Phase I clinical trial of autologous NK cell therapy using novel expansion method in patients with advanced digestive cancer

Table S2. Change in NK population in PBL

Abstract CT214: High purity and activity NK cells therapy in patients with advanced gastrointestinal cancer: Phase I study

Abstract PURPOSE: Our group exploited the novel technique of cultivating high purity and activity NK cells compared with traditional technique from peripheral blood of cancer patients. This phase I study investigated the safety and maximum-tolerated dose of the infusion NK cell number in patients with untreatable advanced gastrointestinal cancer. PATIENTS AND METHODS: Patients with gastrointestinal cancer who previously failed standard chemotherapy regimens were treated with three doses of NK cells infusion. We set three dose levels of NK cells infusion. The numbers of infusion NK cells were 5 X 108 cells as cohort 1, 1 X 109 cells as cohort 2, 2 X 109 cells as cohort 3 and each patient was infused NK cells three times per 1 or 2 weeks. The culture method of NK cells described as below; firstly CH296 (FN-CH296, RetroNctin®) induced T cells (RN-T) were prepared in advance by previously reported method, and processed to use as stimulator cells. NK cells were expanded from peripheral blood mononuclear cells by stimulating with modified RN-T, OK-432 and IL-2, then cultured for 21-22 days. We also established large-scale culture system using gas-permeable culture bag for clinical application. RESULTS: 14 patients were totally enrolled in this study. Of 14 patients, 7 patients were enrolled in cohort 1 study. Of 7 patients, 4 patients couldn't complete NK cells infusion because of some reasons such as bad NK cell proliferation, less purity of active NK cells and worsening of primary cancer. 3 patients could complete in cohort 1. 4 patients were enrolled in cohort 2 study. Of 4 patients, 1 patient couldn't complete the NK cell infusion because of worsening of primary cancer. 3 patients could complete and 1 patient experienced grade 2 pleural effusion accumulation. In cohort 3 study, 3 patients were enrolled. All of 3 patients could complete NK cells infusion, and only 1 patient experienced grade 1 low grade fever. As clinical response, 1 partial response (PR) at cohort 2 and 6 stable diseases (SD) at 4 patients in cohort 1, 1 patient in cohort2 and 1 patient in cohort 3 were observed. In addition, we checked the immune monitoring in detail for all patients. CONCLUSIONS: High purity and activity NK cells therapy was safety and maximum-tolerated dose was 2 X 109 cells. The evaluation is needed to further refine the efficacy and the toxicity of the combination therapy, chemotherapy, IgG1 molecular target drugs and this therapy for using this therapy in clinical in the future. Citation Format: Tetsuya Okayama, Satoshi Kokura, Takeshi ishikawa, Naoyuki Sakamoto, Mitsuko Ideno, Fumiyo Sakai, Akiko Kato, Tatsuji Enoki, Junichi Mineno, Hideyuki Konishi, Yuji Naito, Yoshito Itoh, Toshikazu Yoshikawa. High purity and activity NK cells therapy in patients with advanced gastrointestinal cancer: Phase I study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT214. doi:10.1158/1538-7445.AM2014-CT214</jats:p

Abstract 2791: Advantages and clinical application of fibronectin CH296-stimulated T cells in cancer immunotherapy

Abstract Background: In adoptive T-cell therapy (ACT), the differentiation state of transferred T cells is considered to be crucial to the success of ACT-based approaches. It has been reported that less-differentiated T cells, which have a higher proliferative potential and less prone to apoptosis than more differentiated cells, are ideal for ACT transfer therapy. In this study, we compared the co-stimulation effects of fibronectin CH296 (FN-CH296, RetroNectin®) with that of anti-28Ab or anti-4-1 BB Ab on T-cell expansion and its phenotype. Furthermore, we examined persistence of T cells expanded by these methods in NOG® mice. In addition, we conducted phase 1 clinical study to evaluate the safety and efficacy of FN-CH296 stimulated T cell therapy in patients with advanced cancer. Methods: PBMCs were activated by various stimulation methods and cultured in gas-permeable culture bag CultiLifeTM 215 and CultiLifeTM Eva for 10-14 days. After that, each expanded cells were analyzed for its phenotypes, in vivo persistence and the ability of accumulation in lymph node. In clinical study, patients underwent FN-CH296 stimulated T cell therapy up to six times every two weeks and safety and antitumor activity of the ACT were assessed. In order to determine immune function, whole blood cytokine levels were analyzed prior to ACT and during the follow up. Results: Co-stimulation by FN-CH296 with anti-CD3Ab led to higher T-cell expansion and proportion of CCR7+CD45RA+ T cells than that by the others (anti-28Ab/anti-4-1BB Ab). In the experiment for engraftment of expanded T cells in NOG mice, human CD3+ cells were detected in all groups, but the proportion of CCR7+CD45RA+ and CD8+ T cells was significantly higher specifically in the FN-CH296 co-stimulation condition. In addition, higher accumulation of human CD3+ cells in lymph node of NOG® mice was observed when stimulated by the FN-CH296 co-stimulation condition. In phase 1 clinical trial, nine patients were enrolled and infused 1, 3, or 9×109 of T cells to each cohort group (3 patients in each group). As a result, there was no ACT-related serious adverse event in all doses and one patient achieved CR, one achieved PR, four had SD, and three had PD. The number of less-differentiated cells that was infused showed a strong positive correlation with the change in whole blood IFN-γ level after ACT treatment. Conclusion: These results indicated that FN-CH296 stimulated T cells therapy was very well tolerated with a level of efficacy that is promising and the FN-CH296 stimulation method for ex vivo T-cell expansion is useful as basic technology for adoptive T-cell therapy, as it can be expanded preferentially less-differentiated T cells. Citation Format: Takeshi Ishikawa, Satoshi Kokura, Tetsuya Okayama, Naoyuki Sakamoto, Mitsuko Ideno, Nobuko Muraki, Akiko Kato, Tatsuji Enoki, Junichi Mineno, Yuji Naito, Yoshito Itoh, Toshikazu Yoshikawa. Advantages and clinical application of fibronectin CH296-stimulated T cells in cancer immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2791. doi:10.1158/1538-7445.AM2014-2791</jats:p

Abstract 3137: A novel expansion method for functional natural killer cells and its clinical application

Abstract Background: Natural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. But, it is difficult to generate the sufficient scale and purity of NK cells, and reliable methods to produce large number of functional NK cells have not been established yet. We have developed novel clinical-grade NK cells expansion method to produce the high purity, large scale and functional NK cells using a combination of recombinant human fibronectin fragment (RetroNectin®) -induced T-cells (RN-T cells), OK-432 and IL-2. We subsequently conducted a Phase 1 clinical study to evaluate the safety and efficacy of this NK cell therapy. In this paper, we address the characteristics of the NK cells elicited by this method and the results of immune monitoring in the clinical trial. Methods: To confirm the significance of RN-T cells as stimulator, we compared the stimulation effects on NK cells between RN-T cells and aCD3-T cells, which stimulated by anti-CD3 mAb only. Next, we analyzed expanded NK cells from PBMCs obtained from 31 cancer patients to verify the efficacy of this method. In the Phase 1 trial, patients with unresectable digestive cancer were enrolled. They received weekly intravenous administration of autologous NK cells elicited by the novel method three times to assess the safety of the number of adoptive cells at 0.5 × 109(cohort1), 1 × 109(cohort 2) and 2 × 109 cells (cohort 3) per dose. The phenotype and cytotoxicity of expanded NK cells were analyzed. For immune monitoring, cytotoxicity of PBMCs and whole blood cytokine levels were examined following NK cell infusion. Results: The stimulation by RN-T cells could induce preferable NK cell proliferation rather than the one by aCD3-T. As results of 31 cancer patients, 688±76-fold expansion was achieved in this system with PBMCs, and the NK cell populations were highly purified (84.7±3.6%) and highly expressed functional markers such as NKG2D (97.3±0.6%) and CD16 (96.8±0.7%). In the Phase 1 clinical trial, no NK cell infusion related severe or unexpected toxicities were observed. The response rate and the disease control rate in 10 per protocol patients were 0% and 50.0%, respectively. Although no clinical responses were observed, the cytotoxicity of PBMCs against K-562 targets increased in most patients (80%). The average of cytotoxic activity of PBMCs increased more than two times after the NK cell infusion. Conclusion: Although no patients receiving the NK cell therapy alone experienced a clinical response, adoptive immunotherapy of the NK cells elicited by the novel method is expected to exert considerable ADCC activity in vivo because of their high expression of CD16. Now, we are conducting clinical trial in which we explore the combination of this novel NK therapy with IgG1 antibodies such as trastuzumab and cetuximab. We also address the ongoing clinical trial in this paper. Citation Format: Takeshi Ishikawa, Naoyuki Sakamoto, Tetsuya Okayama, Kaname Oka, Satoshi Kokura, Mitsuko Ideno, Akiko Kato, Tatsuji Enoki, Masanari Kitagawa, Junichi Mineno, Tomoyo Yasuda, Toshifumi Doi, Yuji Naito, Yoshito Itoh, Toshikazu Yoshikawa. A novel expansion method for functional natural killer cells and its clinical application. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3137. doi:10.1158/1538-7445.AM2015-3137</jats:p

Additional file 1: of Phase I clinical trial of autologous NK cell therapy using novel expansion method in patients with advanced digestive cancer

Table S1. Summary of NK cell expansion from 31 cancer patients (supplementary information of reference #25)

Phase I Clinical Trial of Fibronectin CH296-Stimulated T Cell Therapy in Patients with Advanced Cancer

<div><p>Background</p><p>Previous studies have demonstrated that less-differentiated T cells are ideal for adoptive T cell transfer therapy (ACT) and that fibronectin CH296 (FN-CH296) together with anti-CD3 resulted in cultured cells that contain higher amounts of less-differentiated T cells. In this phase I clinical trial, we build on these prior results by assessing the safety and efficacy of FN-CH296 stimulated T cell therapy in patients with advanced cancer.</p><p>Methods</p><p>Patients underwent fibronectin CH296-stimulated T cell therapy up to six times every two weeks and the safety and antitumor activity of the ACT were assessed. In order to determine immune function, whole blood cytokine levels and the number of peripheral regulatory T cells were analyzed prior to ACT and during the follow up.</p><p>Results</p><p>Transferred cells contained numerous less-differentiated T cells greatly represented by CD27+CD45RA+ or CD28+CD45RA+ cell, which accounted for approximately 65% and 70% of the total, respectively. No ACT related severe or unexpected toxicities were observed. The response rate among patients was 22.2% and the disease control rate was 66.7%.</p><p>Conclusions</p><p>The results obtained in this phase I trial, indicate that FN-CH296 stimulated T cell therapy was very well tolerated with a level of efficacy that is quite promising. We also surmise that expanding T cell using CH296 is a method that can be applied to other T- cell-based therapies.</p><p>Trial Registration</p><p>UMIN <a href="https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&type=summary&recptno=R000002101&language=J" target="_blank">UMIN000001835</a></p></div

Patient characteristics.

<p>ECOG = Eastern Cooperative Oncology Group.</p><p>PS = performance status.</p

Changes in cell-surface phenotype after culture.

<p>PBMCs were stimulated with anti-CD3/CH-296. On day 10, cultured cells were harvested for transfusion. Cell-surface phenotypes of PBMCs or cultured cells were analyzed by flow cytometry. The average results from nine subjects are shown. In all panels, the lines represent the mean or standard deviation. *P<0.05.</p