The great peculiarity of such effector cells is the major histocompatibility complex (MHC)-unrestricted lytic capacity towards a wide range of hematological (21, 25, 29–33) and solid (27, 34–38) tumor cells, without the need of prior antigen exposure or priming. CIK cell cytotoxic capacity is exerted by the engagement of natural killer group 2 member D (NKG2D) molecules, which recognize specific ligands known as UL16-binding protein family members (ULBP1–6) and MHC class I-related molecules A and B (MIC A/B) widely expressed on tumor cells (39–42). To better define the functional characteristic of the different cell fractions present in bulk cultures, in a preclinical study the CD3⁺CD56⁺ and CD3⁺CD56^- subpopulations were sorted, and their cytotoxicity tested both in vitro and in vivo against hematological malignancies, to be compared with unsorted bulk CIK cells (35). Results showed that the CD3⁺CD56⁺ cells retain the killing activity due to the high expression of NKG2D, and are endowed with low proliferative capacity. The CD3⁺CD56^- subpopulation instead showed a low anti-tumor capacity while expressing NKG2D, suggesting that the latter cannot be the only player accounted for CIK cytotoxicity (35). Moreover, this population is highly proliferative, since even the smallest amount of CD3⁺CD56^- cells that remained in culture after cell selection were able to proliferate in the tumor of the injected mice (32). Nevertheless, studies have shown that each subpopulation is required for proper differentiation, proliferation, and ultimately tumor killing, emphasizing the benefit of infusing the bulk CIK cell culture rather than sorted subpopulations (28, 43). Similar conclusions were drawn in the context of solid tumors, such as colorectal cancer, by Sangiolo and colleagues (34, 35), underlying the different roles of the distinct CIK cell subpopulations.

In addition to NKG2D, other NK-specific receptors including NKp30 and DNAM-1 are expressed, albeit at lower levels when compared to NK cells (28). The co-stimulatory molecule DAP10 and the ICAM-1 ligand CD244 (2B4) could also in part be responsible for the CIK cytotoxicity (39, 42), but more detailed studies are needed. Indeed, Wu et al. showed that 2B4 alone is not able to stimulate CIK cell cytolytic activity but can synergize with NKG2D in certain circumstances, and, in turn, induce LFA-1 expression on CIK effectors (39). The crucial role of LFA-1 in CIK cells for functional binding and cytotoxicity is clear, but it has been hypothesized that also other surface molecules are involved in binding, since its surface expression does not correlate with the cytotoxic activity (44).

Strikingly, CIK cells express T-cell receptor (TCR) in a polyclonal fashion and with a similar proportion of αβ and γδ chains as in peripheral blood T cells (28, 44). Nevertheless, a seminal work from Negrin’s lab showed that the inhibition of TCR downstream activation does not affect CIK cell cytotoxicity, demonstrating that their lytic activity mainly relies on a non-MHC restricted killing mechanism, rather than a TCR/MHC-restricted modality (44, 45). Finally, CIK cell activation and engagement with the target cell leads to the release of granzymes and perforins that provoke cell lysis (45).

Moreover, it has been reported that CIK cells express the FcγRIIIa (CD16a) receptor. Cappuzzello et al. showed that the CD16a expression is highly donor-dependent, and remains stable during the entire period of the expansion protocol (27, 33, 36). The CD16a receptor can be easily engaged by monoclonal antibodies (mAb), becoming, therefore, a powerful mediator for triggering a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against tumor cells (27). Thus, using mAbs and other more recently developed immunotools, CIK cells can be readily redirected toward diverse tumor types and their cytotoxic capacity can be further amplified (27, 33, 36, 46). These combination strategies involving CIK cells will be further described in this review.

The great potential of CIK cells when compared to other cellular therapy products lies in the very limited ability to induce graft-versus-host disease (GvHD) in the allogeneic setting (47). On one hand, the expression of chemokine receptors and adhesion molecules empower the CIK cells with strong homing capacity towards the targeted tumor tissue, lymph nodes, and spleen. On the other hand, the homing and infiltration into healthy organs is minor, therefore limiting a GvHD reaction. Nishimura et al. demonstrated in Balb/c mice that both splenocytes and CIK cells are able to traffic to the GvHD target organs such as the liver, spleen and gastrointestinal tract, but CIK cells showed a lower proliferation rate, a higher number of early apoptotic events, and a higher and more sustained IFN-γ production when compared to splenocytes (47). Indeed, the IFN-γ produced by CIK cells is crucial in the protection from GvHD as CIK cells obtained from IFN-γ knock-out mice cause a lethal acute GvHD (aGvHD) (48–50).

Furthermore, it has been shown that CIK cells exhibit very low alloreactivity across human leukocyte antigen (HLA) barriers when compared to conventional donor lymphocyte infusion (DLI) (35, 51), as confirmed by preclinical and phase I/II studies where the infusion of bulk CIK cells population was well-tolerated (43, 52–56).

Altogether, a deeper insight into the molecular patterns involved in CIK cell activity not only during expansion but also during interaction with target cells, would improve our understanding of their behavior and provide opportunities to enhance their immunotherapeutic potential.

The EMA defines as “Advanced Therapy Medicinal Products” (ATMPs) any drug for human use, which are based on genes, tissues, or cells. The European Union (EU) Regulation 1394/2007 defines the guidelines for their centralized marketing authorization, supervision and pharmacovigilance, and establishes that only accredited and authorized facilities can manipulate tissues or cell therapies according to Good Manufacturing Practice (GMP) (57, 58). In particular, CAR-T cells are classified as gene therapy medicinal products and CIK cells as a cellular therapy medicinal product. The understanding of the distinctive features of CIK cells and the experience acquired with this cell therapy in clinical trials underline the extremely compelling potential of this effector cell population, placing them as an alternative therapeutic approach to CAR-T cells in specific clinical settings ( Figure 1 ).

It is well known that the manufacturing process of autologous CAR-T cells presents clinical and logistical hurdles. For the latter, the current logistic organization follows a centralized manufacturing model where the starting material, namely the patient’s cells, is collected by leukapheresis at the hospital and shipped frozen to the manufacturing site. Once produced, the ATMP is shipped back cryopreserved to the clinic and administered to the patients (11, 59). Only a few manufacturing sites are authorized for the CAR-T cell production, which results in a shortage of manufacturing slots and cumbersome logistics, leading to extremely high costs (11, 20). To move away from this centralized scheme, a point-of-care manufacturing model should be envisaged, so that the geographical proximity of the manufacturing site and of the point-of-care would limit the costs of the shipment across international borders of both the starting material and the ATMP (59). To this end, the use of an ATMP that can be conveniently manufactured in local GMP facilities would allow a more accessible therapy for patients.

Actually, CIK cells are produced in academic GMP facilities, authorized by the National competent authorities, and CIK cell treatment is now licensed in many countries (22, 59). Moreover, simple and extremely efficient expansion protocols have been recently developed, and they will be further described in this review (60, 61).

Besides the shortage of manufacturing slots, the accessibility to CAR-T cell therapy is restricted to patients who have a higher probability to achieve a clinical response and a lower risk of treatment-related toxicities or relapse. Often these are heavily pre-treated patients, and it is not always possible to generate clinically relevant doses of CAR-T cells. Thus, manufacturing failures of CAR-T cells have to be foreseen, since, in this case, alternative and bridge therapies need to be considered (62). Conversely, the expansion of CIK cells, even from heavily pre-treated patients, has been already described, reaching the clinically relevant doses with standard culturing protocols (22).

Patients are also evaluated for their risk of developing and tolerating therapy-related toxicities, which are one of the most challenging factors of CAR-T cell therapy. Upon infusion, CAR-T cells can undergo rapid in vivo proliferation that can be associated with severe and life-threatening adverse events, such as CRS, ICANS, prolonged cytopenia, and bacterial, fungal, and viral infections. CRS represents the most common side effect with a range of incidence of 50-90% among the different clinical studies (16, 17, 63–68). Thus, patients are generally admitted to the inpatient unit during the pre- and early post-infusion period to identify the onset of toxicities and manage them in the shortest time. Reference centers are required for the infusion of CAR-T cells to handle the complex scheduling logistics and patient care needs, which include the availability of an on-site hematology unit, an intensive care unit, neurological and emergency departments, a pharmacy, and blood transfusion center, in which all categories of personnel including scientists, nurses, and physicians have to be appropriately trained, thus limiting the number of hospitals that could safely administer such treatments (65, 69, 70). The scientific community is working to develop strategies to reduce toxicities and thus move toward an outpatient administration of cellular therapies, where infused patients do not require overnight hospitalization, reducing the time and costs associated with inpatient stays and patient monitoring (71). The related toxicities observed for CAR-T cells are not present in CIK cell-treated patients. Clinical trials occasionally reported the incidence of only minor side effects, which include mild hypotension, fever and chills, headaches, nausea, and vomiting that do not require intensive medical interventions (72). The use of an effector cell population with a safer profile, such as CIK cells, would thus foster the delivery of cell therapies in the outpatient setting.

An additional limitation of CAR-T cell therapy is the pricing, since a single infusion is estimated at US$ 373,000 for axi-cel and US$ 475,000 for tisa-cel, with similar prices for other authorized products (14). Moreover, considering the supportive care required in most cases to manage the therapy-associated toxicity and the fact that many patients do not remain in remission after CAR-T cell therapy, such costs are certainly much higher (14). Indeed, approximately 30-50% of patients who achieve a complete response following tisa-cel or axi-cel do not achieve long-term remission and ultimately relapse (73, 74).

On the contrary, the expansion of CIK cells requires a drastically lower financial commitment, as the manufacturing does not involve the engineering with expensive GMP-grade viral vectors in centralized manufacturing facilities (75, 76). We estimated that the cost of consumables and reagents, which include GMP-grade culture medium, plasticware, cytokines, freezing media, and antibodies, is less than €5,000 per batch. This cost has to be integrated with the costs of the personnel, quality control, GMP facility--related costs, and plant depreciation, which can vary from one academic facility to another. Although it is not possible to thoroughly compare the price of an ATMP produced in academic cell factories, such as CIK cells, and an ATMP with a global market size, such as CAR-T cells, we hypothesize that the overall cost for the expansion of a batch of CIK cells is of an order of magnitude less than CAR-T cells.

Moreover, the manufacturing of CIK cells leads to a massive expansion of the effector cells, which allows multiple doses to be prepared and patients to be treated with multiple infusions rather than a single infusion, as it is the case with CAR-T cells. Even using multiple infusions of CIK cells, the dose-limiting toxicity was not reached in clinical trials, suggesting that higher doses may actually be used (54). The administration of several cycles of CIK cells could help tailor the therapy to the patient by adjusting the cell dose, improving the control of the disease in the long term, and consolidating the clinical response avoiding relapses and related costs.

For the expansion of CAR-T cells, it is necessary to perform a lymphapheresis, a procedure that takes hours, processes volumes ranging from 3 to 25 liters, and aims at collecting a minimum of 0.6x10⁹ and a target of 2x10⁹ CD3⁺ cells (77). The status of the patient, who is often heavily pre-treated and cytopenic, the quantity and the quality of CD3⁺ cells collected, and the purity of the apheresis, in particular the quantity of contaminating myeloid cells, are all factors that affect the successful transduction and expansion of CAR-T cells, potentially leading to manufacturing failures and thus the withdrawal of the patient from the treatment (59, 77, 78). On the contrary, apheresis is not necessary for the expansion of CIK cells, since as low as 30 ml of peripheral blood is sufficient to produce enough CIK cells to repeatedly treat a 70-kg patient when administered at a concentration of about 5x10⁶ cells/kg, as already performed in previous clinical trials (54, 60).

The first preclinical studies on human CIK cells were conducted starting from peripheral blood-circulating mononuclear cells (PBMCs) of healthy donors, subsequently, from PBMCs obtained from leukemia patients (26, 79), and later from heavily pre-treated patients (60). The use of peripheral blood represents an undoubtedly easier procedure to access the starting material, and extremely smaller volumes need to be manipulated compared to apheresis, still allowing to obtain clinically relevant doses of CIK cells (60). Details on the expansion protocols will be described in a dedicated section below.

To overcome the risk of blast contamination in the drug product, and to overcome the pitfalls related to the manufacturing of autologous products, scientists have been investigating allogeneic, “off-the-shelf” CAR-T cells from healthy donors, in order to allow large-scale production, reduce costs and time for the therapy availability, and finally achieve a higher level of standardization (11, 20). The main risk of allogeneic products is the development of GvHD, due to the HLA mismatch between donor and recipient. Additionally, there could be a risk of reduced persistence and therefore efficacy of the CAR-T cell therapy whether it occurs the rejection of the allogeneic CAR-T cells (11). Two main approaches have been evaluated for B-cell malignancies, namely the use of CAR-T cells modified by TCR α-chain knockout, or the use of alternative cell populations lacking allogeneic reactivity, such as NK or γδ T cells; both strategies, however, have their own manufacturing challenges, including prolonged culture times and relative resistance to transduction (57, 80). In terms of product manufacturing, CIK cells appear to have advantages over these alternative cell populations, since they do not require selection during or after expansion, as it occurs for CAR-NK or CAR-γδ T cells, and have a higher proliferation capacity that allows to easily reach clinical relevant doses (51). Moreover, it has been reported that at the end of the expansion period, malignant cells are not present in CIK cell cultures (26, 33).

As previously described in this review, one of the most distinctive features of CIK cells when compared to other cell therapy products, is the very limited ability to induce GvHD in an allogeneic setting (47), as confirmed by several clinical trials treating patients with HLA-matched, haploidentical or unmatched CIK cells (52–56). In the first published phase I study with allogeneic CIK cells, the infusion of donor-derived CIK cells demonstrated some clinical activity in half of the patients, with aGvHD that never exceeded grade 2 (52) and even lower incidences in subsequent studies (54, 55, 81). Moreover, CIK cells have been isolated and clinically tested also when derived from umbilical cord blood (UCB) (24, 82), which represent an optimal starting material for the expansions of cells for allogeneic immunotherapy due to the lower immunogenicity; thus, a higher degree of HLA-mismatch between the recipient and donor can be tolerated (53). UCB has also the potential to provide products with “off-the-shelf” availability by cryopreservation of ex vivo expanded UCB cells (83). Several studies demonstrated that CIK cells can be efficiently expanded from small volumes of freshly collected UCB (10-15 ml), or even from the washouts of bags used for UCB transplant. UCB-derived CIK cells showed a phenotype and an antitumor activity comparable to their peripheral blood counterparts (24). Starting from a very small percentage of total nucleated cells, it was possible to expand enough cells to treat patients with multiple infusions of CIK cells (53).

The ATMP regulation requires GMP manufacturing of cells for immunotherapy. The expansion of millions or billions of cells, which is the number necessary to treat a patient, would require the use of roughly one hundred of conventional T175 flasks. To handle such a huge number of flasks is cumbersome and exhibits numerous limitations in the GMP setting. Thus, several closed systems have been developed that drastically reduce cell manipulation, thus limiting the risk of microbial contamination, as well as ensuring a higher level of standardization. Bioreactors, such as Xuri or Prodigy, rely on the mechanical rocking of culture vessels to guarantee adequate distribution of nutrients and gas exchange during cell expansion (84, 85). However, bioreactors are expensive, require large amounts of fresh medium, frequent cell density adjustments, and usually allow only one batch of cells to be expanded at a time; thus, GMP facilities need to have several bioreactors to guarantee the production of more than one batch. Additionally, bioreactors must be subjected to periodical qualification. Hence, alternative systems for GMP-compliant expansion of cells have been developed, such as Gas-permeable rapid expansion G-Rex devices (Wilson Wolf), which are disposable, GMP-compliant, quite simple closed-culture vessels that can be accommodated in standard incubators. They are provided with a flat, gas-permeable silicone membrane at the base of the vessel that allows an efficient gas exchange. The structure of the G-Rex flasks allows an increased depth of the medium above cells, thus optimizing cell proliferation and survival. Moreover, cells in G-Rex grow statically, favoring the proliferation of lymphocytes, which tend to grow in clusters (86). G-Rex culture devices have been used for the ex vivo expansion of many cell types, such as cytotoxic T cells (87, 88), tumor-infiltrating lymphocytes (89, 90), regulatory T cells (91), NK cells (92) and also CAR-T cells (93, 94).

Two reports have been published on the expansion of CIK cells in G-Rex devices. Palmerini et al. demonstrated for the first time the efficacy and feasibility of the expansion of CIK cells from a small volume of healthy donor peripheral blood using a serum-free protocol, and compared the results to the standard cultures in conventional T-flasks (60). The culture of CIK cells in G-Rex led to a significant more efficient cell expansion, with a 752-fold increase in cell number, an enrichment in CD3⁺CD56⁺ CIK cells, a higher proportion of CD8⁺ cells, and a less differentiated phenotype, which could contribute to long-lasting therapeutic responses and in vivo persistence. Notably, in the protocol described by Palmerini et al., differently from other protocols, CIK cells are efficiently expanded using a serum-free medium, which eliminates the risk of viral infection and the batch-to-batch variability, since the composition of supplements such as AB serum, frozen plasma or platelet lysate (95) is highly batch-dependent. The protocol described was upgraded to successfully expand in G-Rex CIK cells from small amounts of peripheral blood from B-cell malignancy patients with extremely low CD3⁺ counts and high tumor burden. Indeed, the early addition in the culture of Blinatumumab, a bispecific antibody that simultaneously targets CD3 on effectors and CD19 on target cells, led to the concomitant expansion of clinically relevant numbers of CIK cells, and the complete elimination of the malignant B cell fraction, without any magnetic selection or cell sorting at the beginning of the expansion (33).

Gotti et al. confirmed the feasibility of CIK cell expansion in G-Rex devices developing a protocol that further reduces culture manipulation, using lactate as an indicator of cell growth (61). CIK cells, which were expanded starting from both peripheral blood and cord blood, showed a similar phenotype for most activation markers, adhesion molecules, and checkpoint inhibitors, and a T cell subset composition similar to that of CIK cells cultured from flasks. Gotti and colleagues also demonstrated the therapeutic activity in vivo in an orthotopic model of pre-B acute leukemia, and confirmed the lack of GvHD in contrast to unmanipulated mononuclear cells (61).

Allogeneic-hematopoietic stem cell transplantation (allo-HSCT) represents a curative treatment for patients with hematologic malignancies, but relapse remains one of the leading causes of treatment failure. Strategies to induce remission after relapse include DLI, which however often induces aGvHD and, in turn, treatment failure. A phase I dose-escalating trial showed how CIK cells could be safely administered in the relapsed allo-HSCT setting, being associated with a low incidence of aGvHD. Indeed, in a cohort of 18 patients, aGvHD of grade 1 or 2 was observed in two patients, and five patients achieved or maintained a CR for more than one year after CIK infusion (81). Furthermore, in 2019, the same group proposed CIK cells as early post-transplant consolidation therapy. They showed that this treatment regimen is well tolerated, has antitumor activity, and promotes early donor chimerism without significantly affecting the rates of aGvHD in a cohort of 44 patients (NCT01392989) (97). Indeed, CIK cell therapy showed similar, or even improved, graft-versus-leukemia (GVL) effects when compared to DLI treatment in patients with a low tumor burden, observing a significantly reduced 6-month cumulative incidence of relapse (CIR) following allogenic HSCT (98).

Interestingly, Introna et al. in a phase IIA study investigated the therapeutic potential of the sequential infusion of a small amount of DLI followed by CIK cells in relapsing allo-HSCT patients. The authors demonstrated that this combinatorial approach is safe, and shows significant efficacy in patients with low tumor burden (CR: 26%) with a remarkably low severe acute and chronic GvHD incidence. In most cases, aGvHD developed after DLI infusion, and subsequent administration of CIK cells did not induce further aGvHD. In this regard, they also found that haploidentical CIK cells did not cause aGvHD even at higher doses, highlighting a significant difference compared to standard DLI treatment (54).

CIK cells have demonstrated their safety and efficacy in several clinical trials where patients with solid tumors were enrolled. According to the IRCC reports, the tumor entities investigated in most CIK cell studies are lung cancer (26.4%), hepatocellular carcinoma (HCC, 18.9%), renal cell carcinoma (RCC, 15.1%) and lymphoma (15%) (72, 75, 111).

In the study from Wang et al., non-small cell Lung Cancer (NSCLC) patients were treated at different stages with either autologous or allogeneic CIK cells, observing no significant differences in the AE between treated and control groups, with a mOS of 11 months and 8 months, respectively (56).

Moreover, in a multicenter, randomized, open-label, phase III trial with 114 HCC patients (NCT00699816), Lee et al. showed that CIK cell treatment induced a 14-months median disease-free survival (mDFS) improvement compared to control group, which did not receive any CIK cells infusions (44 months vs 30 months). The CIK cell immunotherapy reduced all types of tumor recurrence and, moreover, the reported AEs were mild to moderate (grade 1 or 2) (99).

The efficacy of CIK cells for the treatment of HCC was confirmed also by Chen et al. in a retrospective study on 102 patients who received CIK treatment after curative resection, observing a significant improvement in survival. Specifically, the 1-, 3-, and 5-year disease-free survival (DFS) rates were 85.3%, 68.2%, and 60.4% respectively, with a marginal significant difference between CIK and control group (P=0.055). Also, the CIK group exhibited significantly higher OS than the control group, underlying the efficacy of CIK cell therapy (100).

The low toxicity and high feasibility of CIK cell treatment have encouraged the study of novel combination therapies, which are showing higher efficacy compared to monotherapy. The effects of the combination of CIK cells and chemotherapy on metastatic Nasopharyngeal Carcinoma (NPC) was investigated in a retrospective study with 222 patients, among whom 112 received gemcitabine plus cisplatin (GC) regimen chemotherapy followed by at least 4 cycles of CIK immunotherapy. Over the 3-year follow-up period, the GC plus CIK therapy group had significantly higher survival rates than the GC alone group (mPFS 21 months vs 15 months; mOS 32 months vs 23 months), which indicated that CIK adjuvant immunotherapy could effectively maintain disease stability and prolonged survival in advanced metastatic NPC patients (101).

Similarly, ovarian cancer patients treated with CIK cells combined with a chemotherapeutic regimen exhibited a significantly more favorable OS and PFS than control group who received only chemotherapy (mOS, 63.6 vs 39.6 months and mPFS, 41.6 vs 26.1 months) (102).

In addition, a study conducted by Wang et al. revealed a significant improvement in disease-control rate (DCR) in advanced pancreatic cancer patients (68.0%) treated with a combination of CIK cells and gemcitabine and/or the oral chemotherapy S-1, compared with the control chemotherapy group (29.8%) (103).

In a multi‐center, Phase III trial on a total of 180 patients with newly diagnosed glioblastoma, autologous CIK cells infusion combined with standard radiotherapy‐temozolomide chemoradiotherapy significantly prolonged the mPFS of 8.1 months in the CIK group compared with 5.8 months in the control group. Grade 3 or higher adverse events, health‐related quality of life and performance status did not differ between the two groups (104).

Immunotherapy with anti-PD-1 antibody is revolutionizing the treatment of many cancers; however, many patients do not have tumor responses to PD-1/PD-L1 inhibitors in part due to inadequate intra-tumoral T cell infiltration. CIK cells can selectively infiltrate the tumor tissue and secrete INF-γ (47), thus inducing a local inflammatory microenvironment and ultimately anti-PD-1 therapy effectiveness (112). The efficacy of CIK cells combined with anti-PD-1 therapy was investigated in metastatic RCC (105) and NSCLC (106) patients observing an mPFS of 15 and 19.3 months, respectively.

To improve CIK cell-based therapies, preclinical studies have shown that the combination with pulsed dendritic cells (DC) leads to a significant improvement in antitumor activity when compared with CIK or DC treatment alone. Indeed, DC-CIK immunotherapy allows delivery of both DCs, which have potent capacity for antigen presentation and induction of adaptive immune responses, and CIK cells with cytotoxic activity. Moreover, the DC-CIK interaction stimulates the proliferation and antitumor activity of CIK cells through the secretion of IL-12, IFN-γ, and other cytokines (113, 114). Indeed, in a 10 year follow-up study, Lin et al. showed that DC-CIK treatment can significantly improve DFS and OS compared to the control group, in patients with stage IV breast cancer who only rely on chemotherapy regimen option (5-year DFS 42% vs 30% and 5-year mOS 44% vs 29%). Additionally, multivariate analysis confirmed that DC-CIK therapy significantly and independently reduced the risk of post-operative disease progression and patient death (107). Also, a comparable improvement in patient survival and therapeutic efficacy was highlighted in a prospective study in advanced pancreatic cancer patients (NCT01781520), where the combination of oral chemotherapy S-1 and DC-CIK immunotherapy resulted in circulating immune effectors modulation and prolonged 6-month OS and PFS (62.2% and 41.6%) when compared with the DC-CIK (18.2% and 9.09%) and chemotherapy (25% and 0%) groups (108). Interestingly, Li et al. analyzed 70 studies involving 6743 colorectal cancer (CRC) patients in a systematic review and meta-analysis. Results indicate that CIK or DC-CIK therapy improves OS, PFS, and ORR compared to standard treatment, without increasing toxicity. Overall, the study from Li et al. reported that, co-treatment with DCs did not improve clinical outcomes over CIK therapy alone. These findings underline the intrinsic therapeutic efficacy of CIK cells and suggest that in this setting the combination with DC therapy may not provide additional benefit (109).

CIK cells have been also used as a promising platform for genetic modification with CAR molecules targeting CD123⁺/CD33⁺ acute myeloid leukemia (AML) (115), CD19⁺ acute lymphoblastic leukemia (ALL) (116), ErbB2⁺ rhabdomyosarcoma (RMS) (117), CSPG4⁺ (118) and CD44v6⁺ (119) soft-tissue sarcomas (STS), showing an increased cytotoxicity compared to untransduced CIK cells. CAR-modified CIK cells would exert anti-tumor activity both by intrinsic NKG2D-mediated and CAR-specific targeting (120). In 2020, Magnani et al. reported for the first time the administration of CAR-engineered CIK cells in a clinical setting (110). The multicentric phase I/II clinical trial (NCT03389035) aimed to assess the safety and feasibility of infusing allogeneic CARCIK-CD19 in patients with Acute B Lymphoblastic Leukemia (B-ALL) relapsed after HSCT. Four pediatric and nine adult patients were infused with a single dose of CAR-CIK cells. Six out of seven patients who received the highest doses achieved CR and CR with Incomplete blood Count Recovery (CRi) at day 28. Grade 1 and grade 2 CRS toxicities were reported only at the highest CARCIK-CD19 dose. Impressively, no GvHD, neurotoxicity, or dose-limiting toxicities were observed (110). This study demonstrated that even when modified with CAR molecules, CIK cells show a better safety profile when compared to CAR-T lymphocytes.