we engineered conventional CD33.CAR-cytokine-induced killer cells (CIKs) with the wild-type (wt) CXCR4 and the variant CXCR4R334X, responsible for leukocyte sequestration in the BM of patients with warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis syndrome. Overexpression of both CXCR4wt and CXCR4mut in CD33.CAR-CIKs resulted in significant improvement of chemotaxis toward recombinant CXCL12 or BM stromal cell-conditioned medium, with no observed impairment of cytotoxic potential in vitro. Moreover, CXCR4-overexpressing CD33.CAR-CIKs showed enhanced in vivo BM homing, associated with a prolonged retention for the CXCR4R334X variant. However, only CD33.CAR-CIKs coexpressing CXCR4wt but not CXCR4mut exerted a more sustained in vivo antileukemic activity and extended animal survival, suggesting a noncanonical role for CXCR4 in modulating CAR-CIK functions independent of BM homing. Taken together, these data suggest that arming CAR-CIKs with CXCR4 may represent a promising strategy for increasing their therapeutic potential for AML.

IL-3-zetakine combined with a CD33 costimulatory receptor as a dual CAR approach for safer and selective targeting of AML.

Acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric patients. Adoptive cell therapy by chimeric antigen receptor (CAR)-engineered T cells demonstrated a high therapeutic potential, but further development is required to ensure a safe and durable disease remission in AML, especially in elderly patients. To date, translation of CAR T-cell therapy in AML is limited by the absence of an ideal tumor-specific antigen. CD123 and CD33 are the 2 most widely overexpressed leukemic stem cell biomarkers but their shared expression with endothelial and hematopoietic stem and progenitor cells increases the risk of undesired vascular and hematologic toxicities. To counteract this issue, we established a balanced dual-CAR strategy aimed at reducing off-target toxicities while retaining full functionality against AML. Cytokine-induced killer (CIK) cells, coexpressing a first-generation low affinity anti-CD123 interleukin-3-zetakine (IL-3z) and an anti-CD33 as costimulatory receptor without activation signaling domains (CD33.CCR), demonstrated a powerful antitumor efficacy against AML targets without any relevant toxicity on hematopoietic stem and progenitor cells and endothelial cells. The proposed optimized dual-CAR cytokine-induced killer cell strategy could offer the opportunity to unleash the potential of specifically targeting CD123+/CD33+ leukemic cells while minimizing toxicity against healthy cells.

Lenalidomide enhances CD23.CAR T cell therapy in chronic lymphocytic leukemia.

Chimeric antigen receptors (CAR)-modified T cells are an emerging therapeutic tool for chronic lymphocytic leukemia (CLL). However, in patients with CLL, well-known T-cell defects and the inhibitory properties of the tumor microenvironment (TME) hinder the efficacy of CAR T cells. We explored a novel approach combining CARs with lenalidomide, an immunomodulatory drug that tempers the immunosuppressive activity of the CLL TME. T cells from patients with CLL were engineered to express a CAR specific for CD23, a promising target antigen. Lenalidomide maintained the in vitro effector functions of CD23.CAR+ T cells effector functions in terms of antigen-specific cytotoxicity, cytokine release and proliferation. Overall, lenalidomide preserved functional CAR T-CLL cell immune synapses. In a Rag2-/-γc-/--based xenograft model of CLL, we demonstrated that, when combined with low-dose lenalidomide, CD23.CAR+ T cells efficiently migrated to leukemic sites and delayed disease progression when compared to CD23.CAR+ T cells given with rhIL-2. These observations underline the therapeutic potential of this novel CAR-based combination strategy in CLL.

Optimization of therapeutic T cell expansion in G-Rex device and applicability to large-scale production for clinical use.

Our center performs experimental clinical studies with advanced therapy medicinal products (ATMPs) based on polyclonal T cells, all of which are currently expanded in standard T-flasks. Given the need to increase the efficiency and safety of large-scale T cell expansion for clinical use, we have optimized the method to expand in G-Rex devices both cytokine-induced killer cells (CIKs) from peripheral or cord blood and blinatumomab-expanded T cells (BETs). We show that the G-Rex reproducibly allowed the expansion of >30 × 106 CD3+ cells/cm2 of gas-permeable membrane in a mean of 10 to 11 days in a single unit, without manipulation, except for addition of cytokines and sampling of supernatant for lactate measurement every 3 to 4 days. In contrast, 21 to 24 days, twice-weekly cell resuspension and dilution into 48 to 72 T-flasks were required to complete expansions using the standard method. We show that the CIKs produced in G-Rex (CIK-G) were phenotypically very similar, for a large panel of markers, to those expanded in T-flasks, although CIK-G products had lower expression of CD56 and higher expression of CD27 and CD28. Functionally, CIK-Gs were strongly cytotoxic in vitro against the NK cell target K562 and the REH pre-B ALL cell line in the presence of blinatumomab. CIK-Gs also showed therapeutic activity in vivo in the Ph+ pre-B ALL-2 model in mice. The expansion of both CIKs and BETs in G-Rex was validated in good manufacturing practices (GMP) conditions, and we plan to use G-Rex for T cell expansion in future clinical studies.

Catch me if you can: how AML and its niche escape immunotherapy.

In spite of the remarkable progress in basic and preclinical studies of acute myeloid leukemia (AML), the five-year survival rate of AML patients remains poor, highlighting the urgent need for novel and synergistic therapies. Over the past decade, increased attention has been focused on identifying suitable immunotherapeutic strategies for AML, and in particular on targeting leukemic cells and their progenitors. However, recent studies have also underlined the important contribution of the leukemic microenvironment in facilitating tumor escape mechanisms leading to disease recurrence. Here, we describe the immunological features of the AML niche, with particular attention to the crosstalk between the AML blasts and the cellular components of the altered tumor microenvironment (TME) and the mechanisms of immune escape that hamper the therapeutic effects of the most advanced treatments. Considering the AML complexity, immunotherapy approaches may benefit from a rational combination of complementary strategies aimed at preventing escape mechanisms without increasing toxicity.

Sleeping Beauty-engineered CAR T cells achieve antileukemic activity without severe toxicities.

BACKGROUNDChimeric antigen receptor (CAR) T cell immunotherapy has resulted in complete remission (CR) and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability.METHODSWe report the early results of a phase I/II trial in B cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine-induced killer (CIK) cells.RESULTSThe cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of CD3+ lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were 2 grade I and 1 grade II cytokine-release syndrome (CRS) cases at the highest dose in the absence of graft-versus-host disease (GVHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients receiving the highest doses achieved CR and CR with incomplete blood count recovery (CRi) at day 28. Five out of 6 patients in CR were also minimal residual disease negative (MRD-). Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion.CONCLUSIONSB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Antileukemic activity was achieved without severe toxicities.TRIAL REGISTRATIONClinicalTrials.gov NCT03389035.FUNDINGThis study was supported by grants from the Fondazione AIRC per la Ricerca sul Cancro (AIRC); Cancer Research UK (CRUK); the Fundación Científica de la Asociación Española Contra el Cáncer (FC AECC); Ministero Della Salute; Fondazione Regionale per la Ricerca Biomedica (FRRB).

Targeting CD33 in Chemoresistant AML Patient-Derived Xenografts by CAR-CIK Cells Modified with an Improved SB Transposon System.

The successful implementation of chimeric antigen receptor (CAR)-T cell therapy in the clinical context of B cell malignancies has paved the way for further development in the more critical setting of acute myeloid leukemia (AML). Among the potentially targetable AML antigens, CD33 is insofar one of the main validated molecules. Here, we describe the feasibility of engineering cytokine-induced killer (CIK) cells with a CD33.CAR by using the latest optimized version of the non-viral Sleeping Beauty (SB) transposon system "SB100X-pT4." This offers the advantage of improving CAR expression on CIK cells, while reducing the amount of DNA transposase as compared to the previously employed "SB11-pT" version. SB-modified CD33.CAR-CIK cells exhibited significant antileukemic activity in vitro and in vivo in patient-derived AML xenograft models, reducing AML development when administered as an "early treatment" and delaying AML progression in mice with established disease. Notably, by exploiting an already optimized xenograft chemotherapy model that mimics human induction therapy in mice, we demonstrated for the first time that CD33.CAR-CIK cells are also effective toward chemotherapy resistant/residual AML cells, further supporting its future clinical development and implementation within the current standard regimens.

Transposon-Based CAR T Cells in Acute Leukemias: Where are We Going?

Chimeric Antigen Receptor (CAR) T-cell therapy has become a new therapeutic reality for refractory and relapsed leukemia patients and is also emerging as a potential therapeutic option in solid tumors. Viral vector-based CAR T-cells initially drove these successful efforts; however, high costs and cumbersome manufacturing processes have limited the widespread clinical implementation of CAR T-cell therapy. Here we will discuss the state of the art of the transposon-based gene transfer and its application in CAR T immunotherapy, specifically focusing on the Sleeping Beauty (SB) transposon system, as a valid cost-effective and safe option as compared to the viral vector-based systems. A general overview of SB transposon system applications will be provided, with an update of major developments, current clinical trials achievements and future perspectives exploiting SB for CAR T-cell engineering. After the first clinical successes achieved in the context of B-cell neoplasms, we are now facing a new era and it is paramount to advance gene transfer technology to fully exploit the potential of CAR T-cells towards next-generation immunotherapy.

Preclinical Efficacy and Safety of CD19CAR Cytokine-Induced Killer Cells Transfected with Sleeping Beauty Transposon for the Treatment of Acute Lymphoblastic Leukemia.

Infusion of patient-derived CD19-specific chimeric antigen receptor (CAR) T cells engineered by viral vectors achieved complete remission and durable response in relapsed and refractory (r/r) B-lineage neoplasms. Here, we expand on those findings by providing a preclinical evaluation of allogeneic non-viral cytokine-induced killer (CIK) cells transfected with the Sleeping Beauty (SB) transposon CD19CAR (CARCIK-CD19). Specifically, thanks to a large-scale 18-day manufacturing process, it was possible to achieve stable CD19CAR expression (62.425 ± 6.399%) and efficient T-cell expansion (23.36 ± 3.00-fold). Frozen/thawed CARCIK-CD19 remained fully functional both in vitro and in an established patient-derived xenograft (PDX) of MLL-ENL rearranged acute lymphoblastic leukemia (ALL). CARCIK-CD19 showed a dose-dependent antitumor response and prolonged persistence in a PDX, bearing the feature of a Philadelphia-like ALL with PAX5/AUTS2 translocation, and in a survival model of lymphoma, achieving complete eradication of disseminated tumors. Finally, the infusion of CARCIK-CD19 proved to be safe and well tolerated in a biodistribution and toxicity model. The infused cells persisted in the hematopoietic and post-injection perfused organs until the end of the study and consisted of CD8+, CD56+, and CAR+ T cells. Overall, these findings provide important implications for non-viral technology and the proof-of-concept that donor-derived CARCIK-CD19 are indeed effective against relapsed ALL, a possibility that will be tested in Phase I/II clinical trials after allogeneic hematopoietic stem-cell transplantation.

Redirecting T cells with Chimeric Antigen Receptor (CAR) for the treatment of childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Nowadays the survival rate is around 85%. Nevertheless, an urgent clinical need is still represented by primary refractory and relapsed patients who do not significantly benefit from standard approaches, including chemo-radiotherapy and hematopoietic stem cell transplantation (HSCT). For this reason, immunotherapy has so far represented a challenging novel treatment opportunity, including, as the most validated therapeutic options, cancer vaccines, donor-lymphocyte infusions and tumor-specific immune effector cells. More recently, unexpected positive clinical results in ALL have been achieved by application of gene-engineered chimeric antigen expressing (CAR) T cells. Several CAR designs across different trials have generated similar response rates, with Complete Response (CR) of 60-90% at 1 month and an Event-Free Survival (EFS) of 70% at 6 months. Relevant challenges anyway remain to be addressed, such as amelioration of technical, cost and feasibility aspects of cell and gene manipulation and the necessity to face the occurrence of relapse mechanisms. This review describes the state of the art of ALL immunotherapies, the novelties in terms of gene manipulation approaches and the problems emerged from early clinical studies. We describe and discuss the process of clinical translation, including the design of a cell manufacturing protocol, vector production and regulatory issues. Multiple antigen targeting and combination of CAR T cells with molecular targeted drugs have also been evaluated as latest strategies to prevail over immune-evasion.

Balance of Anti-CD123 Chimeric Antigen Receptor Binding Affinity and Density for the Targeting of Acute Myeloid Leukemia.

Chimeric antigen receptor (CAR)-redirected T lymphocytes are a promising immunotherapeutic approach and object of pre-clinical evaluation for the treatment of acute myeloid leukemia (AML). We developed a CAR against CD123, overexpressed on AML blasts and leukemic stem cells. However, potential recognition of low CD123-positive healthy tissues, through the on-target, off-tumor effect, limits safe clinical employment of CAR-redirected T cells. Therefore, we evaluated the effect of context-dependent variables capable of modulating CAR T cell functional profiles, such as CAR binding affinity, CAR expression, and target antigen density. Computational structural biology tools allowed for the design of rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity without affecting the overall CAR design. We defined both lytic and activation antigen thresholds, with early cytotoxic activity unaffected by either CAR expression or CAR affinity tuning but later effector functions impaired by low CAR expression. Moreover, the anti-CD123 CAR safety profile was confirmed by lowering CAR binding affinity, corroborating CD123 is a good therapeutic target antigen. Overall, full dissection of these variables offers suitable anti-CD123 CAR design optimization for the treatment of AML.

Acute Myeloid Leukemia Targeting by Chimeric Antigen Receptor T Cells: Bridging the Gap from Preclinical Modeling to Human Studies.

Acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients, thus demanding advanced and personalized therapies. In this regard, different targeted immunotherapeutic approaches are available, ranging from naked monoclonal antibodies (mAb) to conjugated and multifunctional mAbs (i.e., BiTEs and DARTs). Recently, researchers have focused their attention on novel techniques of genetic manipulation specifically to redirect cytotoxic T cells endowed with chimeric antigen receptors (CARs) toward selected tumor associated antigens. So far, CAR T cells targeting the CD19 antigen expressed by B-cell origin hematological cancers have gained impressive clinical results, leading to the possibility of translating the CAR platform to treat other hematological malignancies such as AML. However, one of the main concerns in the field of AML CAR immunotherapy is the identification of an ideal target cell surface antigen, being highly expressed on tumor cells but minimally present on healthy tissues, together with the design of an anti-AML CAR appropriately balancing efficacy and safety profiles. The current review focuses mainly on AML target antigens and the related immunotherapeutic approaches developed so far, deeply dissecting methods of CAR T cell safety improvements, when designing novel CARs approaching human studies.

Immunotherapy of acute leukemia by chimeric antigen receptor-modified lymphocytes using an improved Sleeping Beauty transposon platform.

Chimeric antigen receptor (CAR)-modified T-cell adoptive immunotherapy is a remarkable therapeutic option proven effective in the treatment of hematological malignancies. In order to optimize cell manufacturing, we sought to develop a novel clinical-grade protocol to obtain CAR-modified cytokine-induced killer cells (CIKs) using the Sleeping Beauty (SB) transposon system. Administration of irradiated PBMCs overcame cell death of stimulating cells induced by non-viral transfection, enabling robust gene transfer together with efficient T-cell expansion. Upon single stimulation, we reached an average of 60% expression of CD123- and CD19- specific 3rd generation CARs (CD28/OX40/TCRzeta). Furthermore, modified cells displayed persistence of cell subsets with memory phenotype, specific and effective lytic activity against leukemic cell lines and primary blasts, cytokine secretion, and proliferation. Adoptive transfer of CD123.CAR or CD19.CAR lymphocytes led to a significant anti-tumor response against acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) disseminated diseases in NSG mice. Notably, we found no evidence of integration enrichment near cancer genes and transposase expression at the end of the differentiation. Taken all together, our findings describe a novel donor-derived non-viral CAR approach that may widen the repertoire of available methods for T cell-based immunotherapy.

CD123 AML targeting by chimeric antigen receptors: A novel magic bullet for AML therapeutics?

Chimeric antigen receptor (CAR) modified T cells have emerged as powerful tools for controlling leukemias. We recently showed that anti-CD123 CAR-expressing cytokine-induced killer T cell treatment is an effective immunotherapeutic approach to eradicate Acute Myeloid Leukemia (AML) cells. Here, we discuss how this genetically modified cell-based strategy could be relevant to the field of AML therapeutics.

Acute myeloid leukemia and novel biological treatments: monoclonal antibodies and cell-based gene-modified immune effectors.

In the context of acute myeloid leukemia (AML) treatment, the interface between chemotherapy and immunotherapy is at present getting closer as never before. Scientific research is oriented in overcoming the main limits of actual chemotherapeutic regimens against AML, which still accounts for a considerable number of relapsed or resistant forms. A lot of investments have been done in the use of monoclonal antibodies (mAbs) and recently gene-modified immune cells have been considered as an alternative approach whenever chemotherapy fails to eradicate the disease. In this sense, AML is a potential suitable target for immunotherapeutic approaches, due to overexpression of several tumor antigens. Here we describe the state of the art of mAbs and cellular therapies employing engineered immune effectors, developed against specific AML antigens, in a window embracing preclinical research and translational studies to the clinical setting.

Advanced targeted, cell and gene-therapy approaches for pediatric hematological malignancies: results and future perspectives.

Despite the survival of pediatric patients affected by hematological malignancies being improved in the last 20 years by chemotherapy and hematopoietic stem cell transplantation, a significant amount of patients still relapses. Treatment intensification is limited by toxic side effects and is constrained by the plateau of efficacy, while the pipeline of new chemotherapeutic drugs is running short. Therefore, novel therapeutic strategies are essential and researchers around the world are testing in clinical trials immune and gene-therapy approaches as second-line treatments. The aim of this review is to give a glance at these novel promising strategies of advanced medicine in the field of pediatric leukemias. Results from clinical protocols using new targeted "smart" drugs, immunotherapy, and gene therapy are summarized, and important considerations regarding the combination of these novel approaches with standard treatments to promote safe and long-term cure are discussed.

Targeting of acute myeloid leukaemia by cytokine-induced killer cells redirected with a novel CD123-specific chimeric antigen receptor.

Current therapeutic regimens for acute myeloid leukaemia (AML) are still associated with high rates of relapse. Immunotherapy with T-cells genetically modified to express chimeric antigen receptors (CARs) represents an innovative approach. Here we investigated the targeting of the interleukin three receptor alpha (IL3RA; CD123) molecule, which is overexpressed on AML bulk population, CD34(+) leukaemia progenitors, and leukaemia stem cells (LSC) compared to normal haematopoietic stem/progenitor cells (HSPCs), and whose overexpression is associated with poor prognosis. Cytokine-induced killer (CIK) cells were transduced with SFG-retroviral-vector encoding an anti-CD123 CAR. Transduced cells were able to strongly kill CD123(+) cell lines, as well as primary AML blasts. Interestingly, secondary colony experiments demonstrated that anti-CD123.CAR preserved in vitro HSPCs, in contrast to a previously generated anti-CD33.CAR, while keeping an identical cytotoxicity profile towards AML. Furthermore, limited killing of normal monocytes and CD123-low-expressing endothelial cells was noted, thus indicating a low toxicity profile of the anti-CD123.CAR. Taken together, our results indicate that CD123-specific CARs strongly enhance anti-AML CIK functions, while sparing HSPCs and normal low-expressing antigen cells, paving the way to develop novel immunotherapy approaches for AML treatment.