Genetic engineering of human NK cells to express CXCR2 improves migration to renal cell carcinoma

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Abstract

Background

Adoptive natural killer (NK) cell transfer is being increasingly used as cancer treatment. However, clinical responses have so far been limited to patients with hematological malignancies. A potential limiting factor in patients with solid tumors is defective homing of the infused NK cells to the tumor site. Chemokines regulate the migration of leukocytes expressing corresponding chemokine receptors. Various solid tumors, including renal cell carcinoma (RCC), readily secrete ligands for the chemokine receptor CXCR2. We hypothesize that infusion of NK cells expressing high levels of the CXCR2 chemokine receptor will result in increased influx of the transferred NK cells into tumors, and improved clinical outcome in patients with cancer.

Methods

Blood and tumor biopsies from 14 primary RCC patients were assessed by flow cytometry and chemokine analysis. Primary NK cells were transduced with human CXCR2 using a retroviral system. CXCR2 receptor functionality was determined by Calcium flux and NK cell migration was evaluated in transwell assays.

Results

We detected higher concentrations of CXCR2 ligands in tumors compared with plasma of RCC patients. In addition, CXCL5 levels correlated with the intratumoral infiltration of CXCR2-positive NK cells. However, tumor-infiltrating NK cells from RCC patients expressed lower CXCR2 compared with peripheral blood NK cells. Moreover, healthy donor NK cells rapidly lost their CXCR2 expression upon in vitro culture and expansion. Genetic modification of human primary NK cells to re-express CXCR2 improved their ability to specifically migrate along a chemokine gradient of recombinant CXCR2 ligands or RCC tumor supernatants compared with controls. The enhanced trafficking resulted in increased killing of target cells. In addition, while their functionality remained unchanged compared with control NK cells, CXCR2-transduced NK cells obtained increased adhesion properties and formed more conjugates with target cells.

Conclusions

To increase the success of NK cell-based therapies of solid tumors, it is of great importance to promote their homing to the tumor site. In this study, we show that stable engineering of human primary NK cells to express a chemokine receptor thereby enhancing their migration is a promising strategy to improve anti-tumor responses following adoptive transfer of NK cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s40425-017-0275-9) contains supplementary material, which is available to authorized users.

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Most cited references 35

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Expression of a functional CCR2 receptor enhances tumor localization and tumor eradication by retargeted human T cells expressing a mesothelin-specific chimeric antibody receptor.

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Adoptive T-cell immunotherapy with tumor infiltrating lymphocytes or genetically-modified T cells has yielded dramatic results in some cancers. However, T cells need to traffic properly into tumors to adequately exert therapeutic effects. The chemokine CCL2 was highly secreted by malignant pleural mesotheliomas (MPM; a planned tumor target), but the corresponding chemokine receptor (CCR2) was minimally expressed on activated human T cells transduced with a chimeric antibody receptor (CAR) directed to the MPM tumor antigen mesothelin (mesoCAR T cells). The chemokine receptor CCR2b was thus transduced into mesoCAR T cells using a lentiviral vector, and the modified T cells were used to treat established mesothelin-expressing tumors. CCR2b transduction led to CCL2-induced calcium flux and increased transmigration, as well as augmentation of in vitro T-cell killing ability. A single intravenous injection of 20 million mesoCAR + CCR2b T cells into immunodeficient mice bearing large, established tumors (without any adjunct therapy) resulted in a 12.5-fold increase in T-cell tumor infiltration by day 5 compared with mesoCAR T cells. This was associated with significantly increased antitumor activity. CAR T cells bearing a functional chemokine receptor can overcome the inadequate tumor localization that limits conventional CAR targeting strategies and can significantly improve antitumor efficacy in vivo.

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Natural killer (NK) cells have a spontaneous cytotoxic capacity-against tumor cells. These cells represent a small proportion of human colon carcinoma-infiltrating lymphocytes. Their prognostic significance in these tumors has yet to be determined. One hundred and fifty-seven patients who each had a colectomy for large bowel adenocarcinoma were studied. No patient received adjuvant therapy. Immunohistochemical stains were performed for NK cells using the monoclonal antibody CD57. The number of NK cells was counted using a MICRON image analyzer. The total area studied for each tumor was 1 cm2. In this area, 50 intratumoral fields of 0.173 mm2 were selected. The degree of NK infiltration was classified as little ( 150 NK cells). The Kaplan-Meier method was used to obtain survival figures. Multivariate analyses were performed using the Cox regression model. At 5 years, patients with little and moderate NK infiltration showed significantly shorter survival rates (overall and disease free survival) than those with extensive infiltration (P < 0.01). Three significant factors affecting survival were selected in a stepwise fashion in increasing order as follows: TNM stage, NK infiltration, and lymphocytic infiltration. Patients with TNM Stage III disease and extensive NK infiltration showed significantly longer survival rates than those with little or moderate infiltration (P < 0.001). In these patients, multivariate analysis using the Cox regression model identified two significant variables: number of involved lymph nodes and NK cells infiltration. In patients with colorectal carcinoma, an extensive intratumoral infiltration of NK cells is associated with a favorable tumor outcome. Intratumoral infiltration of NK cells can be used as a variable with prognostic value, especially in patients with TNM Stage III disease.

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Immunotype and immunohistologic characteristics of tumor-infiltrating immune cells are associated with clinical outcome in metastatic melanoma.

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Immune cells infiltrating the microenvironment of melanoma metastases may either limit or promote tumor progression, but the characteristics that distinguish these effects are obscure. In this study, we systematically evaluated the composition and organization of immune cells that infiltrated melanoma metastases in human patients. Three histologic patterns of immune cell infiltration were identified, designated immunotypes A, B, and C. Immunotype A was characterized by no immune cell infiltrate. Immunotype B was characterized by infiltration of immune cells limited only to regions proximal to intratumoral blood vessels. Immunotype C was characterized by a diffuse immune cell infiltrate throughout a metastatic tumor. These immunotypes represented 29%, 63%, and 8% of metastases with estimated median survival periods of 15, 23, and 130 months, respectively. Notably, from immunotypes A to C, there were increasing proportions of B cells and decreasing proportions of macrophages. Overall, the predominant immune cells were T cells (53%), B cell lineage cells (33%), and macrophages (13%), with natural killer and mature dendritic cells only rarely present. Whereas higher densities of CD8(+) T cells correlated best with survival, a higher density of CD45(+) leukocytes, T cells, and B cells also correlated with increased survival. Together, our findings reveal striking differences in the immune infiltrate in melanoma metastases in patients, suggesting microenvironmental differences in immune homing receptors and ligands that affect immune cell recruitment. These findings are important, not only by revealing how the immune microenvironment can affect outcomes but also because they reveal characteristics that may help improve individualized therapy for patients with metastatic melanoma.

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Author and article information

Contributors

Veronika Kremer: veronika.kremer@ki.se

Maarten Ligtenberg: marten.ligtenberg@gmail.com

Rosa Zendehdel: rosa.zendehdel@hotmail.com

Christina Seitz: Christina.seitz@ki.se

Annet Duivenvoorden: annet.duivenvoorden@student.uva.nl

Erik Wennerberg: erw2010@med.cornell.edu

Eugenia Colón: Eugenia.Colon@ki.se

Ann-Helén Scherman-Plogell: ann-helen.scherman-plogell@sll.se

Andreas Lundqvist:

ORCID: http://orcid.org/0000-0002-9709-2970

+46851776859 , Andreas.lundqvist@ki.se

Journal

Journal ID (nlm-ta): J Immunother Cancer

Journal ID (iso-abbrev): J Immunother Cancer

Title: Journal for Immunotherapy of Cancer

Publisher: BioMed Central (London )

ISSN (Electronic): 2051-1426

Publication date (Electronic): 19 September 2017

Publication date PMC-release: 19 September 2017

Publication date Collection: 2017

Volume: 5

Electronic Location Identifier: 73

Affiliations

[1 ]ISNI 0000 0004 1937 0626, GRID grid.4714.6, Department of Oncology-Pathology, , Karolinska Institutet, ; Stockholm, Sweden

[2 ]GRID grid.430814.a, Department of Molecular Oncology, , The Netherlands Cancer Institute, ; Amsterdam, Netherlands

[3 ]ISNI 0000 0004 1937 0626, GRID grid.4714.6, Department of Medicine Solna, , Karolinska Institutet, ; Stockholm, Sweden

[4 ]ISNI 000000041936877X, GRID grid.5386.8, Department of Radiation Oncology, , Weill Cornell Medicine, ; New York, NY USA

[5 ]ISNI 0000 0000 8986 2221, GRID grid.416648.9, Department of Oncology-Pathology, , Stockholm South General Hospital, ; Stockholm, Sweden

[6 ]ISNI 0000 0004 1937 0626, GRID grid.4714.6, Department of Woman and Child Health, , Karolinska Institutet, ; Stockholm, Sweden

[7 ]ISNI 0000 0000 8986 2221, GRID grid.416648.9, Department of Urology, , Stockholm South General Hospital, ; Stockholm, Sweden

[8 ]ISNI 0000 0001 2168 8324, GRID grid.261241.2, Cell Therapy Institute, Nova Southeastern University, ; Fort Lauderdale, FL USA

Author information

Andreas Lundqvist http://orcid.org/0000-0002-9709-2970

Article

Publisher ID: 275

DOI: 10.1186/s40425-017-0275-9

PMC ID: 5604543

PubMed ID: 28923105

SO-VID: c68c6161-3766-46e7-93d3-fd3c8cbdde7d

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 8 February 2017

Date accepted : 14 August 2017