Endogenous IL-21 restricts CD8+ T cell expansion and is not required for tumor immunity.

IL-21 has antitumor activity through actions on NK cells and CD8(+) T cells, and is currently in clinical development for the treatment of cancer. However, no studies have addressed the role of endogenous IL-21 in tumor immunity. In this study, we have studied both primary and secondary immune responses in IL-21(-/-) and IL-21R(-/-) mice against several experimental tumors. We found intact immune surveillance toward methylcholanthrene-induced sarcomas in IL-21(-/-) and IL-21R(-/-) mice compared with wild-type mice and B16 melanomas showed equal growth kinetics and development of lung metastases. IL-21R(-/-) mice showed competent NK cell-mediated rejection of NKG2D ligand (Rae1beta) expressing H-2b(-) RMAS lymphomas and sustained transition to CD8(+) T cell-dependent memory against H-2b(+) RMA lymphomas. alpha-Galactosylceramide stimulation showed equal expansion and activation of NKT and NK cells and mounted a powerful antitumor response in the absence of IL-21 signaling, despite reduced expression of granzyme B in NKT, NK, and CD8(+) T cells. Surprisingly, host IL-21 significantly restricted the expansion of Ag-specific CD8(+) T cells and inhibited primary CD8(+) T cell immunity against OVA-expressing EG7 lymphomas, as well as the secondary expansion of memory CD8(+) T cells. However, host IL-21 did not alter the growth of less immunogenic MC38 colon carcinomas with dim OVA expression. Overall, our results show that endogenous IL-21/IL-21R is not required for NK, NKT, and CD8(+) T cell-mediated tumor immunity, but restricts Ag-specific CD8(+) T cell expansion and rejection of immunogenic tumors, indicating novel immunosuppressive actions of this cytokine.

Cytokine requirements for acute and Basal homeostatic proliferation of naive and memory CD8+ T cells.

Both naive and memory T cells undergo antigen-independent proliferation after transfer into a T cell-depleted environment (acute homeostatic proliferation), whereas only memory T cells slowly divide in a full T cell compartment (basal proliferation). We show, first, that naive and memory CD8+ T cells have different cytokine requirements for acute homeostatic proliferation. Interleukin (IL)-7 receptor(R)alpha-mediated signals were obligatory for proliferation of naive T cells in lymphopenic hosts, whereas IL-15 did not influence their division. Memory T cells, on the other hand, could use either IL-7Ralpha- or IL-15-mediated signals for acute homeostatic proliferation: their proliferation was delayed when either IL-7Ralpha was blocked or IL-15 removed, but only when both signals were absent was proliferation ablated. Second, the cytokine requirements for basal and acute homeostatic proliferation of CD8+ memory T cells differ, as basal division of memory T cells was blocked completely in IL-15-deficient hosts. These data suggest a possible mechanism for the dearth of memory CD8+ T cells in IL-15- and IL-15Ralpha-deficient mice is their impaired basal proliferation. Our results show that naive and memory T lymphocytes differ in their cytokine dependence for acute homeostatic proliferation and that memory T lymphocytes have distinct requirements for proliferation in full versus empty compartments.

Interleukin-21 signaling: functions in cancer and autoimmunity.

Interleukin-21 (IL-21) is a cytokine with structural and sequence homology to IL-2 and IL-15, yet possesses several biological properties distinct from these cytokines. IL-21 is produced mainly by activated CD4(+) T cells and natural killer T cells and mediates its activity by binding to the IL-21 receptor (IL-21R), consisting of an IL-21-specific alpha chain (IL-21Ralpha; JAK/STAT) that heterodimerizes with the common gamma chain (CD132). Intracellular signaling occurs through the Janus-activated kinase/signal transducer and activator of transcription pathways. Physiologic expression of IL-21R is restricted to lymphoid tissues and peripheral blood mononuclear cells; however, other tissues such as epithelium, synovium, or transformed cells can acquire expression of both components of IL-21R heterodimer. IL-21 has complex activities on a wide variety of cell types, leading to enhancement of adaptive T-cell immunity, antibody production, activation of natural killer cell subtypes, and opposition to suppressive effects mediated by regulatory T cells. Functionally, these activities promote immune responses and point to a physiologic role of IL-21 in autoimmunity and immune enhancement. Therapeutic manipulation of IL-21 activity may allow improved immunotherapy for cancer as well as insights into autoimmune disease. Recently conducted phase 1 trials in metastatic melanoma and renal cell carcinoma have shown that recombinant IL-21 has a favorable safety profile and support its continued investigation as a potential anticancer drug.

Comparison of vascular leak syndrome in mice treated with IL21 or IL2.

Interleukin 21 (IL21) is a T-cell-derived 4-helix-bundle cytokine that has sequence homology to the IL2 family. Recombinant human interleukin 2 (rIL2) is approved for the treatment of metastatic melanoma and renal cell carcinoma. However, toxicity of rIL2, including induction of vascular leak syndrome (VLS), has limited use of this cytokine to a small proportion of eligible patients. Both rIL2 and murine IL21 (mIL21) have potent antitumor efficacy in murine models. The purpose of the current study was to compare the ability of mIL21 and rIL2 to induce vascular leakage in a mouse model. Pulmonary and hepatic uptake of Evans blue dye, serum cytokine levels, spleen cell immunophenotype, and histologic changes in lung and liver were evaluated to detect VLS. High-dose (200 µg) rIL2 treatment induced vascular leakage in mice, evidenced by inflammatory cell infiltration and fluid extravasation into the lung and liver and increased levels of TNFα, IFNγ, IL5, MCP1, and IL6 in serum. In contrast, an equivalent dose of mIL21 resulted in minimal vascular leakage with no evidence of cytopenia or cytokine production. These results support the use of IL21 as a cancer immunotherapeutic agent, potentially providing an antitumor response without induction of VLS.

Interleukin-21 enhances rituximab activity in a cynomolgus monkey model of B cell depletion and in mouse B cell lymphoma models.

Rituximab, a monoclonal antibody targeting CD20 on B cells, is currently used to treat many subtypes of B cell lymphomas. However, treatment is not curative and response rates are variable. Recombinant interleukin-21 (rIL-21) is a cytokine that enhances immune effector function and affects both primary and transformed B cell differentiation. We hypothesized that the combination of rIL-21 plus rituximab would be a more efficacious treatment for B cell malignancies than rituximab alone. We cultured human and cynomolgus monkey NK cells with rIL-21 and found that their activity was increased and proteins associated with antibody dependent cytotoxicity were up-regulated. Studies in cynomolgus monkeys modeled the effects of rIL-21 on rituximab activity against CD20 B cells. In these studies, rIL-21 activated innate immune effectors, increased ADCC and mobilized B cells into peripheral blood. When rIL-21 was combined with rituximab, deeper and more durable B cell depletion was observed. In another series of experiments, IL-21 was shown to have direct antiproliferative activity against a subset of human lymphoma cell lines, and combination of murine IL-21 with rituximab yielded significant survival benefits over either agent alone in xenogeneic mouse tumor models of disseminated lymphoma. Therefore, our results do suggest that the therapeutic efficacy of rituximab may be improved when used in combination with rIL-21.

A dual-targeting PDGFRbeta/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo.

Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VEGF and PDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments. The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting PDGFRbeta and VEGF-A were selected for superior stability. The scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. The resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. The assembly of a bsAb using stable monomeric units allowed development of an anti-PDGFRB/VEGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules.

Interleukin 21: combination strategies for cancer therapy.

In the past 20 years researchers have attempted to activate the host immune defence system to kill tumour cells and eradicate cancer. In some cases, the response of patients to immunotherapy has been extremely successful; however, other trials have shown disappointing results, and so there is a clear need for more effective therapies that can effectively adjunct conventional approaches. Interleukin 21 (IL21) is a new immune-stimulating cytokine that has demonstrated antitumour activity in several preclinical models, and has recently undergone Phase I trials in metastatic melanoma and renal cell carcinoma. Here, we provide an overview of the antitumour effects of IL21 and describe strategies to combine IL21 with other drugs for future cancer therapies.

Interleukin 21 enhances antibody-mediated tumor rejection.

Interleukin-21 (IL-21) is a cytokine with structural and sequence homology to IL-2 and IL-15 that has antitumor activity alone in mouse experimental tumor models and a tolerable safety profile in phase I trials in patients with metastatic melanoma and renal cell carcinoma. Several monoclonal antibodies (mAb) targeted at tumor-associated antigens also have improved antitumor activities in mice when used in combination with IL-21. Recently, we described a rational three antibody-based approach (triple mAb, TrimAb) to eradicating established mouse tumors that required the generation of tumor-reactive CD8(+) T cells and IFN-gamma. Herein, we show that sequentially combining TrimAb with recombinant IL-21 can significantly improve the antitumor activity of this combination against very advanced disease. These data further support the use of IL-21 in adjuvant settings where strong T cell-mediated immune responses to tumors can be generated.

IL-21 enhances tumor rejection through a NKG2D-dependent mechanism.

IL-21 is a cytokine that can promote the anti-tumor responses of the innate and adaptive immune system. Mice treated with IL-21 reject tumor cells more efficiently, and a higher percentage of mice remain tumor-free compared with untreated controls. In this study, we demonstrate that in certain tumor models IL-21-enhanced tumor rejection is NKG2D dependent. When engagement of the NKG2D receptor was prevented, either due to the lack of ligand expression on the tumor cells or due to direct blocking with anti-NKG2D mAb treatment, the protective effects of IL-21 treatment were abrogated or substantially diminished. Specifically, IL-21 only demonstrated a therapeutic effect in mice challenged with a retinoic acid early inducible-1delta-bearing lymphoma but not in mice bearing parental RMA tumors lacking NKG2D ligands. Furthermore, treatment with a blocking anti-NKG2D mAb largely prevented the therapeutic effect of IL-21 in mice challenged with the 4T1 breast carcinoma, the 3LL lung carcinoma, and RM-1 prostate carcinoma. By contrast, IL-21 did mediate beneficial effects against both the parental DA3 mammary carcinoma and DA3 tumors transfected with H60, a NKG2D ligand. We also observed that IL-21 treatment could enhance RMA-retinoic acid early inducible-1delta tumor rejection in RAG-1(-/-) deficient mice, thereby demonstrating that the IL-21-induced protective effect can be mediated by the innate immune system and that, in this case, IL-21 does not require the adaptive immune response. Collectively, these findings suggest that IL-21 therapy may work optimally against tumors that can elicit a NKG2D-mediated immune response.

Interleukin-21 is a T-helper cytokine that regulates humoral immunity and cell-mediated anti-tumour responses.

Cytokines and their receptors represent key targets for therapeutic intervention. Ligands are being used to supplement cell numbers that become depleted as a result of disease (organ failure, infection) or subsequent disease treatments (i.e. chemotherapy). Conversely, the inhibition of target cell binding by cytokines is an established strategy for abrogating pathologic cellular activities common to many immunological diseases. Considerable effort in biomedical research is being focused on the cytokine families that play a dominant role in regulating immunity and then prioritizing each member for its therapeutic potential. Currently, the interleukin-2 (IL-2) family of cytokines is widely recognized for its central involvement in controlling lymphocyte function and is the most explored for medical utility. Collectively, these proteins (or their antagonists) are either marketed drugs or have received advanced testing for an impressive array of indications including cancer, infectious disease, transplantation, inflammation and allergic asthma. Here we review the current understanding of IL-21, the most recent member of this cytokine family to be discovered. As will be discussed, IL-21 shares many of the same attributes as its relatives in that it has broad immunoregulatory activity and can modulate both humoral and cell-mediated responses. Its ability to stimulate durable anti-tumour responses in mice defines one therapeutic indication that merits clinical development.