Combinatorial immunotherapy of N-803 (IL-15 superagonist) and dinutuximab with ex vivo expanded natural killer cells significantly enhances in vitro cytotoxicity against GD2 <sup>+</sup> pediatric solid tumors and in vivo survival of xenografted immunodeficient NSG mice

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Abstract

Background

Children with recurrent and/or metastatic osteosarcoma (OS), neuroblastoma (NB) and glioblastoma multiforme (GBM) have a dismal event-free survival (<25%). The majority of these solid tumors highly express GD2. Dinutuximab, an anti-GD2 monoclonal antibody, significantly improved event-free survival in children with GD2 ⁺ NB post autologous stem cell transplantation and enhanced natural killer (NK) cell-mediated antibody-dependent cell cytotoxicity. Thus, approaches to increase NK cell number and activity, improve persistence and trafficking, and enhance tumor targeting may further improve the clinical benefit of dinutuximab. N-803 is a superagonist of an interleukin-15 (IL-15) variant bound to an IL-15 receptor alpha Su-Fc fusion with enhanced biological activity.

Methods

The anti-tumor combinatorial effects of N-803, dinutuximab and ex vivo expanded peripheral blood NK cells (exPBNK) were performed in vitro using cytoxicity assays against GD2 ⁺ OS, NB and GBM cells. Perforin and interferon (IFN)-γ levels were measured by ELISA assays. Multiple cytokines/chemokines/growth factors released were measured by multiplex assays. Human OS, GBM or NB xenografted NOD/SCID/IL2rγnull (NSG) mice were used to investigate the anti-tumor combinatorial effects in vivo.

Results

N-803 increased the viability and proliferation of exPBNK. The increased viability and proliferation are associated with increased phosphorylation of Stat3, Stat5, AKT, p38MAPK and the expression of NK activating receptors. The combination of dinutuximab and N-803 significantly enhanced in vitro cytotoxicity of exPBNK with enhanced perforin and IFN-γ release against OS, GBM and NB. The combination of exPBNK+N-803+dinutuximab significantly reduced the secretion of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), platelet-derived growth factor-BB (PDGF-BB), and stem cell growth factor beta (SCGF-β) from OS or GBM tumor cells. Furthermore, OS or GBM significantly inhibited the secretion of regulated on activation, normal T cell expressed and presumably secreted (RANTES) and stromal cell-derived factor-1 alpha (SDF-1α) from exPBNK cells (p<0.001) but significantly enhanced monokine induced by gamma interferon (MIG) secretion from exPBNK cells (p<0.001). N-803 combined with dinutuximab and exPBNK cells significantly extended the survival of OS, GBM or NB xenografted NSG mice.

Conclusions

Our results provide the rationale for the development of a clinical trial of N-803 in combination with dinutuximab and ex vivo exPBNK cells in patients with recurrent or metastatic GD2 ⁺ solid tumors.

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Most cited references 52

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Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma.

Alice L Yu, Andrew L Gilman, M. Ozkaynak … (2010)

Preclinical and preliminary clinical data indicate that ch14.18, a monoclonal antibody against the tumor-associated disialoganglioside GD2, has activity against neuroblastoma and that such activity is enhanced when ch14.18 is combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-2. We conducted a study to determine whether adding ch14.18, GM-CSF, and interleukin-2 to standard isotretinoin therapy after intensive multimodal therapy would improve outcomes in high-risk neuroblastoma. Patients with high-risk neuroblastoma who had a response to induction therapy and stem-cell transplantation were randomly assigned, in a 1:1 ratio, to receive standard therapy (six cycles of isotretinoin) or immunotherapy (six cycles of isotretinoin and five concomitant cycles of ch14.18 in combination with alternating GM-CSF and interleukin-2). Event-free survival and overall survival were compared between the immunotherapy group and the standard-therapy group, on an intention-to-treat basis. A total of 226 eligible patients were randomly assigned to a treatment group. In the immunotherapy group, a total of 52% of patients had pain of grade 3, 4, or 5, and 23% and 25% of patients had capillary leak syndrome and hypersensitivity reactions, respectively. With 61% of the number of expected events observed, the study met the criteria for early stopping owing to efficacy. The median duration of follow-up was 2.1 years. Immunotherapy was superior to standard therapy with regard to rates of event-free survival (66±5% vs. 46±5% at 2 years, P=0.01) and overall survival (86±4% vs. 75±5% at 2 years, P=0.02 without adjustment for interim analyses). Immunotherapy with ch14.18, GM-CSF, and interleukin-2 was associated with a significantly improved outcome as compared with standard therapy in patients with high-risk neuroblastoma. (Funded by the National Institutes of Health and the Food and Drug Administration; ClinicalTrials.gov number, NCT00026312.)

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Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells

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NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy.

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Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells.

Chihaya Imai, Shotaro Iwamoto, Dario Campana (2005)

Natural killer (NK) cells hold promise for improving the therapeutic potential of allogeneic hematopoietic transplantation, but their effectiveness is limited by inhibitory HLA types. We sought to overcome this intrinsic resistance by transducing CD56+CD3- NK cells with chimeric receptors directed against CD19, a molecule widely expressed by malignant B cells. An abundance of NK cells for transduction was secured by culturing peripheral blood mononuclear cells with K562 cells expressing the NK-stimulatory molecules 4-1BB ligand and interleukin 15, which yielded a median greater than 1000-fold expansion of CD56+CD3- cells at 3 weeks of culture, without T-lymphocyte expansion. Expression of anti-CD19 receptors linked to CD3zeta overcame NK resistance and markedly enhanced NK-cell-mediated killing of leukemic cells. This result was significantly improved by adding the 4-1BB costimulatory molecule to the chimeric anti-CD19-CD3zeta receptor; the cytotoxicity produced by NK cells expressing this construct uniformly exceeded that of NK cells whose signaling receptors lacked 4-1BB, even when natural cytotoxicity was apparent. Addition of 4-1BB was also associated with increased cell activation and production of interferon gamma and granulocyte-macrophage colony-stimulating factor. Our findings indicate that enforced expression of signaling receptors by NK cells might circumvent inhibitory signals, providing a novel means to enhance the effectiveness of allogeneic stem cell transplantation.

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Author and article information

Journal

Journal ID (nlm-ta): J Immunother Cancer

Journal ID (iso-abbrev): J Immunother Cancer

Journal ID (hwp): jitc

Journal ID (publisher-id): jitc

Title: Journal for Immunotherapy of Cancer

Publisher: BMJ Publishing Group (BMA House, Tavistock Square, London, WC1H 9JR )

ISSN (Electronic): 2051-1426

Publication date Collection: 2021

Publication date (Electronic): 8 July 2021

Volume: 8

Issue: 7

Electronic Location Identifier: e002267

Affiliations

[1 ]departmentDepartment of Pediatrics , New York Medical College , Valhalla, New York, USA

[2 ]ImmunityBio, Inc , Culver City, California, USA

[3 ]NantKwest , Culver City, California, USA

[4 ]departmentDepartment of Hem/Onc/BMT , Nationwide Children’s Hospital , Columbus, Ohio, USA

[5 ]departmentDepartment of Medicine , New York Medical College , Valhalla, New York, USA

[6 ]departmentDepartment of Pathology , New York Medical College , Valhalla, New York, USA

[7 ]departmentDepartment of Microbiology and Immunology , New York Medical College , Valhalla, New York, USA

[8 ]departmentDepartment of Cell Biology and Anatomy , New York Medical College , Valhalla, New York, USA

Author notes

[Correspondence to ] Dr Mitchell S. Cairo; mitchell_cairo@ 123456nymc.edu ; Dr Yaya Chu; yaya_chu@ 123456nymc.edu

Author information

Mitchell S. Cairo http://orcid.org/0000-0002-2075-434X

Article

Publisher ID: jitc-2020-002267

DOI: 10.1136/jitc-2020-002267

PMC ID: 8268924

PubMed ID: 34244307

SO-VID: 1f7ee275-c4de-4c09-8d77-b8ab4b480df5

License:

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

History

Date accepted : 23 May 2021

Funding

Funded by: New York Medical College School of Medicine Translational Science Institute Children Health Translational Research Award;

Funded by: http://dx.doi.org/10.13039/100000902, Pediatric Cancer Research Foundation;

Funded by: http://dx.doi.org/10.13039/100000002, National Institutes of Health;

Award ID: 1U54 CA232561-01A1

Funded by: Department of Defense;

Award ID: W81XWH-16-PRCRP-TTSA #CA160461

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Keywords: immunotherapy,killer cells,natural,sarcoma,neuroblastoma,brain neoplasms,osteosarcoma,glioblastoma,dinutuximab,expanded natural killer cells,targeted immunotherapy,adcc

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Keywords: immunotherapy, killer cells, natural, sarcoma, neuroblastoma, brain neoplasms, osteosarcoma, glioblastoma, dinutuximab, expanded natural killer cells, targeted immunotherapy, adcc

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