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Title: REVISITING INTERLEUKIN-12 AS A CANCER IMMUNOTHERAPY

AGENT

Authors: Pedro Berraondo1,2,3*

, Iñaki Etxeberria1,2

, Mariano Ponz-Sarvise2,4

, Ignacio

Melero1,2,3,4

1 Immunology and Immunotherapy Program, Center for Applied Medical Research,

CIMA, University of Navarra, Pamplona, Spain.

2 Navarra Institute for Health Research (IDISNA), Pamplona, Spain.

3 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Spain.

4 Departments of Oncology and immunology, University Clinic of Navarra, Pamplona,

Spain.

Correspondence:

Ignacio Melero MD PhD.

CIMA and CUN

Av Pio XII,55

31008 Pamplona. Spain

imelero@unav.es

Running title: IL-12 back to the future

Conflicts of interest: IM is a consultant for Bristol-MyersSquibb, Roche-Genentech

Bayer, Medimmune, Merck Serono, Genmab, F-Star, TUSK, Alligator, Bioncotech and

receives research grants from Roche Genentech, Bristol Myers Squibb and Alligator.

Financial support: I. Melero is supported by MINECO (SAF2014-52361-R and

SAF2017-83267-C2-1R), European Commission VII Framework and Horizon 2020

programs (IACT and PROCROP), Cancer Research Institute (CRI) CLIP Grant 2017,

Fundación de la Asociación Española Contra el Cáncer (AECC) and Fundación BBVA.

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ABSTRACT

Interleukin-12 antitumor activities are mediated by the activation of T and NK

lymphocytes to produce IFNγ. Systemically, recombinant interleukin-12 has a narrow

therapeutic window that favors local delivery, i.e., by gene therapy approaches.

Interleukin-12 is a powerful partner in immunotherapy combinations with checkpoint

inhibitors and adoptive T-cell transfer.

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TEXT

In this issue of Clinical Cancer Research, Hu et al. report on the plasmid gene transfer

of an interleukin-12 (IL-12) gene expression cassette to mouse transplantable tumors

and to xenografted human tumors in NSG mice (1). IL-12 local expression is attained

by injecting plasmid DNA and performing in vivo electroporation inserting a needle-

shaped electrode (1). This strategy is currently under clinical trials as a single agent

(NCT01579318, NCT00323206, NCT01502293, and NCT02345330) and in

combination with pembrolizumab (NTC02493361 and NTC03132675). In these mice,

IL-12 is combined with doxorubicin, a chemotherapy agent with multiple effects on the

tumor tissue microenvironment that can help the antitumor immune response such as

immunogenic cell death of a fraction of tumor cells (2) and reduction of T regulatory

cells and myeloid-derived suppressor cells (3,4). Indeed, under treatment, tumors

accumulated infiltrating cytotoxic T cells (CD8+ NKG2D

+). If exogenous T cells

recognizing tumor antigens are infused, these adoptively transferred cells extravasate

and infiltrate the tumor much more efficiently.

Adoptive T cell therapy is revolutionizing cancer therapy mainly for hematological

malignancies. Chimeric antigen receptor-transduced T cells, tumor-infiltrating

lymphocytes (TILs), and T cell receptor-transduced cells are in the limelight of clinical

development. Early observations suggested that IL-12 local gene therapy can be

synergistically combined with adoptive T cell transfer (5). Moreover, attempts to

engineer T cells to produce IL-12 in a controlled fashion have been shown to be

remarkably efficacious in pre-clinical models, although toxic due to leaky expression of

the retroviral construct (6). Therefore, safer modes of conferring T cells the ability to

produce IL-12 in an autocrine fashion are needed.

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A remarkable function of IL-12 is its ability to induce IFNγ release from NK cells as

well as CD4+ and CD8

+ T cells. In fact, IL-12 signaling via STAT-4 is critical for Th1

differentiation and acquisition of cytolytic functions by CD8+ T cells (7). IFNγ in turn

strongly modifies the tumor microenvironment. The best studied beneficial mechanisms

are (Figure 1):

(i) Enhancing major histocompatibility class I antigen presentation in tumor cells.

(ii) Inducing the expression of CXCL9, 10 and 11 chemokines to attract NK, Th1

and CD8+ T cells.

(iii) Transforming M2 macrophages into activated antitumor M1 macrophages.

(iv) Acting on endothelial cells to mediate anti-angiogenesis in a CXCR3-dependent

fashion, while enhancing the expression of homing receptors for T cell

recruitment.

Unfortunately, IFNγ is also the main mediator of the toxic effects of IL-12 and over

time turns on immunoregulatory mechanisms such as PD-L1 and IDO-1 expression

which mediate adaptive resistance to immunotherapy.

In the era of checkpoint inhibitors and adoptive T cell therapy, we must revisit IL-12 as

an antitumor agent. On the one hand, IL-12 can be synergistic with PD-1/PD-L1

blockade (8) and on the other hand, it might help T cell therapy in several ways,

adoptive chiefly by including attraction and homing to the tumor tissue as reported by

Hu et al. in mouse models (1). The foreseen importance of these mechanisms is that IL-

12 might be a key tool to translate the efficacy of adoptive T cell therapy to a wider

spectrum of tumors aside from B cell malignancies, melanoma, and synovial sarcoma.

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IL-12 is neglected to some extent in clinical development with some exceptions: Merck

Serono is testing a fusion protein encompassing single chain IL-12 coupled to an

antibody that binds extracellular dsDNA (9). There are also attempts by Moderna

Therapeutics/MedImmune to transfer mRNA encoding IL-12 as well as the intratumoral

gene-electroporation strategies by OncoSec Medical (NTC03132675) already

commented on.

It must be said that IL-12 is a powerful wild horse difficult to harness (10). Systemic

treatment has a narrow therapeutic window due to circulating IFNγ levels that can be

fatal. Permanent retroviral gene-transfer of IL-12 into T cells has serious safety

problems (11). The route to clinical success of IL-12-based immunotherapy must

contemplate three key concepts:

i) Transient exposure or expression.

ii) Combination with other agents chiefly including adoptive T cell therapy and

checkpoint inhibitors.

iii) If possible, targeting the cytokine or its function or its expression to the tumor

microenvironment.

The potential of IL-12 as a partner in combination immunotherapy strategies is

promising and in need of improvements based on biotechnology, gene therapy and cell

therapy. The promotion of T-cell infiltration into tumors by IL-12 is certainly an

exciting feature. IL-12 is “back to the future.”

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REFERENCES

1. Hu J, Sun C, Bernatchez C, Xia X, Hwu P, Dotti G, et al. T cell homing therapy

for reducing regulatory T cells and preserving effector T cell function in large

solid tumors. Clinical cancer research : an official journal of the American

Association for Cancer Research 2018 doi 10.1158/1078-0432.CCR-17-1365.

2. Casares N, Pequignot MO, Tesniere A, Ghiringhelli F, Roux S, Chaput N, et al.

Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death.

The Journal of experimental medicine 2005;202(12):1691-701 doi

10.1084/jem.20050915.

3. Hsu FT, Chen TC, Chuang HY, Chang YF, Hwang JJ. Enhancement of adoptive

T cell transfer with single low dose pretreatment of doxorubicin or paclitaxel in

mice. Oncotarget 2015;6(42):44134-50 doi 10.18632/oncotarget.6628.

4. Alizadeh D, Trad M, Hanke NT, Larmonier CB, Janikashvili N, Bonnotte B, et

al. Doxorubicin eliminates myeloid-derived suppressor cells and enhances the

efficacy of adoptive T-cell transfer in breast cancer. Cancer research

2014;74(1):104-18 doi 10.1158/0008-5472.CAN-13-1545.

5. Mazzolini G, Qian C, Narvaiza I, Barajas M, Borras-Cuesta F, Xie X, et al.

Adenoviral gene transfer of interleukin 12 into tumors synergizes with adoptive

T cell therapy both at the induction and effector level. Human gene therapy

2000;11(1):113-25 doi 10.1089/10430340050016201.

6. Kerkar SP, Muranski P, Kaiser A, Boni A, Sanchez-Perez L, Yu Z, et al. Tumor-

specific CD8+ T cells expressing interleukin-12 eradicate established cancers in

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lymphodepleted hosts. Cancer research 2010;70(17):6725-34 doi 10.1158/0008-

5472.CAN-10-0735.

7. Carter LL, Murphy KM. Lineage-specific requirement for signal transducer and

activator of transcription (Stat)4 in interferon gamma production from CD4(+)

versus CD8(+) T cells. The Journal of experimental medicine 1999;189(8):1355-

60.

8. Quetglas JI, Labiano S, Aznar MA, Bolanos E, Azpilikueta A, Rodriguez I, et al.

Virotherapy with a Semliki Forest Virus-Based Vector Encoding IL12

Synergizes with PD-1/PD-L1 Blockade. Cancer immunology research

2015;3(5):449-54 doi 10.1158/2326-6066.CIR-14-0216.

9. Fallon JK, Vandeveer AJ, Schlom J, Greiner JW. Enhanced antitumor effects by

combining an IL-12/anti-DNA fusion protein with avelumab, an anti-PD-L1

antibody. Oncotarget 2017;8(13):20558-71 doi 10.18632/oncotarget.16137.

10. Sangro B, Melero I, Qian C, Prieto J. Gene therapy of cancer based on

interleukin 12. Current gene therapy 2005;5(6):573-81.

11. Zhang L, Morgan RA, Beane JD, Zheng Z, Dudley ME, Kassim SH, et al.

Tumor-infiltrating lymphocytes genetically engineered with an inducible gene

encoding interleukin-12 for the immunotherapy of metastatic melanoma.

Clinical cancer research : an official journal of the American Association for

Cancer Research 2015;21(10):2278-88 doi 10.1158/1078-0432.CCR-14-2085.

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FIGURE LEGEND

Figure 1. Mechanisms of action of interleukin 12. Different cells derived from

myeloid precursors release interleukin 12 upon activation. Interleukin 12 induces the

release of interferon gamma by NK cells, CD4+ T lymphocytes, and CD8

+ T

lymphocytes. Interferon gamma is the main mediator of the immunostimulatory

properties of interleukin 12 acting on tumor cells, macrophages, lymphocytes and

endothelial cells.

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Figure 1:

© 2018 American Association for Cancer Research

Interleukin-12

NK cells CD4+ T lymphocytes CD8+ T lymphocytes

IFN

Increased MHCIon tumor cells

M2 to M1 macrophagesconversion

CXCL 9, 10, and 11on lymphocytes

Antiangiogenesis andT-cell recruitment

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Published OnlineFirst March 16, 2018.Clin Cancer Res   Pedro Berraondo, Inaki Etxeberria, Mariano Ponz-Sarvise, et al.   IMMUNOTHERAPY AGENTREVISITING INTERLEUKIN-12 AS A CANCER

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