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 Table of Contents  
Year : 2020  |  Volume : 16  |  Issue : 2  |  Page : 393-396

Robo1-specific chimeric antigen receptor natural killer cell therapy for pancreatic ductal adenocarcinoma with liver metastasis

1 Department of Interventional Radiology; Department of Radiology, Luwan Branch, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
2 Department of Radiology, Luwan Branch, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
3 Center of Systems Medicine, Chinese Academy of Medical Sciences, Suzhou Institute of Systems Medicine; Asclepius (Soochow) Technology Company Group, Suzhou, Jiangsu, China

Date of Submission18-Feb-2020
Date of Decision06-Mar-2020
Date of Acceptance31-Mar-2020
Date of Web Publication28-May-2020

Correspondence Address:
Zhongmin Wang
Department of Interventional Radiology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 197 2nd Ruijin Road, Shanghai 200000
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_190_20

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Keywords: Chimeric antigen receptor natural killer, Robo1, pancreatic ductal adenocarcinoma, liver metastasis

How to cite this article:
Li C, Yang N, Li H, Wang Z. Robo1-specific chimeric antigen receptor natural killer cell therapy for pancreatic ductal adenocarcinoma with liver metastasis. J Can Res Ther 2020;16:393-6

How to cite this URL:
Li C, Yang N, Li H, Wang Z. Robo1-specific chimeric antigen receptor natural killer cell therapy for pancreatic ductal adenocarcinoma with liver metastasis. J Can Res Ther [serial online] 2020 [cited 2021 Sep 24];16:393-6. Available from: https://www.cancerjournal.net/text.asp?2020/16/2/393/285180

 > Introduction Top

Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, is one of the highly malignant cancers with a low 5-year survival rate.[1] Due to its increasing incidence, PDAC is expected to be the second leading cause of cancer-related deaths in the United States by 2030.[2] The tumor is unresectable by surgical treatment in most PDAC patients. With advancement in systemic chemotherapy for metastatic PDAC, there has been some improvement in response rates and outcomes.[3] Unfortunately, its median overall survival is poor (<1 year), and the therapy for these patients meets new challenges.

Chimeric antigen receptor-modified T-cell (CAR-T) therapy is a recently developed immunotherapeutic strategy for cancer. Although CAR-T therapy has achieved a breakthrough in blood cancers,[4],[5] it showed less impressive therapeutic efficacy in solid tumors. Further, its severe toxic effects involving cytokine storm and high cost have limited its widespread use.

Nature killer (NK) cell is another kind of immune effect cell contributing to the body's immune defenses. Clinically, NK cells serve as an effective and safe alternative immunotherapeutic strategy to CAR-T cells. However, there have been fewer clinical studies and little clinical data published for CAR-NK cells,[6],[7] compared to those for CAR-T cells.

We conducted research using Robo1 CAR-NK cells, showing great synergistic efficacy on pancreatic cancer in an orthotopic nude mouse model.[8] This case report describes Robo1-specific CAR-NK cell therapy on a patient with PDAC liver metastasis.

 > Case Report Top

A 46-year-old male presented with epigastric pain and increased CA19-9 level. computed tomography and magnetic resonance imaging scan imaging showed pancreatic lesion (about 6 cm × 2.5 cm × 3 cm) with lymph infiltration and several liver hypermetabolic lesions [Figure 1]a and [Figure 1]b. The diameter of the largest metastatic lesion was about 3.5 cm. The patient was diagnosed with PDAC with liver metastasis (T4N1M1, Stage IV). Through pancreatic tumor puncture biopsy, Robo1 was found to be present in the pancreas respectively. The tumor was unresectable by surgical treatment and the patient rejected chemotherapy. The patient was treated with Robo1-specific CAR-NK cell infusion. Liver metastasis was treated with percutaneous Robo1-specific CAR-NK cell injection [Figure 2].
Figure 1: (a and b) Before the therapy, pancreatic lesion (about 6 cm × 2.5 cm × 3 cm) with lymph infiltration and several liver hypermetabolic lesions. (c and d) Pancreatic lesion and liver metastasis were controlled within 5 months

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Figure 2: (a and b) Robo1-specific CAR-NK cells mixed with isotonic contrast medium (iodixanol) were injected into Liver metastasis. (c and d) About 10 ml was injected and dosage was respectively 1 × 109 cells

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We used CAR lentiviral construct [Figure 3]a containing CAR of anti-Robo1 human antibody sequence, extracellular domain (Robo1 single chain variable fragment), transmembrane domain (CD8), intracellular signal area, and both CD3 ζ and 4-1BB costimulatory molecules. NK-92 cells were transduced with the Robo1-CAR construct. The transduction efficiency was determined through flow cytometry. After confirming that transduction efficiency was over 90%, the transduced cells were expanded. All cell processes and transduction protocols were undertaken in a good manufacturing practice facility. Robo1-specific CAR-NK cells were supported by Asclepius Technology Company Group. The clinical trial identification number for our study is NCT03941457. All human experiments were approved by Ruijin Hospital Institutional Review Board. The CAR lentiviral construct is shown in [Figure 3]a. NK-92 cells were transduced with the above-mentioned Robo1-CAR vector with efficiency over 90% [Figure 3]b.
Figure 3: (a) CAR lentiviral construct. (b) NK-92 cells were transduced with the above-mentioned Robo1-CAR vector with efficiency over 90%

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In the 1st month, the patient received Robo1-CAR-NK intravenous injection on days 1 and 3 and percutaneous injection on days 2 and 4 every week, respectively [Figure 4]. The dosage was respectively 1 × 109 cells. Robo1-specific CAR-NK cells were mixed with isotonic contrast medium (iodixanol), and about 10 ml was injected into the liver metastasis [Figure 2]. The patient continuously received Robo1-CAR-NK intravenous injection once a week and evaluation was conducted every 2 weeks.
Figure 4: Timetable of Robo1-chimeric antigen receptor natural killer -92 cells for the treatment

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The patient had moderate fever (about 38.5°C) 2 h after intravenous injection on the 2nd and 3rd week, which quickly resolved in 24 h. There was no significant body temperature change after percutaneous liver metastasis injection. Pancreatic lesion and liver metastasis were controlled within 5 months [Figure 1]c and [Figure 1]d. The therapy was maintained for 5 months, and no further treatment was administered. Karnofsky Performance Score and child stage of the patient did not change greatly during the therapy [Figure 5] and [Figure 6].
Figure 5: Child stage during the therapy

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Figure 6: Karnofsky Performance Score during the therapy

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Two months following CAR-NK cell treatment, the patient had complications of worsening liver function and excessive ascites. Unfortunately, the patient died of multiple organ failure due to tumor progression 1 month later. The overall survival time of the patient was 8 months.

 > Discussion Top

We described Robo1-CAR-NK -92 cell infusions and percutaneous injection at a dose of 1×109 cells, respectively.

Because of immunosuppressive microenvironment and inability of NK cells to enter the tumor tissue, the efficacy of NK cell therapy on solid tumors was limited. Through percutaneous injection, Robo1-CAR-NK cells could kill tumor cells directly.[9] Furthermore, percutaneous injection did not cause cytokine release syndromes (CRS), and it was safely applied on patients without substantial adverse effects. Systemic CAR-T infusions are associated with severe CRS, among other toxicities. An interventional method adopted to solve these pitfalls of cellular immunotherapy through transcatheter hepatic artery chemotherapy to deliver CAR-T was reported.[10]

The mechanism of CAR-mediated therapies (both T and NK cells) depends on the associated antigens of the cells. Robo1 is a potential target and spectacular paradigm in the treatment of solid tumors. It has also been reported to play a role in modulating T cells chemotaxis and tumor angiogenesis. Slit/Robo pathway can be considered as a master regulator of multiple oncogenic signaling pathways.[11] Increased expression of Robo1 has been found in pancreatic cancer. In our preclinical investigation, Robo1-CAR-NK cells showed moderate efficacy in vitro.

Combined CAR-mediated therapy, in which multiple antigens are simultaneously targeted in patients through their corresponding CARs, may exert synergistic effect and induce improved clinical response. CAR-NK cells are target-dependent and mainly kill cells with high expression of specific antigens. Antigens can also be expressed in normal tissues and cells, and there are no definite data to make an assessment for the off-target toxicity of CAR-NK cells once it occurs. Dose control and other factors (introducing suicide genes) are necessary to be considered to reduce risk.[12]

For CAR-NK therapy to be suitable for better clinical treatment, there are still some problems needed to be solved. For example, irradiation of CAR-NK-92 cells prior to infusion is required. After irradiation, NK-92 cells have a short life cycle and survivein vivo for a short period, avoiding some off-target effects.[13] This method reduces the toxicity of CAR-NK cells in the body. Accordingly, it also reduces the antitumor effects. Our therapy requires multiple injections, leading to large emotional burden for the patient.

Here, we presented a case of Robo1-CAR-NK cell therapy for PDAC with liver metastasis. The safety and clinical efficiency of CAR-NK cell therapy for solid tumor need to be assessed in further studies.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

This work was supported by the National Natural Science Foundation of China (Grant No. 81771949), the Project of Medical Key Specialty of Shanghai Municipal Health Commission (Grant No. ZK2019A02) the project of Shanghai Municipal Health Commission (Grant No. 201840058) and Shanghai Municipal key Clinical Specialty (Grant No. shslczdzk06002 and No. shslczdzk07002).

Conflicts of interest

There are no conflicts of interest.

 > References Top

Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014;64:9-29.  Back to cited text no. 1
Rahib L, Smith BD, Aizenberg R, Rosenzweig AB, Fleshman JM, Matrisian LM. Projecting cancer incidence and deaths to 2030: The unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Res 2014;74:2913-21.  Back to cited text no. 2
Katz MH, Shi Q, Ahmad SA, Herman JM, Marsh Rde W, Collisson E, et al. Preoperative modified FOLFIRINOX treatment followed by capecitabine-based chemoradiation for borderline resectable pancreatic cancer: Alliance for clinical trials in oncology trial A021101. JAMA Surg 2016;151:e161137.  Back to cited text no. 3
Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 2011;365:725-33.  Back to cited text no. 4
Brown CE, Alizadeh D, Starr R, Weng L, Wagner JR, Naranjo A, et al. Regression of glioblastoma after chimeric antigen receptor T-cell therapy. N Engl J Med 2016;375:2561-9.  Back to cited text no. 5
Mehta RS, Rezvani K. Chimeric antigen receptor expressing natural killer cells for the immunotherapy of cancer. Front Immunol 2018;9:283.  Back to cited text no. 6
Tang X, Yang L, Li Z, Nalin AP, Dai H, Xu T, et al. First-in-man clinical trial of CAR NK-92 cells: Safety test of CD33-CAR NK-92 cells in patients with relapsed and refractory acute myeloid leukemia. Am J Cancer Res 2018;8:1083-9.  Back to cited text no. 7
Xia N, Haopeng P, Gong JU, Lu J, Chen Z, Zheng Y, et al. Robo1-specific CAR-NK immunotherapy enhances efficacy of (125)I seed brachytherapy in an orthotopic mouse model of human pancreatic carcinoma. Anticancer Res 2019;39:5919-25.  Back to cited text no. 8
Böttcher JP, Bonavita E, Chakravarty P, Blees H, Cabeza-Cabrerizo M, Sammicheli S, et al. NK cells stimulate recruitment of cDC1 into the tumor microenvironment promoting cancer immune control. Cell 2018;172:1022-37.  Back to cited text no. 9
Katz SC, Hardaway J, Prince E, Guha P, Cunetta M, Moody A, et al. HITM-SIR: Phase Ib trial of intraarterial chimeric antigen receptor T-cell therapy and selective internal radiation therapy for CEA(+) liver metastases. Cancer Gene Ther 2019. doi: 10.1038/s41417-019-0104-z.  Back to cited text no. 10
Gara RK, Kumari S, Ganju A, Yallapu MM, Jaggi M, Chauhan SC. Slit/Robo pathway: A promising therapeutic target for cancer. Drug Discov Today 2015;20:156-64.  Back to cited text no. 11
Chen ZH, Yu YP, Zuo ZH, Nelson JB, Michalopoulos GK, Monga S, et al. Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene. Nat Biotechnol 2017;35:543-50.  Back to cited text no. 12
Zhang C, Oberoi P, Oelsner S, Waldmann A, Lindner A, Tonn T, et al. Chimeric antigen receptor-engineered NK-92 cells: An off-the-shelf cellular therapeutic for targeted elimination of cancer cells and induction of protective antitumor immunity. Front Immunol 2017;8:533.  Back to cited text no. 13


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]


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