efficient-rapid-generation-car-t-cytokine-induced-killer-cells-gmp

Efficient and Rapid Generation of CAR-T and Cytokine-Induced Killer Cells in GMP-scalable Devices

Adoptive cell therapies (ACT) have progressed rapidly in recent years, playing a pivotal role in cancer immunotherapy and significantly influencing ...

Veneto Institute of Oncology IOV - IRCCS

University of Padua

Triple-negative breast cancer (TNBC) represents the most aggressive breast tumor subtype. However, the molecular determinants responsible for the metastatic TNBC phenotype are only partially understood. We here show that expression of the mitochondrial calcium uniporter (MCU), the selective channel responsible for mitochondrial Ca(2+) uptake, correlates with tumor size and lymph node infiltration, suggesting that mitochondrial Ca(2+) uptake might be instrumental for tumor growth and metastatic formation. Accordingly, MCU downregulation hampered cell motility and invasiveness and reduced tumor growth, lymph node infiltration, and lung metastasis in TNBC xenografts. In MCU-silenced cells, production of mitochondrial reactive oxygen species (mROS) is blunted and expression of the hypoxia-inducible factor-1α (HIF-1α) is reduced, suggesting a signaling role for mROS and HIF-1α, downstream of mitochondrial Ca(2+) Finally, in breast cancer mRNA samples, a positive correlation of MCU expression with HIF-1α signaling route is present. Our results indicate that MCU plays a central role in TNBC growth and metastasis formation and suggest that mitochondrial Ca(2+) uptake is a potential novel therapeutic target for clinical intervention.

The mitochondrial calcium uniporter regulates breast cancer progression via HIF-1α.

Cytokine-induced Killer (CIK) cells are a heterogeneous population of ex vivo expanded T lymphocytes capable of MHC-unrestricted antitumor activity, which share phenotypic and functional features with both NK and T cells. Preclinical data and initial clinical studies demonstrated their high tolerability in vivo, supporting CIK cells as a promising cell population for adoptive cell immunotherapy. In this study, we report for the first time that CIK cells display a donor-dependent expression of CD16, which can be engaged by trastuzumab or cetuximab to exert a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian and breast cancer cell lines, leading to an increased lytic activity in vitro, and an enhanced therapeutic efficacy in vivo. Thus, an efficient tumor antigen-specific retargeting can be achieved by a combination therapy with clinical-grade monoclonal antibodies already widely used in cancer therapy, and CIK cell populations that are easily expandable in very large numbers, inexpensive, safe and do not require genetic manipulations. Overall, these data provide a new therapeutic strategy for the treatment of Her2 and EGFR expressing tumors by adoptive cell therapy, which could find wide implementation and application, and could also be expanded to the use of additional therapeutic antibodies.

Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies.

Cytokine-Induced killer (CIK) cells are raising growing interest in cellular antitumor therapy, as they can be easily expanded with a straightforward and inexpensive protocol, and are safe requiring only GMP-grade cytokines to obtain very high amounts of cytotoxic cells. CIK cells do not need antigen-specific stimuli to be activated and proliferate, as they recognize and destroy tumor cells in an HLA-independent fashion through the engagement of NKG2D. In several preclinical studies and clinical trials, CIK cells showed a reduced alloreactivity compared to conventional T cells, even when challenged across HLA-barriers; only in a few patients, a mild GVHD occurred after treatment with allogeneic CIK cells. Additionally, their antitumor activity can be redirected and further improved with chimeric antigen receptors, clinical-grade monoclonal antibodies or immune checkpoint inhibitors. The evidence obtained from a growing body of literature support CIK cells as a very promising cell population for adoptive immunotherapy. In this review, all these aspects will be addressed with a particular emphasis on the role of the cytokines involved in CIK cell generation, expansion and functionalization.

Cytokines for the induction of antitumor effectors: The paradigm of Cytokine-Induced Killer (CIK) cells.

Plasmonic nanostructures show important properties for biotechnological applications, but they have to be guided on the target for exploiting their potentialities. Antibodies are the natural molecules for targeting. However, their possible adverse immunogenic activity and their cost have suggested finding other valid substitutes. Small molecules like peptides can be an alternative source of targeting agents, even if, as single molecules, their binding affinity is usually not very good. GE11 is a small dodecapeptide with specific binding to the epidermal growth factor receptor (EGFR) and low immunogenicity. The present work shows that thousands of polyethylene glycol (PEG) chains modified with lysines and functionalized with GE11 on clusters of naked gold nanoparticles, obtained by laser ablation in water, achieves a better targeting activity than that recorded with nanoparticles decorated with the specific anti-EGFR antibody Cetuximab (C225). The insertion of the cationic spacer between the polymeric part of the ligand and the targeting peptide allows for a proper presentation of GE11 on the surface of the nanosystems. Surface enhanced resonance Raman scattering signals of the plasmonic gold nanoparticles are used for quantifying the targeting activity. Molecular dynamic calculations suggest that subtle differences in the exposition of the peptide on the PEG sea are important for the targeting activity.

Enhanced EGFR Targeting Activity of Plasmonic Nanostructures with Engineered GE11 Peptide.

Cytokine-Induced (CIK) cells represent an attractive approach for cell-based immunotherapy, as they show several advantages compared with other strategies. Here we describe an original serum-free protocol for CIK cell expansion that employs G-Rex devices and compare the resulting growth, viability, phenotypic profile and cytotoxic activity with conventional culture in tissue flasks. CIK cells were obtained from buffy coats, seeded in parallel in G-Rex and tissue flasks, and stimulated with clinical-grade IFN-γ, anti-CD3 antibody and IL-2. G-Rex led to large numbers of CIK cells, with a minimal need for technical interventions, thus reducing the time and costs of culture manipulation. CIK cells generated in G-Rex showed a less differentiated phenotype, with a significantly higher expression of naive-associated markers such as CD62L, CD45RA and CCR7, which correlates with a remarkable expansion potential in culture and could lead to longer persistence and a more sustained anti-tumor response in vivo. The described procedure can be easily translated to large-scale production under Good Manufacturing Practice. Overall, this protocol has strong advantages over existing procedures, as it allows easier, time-saving and cost-effective production of CIK effector cells, fostering their clinical application.

A serum-free protocol for the ex vivo expansion of Cytokine-Induced Killer cells using gas-permeable static culture flasks.

TP53 missense mutations leading to the expression of mutant p53 oncoproteins are frequent driver events during tumorigenesis. p53 mutants promote tumor growth, metastasis and chemoresistance by affecting fundamental cellular pathways and functions. Here, we demonstrate that p53 mutants modify structure and function of the Golgi apparatus, culminating in the increased release of a pro-malignant secretome by tumor cells and primary fibroblasts from patients with Li-Fraumeni cancer predisposition syndrome. Mechanistically, interacting with the hypoxia responsive factor HIF1α, mutant p53 induces the expression of miR-30d, which in turn causes tubulo-vesiculation of the Golgi apparatus, leading to enhanced vesicular trafficking and secretion. The mut-p53/HIF1α/miR-30d axis potentiates the release of soluble factors and the deposition and remodeling of the ECM, affecting mechano-signaling and stromal cells activation within the tumor microenvironment, thereby enhancing tumor growth and metastatic colonization.

Mutant p53 induces Golgi tubulo-vesiculation driving a prometastatic secretome.

Cytokine-Induced Killer (CIK) cells share several functional and phenotypical properties of both T and natural killer (NK) cells. They represent an attractive approach for cell-based immunotherapy, as they do not require antigen-specific priming for tumor cell recognition, and can be rapidly expanded . Their relevant expression of FcγRIIIa (CD16a) can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their lytic activity in an antigen-specific manner. Here, we report the efficacy of this combined approach against triple negative breast cancer (TNBC), an aggressive tumor that still requires therapeutic options. Different primitive and metastatic TNBC cancer mouse models were established in NSG mice, either by implanting patient-derived TNBC samples or injecting MDA-MB-231 cells orthotopically or intravenously. The combined treatment consisted in the repeated intratumoral or intravenous injection of CIK cells and cetuximab. Tumor growth and metastasis were monitored by bioluminescence or immunohistochemistry, and survival was recorded. CIK cells plus cetuximab significantly restrained primitive tumor growth in mice, either in patient-derived tumor xenografts or MDA-MB-231 cell line models. Moreover, this approach almost completely abolished metastasis spreading and dramatically improved survival. The antigen-specific mAb favored tumor and metastasis tissue infiltration by CIK cells, and led to an enrichment of the CD16a subset.Data highlight the potentiality of this novel immunotherapy strategy where a nonspecific cytotoxic cell population can be converted into tumor-specific effectors with clinical-grade antibodies, thus providing not only a therapeutic option for TNBC but also a valid alternative to more complex approaches based on chimeric antigen receptor-engineered cells.

Adoptive cell therapy of triple negative breast cancer with redirected cytokine-induced killer cells.

Patients affected by aggressive B-cell malignancies who are resistant to primary or salvage chemoimmunotherapy have an extremely poor prognosis and limited therapeutic options. Promising therapeutic success has been achieved with the infusion of CD19 chimeric antigen receptor-T cells, but several limits still restrain the administration to a limited proportion of patients. This unmet clinical need might be fulfilled by an adoptive immunotherapy approach that combines cytokine-induced killer (CIK) cells and monoclonal antibodies (mAb) to the CD20 antigen. Indeed, CIK cells are an effector population endowed with antitumor activity, which can be further improved and antigen-specifically redirected by clinical-grade mAb triggering antibody-dependent cell-mediated cytotoxicity.

Innovative therapeutic strategy for B-cell malignancies that combines obinutuzumab and cytokine-induced killer cells.

The successful treatment of patients affected by B-cell malignancies with Chimeric Antigen Receptor (CAR)-T cells represented a breakthrough in the field of adoptive cell therapy (ACT). However, CAR-T therapy is not an option for every patient, and several needs remain unmet. In particular, the production of CAR-T cells is expensive, labor-intensive and logistically challenging; additionally, the toxicities deriving from CAR-T cells infusion, such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), have been documented extensively. Alternative cellular therapy products such as Cytokine-induced killer (CIK) cells have the potential to overcome some of these obstacles. CIK cells are a heterogeneous population of polyclonal CD3CD56 T cells with phenotypic and functional properties of NK cells. CIK cell cytotoxicity is exerted in a major histocompatibility complex (MHC)-unrestricted manner through the engagement of natural killer group 2 member D (NKG2D) molecules, against a wide range of hematological and solid tumors without the need for prior antigen exposure or priming. The foremost potential of CIK cells lies in the very limited ability to induce graft-versus-host disease (GvHD) reactions in the allogeneic setting. CIK cells are produced with a simple and extremely efficient expansion protocol, which leads to a massive expansion of effector cells and requires a lower financial commitment compared to CAR-T cells. Indeed, CAR-T manufacturing involves the engineering with expensive GMP-grade viral vectors in centralized manufacturing facilities, whereas CIK cell production is successfully performed in local academic GMP facilities, and CIK cell treatment is now licensed in many countries. Moreover, the toxicities observed for CAR-T cells are not present in CIK cell-treated patients, thus further reducing the costs associated with hospitalization and post-infusion monitoring of patients, and ultimately encouraging the delivery of cell therapies in the outpatient setting. This review aims to give an overview of the limitations of CAR-T cell therapy and outline how the use of CIK cells could overcome such drawbacks thanks to their unique features. We highlight the undeniable advantages of using CIK cells as a therapeutic product, underlying the opportunity for further research on the topic.

How can Cytokine-induced killer cells overcome CAR-T cell limits.

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.

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.

A rescue approach in refractory diffuse large B-cell lymphoma with obinutuzumab-redirected cytokineinduced killer cells: a first-in-human case report.

From 17 to 19th October 2024, the XXI Italian Network for Bio-Immunotherapy of Tumors Meeting (NIBIT) took place in Palermo, in the marvelous historical location of Teatro Politeama, under the auspices of the Italian Association of Medical Oncology (AIOM), Italian Association of Cancer Research (AIRC), Fondazione Pezcoller, Italian Alliance against Cancer (ACC), Italian Lymphoma Foundation (FIL), Grazia Focacci Foundation and Melagioco Foundation. The conference covered a spectrum of topics ranging from target discovery to therapeutic advances in immuno-oncology, bringing world-renowned experts to present groundbreaking innovations in basic, translational, and clinical cancer research. Six sessions focused on cellular therapies, digital pathology, vaccines, tertiary lymphoid structures, and microenvironment in order to get deep insights on how to personalize diagnosis and therapies in the clinical setting. Young investigators had the opportunity to meet and greet their mentors, promoting the synergy of the academic and industrial sectors within the national and international panorama, discussing the application of artificial intelligence on multi-specific antibodies, drug conjugates, and antibody fusion proteins that are advancing the efficacy of precision medicine and minimizing off-target effects.

Highlighting recent achievements to advance more effective cancer immunotherapy.

Vaccines based on mRNA have been fundamental in facing the COVID-19 pandemic, however, they still raise concerns about stability and long-term efficacy. Thus, protein-based vaccines remain valid options and hence the study of effective adjuvants is crucial. Here, we developed a COVID-19 vaccine based on the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein, which is covalently conjugated to the natural polymer hyaluronan (HA) that acts as an immunological adjuvant. Vaccination of K18-hACE2 mice with HA-RBD was well tolerated, and elicited high and sustained titres of RBD-binding antibodies and SARS-CoV-2-neutralizing antibodies, without the addition of other immunostimulatory compounds. Most importantly, HA-RBD vaccination conferred long-term protection to K18-hACE2 mice after challenge with SARS-CoV-2, also in the case of two consequent infections driven by different variants. These findings demonstrate the efficacy of HA-based vaccination against COVID-19 disease, and support the promising use of HA as an efficient and well tolerated adjuvant.

Article	Year
The mitochondrial calcium uniporter regulates breast cancer progression via HIF-1α. EMBO molecular medicine\| PubMed ID: 27138568	2016
Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies. Oncoimmunology\| PubMed ID: 27622068	2016
Cytokines for the induction of antitumor effectors: The paradigm of Cytokine-Induced Killer (CIK) cells. Cytokine & growth factor reviews\| PubMed ID: 28629761	2017
Enhanced EGFR Targeting Activity of Plasmonic Nanostructures with Engineered GE11 Peptide. Advanced healthcare materials\| PubMed ID: 28945012	2017
A serum-free protocol for the ex vivo expansion of Cytokine-Induced Killer cells using gas-permeable static culture flasks. Cytotherapy\| PubMed ID: 32631696	2020
Mutant p53 induces Golgi tubulo-vesiculation driving a prometastatic secretome. Nature communications\| PubMed ID: 32770028	2020
Adoptive cell therapy of triple negative breast cancer with redirected cytokine-induced killer cells. Oncoimmunology\| PubMed ID: 32923140	2020
Innovative therapeutic strategy for B-cell malignancies that combines obinutuzumab and cytokine-induced killer cells. Journal for immunotherapy of cancer\| PubMed ID: 34272306	2021
How can Cytokine-induced killer cells overcome CAR-T cell limits. Frontiers in immunology\| PubMed ID: 37675107	2023
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. Cancer immunology, immunotherapy : CII\| PubMed ID: 38279995	2024
A rescue approach in refractory diffuse large B-cell lymphoma with obinutuzumab-redirected cytokineinduced killer cells: a first-in-human case report. Haematologica\| PubMed ID: 38721751	2024
Highlighting recent achievements to advance more effective cancer immunotherapy. Journal of experimental & clinical cancer research : CR\| PubMed ID: 39966867	2025
On the adjuvanticity of hyaluronan: The case of a SARS-CoV-2 vaccine. Journal of controlled release : official journal of the Controlled Release Society\| PubMed ID: 40164435	2025

Roberta Sommaggio

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