Trial Watch—Immunostimulation with cytokines in cancer therapy

ABSTRACT

During the past decade, great efforts have been dedicated to the development of clinically relevant interventions that would trigger potent (and hence potentially curative) anticancer immune responses. Indeed, developing neoplasms normally establish local and systemic immunosuppressive networks that inhibit tumor-targeting immune effector cells, be them natural or elicited by (immuno)therapy. One possible approach to boost anticancer immunity consists in the (generally systemic) administration of recombinant immunostimulatory cytokines. In a limited number of oncological indications, immunostimulatory cytokines mediate clinical activity as standalone immunotherapeutic interventions. Most often, however, immunostimulatory cytokines are employed as immunological adjuvants, i.e., to unleash the immunogenic potential of other immunotherapeutic agents, like tumor-targeting vaccines and checkpoint blockers. Here, we discuss recent preclinical and clinical advances in the use of some cytokines as immunostimulatory agents in oncological indications.

Abbreviations

5-FU

5-fluorouracil

AML

acute myeloid leukemia

CML

chronic myelogenous leukemia

DC

dendritic cell

ERBB2

erb-b2 receptor tyrosine kinase 2

FDA

Food and Drug Administration

FLT3LG

fms-related tyrosine kinase 3 ligand

ICD

immunogenic cell death

IFN

interferon

IL

interleukin

i.v.

intra venam

G-CSF

granulocyte colony-stimulating factor GM-CSF, granulocyte monocyte colony-stimulating factor

mAb

monoclonal antibody

MTOR

mechanistic target of rapamycin

NK

natural killer

NSCLC

non-small cell lung carcinoma

peg

pegylated

RCC

renal cell carcinoma s.c., sub cutem

TLR

Toll-like receptor

TNF

tumor necrosis factor

Introduction

Cytokines are a large and very heterogeneous family of small and generally soluble glycoproteins that regulate nearly all biological functions via autocrine, paracrine or endocrine circuitries.^1-4 At least in part, such a central position in the biology of mammalian organisms stems from the extreme pleiotropism of cytokine signaling.^5-8 Indeed, (1) virtually all mammalian cell types synthesize at least one cytokine; (2) one single cytokine can bind to, hence activating, distinct receptors or receptor isoforms, which normally exhibit differential binding and expression patterns; (3) cytokines often elicit the secretion of other mediators, including additional cytokines and (4) the biological outcome of cytokine signaling exhibit an elevated degree of context dependency, as it is profoundly influenced by contextual variables like local concentration, receptor type/isoform, cell type and differentiation state and presence of additional mediators.^5-8 The cytokine system is so pleiotropic that previous attempts to classify cytokines based on structural or functional aspects of their biology are now considered relatively reductionistic, imprecise and outdated.^5,6 Indeed, novel functions for members of the cytokine family as well as new cytokine-like proteins are discovered every year.^9-12

Owing to their capacity to regulate various cellular responses including proliferation, differentiation, activation and regulated cell death, cytokines orchestrate complex organismal functions as different as hematopoiesis, angiogenesis, wound healing and inflammation.^13-19 Moreover, some cytokines play a critical role in the initiation, propagation and extinction of innate and adaptive immune responses, irrespective of whether such responses are initiated by microbial challenges or endogenous states of danger.^20-22 As a standalone example, Type I interferon (IFN) is not only a key determinant of antiviral immunity,^21,22 but also an important mediator of immune responses elicited by cancer cells undergoing so-called “immunogenic cell death” (ICD).^20,23-29 The immunostimulatory activity of some cytokines is so pronounced that (similar to other immunotherapeutics) they mediate clinical activity as standalone interventions in patients affected by particularly immunogenic neoplasms,^30-32 like melanoma and renal cell carcinoma (RCC).^33-35 In line with this notion, three cytokines are currently licensed by the US Food and Drug Administration (FDA) and equivalent regulatory agencies worldwide for use as immunostimulatory interventions in cancer patients, namely recombinant IFN-α2a (Roferon-A^→), recombinant IFN-α2b (Intron A^→) and recombinant interleukin (IL)-2 (aldesleukin, Proleukin^→) (sources www.fda.gov and http://www.ema.europa.eu/ema/). Recombinant IFN-α2a is employed for the treatment of hairy cell leukemia and chronic phase, Philadelphia chromosome-positive chronic myelogenous leukemia (CML), upon minimal pretreatment (within 1 y of diagnosis); recombinant IFN-α2b is approved for use in patients with follicular lymphoma, multiple myeloma, hairy cell leukemia, AIDS-related Kaposi's sarcoma, melanoma, genital warts (Condyloma acuminata) and cervical intraepithelial neoplasms; and recombinant IL-2 is employed for the therapy of metastatic forms of melanoma and RCC (sources www.fda.gov and http://www.ema.europa.eu/ema/).

Of note, at least three other cytokines are approved by various regulatory agencies worldwide for use in cancer patients, namely recombinant granulocyte colony-stimulating factor (G-CSF, also known as Filgrastim, Lenograstim or Neupogen^→), recombinant granulocyte monocyte colony-stimulating factor (GM-CSF, also known as Molgramostim, Sargramostim, Leukomax®, Mielogen® or Leukine®), and recombinant tumor necrosis factor (TNF) (sources www.fda.gov and http://www.ema.europa.eu/ema/). However, these molecules are not employed to boost tumor-targeting immune responses, but rather as immunoreconstituting (G-CSF, GM-CSF)^36-43 or oncotoxic (TNF) factors.^44-55

Importantly, cytokines can cause relatively severe adverse effects (especially upon systemic administration), which de facto reflect their robust immunostimulatory activity and/or their biological pleiotropism. In particular, cytokines can (1) trigger an acute, potentially lethal systemic response that involves the release of pyrogenic and cytotoxic mediators (including additional cytokines) into the bloodstream;^56-60 (2) establish or perpetuate foci of chronic inflammation that may sustain oncogenesis or tumor progression;^61-63 or (3) promote proliferation among otherwise non-proliferating cells, thereby promoting the fixation of potentially oncogenic genetic or epigenetic defects.^64-67

Owing to such non-negligible side effects as well to generally low-objective response rates, the interest of oncologists in using recombinant cytokines as systemic standalone anticancer agents had dropped. Relatively safe and efficient treatments are indeed available for all the indications mentioned above, perhaps with the single exception of Stage II–III melanoma, which is still treated with surgery followed by IFN-α2b-based immunotherapy.^35,68 Rather, investigators and practitioners are focusing their efforts on the possibility to use recombinant cytokines (at low doses and locally) to boost the anticancer activity of other immunotherapeutic regimens, including checkpoint blockers,^69-72 adoptive cell transfer,^73-76 oncolytic virotherapy,^77-81 DNA- and peptide-based vaccines,^82–87 dendritic cell (DC)-based interventions,^88-92 as well as other immunostimulatory agents like Toll-like receptor (TLR) agonists.^93-97 Here, we discuss recent preclinical and clinical advances in the development of recombinant cytokines for use as immunological adjuvants in cancer patients.

Update on the development of recombinant cytokines as immunostimulants for cancer therapy

Completed clinical studies

Since the submission of our latest Trial Watch dealing with the use of recombinant cytokines as immunological adjuvants in experimental or off-label oncological indications (May 2014),⁶ the results of 17 clinical trials investigating this immunotherapeutic paradigm have been published in the peer-reviewed scientific literature (source http://www.ncbi.nlm.nih.gov/pubmed). Of note, 16 of these studies assessed the safety and efficacy of FDA-approved cytokines (i.e., G-CSF, GM-CSF, IFN-α2a, IFN-α2b and IL-2) as off-label immunostimulatory interventions,^98-113 while only one trial tested the clinical profile of a hitherto experimental cytokine (i.e., IL-15).¹¹⁴

The safety and efficacy of G-CSF have been assessed in 11 breast carcinoma patients, who were treated with subcutaneous G-CSF in combination with 5-fluorouracil (5-FU), epirubicin, cyclophosphamide and paclitaxel (NCT02225652).⁹⁸ The clinical profile of GM-CSF has been investigated in (1) 58 patients with newly diagnosed, treatment-naive CML, who received subcutaneous GM-CSF in combination with IFN-α,¹⁰¹ (2) two cohorts of 125 and 11 subjects with castration-resistant prostate carcinoma, who were treated with GM-CSF s.c. as a maintenance therapy upon successful docetaxel- and prednisone-based chemotherapy,¹¹⁵ or in combination with thalidomide,^116,117 respectively;^103,111 (3) 32 individuals with metastatic melanoma, receiving subcutaneous GM-CSF along with the FDA-approved, cytotoxic T lymphocyte-associated protein 4 (CTLA4)-blocking monoclonal antibody (mAb) ipilimumab;¹⁰⁹ and (4) 41 patients affected with advanced solid neoplasms, who were treated with GM-CSF s.c. in combina-tion with local radiotherapy,^118-120 with the specific aim to provoke systemic tumor-targeting immune responses (NCT02474186).¹¹⁰ Taken together, the results of these studies confirm that the subcutaneous administration of GM-CSF to cancer patients is safe and may boost the therapeutic activity of other treatments, at least in a proportion of individuals. Conversely, the combination of G-CSF with aggressive, multimodal chemotherapy was associated with severe side effects, resulting in the premature closure of the study.⁹⁸

The safety and efficacy of IFN-α2a and IFN-α2b have been evaluated in (1) five cohorts of 791, 365, 53, 51 and 40 subjects with advanced RCC, who received IFN-α2a in combination with the FDA-approved tumor-targeting antibody bevacizumab (which neutralizes vascular endothelial growth factor A, VEGFA)^121-123 (NCT00719264), along with the multitarget tyrosine kinase inhibitor sorafenib (UMIN000002466),^124,125 in the context of a multimodal treatment involving potentially immunogenic chemotherapy plus bevacizumab i.v. and low dose IL-2 s.c.,^126-128; or optionally combined with a trophoblast glycoprotein (TPBG)-redirected variant of staphylococcal enterotoxin A (naptumomab estafenatox);^{100,105,108,113,129} (2) 313 patients with symptomatic indolent B-cell lymphoma, receiving IFN-α2a in combination with the CD20-targeting mAb rituximab^130,131 (NCT01609010);¹⁰⁷ (3) 236 subjects with recurrent non-muscle-invasive bladder carcinoma, who were treated with intravesical IFN-α2b after intravesical mitomycin C-based chemotherapy;¹⁰⁶ (4) 23 individuals with recurrent epithelial ovarian carcinoma, receiving pegylated (peg)-IFN-α2b s.c. in the context of carboplatin- and doxorubicin-based chemotherapy plus a mAb targeting interleukin 6 receptor (IL6R) (NCT01637532);¹¹² and (5) 20 patients with advanced intrahepatic cholangiocarcinoma, who received subcutaneous peg-IFN-α2b along with hepatic arterial infusion 5-FU-based chemotherapy.⁹⁹ Cumulatively, these studies confirm that IFN-α2a and IFN-α2b can be safely administered to cancer patients as off-label indications. IFN-α2a improved the clinical efficacy of sorafenib-based chemotherapy for advanced RCC,¹¹³ but failed to do so better than the mechanistic target of rapamycin (MTOR) inhibitors everolimus or temsirolimus^132,133 (although it was associated with a lower incidence of side effects) in patients with metastatic RCC co-treated with bevacizumab,^108,129 as it failed to ameliorate the therapeutic profile of rituximab in indolent B-cell lymphoma patients.¹⁰⁷ Moreover, the intravesical administration of IFN-α2b after mitomycin C-based chemotherapy was associated with an increased frequency of recurrence among patients with non-muscle-invasive bladder carcinoma as compared to the post-chemotherapy instillation of the Bacillus Calmette-Guérin (BCG).¹⁰⁶ These findings indicate that the use of IFN-α2a and IFN-α2b as immunological adjuvants may not be optimal for all oncological indications.

Finally, subcutaneous low-dose IL-2 has been tested as an adjuvant to autologous DCs in 10 patients with ovarian carcinoma;¹⁰² the safety and efficacy of F16-IL2 (a variant of IL-2 retargeted to the extracellular domain A1 of tenascin C, TNC)¹³⁴ administered i.v. in combination with doxorubicin-based chemotherapy have been evaluated first in 10 subjects with advanced solid tumors (to identify a recommended dose) and subsequently in 19 breast carcinoma patients (at the recommended dose);¹⁰⁴ and the clinical profile of recombinant IL-15 given i.v. or s.c. has been assessed in 18 individuals with advanced melanoma or RCC.¹¹⁴ These latter two studies identified doses of F16-IL2 and IL-15 that are suitable for further investigation.^104,114 In addition, IL-15 was found to markedly alter the homeostasis of various circulating lymphocyte subsets, notably natural killer (NK), γδ T and memory CD8⁺ T cells.¹¹⁴

Preclinical and translational advances

Among recent preclinical and translational studies focusing on immunostimulation by cytokines, we found of particular interest the works of (1) Finisguerra and colleagues (Vesalius Research Center, Leuven, Belgium), who demonstrated that the proto-oncogene MET and its cognate cytokine hepatocyte growth factor (HGF) are required for the infiltration of neoplastic lesions by neutrophils with tumor-suppressive functions;¹³⁵ (2) Marçais and co-authors (International Center for Infectiology Research, Lyon, France), who found that MTOR is essential for the development and activation of NK cells in response to IL-15;¹³⁶ (3) O'Sullivan and collaborators (University of California San Diego, La Jolla, CA, US), who reported that the secretion of IL-17D by malignant cells stimulates the recruitment of NK cells to the tumor bed, and the consequent initiation of a tumor-targeting immune response that partially relies on M1 macrophages;¹³⁷ (4) Zhu et al. (Massachusetts Institute of Technology, Cambridge, MA, US), who found that the efficacy of a tumor-targeting mAb is significantly boosted by the co-administration of an IL-2 variant with extended serum half-life;¹³⁸ (5) Ruffell and colleagues (Oregon Health & Science University, Portland, OR, US), who characterized an IL-10-dependent mechanism whereby immunosuppressive macrophages block chemotherapy-driven anticancer immune responses at the level of IL-12 production by DCs;¹³⁹ (6) Hou and co-authors (Chinese Academy of Medical Sciences, Beijing, China), who demonstrated that the expression levels of DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 (DDX58, best known as RIG-I) in malignant cells predict the response of hepatocellular carcinoma patients to IFN-α;¹⁴⁰ (7) Litvin and collaborators (Columbia University, New York, NY, US), who found that recombinant Type I IFN robustly enhances the efficacy of mitogen-activated protein kinase (MAPK) inhibitors in melanoma cells, but only when IFN signaling in baseline conditions is reduced;¹⁴¹ (8) Escobar et al. (Vita-Salute San Raffaele University, Milan, Italy), who engineered the hematopoietic system of human hematochimeric mice so that IFN-α would be expressed selectively by differentiated monocytes, thereby endowing these animals with a superior ability to control the progression of experimental breast carcinomas;¹⁴² and (9) Bald and colleagues (University of Bonn, Bonn, Germany), who demonstrated that melanoma lesions with limited immune infiltrate respond to the local administration of immunostimulatory RNA (which activates Type I IFN signaling) in combination with a mAb blocking programmed cell death 1 (PDCD1, best known as PD-1).¹⁴³ Moreover, our laboratories demonstrated that cancer cells succumbing to ICD inducers produce Type I IFN upon the activation of TLR3, driving an autocrine/paracrine signal transduction pathway that ultimately leads to the secretion of chemokine (C-X-C motif) ligand 10 (CXCL10).¹⁴⁴ Thus, Type I IFN stands out as a potent immunostimulatory cytokine in several distinct experimental settings. However, results from recent clinical studies indicate that IFN-α2a or IFN-α2b may not universally ameliorate the efficacy of other anticancer interventions (see above). Large, randomized clinical trials are urgently awaited to understand whether recombinant IFN-α2a or IFN-α2b might safely improve the therapeutic activity of specific FDA-approved chemo- and immune-therapeutics, including targeted anticancer agents like the BRAF inhibitor vemurafenib and checkpoint-blocking mAbs like ipilimumab, nivolumab and pembrolizumab.

Recently initiated clinical trials

During the past 17 mo, no less than 60 clinical trials have been launched to test recombinant cytokines as on-label immunoreconstituting or immunostimulatory interventions in cancer patients, but these studies will not be discussed here. In the same period, at least 59 additional trials have been initiated to test the safety and efficacy of recombinant cytokines as immunostimulatory agents in off-label oncological indications. Of these studies, eight involve G-CSF, 20 GM-CSF, 10 IFN-α, 13 IL-2, and the remaining eight other cytokines including fms-related tyrosine kinase 3 ligand (FLT3LG), IFNγ, IL-8, IL-12, IL-15 and IL-18 (Table 1) (source www.clinicaltrials.org).

Table 1.

Clinical trials recently started to investigate the safety and efficacy of immunostimulatory cytokines as off-label interventions for cancer therapy.^*

Molecule	Indication(s)	Phase	Status	Route	Notes	Ref.
FLT3LG	Melanoma	II	Recruiting	s.c.	Combined with a peptide-based vaccine targeted to DCs in vivo	NCT02129075
G-CSF	Bladder carcinoma	II	Recruiting	i.v.	In the context of dense multicomponent chemotherapy	NCT02177695
	Breast carcinoma	II	Completed	s.c.	Combined with 5-FU, epirubicin, cyclophosphamide and paclitaxel	NCT02225652
	Gastric adenocarcinoma	I	Recruiting	i.v.	In the context of the SIRINOX regimen	NCT02387138
	HNC	II	Recruiting	s.c.	Combined with mitomycin C-based chemotherapy	NCT02369458
	Ovarian carcinoma	II	Terminated	n.a.	Combined with docetaxel and carboplatin	NCT02469116
	Prostate carcinoma	I/II	Recruiting	n.a.	Combined with multimodal chemotherapy	NCT02494921
		II	Not yet recruiting	n.a.	Combined with multimodal chemotherapy and androgen ablation	NCT02543255
	Solid tumors	II	Recruiting	s.c.	Formulated as Tbo-filgrastim	NCT02190721
GM-CSF	Brain tumors	0	Not yet recruiting	n.a.	Combined with a personalized peptide-based vaccine and polyICLC after temozolomide	NCT02510950
		I	Recruiting	s.c.	Combined with replication competent reovirus	NCT02444546
		II	Not yet recruiting	s.c.	Combined with a DC-based vaccine after temozolomide-based chemotherapy	NCT02465268
	Breast carcinoma	II	Recruiting	s.c.	Combined with an ERBB2-targeting vaccine and/or trastuzumab	NCT02297698
	Colorectal carcinoma	II	Recruiting	s.c.	In the context of the XELOX regimen	NCT02466906
	Glioblastoma	I	Recruiting	s.c.	Combined with a personalized peptide-based vaccine and polyICLC after temozolomide	NCT02149225
		II	Recruiting	s.c.	Combined with a personalized peptide-based vaccine after temozolomide	NCT02455557
	Gynecological malignancies	I	Recruiting	s.c.	Combined with autologous T cells armed with a BiTE plus subcutaneous IL-2	NCT02470559
	Hematological malignancies	I	Recruiting	s.c.	Combined with multipeptide-based vaccination	NCT02240537
	Melanoma	I/II	Recruiting	s.c.	Combined with a TERT-targeting vaccine and ipilimumab	NCT02275416
		IV	Recruiting	n.a.	As standalone immunotherapeutic agent	NCT02451488
	Neuroblastoma	II	Not yet recruiting	n.a.	Combined with dinutuximab, isotretinoin and IL-2	NCT02169609
	Neuroblastoma Osteosarcoma Soft tissue sarcoma	I/II	Recruiting	s.c.	Combined with autologous T cells armed with a GD2-targeting BiTE plus IL-2	NCT02173093
	Osteosarcoma	II	Not yet recruiting	s.c.	Combined with dinutuximab	NCT02484443
			Recruiting	s.c.	Combined with a humanized anti-GD2 mAb	NCT02502786
	Prostate carcinoma	I	Not yet recruiting	s.c.	Combined with cryotherapy	NCT02250014
		I/II	Active not recruiting	i.d.	Combined with peptide-based vaccination	NCT02452307
	RCC	I/II	Active not recruiting	i.d.	Combined with peptide-based vaccination	NCT02429440
	Solid tumors	I	Recruiting	n.a.	Combined with an ERBB2-targeting vaccine	NCT02276300
		II	Active not recruiting	n.a.	Combined with radiochemotherapy	NCT02474186
IFNα-2a	AML	I/II	Recruiting	s.c.	In the context of HSCT and methotrexate-based chemotherapy	NCT02328755
	Solid tumors	I	Recruiting	s.c.	Combined with a PD-L1-targeing mAb	NCT02174172
		II	Terminated has results	s.c.	As standalone immunotherapeutic agent	NCT02159482
IFNα-2b	Astrocytomas Gliomas	II	Recruiting	s.c.	As standalone immunotherapeutic agent	NCT02343224
	Ovarian carcinoma	I/II	Not yet recruiting	i.p.	Combined with cisplatin plus DC-based vaccination	NCT02432378
	Peritoneal surface malignancies	I/II	Recruiting	i.v.	Combined with DC-based vaccination	NCT02151448
	RCC	I/II	Not yet recruiting	n.a.	Combined with a proteasomal inhibitor	NCT02447887
	Skin SCC	II	Recruiting	s.c.	Combined with capecitabine	NCT02218164
	Solid tumors	I	Not yet recruiting	s.c.	As single agent with specific formulation	NCT02464007
			Recruiting	s.c.	As single agent with specific formulation	NCT02387307
IFNγ	Glioblastoma Gliosarcoma	I	Recruiting	i.t.	Combined with a replicative adenovirus	NCT02197169
IL-2	AML	n.a.	Recruiting	s.c.	Combined with adoptively transferred NK cells and decitabine	NCT02316964
	Gynecological malignancies	I	Recruiting	s.c.	Combined with autologous T cells armed with a BiTE plus subcutaneous GM-CSF	NCT02470559
		II	Not yet recruiting	n.a.	Combined with adoptively transferred NK cells and cytarabine	NCT02229266
	Hematological malignancies	I	Recruiting	n.a.	Combined with adoptively transferred NK cells	NCT02123836
		I/II	Recruiting	n.a.	Combined with adoptively transferred T cells and rituximab	NCT02315118
	HNC	I/II	Recruiting	s.c.	Combined with adoptively transferred NK cells and cetuximab	NCT02507154
	MesotheliomaNSCLC	I/II	Recruiting	s.c.	Combined with cyclophosphamide and genetically modified T cells	NCT02408016
	Neuroblastoma	II	Not yet recruiting	n.a.	Combined with dinutuximab, isotretinoin and GM-CSF	NCT02169609
	NeuroblastomaOsteosarcomaSoft tissue sarcoma	I/II	Recruiting	s.c.	Combined with autologous T cells armed with a GD2-targeting BiTE plus GM-CSF	NCT02173093
	Non-Hodgkin's lymphoma	I/II	Enrolling by invitation	s.c.	As immunocytokine targeted to CD20	NCT02151903
	Ovarian carcinoma	I	Recruiting	i.v.	Combined with adoptively transferred TILs	NCT02482090
	Pleural mesothelioma	I/II	Not yet recruiting	s.c.	Combined with adoptively transferred TILs	NCT02414945
	Solid tumors Lymphoma	I	Recruiting	s.c.	Combined with adoptively transferred haploidentical NK cells	NCT02130869
IL-8	Solid tumors	I	Recruiting	i.v.	As standalone immunotherapeutic agent	NCT02536469
IL-12	AML	I	Not yet recruiting	i.v.	As standalone immunotherapeutic agent	NCT02483312
	CTCL	II	Not yet recruiting	s.c.	Combined with low-dose total skin electron beam therapy	NCT02542124
	DLBCL	II	Not yet recruiting	s.c.	As single agent (upon completion of chemotherapy)	NCT02544724
IL-15	AML	II	Recruiting	s.c.	Combined with adoptively transferred haploidentical NK cells	NCT02395822
IL-18	Gynecological malignancies	I	Completed	n.a.	Combined with adoptively transfer of vaccine-primed, CD3/CD28-costimulated T cells	NCT02277392

Abbreviations: 5-FU, 5-fluorouracil; AML, acute myeloid leukemia; BiTE, bispecific T-cell engager; CTCL, cutaneous T cell lymphoma; DC, dendritic cell; DLBCL, diffuse large B-cell lymphoma; FLT3LG, fms-related tyrosine kinase 3 ligand; G-CSF; granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage-colony stimulating factor; HNC, head and neck cancer; HSCT, hematopoietic stem cell transplantation; i.d., intra dermam; IFN, interferon; IL, interleukin; i.p., intra peritoneum; i.t., intra tumorem; i.v., intra venam; mAb, monoclonal antibody; n.a., not available; NK, natural killer; NSCLC, non-small cell lung carcinoma; poly-ICLC, polyinosinic:polycytidylic acid; RCC, renal cell carcinoma; s.c., sub cutem; SCC, squamous cell carcinoma; SIRINOX, S-1 + irinotecan + oxaliplatin; TIL, tumor infiltrating lymphocyte; XELOX, capecitabine + oxaliplatin.

^*initiated between 2014, May 1st and 2015, Sept 30th.

The safety and efficacy of G-CSF are being assessed in (1) bladder carcinoma patients, who receive G-CSF i.v. in the context of dense multicomponent chemotherapy (NCT02177695); (2) breast carcinoma patients,¹⁴⁵ receiving subcutaneous G-CSF in combination with 5-FU, epirubicin, cyclophosphamide and paclitaxel (NCT02225652); (3) subjects with gastric adenocarcinoma, receiving G-CSF i.v. in the context of the so-called SIRINOX chemotherapeutic regimen (S-1 plus irinotecan plus oxaliplatin) (NCT02387138); (4) individuals with head and neck squamous cell carcinoma, who are treated with subcutaneous G-CSF together with mitomycin C-based chemotherapy (NCT02369458); (5) ovarian carcinoma patients, receiving G-CSF in combination with docetaxel and carboplatin (NCT02469116); (6) subjects with prostate carcinoma, who are treated with G-CSF in the context of chemotherapy alone (NCT02494921) or chemotherapy plus androgen ablation therapy (NCT02543255); and (7) patients with advanced solid malignancies without bone marrow involvement, who receive Tbo-filgrastim (a short-acting form of G-CSF) as standalone immunotherapeutic intervention (NCT02190721). All these studies are ongoing with the exception of NCT02225652, which has been completed (see above), and NCT02469116, which has been terminated owing to the withdrawal of financial support (source www.clinicaltrials.org).

The immunostimulatory activity of GM-CSF is being tested in (1) patients with various malignancies of the central nervous system, who are treated with GM-CSF s.c. in combination with a personalized peptide-based vaccine and optionally a TLR3 agonist after temozolomide-based chemotherapy (NCT02149225; NCT02455557; NCT02510950), a DC-based vaccine after temozolomide-based chemotherapy (NCT02465268), or a replication competent oncolytic reovirus (NCT02444546); (2) women with breast carcinoma, who receive subcutaneous GM-CSF together with the erb-b2 receptor tyrosine kinase 2 (ERBB2)-specific mAb trastuzumab^146,147 and an ERBB2-targeting vaccine (NCT02297698); (3) subjects with colorectal cancer, receiving GM-CSF s.c. in the context of the so-called XELOX chemotherapeutic regimen (capecitabine plus oxaliplatin) (NCT02466906); (4) patients with gynecological malignancies, who are treated with subcutaneous GM-CSF and IL-2 as a support to autologous T cells armed with a bispecific T-cell engager (BiTE)^148-150 targeting ERBB2 (NCT02470559); (5) individuals with hematological neoplasms, who receive GM-CSF s.c. in support of a multipeptide-based vaccine (NCT02240537); (6) melanoma patients, who are treated with GM-CSF as a standalone immunotherapeutic intervention (NCT02451488) or in combination with ipilimumab and peptide-based vaccination targeting telomerase reverse transcriptase (TERT)^151,152 (NCT02275416); (7) subjects with neuroblastoma, osteosarcoma or soft tissue sarcoma, receiving subcutaneous GM-CSF and IL-2 along with autologous T cells armed with a BiTE targeting ganglioside GD2 (NCT02173093); (8) individuals with neuroblastoma, who are treated with subcutaneous GM-CSF plus IL-2 i.v. and dinutuximab (a chimeric ganglioside GD2-specific mAb)^153,154 (NCT02169609); (9) osteosarcoma patients, who receive GM-CSF s.c. plus dinutuximab or a humanized ganglioside GD2-specific mAb (NCT02484443; NCT02502786); (10) individuals with prostate carcinoma, who are treated with subcutaneous or intradermal GM-CSF in combination with cryotherapy¹⁵⁵ (NCT02250014) or a peptide-based vaccine (NCT02452307), respectively; (11) RCC patients, receiving intradermal GM-CSF as an adjuvant to peptide-based vaccination (NCT02429440); and (12) subjects with metastatic solid tumors, who receive GM-CSF in combination with radio- and chemo-therapy (NCT02474186) or with a ERBB2-targeting vaccine plus cyclophosphamide (NCT02276300). All these studies are ongoing (source www.clinicaltrials.org).

The clinical profile of IFN-α2a is being investigated in (1) acute myeloid leukemia (AML) patients, who receive peg-IFN-α2a in the context of hematopoietic stem cell transplantation (not as an immunoreconstituting agent) and methotrexate-based therapy (NCT02328755); (2) patients with advanced solid tumors, who are treated with IFN-α2a as standalone immunotherapeutic intervention (NCT02159482) or together with a mAb specific for the PD-1 ligand CD274 (best known as PD-L1)¹⁵⁶ (NCT02174172). Recombinant IFN-α2b is being tested in (1) subjects with astrocytomas or optic pathway gliomas, who receive peg-IFN-α2b s.c. as standalone immunotherapeutic agent (NCT02343224); (2) individuals with ovarian carcinoma or peritoneal surface malignancies, who are treated with intraperitoneal or intravenous IFN-α2b, respectively, as adjuvant to cisplatin plus DC-based vaccination (NCT02432378) or DC-based vaccination alone (NCT02151448); (3) RCC patients, receiving peg-IFN-α2b in combination with the proteasomal inhibitor ixazomib (NCT02447887); and (4) subjects with squamous cell carcinomas of the skin, who receive peg-IFN-α2b s.c. in together with capecitabine-based chemotherapy (NCT02218164). Finally, two clinical studies are assessing the safety and efficacy of solubilized form of crystalline recombinant IFN-α2b (known as rSIFN-co) administered s.c. as standalone immunotherapeutic agent to patients with advanced solid neoplasms (NCT02387307; NCT02464007). With the exception of NCT02159482, which has already been terminated owing to low accrual, all these studies are ongoing. Of 11 patients enrolled in NCT02159482, only 6 completed the vaccination protocol. Five of these individuals (83.33%) experienced moderate adverse effects, the most common of which were fatigue and fever. Moreover, fiveof these patients exhibited an increase in the magnitude of the tumor antigen-specific immune responses, as determined by ELISPOT analysis (source www.clinicaltrials.org).

The immunostimulatory activity of IL-2 is being assessed in (1) patients with hematological malignancies including AML, who are treated with IL-2 in combination adoptively transferred NK cells (NCT02123836), adoptively transferred NK cells plus cytarabine- or decitabine-based chemotherapy (NCT02229266; NCT02316964), or adoptively transferred T lymphocytes plus rituximab (NCT02315118) (2) individuals with gynecological malignancies, who receive subcutaneous GM-CSF and IL-2 as a support to autologous T cells armed with an ERBB2-targeting BiTE (NCT02470559); (3) subjects with non-Hodgkin lymphoma, receiving a de-immunized and humanized anti-CD20 mAb (Leu16) fused to IL-2 as standalone immunotherapeutic intervention (NCT02151903); (4) patients with head and neck squamous cell carcinoma or nasopharyngeal carcinoma, who are treated with IL-2 s.c. as adjuvant to the infusion of autologous NK cells and the FDA-approved epidermal growth factor (EGFR)-targeting mAb cetuximab¹⁵⁷ (NCT02507154); (5) individuals with mesothelioma or non-small cell lung carcinoma (NSCLC), receiving subcutaneous IL-2 along with cyclophosphamide-based chemotherapy and T lymphocytes genetically redirected against the tumor-associated antigen Wilms tumor 1 (WT1) (NCT02408016); (6) subjects with neuroblastoma, osteosarcoma or soft tissue sarcoma, who receive subcutaneous GM-CSF and IL-2 along with autologous T cells armed with a BiTE targeting ganglioside GD2 (NCT02173093), or along with dinutuximab-based therapy (NCT02169609); (7) individuals with ovarian carcinoma or pleural mesothelioma patients, who are treated with intravenous or subcutaneous IL-2, respectively combined with the adoptive transfer of autologous tumor-infiltrating lymphocytes (NCT02482090; NCT02414945); and (8) children with high-risk solid tumors or lymphomas, receiving IL-2 s.c. as adjuvant to the adoptive transfer of haploidentical NK cells (NCT02130869). All the studies are ongoing (source www.clinicaltrials.org).

Moreover, (1) intravenous IL-8 is being tested as a standalone immunotherapeutic agent in patients affected by advanced solid tumors (NCT02536469); (2) the safety and efficacy of IL-12 are being investigated in AML patients, who receive IL-12 i.v. as a single immunotherapeutic intervention (NCT02483312), cutaneous T-cell lymphoma, who are treated with subcutaneous IL-12 in combination with low-dose total skin electron beam therapy (NCT02542124), and diffuse large B-cell lymphoma, receiving IL-12 s.c. after standard chemotherapy (NCT02544724); (3) IL-15 is being tested as a standalone immunological adjuvant to haploidentical NK-cell transfer in AML patients (NCT02395822); (4) the clinical profile of IL-18 is being evaluated in subjects with gynecological neoplasms, who are treated with IL-18 in support of vaccine-primed, CD3/CD28-stimulated autologous T cells (NCT02277392); (5) the safety and immunostimulatory activity of FLT3LG are being tested in melanoma patients, who receive FLT3LG s.c. together with a TLR3 agonist as a support to a peptide-based vaccine targeted to DCs in vivo (NCT02129075); and (6) IFNγ is being tested in combination with a replicative oncolytic adenovirus in glioblastoma and gliosarcoma patients (NCT02197169). These clinical trials are ongoing, with the sole exception of NCT02277392, which has already been completed (but for which results are not yet available) (source www.clinicaltrials.org).

Of the clinical studies discussed in our previous Trial Watches dealing with immunostimulatory cytokines in off-label oncological indications,^5,6 the following have changed status during the past 17 mo: NCT01579188, which currently appears as “Not yet recruiting;” NCT01580696, NCT01671774, NCT01784913, NCT01872442, NCT01929239, NCT01989325, NCT02019524, NCT02089685, and NCT02092922, which are nowadays listed as “Active not recruiting;” NCT01727076, NCT01881867, NCT01898793, NCT01903330, NCT01964300, NCT02001818, NCT02070406, and NCT02078648, which are now “Recruiting” participants; NCT01701479, and NCT01806272, whose status is now “Unknown;” NCT01940601, which has been “Withdrawn;” NCT00923351, NCT01767194, and NCT01572493, which have been “Suspended;” NCT00589550, and NCT00784524, which have been “Terminated;” and NCT00631371, NCT00719264, NCT01592045, NCT01609010, NCT01629758, NCT01658813, NCT01673217, NCT01875601, NCT01989572, NCT02076633, and NCT02087176, which have been “Completed” (source www.clinicaltrials.org).

NCT01940601 (a Phase II study testing the pharmacodynamics of human serum albumin-bound G-CSF in pediatric patients with solid tumors) has been withdrawn prior to enrollment for undisclosed reasons. NCT00923351 (a Phase I/II trial testing IL-17 as immunological adjuvant to the adoptive transfer of autologous T cells alone or given in combination with a DC-based vaccine in subjects with neuroectodermal tumors) has been suspended owing to the availability of IL-7, but preliminary results indicate that IL-7 exerts robust immunostimulatory effects in the absence of added toxicity (as compared to adoptive cell transfer alone). NCT01572493 (a Phase I study assessing the safety and efficacy of IL-15 as standalone immunotherapeutic intervention in adults with advanced malignancies) has been suspended for undisclosed reasons, whereas NCT01767194 (a Phase II trial evaluating the clinical profile of subcutaneous or intravenous GM-CSF in neuroblastoma patients receiving chemotherapy plus everolimus or dinutuximab) has been suspended for assessment. Both NCT00784524 (a Phase II study assessing the immunostimulatory activity of subcutaneous IL-2 given as adjuvant to a peptide-based vaccine in breast carcinoma patients) and NCT00589550 (a Phase I trial investigating the clinical profile of subcutaneous peg-IFN-α2b plus sorafenib-based chemotherapy in advanced RCC patients) have been terminated owing to slow accrual (source www.clinicaltrials.org).

We discussed the results of NCT0063137, NCT01609010 and NCT00719264 above. Preliminary results from NCT01592045 (a Phase I/II study comparing the activity of two distinct mAbs targeting ganglioside GD2 adjuvanted with G-CSF and IL-2 in neuroblastoma patients) indicate that this approach is not associated with an increased incidence or a different panel of side effects as compared to the administration of anti-GD2 mAbs alone (based on historical cohorts). Results from NCT02087176 (a large, randomized Phase III trial testing whether subcutaneous GM-CSF increases the clinical efficacy a multipeptide-based vaccine in melanoma patients) suggest that the administration of GM-CSF s.c. does not improve progression-free and overall survival in subjects with melanoma receiving a multipeptide-based vaccine, nor does it change the incidence of side effects. It remains to be clarified whether these negative results reflect the inability of vaccination to initiate tumor-targeting immune responses (irrespective of GM-SCF administration) in this setting. To the best of our knowledge, the results of NCT01629758 (a Phase I trial investigating the safety and efficacy of recombinant IL-21 plus nivolumab in subjects with advanced solid tumors), NCT01658813 (a Phase II study assessing the clinical profile of 5-FU followed by IFN-α2b in patients with metastatic gastrointestinal, kidney or lung cancers), NCT01673217 (a Phase I trial evaluating the safety and efficacy of a peptide-based vaccine adjuvanted with subcutaneous GM-CSF in combination with conventional chemotherapeutics in women with gynecological tumors), NCT01875601 (a Phase I study testing autologous activated NK cells plus intravenous IL-15 in children and young adults with advanced solid tumors), NCT02076633 (a Phase II trial investigating the therapeutic profile of tumor-redirected IL-2 plus tumor-redirected TNF in melanoma patients), and NCT02087176 (a Phase II study testing the efficacy of a targeted anticancer agent given in combination with peg-GM-CSF and placebo or docetaxel in NSCLC patients) have not yet been disseminated (source www.clinicaltrials.org).

Concluding remarks

Preclinical and clinical data accumulating over the past two decades demonstrate that some cytokines, including (but presumably not limited to) G-CSF, GM-CSF, IFN-α2a, IFN-α2b, IL-2, IL-12 and IL-15 mediate robust immunostimulatory effects that may be harnessed to boost natural or therapy-elicited anticancer immune responses in patients. However, using these molecules as standalone immunostimulatory agents is associated with (a relatively low proportion of) objective responses in a limited number of oncological indications (e.g., melanoma, RCC). Recent efforts have therefore been redirected to use immunostimulatory cytokines as adjuvants to other chemo-, radio- or immuno-therapeutic regimens. Moreover, it turned out that—besides being associated with non-negligible side effects—the systemic delivery of some cytokines (like IL-2) actually provides a preferential benefit to immunosuppressive, rather than effector, cells of the immune system.¹⁵⁸ As a consequence, various strategies have been devised to target recombinant cytokines to specific anatomical locations or selected cell populations, including variations in dosage, schedule and administration route as well as genetic/molecular engineering. A majority of recently launched clinical trials involves cytokines that are already licensed by regulatory agencies for use in humans, i.e., G-CSF, GM-CSF, IFN-α2a, IFN-α2b and IL-2. In the era of checkpoint blockade, however, changes in the approval status of these molecules do not seem to stand next door.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

Authors are supported by the Ligue contre le Cancer (équipe labelisée); Agence National de la Recherche (ANR); Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; AXA Chair for Longevity Research; Institut National du Cancer (INCa); Fondation Bettencourt-Schueller; Fondation de France; Fondation pour la Recherche Médicale (FRM); the European Commission (ArtForce); the European Research Council (ERC); the LabEx Immuno-Oncology; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); the SIRIC Cancer Research and Personalized Medicine (CARPEM); and the Paris Alliance of Cancer Research Institutes (PACRI).