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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Biol Blood Marrow Transplant. Author manuscript; available in PMC 2012 November 1.
Published in final edited form as:
PMCID: PMC3175285

Adoptive Immunotherapy with Cytokine Induced Killer Cells For Patients with Relapsed Hematologic Malignancies After Allogeneic Hematopoietic Cell Transplantation


Donor leukocyte infusions induce remissions in some patients (pts) with hematologic malignancies who relapse after allogeneic hematopoietic cell transplantation (HCT). However, graft vs host disease (GVHD) remains the major complication of this strategy. Cytokine induced killer (CIK) cells are a unique population of cytotoxic T lymphocytes that express the CD3+CD56+ phenotype and show marked upregulation of the NK cell receptor, NKG2D (CD314). CIK cells are non-MHC restricted, and NKG2D dependent in target recognition and cytotoxicity. We explored the feasibility of ex vivo expansion of allogeneic CIK cells for pts with relapsed hematologic malignancies after allogeneic HCT. Eighteen patients with a median age of 53 years (range 20–69) received CIK cell infusions based on CD3+cells/kg at escalating doses of 1×107 (n=4), 5×107 (n=6) and 1×108 (n=8). The median expansion of CD3+ cells was 12 fold (range 4–91 fold). CD3+CD56+ cells represented a median of 11% (range 4–44%) of the harvested cells with a median 31 fold (range, 7–515 fold) expansion. Median CD3+CD314+ expression was 53% (range, 32–78%) of harvested cells. Significant cytotoxicity was demonstrated in vitro against a panel of human tumor cell lines. Acute GVHD, grades I–II, were seen in 2 patients and 1 patient has limited chronic GVHD. After a median followup of 20 months (range 1–69 months) from CIK infusion, the median overall survival was 28 months and median event free survival was 4 months. All deaths were due to relapsed disease, however, 5 patients had longer remissions after infusion of CIK cells than from allogeneic transplantation to relapse. This form of adoptive immunotherapy is well tolerated and induces a low incidence of GVHD supporting further investigation as an upfront modality to enhance GVT responses in high risk patient populations.

Keywords: allogeneic, cytokine induced killer, transplantation, hematologic


Allogeneic hematopoietic cell transplantation (HCT) is a curative treatment modality for patients with malignant diseases. Relapse, however, remains one of the leading causes of treatment failure and typically portends a very poor prognosis. Strategies to induce a remission after relapse include withdrawal of immunosuppression medications and/or donor leukocyte infusions (DLI). Such approaches attempt to maximize the graft vs tumor (GVT) effect conferred by donor T cells. Yet, responses are variable and acute graft vs host disease (GVHD) is a major cause of treatment failure.

Various forms of adoptive immunotherapy have been explored to reduce the incidence of acute GVHD associated with donor leukocyte infusions. Such approaches have included escalating doses of T cells, CD8+ depleted DLI, antigen specific CTLs and natural killer (NK) cells13.

Cytokine-induced killer cells (CIK cells) are cytotoxic effector T cells which are readily expandable and express in addition to the T cell marker, CD3+ markers typically associated with NK cells such as CD56+ and NKG2D. CIK cells are generated by the in vitro culture of peripheral blood lymphocytes with IFN-γ, IL-2 and anti-CD3. T cell expansion and activation occurs resulting in cytolytic cells which recognize targets through NKG2D4. NKG2D is an activating receptor expressed on all NK cells and also serves as a T-cell costimulatory molecule which augments cytotoxic and proliferative responses of T cells upon encountering antigen56. CIK cell-mediated cytotoxicity is MHC unrestricted and T cell receptor independent as target killing occurs through NKG2D-mediated recognition. In preclinical studies, CIK cells have shown potent activity against several tumor cell lines and with a markedly reduced capacity of inducing GVHD in murine models4, 7. CIK cells have also been shown to traffic to tumor sites where they persist for 10–14 days and are associated with reduction in tumor burden. CIK cells generated from acute myelogenous leukemia (AML) patients have demonstrated cytotoxic activity against both autologous and allogeneic leukemic blasts8. Additionally, CIK cells show minimal if any cytotoxicity against normal tissues including CD34+ stem cells, do not suppress marrow engraftment in vivo and induce minimal GVHD in allogeneic models9. Compared to CD3CD56+ LAK cells, CIK cells have demonstrated more potency against multi-drug resistant tumor cell lines and are more readily expandable10. Because of these attributes demonstrated in murine model systems, with human cell lines and fresh tumor samples we developed conditions for the expansion of CIK cells on a clinical scale under Good Manufacturing Practice (GMP) conditions. We have previously reported on the use of autologous CIK cells11. Here, we report the results of a phase I feasibility study in which escalating doses of CIK cells derived from HLA matched sibling donors were administered to recipients with hematologic malignancies who relapsed after allogeneic HCT.


Eligibility criteria

Patients with hematologic malignancies who had relapsed disease after undergoing allogeneic hematopoietic cell transplantation from a matched sibling donor were eligible for this study. Patients with chronic myelogenous leukemia were eligible only if persistent disease was demonstrated after prior DLI of at least 1 × 108 cells/kg. Patients could not have active GVHD and must have been either off all immunosuppressive medications or be taking a stable regimen. Adequate organ function was required as defined by 1) serum creatinine of < 2 mg/dl or creatinine clearance of > 50 cc/minute and 2) direct bilirubin of < 3 mg/dl or transaminases < 3 times the upper limit or normal. Exclusion criteria included no active infections. All patients provided written informed consent prior to enrollment on this clinical trial. The protocol was approved by the institutional review board of Stanford University and the conduct of this trial was in accordance with the Declaration of Helsinki.

Study Design

This was a single institution open-label phase I clinical trial to evaluate the feasibility and safety of allogeneic CIK cells. Although efficacy was not a primary objective of this trial all patients were followed for objective outcomes. The hypothesis was that human CIK cells expanded ex vivo will retain anti-tumor activity but reduce the incidence and severity of GVHD associated with unmanipulated DLI. The primary objectives of this trial were 1) to determine the feasibility of expanding allogeneic CIK cells suitable for clinical application 2) to determine the infusional toxicity of ex vivo expanded allogeneic CIK cells in patients with recurrent or refractory disease following allogeneic HCT 3) to determine the incidence of GVHD following infusion of allogeneic CIK cells and 4) to determine the maximum tolerated dose of allogeneic CIK cell infusion.

In this dose escalation study, the starting dose of CIK cells was 1 × 107 CD3+ cells/kg followed by escalation to 5 × 107 CD3+ cells/kg with the highest planned dose being 1 × 108 CD3+ cells/kg. In the usual Phase I design, 3 patients were planned to be enrolled at each cell dose level. If DLT was not seen in the 3 planned patients, then the next higher cell dose level was administered. If a dose limiting toxicity (DLT) was seen, the cohort was expanded to 6 patients. The dose was increased to 5 × 107 expanded cells/kg and 1 × 108 expanded cells/kg in successive escalations based on the absence of significant infusional or other toxicities or GVHD. To progress to the next dose level, there must have been no grade 3 or greater toxicities as defined by the Common Toxicity Criteria and no evidence of acute GVHD beyond grade II.

Donor apheresis and CIK cell expansion

The matched sibling donor who had donated the graft for the earlier allogeneic HCT was used as the CIK cell donor. The donor underwent unmobilized apheresis and up to one liter of autologous plasma was collected from each donor to supplement the expansion culture. Sufficient quantities of cells (>1.6 × 109 cells) were collected to allow inoculation of the CIK cell expansion cultures. The cultures were inoculated on the same day as collection with a minimum of 6.0 × 108 cells and a maximum of 7.5 × 108 cells added to Aim-V medium (Invitrogen, Grand Island, NY) supplemented with 5% autologous (donor) heat-inactivated plasma and 2 × 105 IU IFN-γ (Actimmune®; InterMune, Brisbane, CA) in a continuous perfusion biochamber (Aastrom Biosciences, Ann Arbor, MI) maintained at 37° with 20% O2 and 5% CO2. During the initial activation period of the cultures, medium perfusion did not occur. This continuous perfusion culture was performed for the first 11 patients and thereafter, gas permeable culture bags were used.

The following day (day+1), expansion was initiated with muronmab-CD3 (Orthoclone®,OKT3, Orthobiotech, Raritan, NJ) at 50 ng/mL and interleukin-2 (Proleukin®; Chiron, Emeryville, CA) at 300 IU/m. Both agents were added to the continuous perfusate of the biochamber with AimV medium containing 5% inactivated autologous plasma and 300 U/mL IL-2 which commenced at a rate of 0.104 mL/minute, equivalent to 150 mL/day. This rate of medium flow resulted in a 50% exchange of the culture volume each day. To assess the accuracy of the perfusion rate, the cultures were sampled on every 3rd day of culture beginning on day+4 and lactate levels were measured as indicator of culture health. If the lactate level measured was above 0.8 mg/mL (9 mMol), the medium perfusion rate was increased according to pre-specified guidelines until the lactate level reached below 0.8 mg/mL. The period of expansion with IL-2 was maintained for 21–28 days. The static culture bags were fed every 2–3 days and the volume was increased as needed to maintain cell density at 2–4 × 106/ml.

Samples were removed from each culture bag for Mycoplasma detection by PCR five days prior to harvest. Additional samples were drawn from each bag after the final addition of medium and IL-2 three days prior to culture harvest for sterility testing of bacterial and fungal contaminants. On harvest day, samples were removed for final release tests including WBC count and viability, gram staining, endotoxin testing and T cell content determination. Sterility testing was performed by the Stanford Hospital Clinical Microbiology Laboratory.

Culture conditions and culture harvest

Cultures were maintained in either Aastrom Replicell biochambers or Baxter LifeCell culture bags at 37°C and 5% CO2 over 21–28 days, as indicated. Cultures for patients 1–11 were expanded in Aastrom Replicell biochambers (Aastrom Biosciences, Ann Arbor, MI) modified to allow 10–15 liters of medium perfusion per biochamber over the duration of the culture. Each biochamber was inoculated with 750 × 106 peripheral blood cells collected by apheresis and cytokines added as described above. On day +4 of incubation, continuous perfusion of the culture began with fresh medium supplemented with IL-2 at 300 IU/ml. The infusion rate was gradually increased over the course of the culture based on lactic acid levels in medium perfused from the biochambers. Cultures for patients 12–18 were expanded in LifeCell culture bags (Baxter Healthcare, Deerfield, IL) once Aastrom discontinued the biochambers for this potential indication. Cultures were inoculated in multiple 1 liter LifeCell bags each containing 200 ml of medium with 4 × 108 peripheral blood cells. Cytokines were added as described above. On day +4 and every third day thereafter, cultures were assessed for cell density and viability. Additional culture medium supplemented with sufficient IL-2 to maintain ≥300 IU IL-2/ml was added to LifeCell bags to adjust cell density to ≥ 2 × 106 and 4 × 106 viable cells/ml. If cell density did not require additional volume, IL-2 alone was added at 300 IU/ml of culture volume. When bag volumes exceeded 500 ml, the contents were transferred to 3 liter LifeCell bags and maintained as before until harvest.

After 21 or 28 days, cells cultured in biochambers were collected on the Aastrom Cell Processor and washed with Normosol-R, pH 7.4 (Baxter Healthcare, Deerfield, IL) supplemented with 1% Human serum albumin (CLS Behring, King of Prussia, PA or Telacris, Research Triangle Park, NC). Cells cultured in LifeCell bags, were pooled, volume reduced, and washed with Normosol + 1% HSA. Harvested cells were concentrated to 200–300 ml for infusion and maintained at ambient temperature until infusion. Cells were infused within 6 hours of harvest and were not cryopreserved. Samples were also taken for phenotyping and cytotoxicity assessments. The infused cell dose was based on total CD3+ cell content and adjusted to the cohort dose.

Cell analysis and cytotoxicity assays

Cell counts were performed on an impedance counter (Beckman Coulter) and viability by Trypan blue exclusion. Cell phenotypes were assessed by flow cytometry of inoculating cells and harvested cell cultures using monoclonal antibodies to CD3 (clone HIT-3a), CD56 (clone NCAM) for CIK cell enumeration. Additional phenotyping included CD4 (RPA-T4), CD8 (clones HIT-8A and SK1), and CD314 (clone 1D11). CD314+ designates KLRK1, the gene encoding NKG2D.

Killing of target cell lines including SUDHL-4, OCI-Ly8, DB and Jurkat by CIK cell cultures for patients 1–16 was assessed by chromium release assays as previously described11. For patients 17 & 18, cytotoxicity against the target cell lines was assessed using the ApoLogix carbofluorescein poly-caspase detection assay (Cell Technology, Mountain View, CA) according to the manufacturer's directions.

Infusion of CIK cells

CIK cells harvested from the expansion cultures and meeting the release criteria were infused within 6 hours of the harvest. The cells were infused through a central venous catheter or peripheral intravenous line of at least 18 gauge and were infused over a period of 30 minutes. EKG tracings were obtained prior to the CIK cell infusion and patients had vital signs monitored every 30 minutes following infusion for at least 2 hours.

Study assessments

Post-infusion toxicity evaluations were performed in the outpatient setting on days +1, +3, +7 +14, +21 and +56. VNTR analysis was performed prior to infusion and at days +7, +21 and +56 following infusion of CIK cells. PET-CT scanning and/or bone marrow biopsies were performed at regular intervals following infusion of CIK cells to assess clinical responses. These tests were performed at a minimum at day+30 and +60 following CIK infusion.


Patient characteristics

Eighteen patients received CIK cell infusions at 3 dose levels. See Table 1. The median age was 53 years (range 20–69 years). The diagnoses of these pts included non-Hodgkin lymphoma (n=5,) acute myeloid leukemia (n=3), multiple myeloma (n=3), chronic lymphocytic leukemia (n=2), acute lymphoblastic leukemia (n=2), myelodysplastic syndrome (n=2) and Hodgkin lymphoma (n=1). All patients had relapsed after allogeneic HCT using a matched sibling donor. Twelve patients had received a reduced intensity conditioning (RIC) regimen and 6 patients had received myeloablative regimens. The median time from HCT to relapse was 12 months (range, 3 – 142 months) and the median time from relapse to CIK infusion was 4 months (range, 1–34 months). With the exception of one CLL patient and one MDS patient, all patients received some form of cytoreductive therapy including chemotherapy, corticosteroids, surgical resection or DLI prior to CIK infusion. At the time of CIK infusion, ten patients (56%) were in CR, four patients were in PR, 1 patient with acute promyelocytic leukemia had a molecular relapse that failed to response to DLI and 3 patients had progressive disease.

Table 1
Patient Characteristics

Cell Characterization and Expansion

Table 2 details the viability and cell counts of the 18 harvested products after expansion and activation that were prepared under GMP conditions. The median viability of the cultured products was 86.5% (range 70% – 95%) with a median expansion of CD3+ and CD3+CD56+ cells being 12 fold (range 4 – 91 fold) and 31 fold (range 7 – 515 fold), respectively. The median CD3+CD314+ or NKG2D-expressing population was 53% (range 32% – 78%). Figure 1 is a contour plot from the product of one donor depicting the expansion of CD3+ cells expressing CD56+ and CD314+ over the 21 day culture period.

Figure 1
FACS analysis of PBMCs after 21 days of culture illustrating the increase in number of cells expressing CD3+C56+ and CD3+CD314+ phenotypes.
Table 2
Cell Expansion and Characterization

We performed cytotoxicity assays with the expanded CIK cells against four tumor targets: SUDH-L4, OCI-Ly8, DB and Jurkat. Approximately 40%–60% specific killing at an effector-target ratio of 40:1 was observed against all four cell lines with the greatest percentage of killing seen against Jurkat, a T cell leukemia target (Figures 2a and and2b).2b). Due to ineffective 51Cr labeling or lack of cell expansion, cytotoxicity was not assessed in all donors against all four cell lines and the number of donors tested against each cell line were as follows: SUDH-L4 (n=16), OCI-Ly8 (n=17), DB (n=15) and Jurkat (n=16).

Figure 2a
In vitro cytotoxic activity of CIK cells by specific tumor target. Killing of target cell lines including SUDH-L4, OCI-Ly8, DB and Jurkat at E:T ratio of 40:1
Figure 2b
In vitro cytotoxic activity of CIK cells by each patient against four tumor targets: SUDH-L4, OCI-Ly8, DB and Jurkat at E:T ratio of 40:1

CIK Administration and Clinical Responses

The CIK cell doses administered based on CD3+cells/kg were 1 × 10^7 (n=4), 5 × 10^7 (n=6) and 1 × 10^8 (n=8). The median follow up duration from the time of CIK infusion was 20 months (range 1–69 months) while the median event free survival (EFS) and overall survival (OS) from the time of CIK infusion was 4 months and 28 months, respectively. For the twelve patients who received CIK cells while in a CR, eleven patients have relapsed with a median time to relapse of 6 months (range, 2 –37 months). Patient #3 had a molecular relapse of acute promyelocytic leukemia at day +93 after non-myeloablative HCT. She had previously received 2 DLI infusions without response. This patient achieved a molecular response approximately 4 weeks after CIK infusion. The median follow-up time for all patients from relapse after allogeneic HCT was 29 months, (range, 5–74 months) while the median EFS and OS from the time of relapse after allogeneic HCT was 13 months and 37 months, respectively. See Table 3 for summary of Patient Outcomes.

Table 3
Patient Outcomes

Of the 18 patients enrolled on this trial, five patients achieved or maintained a CR for greater than 1 year after CIK infusion with one patient remaining in CR at 32 months after CIK infusion. Four of these five patients had lymphoid malignancies and one patient had AML. The patient who has sustained longest CR post-CIK infusion is a 33 yo male with multiply relapsed mantle cell lymphoma. This patient underwent a myeloablative allogeneic HCT in 1995 while in CR2. Twelve years later, he had an isolated relapse in a supraglottic node then received involved field radiation therapy followed by CIK infusion. He has maintained a CR for 32 months to date. Another notable case involved a 39 yo male with primary refractory AML who relapsed 3 months after myeloablative allogeneic HCT. He received salvage chemotherapy followed by CIK infusion. His remission lasted 20 months until disease relapse and he expired soon after. A long standing remission was seen in a 56 yo female who underwent autologous HCT in 1995 for relapsed diffuse large cell lymphoma (DLCL). She relapsed 3 years later and received salvage chemotherapy followed by RIC allogeneic HCT. She relapsed in the left axilla 5 months later, underwent excisional biopsy followed by CIK infusion. She sustained a CR for 37 months. A 68 yo female with MDS received a CIK infusion approximately 6 months after RIC allogeneic HCT for persistent disease. A bone marrow biopsy performed 3 months after CIK infusion showed no evidence of MDS.

Four patients, 2 patients with myeloma and two patients with CLL, rapidly progressed within 2 months after CIK infusion. All four patients had active disease at the time of CIK infusion despite receiving cytoreductive therapy prior to CIK infusion.

With a median followup time of 20 months (range, 1– 69 mos) for all patients, ten patients are alive, 2 who remain disease free and eight are alive with relapsed disease after CIK infusion. Eight patients have expired with progressive disease being the primary cause of death.

Chimerism and Serum Immunophenotyping

Eleven patients retained full donor chimerism after relapse from allogeneic HCT and this status did not change after CIK infusion. Five patients exhibited mixed donor chimerism (defined by > 5% and <95% CD3+) at the time of CIK infusion. Of these five patients, one patient with MDS who had mixed chimerism with 82% donor CD3+ chimerism at the time of CIK infusion converted to full donor chimerism showing 100% CD3+ approximately 2 months after CIK infusion. This patient was infused at the highest CIK dose level. The mixed donor chimerism status of the other 4 patients have remained stable. Chimerism data was not obtained on 2 patients.

Graft vs Host Disease and Adverse Events

Acute GVHD was seen in two patients. One patient, at dose level #2, developed grade II skin GVHD approximately 98 days after CIK infusion. The GVHD resolved with topical corticosteroids alone. At the 3rd dose level, one patient developed grade II hepatic GVHD with a concomitant skin rash on the trunk and bilateral upper extremities approximately 7 weeks after CIK infusion. A skin biopsy and liver biopsy were performed and results from both organs were equivocal for either GVHD or a drug reaction. The patient was treated with corticosteroids, tacrolimus and mycophenolate mofetil and achieved partial resolution of GVHD. Chronic GVHD was seen in only one patient. This patient developed limited chronic GVHD on day+ 123 after CIK infusion that manifested as joint stiffness and aches that responded to oral corticosteroids which were eventually discontinued.

One patient at the first dose level experienced a transient grade 3 ventricular arrhythmia that spontaneously resolved without intervention during the infusion of CIK cells. At the 2nd dose level, one patient also experienced syncope approximately 3 weeks after CIK infusion. A comprehensive evaluation for the etiology of the syncope revealed inducible sustained ventricular tachycardia during cardiac electrophysiologic studies which led to placement of an implantable defibrillator one month after CIK infusion. The arrhythmia did not recur after placement of the device. This patient also experienced a transient rise in hepatic transaminases that resolved without intervention. Since these 2 DLTs were seen, the cohort was expanded to a total of 6 patients. No other DLTs were seen at this dose level and we proceeded to the 3rd dose level of 1 × 108 CD3+cells/kg. Eight patients to date have been infused at this dose and no DLTs have been observed at the 3rd dose level.


Relapse remains one of the leading causes of treatment failure after allogeneic HCT and typically carries a poor prognosis. DLI is a strategy that is offered to relapsed patients in this situation but this form of adoptive immunotherapy can incur significant toxicity as acute GVHD and marrow aplasia are the leading causes of nonrelapse mortality after DLI12. Further, although DLI has been extremely effective in the treatment of CML, this treatment modality has been less effective in the treatment of other hematologic malignancies1315.

Our Phase I study demonstrated the feasibility and safety of allogeneic CIK cell infusions in patients with relapsed hematologic malignancies. This Phase I dose escalation trial reached a planned maximal cell dose of 1 × 108 CD3+ cells/kg as the maximum tolerated dose. We observed only 2 cases of acute GVHD, grade II, that responded to topical or systemic corticosteroids and one patient developed limited chronic GVHD. The most serious adverse events seen were at the lowest cell dose whereas 2 patients developed transient ventricular arrhythmias. The etiology of the arrhythmias was never elucidated.

The low incidence of GVHD in this clinical study parallels the low GVHD incidence seen in preclinical models as we have previously demonstrated that the adoptive transfer of allogeneic CIK cells in a rodent model induced minimal GVHD9. With the use of bioluminescence imaging, it was shown that luciferase-expressing CIK cells generated from splenocytes exhibited traffic patterns similar to conventional T cells. However, compared to the conventional T cells, the CIK cells infiltrated GVHD target tissues much less, demonstrated a slower division rate, were less susceptible to apoptosis and produced high amounts of interferon gamma, a cytokine known to confer a protective effect against acute GVHD16.

As this was a Phase I feasibility study in a very heterogeneous population of patients, it is difficult to draw conclusions regarding efficacy. For the 12 patients who received CIK infusions while in CR, the median time to progression was 6 months. This remission duration is notable considering that this was a high risk population with the most durable remissions observed in patients with lymphoid malignancies. From the time of CIK infusion, the median EFS and OS were 4 months and 28 months, respectively. Although almost all patients in this study underwent some form of cytoreduction prior to CIK infusion, five patients had a longer time to progression/relapse after CIK infusion compared to time to progression/relapse immediately after allogeneic HCT. CIK cell infusions had no impact on patients with rapidly progressive disease at the time of infusion. The impact of CIK infusion on donor chimerism was not assessable in most patients since eleven patients already exhibited full donor chimerism at the time of CIK infusion. However, one of 5 patients with mixed chimerism converted to full donor chimerism approximately 2 months after CIK infusion without the development of GVHD.

A previously reported Phase I study of similar design also described clinical responses in patients who had received allogeneic CIK infusions after post-HCT relapse17. Introna et al. administered allogeneic CIK infusions in 11 patients with hematologic malignancies and reported the achievement of a completed remission in 3 patients with 1 remission lasting for over 2 years at the time of the report. Two of the 3 patients converted to full donor chimerism but also developed extensive cutaneous GVHD that commenced soon after the documented clinical response. Unlike our study where patients received only 1 CIK cell infusion, most patients received multiple sequential infusions with cell doses ranging from 3 × 106 to 15 × 106 CD3+cells/kg.

With regards to feasibility of culture and expansion, we generated a median 12 fold expansion of CD3+ cells and 31 fold expansion of CD3+CD56+ cells, similar to the expansion data reported by Introna et al. Additionally, we characterized the percentage and number of CD3+CD314+ cells with 53% of cells expressing this phenotype after culture. In vitro antitumor activity against various tumor targets was also confirmed in our study and as reported previously by our group and others9, 1718.

Other groups have manipulated CIK cells with the intent of improving specificity and enhancing cytotoxicity against various tumor targets. The cytotoxicity of CIK cells was significantly increased against B-NHL targets when co-cultured with the anti-CD20 antibodies, rituximab and GA10119. Other investigators have incorporated bispecific antibodies with the goal of redirecting CIK cells and increasing killing against primary ovarian cancer cells and against B-cell ALL2021. The expansion of cord-blood derived CIK cells represents another promising source of adoptive immunotherapy with potential application for patients with relapsed malignancies after umbilical cord blood transplantation22. We have also utilized CIK cells to deliver an oncolytic virus to the tumor bed in a preclinical model with remarkable efficacy23. This approach is being developed for clinical translation.

In summary, we have presented the feasibility and safety of allogeneic CIK cell infusions in patients with relapsed hematologic malignancies. Although CIK cells require expansion under specific culture conditions, we and other groups have shown the feasibility of this approach and have obtained similar results regarding low observed toxicity and high cell yields. Although it is difficult to assess clinical responses in this setting due to the variable and high risk nature of the patient population under treatment, it should be noted that several responses extended past one year in this high risk patient population. As seen with DLI, CIK cells appear to induce the longest response duration in patients who had minimal residual disease at the time of infusion. The comparable efficacy of CIK cells to DLI in the relapsed setting is difficult to determine but our data and those of others have confirmed lengthy clinical responses accompanied with a low incidence of acute and chronic GVHD compared to DLI17. Thus, our results suggest that CIK cells exerted a biological effect and warrants further investigation. We are conducting two follow-on studies with infusion of CIK cells after allogeneic HCT with reduced intensity conditioning. One study involves high risk MDS patients who receive CIK cells pre-emptively on day +42 and a second trial is eligible to patients with CLL who demonstrate mixed chimerism. In this second trial, both chimerism and molecular disease burden will be assessed.


Supported in part by grant PO1 CA049605 from the National Institutes of Health, Bethesda, MD


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1. Molldrem JJ, Lee PP, Wang C, et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Nat Med. 2000;6:1018–23. [PubMed]
2. Soiffer RJ, Alyea EP, Hochberg E, et al. Randomized trial of CD8+ T-cell depletion in the prevention of graft-versus-host disease associated with donor lymphocyte infusion. Biol Blood Marrow Transplant. 2002;8:625–32. [PubMed]
3. Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science. 2002;295:2097–100. [PubMed]
4. Schmidt-Wolf IG, Negrin RS, Kiem HP, Blume KG, Weissman IL. Use of a SCID mouse/human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cell activity. J Exp Med. 1991;174:139–49. [PMC free article] [PubMed]
5. Groh V, Rhinehart R, Randolph-Habecker J, Topp MS, Riddell SR, Spies T. Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virus-infected cells. Nat Immunol. 2001;2:255–60. [PubMed]
6. Karimi M, Cao TM, Baker JA, Verneris MR, Soares L, Negrin RS. Silencing human NKG2D, DAP10, and DAP12 reduces cytotoxicity of activated CD8+ T cells and NK cells. J Immunol. 2005;175:7819–28. [PubMed]
7. Baker J, Verneris MR, Ito M, Shizuru JA, Negrin RS. Expansion of cytolytic CD8(+) natural killer T cells with limited capacity for graft-versus-host disease induction due to interferon gamma production. Blood. 2001;97:2923–31. [PubMed]
8. Linn YC, Lau LC, Hui KM. Generation of cytokine-induced killer cells from leukaemic samples with in vitro cytotoxicity against autologous and allogeneic leukaemic blasts. Br J Haematol. 2002;116:78–86. [PubMed]
9. Nishimura R, Baker J, Beilhack A, et al. In vivo trafficking and survival of cytokine-induced killer cells resulting in minimal GVHD with retention of antitumor activity. Blood. 2008;112:2563–74. [PubMed]
10. Schmidt-Wolf IG, Lefterova P, Johnston V, et al. Sensitivity of multidrug-resistant tumor cell lines to immunologic effector cells. Cell Immunol. 1996;169:85–90. [PubMed]
11. Leemhuis T, Wells S, Scheffold C, Edinger M, Negrin RS. A phase I trial of autologous cytokine-induced killer cells for the treatment of relapsed Hodgkin disease and non-Hodgkin lymphoma. Biol Blood Marrow Transplant. 2005;11:181–7. [PubMed]
12. Kolb HJ, Schattenberg A, Goldman JM, et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood. 1995;86:2041–50. [PubMed]
13. Porter D, Levine JE. Graft-versus-host disease and graft-versus-leukemia after donor leukocyte infusion. Semin Hematol. 2006;43:53–61. [PubMed]
14. Huff CA, Fuchs EJ, Smith BD, et al. Graft-versus-host reactions and the effectiveness of donor lymphocyte infusions. Biol Blood Marrow Transplant. 2006;12:414–21. [PubMed]
15. Schmid C, Labopin M, Nagler A, et al. Donor lymphocyte infusion in the treatment of first hematological relapse after allogeneic stem-cell transplantation in adults with acute myeloid leukemia: a retrospective risk factors analysis and comparison with other strategies by the EBMT Acute Leukemia Working Party. J Clin Oncol. 2007;25:4938–45. [PubMed]
16. Yang YG, Qi J, Wang MG, Sykes M. Donor-derived interferon gamma separates graft-versus-leukemia effects and graft-versus-host disease induced by donor CD8 T cells. Blood. 2002;99:4207–15. [PubMed]
17. Introna M, Borleri G, Conti E, et al. Repeated infusions of donor-derived cytokine-induced killer cells in patients relapsing after allogeneic stem cell transplantation: a phase I study. Haematologica. 2007;92:952–9. [PubMed]
18. Linn YC, Hui KM. Cytokine-induced killer cells: NK-like T cells with cytotolytic specificity against leukemia. Leuk Lymphoma. 2003;44:1457–62. [PubMed]
19. Pievani A, Belussi C, Klein C, Rambaldi A, Golay J, Introna M. Enhanced killing of human B-cell lymphoma targets by combined use of cytokine-induced killer cell (CIK) cultures and anti-CD20 antibodies. Blood. 2011;117:510–8. [PubMed]
20. Chan JK, Hamilton CA, Cheung MK, et al. Enhanced killing of primary ovarian cancer by retargeting autologous cytokine-induced killer cells with bispecific antibodies: a preclinical study. Clin Cancer Res. 2006;12:1859–67. [PubMed]
21. Marin V, Dander E, Biagi E, et al. Characterization of in vitro migratory properties of anti-CD19 chimeric receptor-redirected CIK cells for their potential use in B-ALL immunotherapy. Exp Hematol. 2006;34:1219–29. [PubMed]
22. Introna M, Franceschetti M, Ciocca A, et al. Rapid and massive expansion of cord blood-derived cytokine-induced killer cells: an innovative proposal for the treatment of leukemia relapse after cord blood transplantation. Bone Marrow Transplant. 2006;38:621–7. [PubMed]
23. Thorne SH, Negrin RS, Contag CH. Synergistic antitumor effects of immune cell-viral biotherapy. Science. 2006;311:1780–4. [PubMed]