Discovery and Preclinical Activity of BMS-986351, an Antibody to SIRPα That Enhances Macrophage-mediated Tumor Phagocytosis When Combined with Opsonizing Antibodies.

In normal cells, binding of the transmembrane protein CD47 to signal regulatory protein-α (SIRPα) on macrophages induces an antiphagocytic signal. Tumor cells hijack this pathway and overexpress CD47 to evade immune destruction. Macrophage antitumor activity can be restored by simultaneously blocking the CD47-SIRPα signaling axis and inducing a prophagocytic signal via tumor-opsonizing antibodies. We identified a novel, fully human mAb (BMS-986351) that binds SIRPα with high affinity. BMS-986351 demonstrated broad binding coverage across SIRPα polymorphisms and potently blocked CD47-SIRPα binding at the CD47 binding site in a dose-dependent manner. In vitro, BMS-986351 increased phagocytic activity against cell lines from solid tumors and hematologic malignancies, and this effect was markedly enhanced when BMS-986351 was combined with the opsonizing antibodies cetuximab and rituximab. A phase I dose-escalation/-expansion study of BMS-986351 for the treatment of advanced solid and hematologic malignancies is underway (NCT03783403). SIGNIFICANCE: Increasing the phagocytotic capabilities of tumor-associated macrophages by modulating macrophage-tumor cell surface signaling via the CD47-SIRPα axis is a novel strategy. Molecules targeting CD47 have potential but its ubiquitous expression necessitates higher therapeutic doses to overcome potential antigen sink effects. The restricted expression pattern of SIRPα may limit toxicities and lower doses of the SIRPα antibody BMS-986351 may overcome target mediated drug disposition while maintaining the desired pharmacology.

Identification of Small Molecule Inhibitors and Ligand Directed Degraders of Calcium/Calmodulin Dependent Protein Kinase Kinase 1 and 2 (CaMKK1/2).

CaMKK2 signals through AMPK-dependent and AMPK-independent pathways to trigger cellular outputs including proliferation, differentiation, and migration, resulting in changes to metabolism, bone mass accrual, neuronal function, hematopoiesis, and immunity. CAMKK2 is upregulated in tumors including hepatocellular carcinoma, prostate, breast, and gastric cancer, and genetic deletion in myeloid cells results in increased antitumor immunity in several syngeneic models. Validation of the biological roles of CaMKK2 has relied on genetic deletion or small molecule inhibitors with activity against several biological targets. We sought to generate selective inhibitors and degraders to understand the biological impact of inhibiting catalytic activity and scaffolding and the potential therapeutic benefits of targeting CaMKK2. We report herein selective, ligand-efficient inhibitors and ligand-directed degraders of CaMKK2 that were used to probe immune and tumor intrinsic biology. These molecules provide two distinct strategies for ablating CaMKK2 signaling in vitro and in vivo.

Integrative network modeling reveals mechanisms underlying T cell exhaustion.

Failure to clear antigens causes CD8+ T cells to become increasingly hypo-functional, a state known as exhaustion. We combined manually extracted information from published literature with gene expression data from diverse model systems to infer a set of molecular regulatory interactions that underpin exhaustion. Topological analysis and simulation modeling of the network suggests CD8+ T cells undergo 2 major transitions in state following stimulation. The time cells spend in the earlier pro-memory/proliferative (PP) state is a fixed and inherent property of the network structure. Transition to the second state is necessary for exhaustion. Combining insights from network topology analysis and simulation modeling, we predict the extent to which each node in our network drives cells towards an exhausted state. We demonstrate the utility of our approach by experimentally testing the prediction that drug-induced interference with EZH2 function increases the proportion of pro-memory/proliferative cells in the early days post-activation.

The IL-20 Cytokine Family in Rheumatoid Arthritis and Spondyloarthritis.

This review describes the IL-20 family of cytokines in rheumatoid arthritis (RA) and spondyloartrhitits (SpA) including psoriatic arthritis. The IL-20 receptor (R) cytokines IL-19, IL-20, and IL-24 are produced in both the peripheral blood and the synovial joint and are induced by Toll-like receptor ligands and autoantibody-associated immune complexes in monocytes. IL-19 seems to have anti-inflammatory functions in arthritis. In contrast, IL-20 and IL-24 increase the production of proinflammatory molecules such as monocyte chemoattractant protein 1 and are associated with bone degradation and radiographic progression. IL-22 is also associated with progression of bone erosions. This suggests that the IL-22RA1 subunit shared by IL-20, IL-22, and IL-24 is important for bone homeostasis. In line with this, the IL-22RA1 has been found on preosteoclasts in early RA. IL-26 is produced in high amounts by myofibroblasts and IL-26 stimulation of monocytes is an important inducer of Th17 cells in RA. This indicates a role for IL-26 as an important factor in the interactions between resident synovial cells and infiltrating leukocytes. Clinical trials that investigate inhibitors of IL-20 (fletikumab) and IL-22 (fezakinumab) in psoriasis and RA have been terminated. Instead, it seems that the strategy for modulating the IL-20 cytokine family should take the overlap in cellular sources and effector mechanisms into account. The redundancy encourages inhibition of more than one cytokine or one of the shared receptors. All IL-20 family members utilize the Janus kinase signaling pathway and are therefore potentially inhibited by drugs targeting these enzymes. Effects and adverse effects in ongoing clinical trials with inhibitors of IL-22 and the IL-22RA1 subunit and recombinant IL-22 fusion proteins will possibly provide important information about the IL-20 subfamily of cytokines in the future.

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.

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.

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.

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.

IL-21 blockade reduces graft-versus-host disease mortality by supporting inducible T regulatory cell generation.

Interleukin-21 (IL-21) enhances T helper 1 (Th1) and Th17 differentiation while inhibiting the conversion of inducible regulatory T cells (Tregs) from naive T cells. To determine the role of IL-21 in graft-versus-host disease (GVHD), anti-IL-21 antibody (Ab) was given to recipients of CD25(-)CD4(+) or CD4(+) and CD8(+) T-effectors. IL-21 neutralization attenuated GVHD-related weight loss and prolonged survival. Likewise, a majority of mice receiving IL-21(-/-) CD25(-) T-effectors survived long term, whereas those receiving wild-type T cells died. The latter recipients had higher grades of GVHD in the ileum and colon. Surprisingly, disruption of IL-21 signaling did not affect IL-17 production, although colon-infiltrating T-effector cells had decreased interferon gamma (IFNgamma) and increased IL-4 production. FoxP3(+) Tregs were increased in colons of anti-IL-21 Ab-treated recipients of FoxP3(-) IL-21(-/-) T cells, indicating Treg conversion. Recipients of FoxP3-deficient T-effectors isolated from chimeras were resistant to the GVHD protective effects of IL-21 blockade. Whereas graft-versus-leukemia (GVL) can occur in the absence of IL-21, loss of both IL-21 and perforin expression abrogated GVL. Together, these data indicate that IL-21 suppresses inducible Treg conversion and further suggest that IL-21 blockade is an attractive strategy to reduce GVHD-induced injury.

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.

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 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.

Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes.

Interleukin-28A (IL-28A), IL-28B and IL-29 are a family of class II cytokines that stimulate antiviral responses through a heterodimeric receptor that is distinct from the type I interferon (IFN) receptor. To better understand how this newly described family of cytokines regulates the antiviral state, we compared various cellular responses elicited by IL-29 and IFN-alpha. Here we show that these cytokines stimulate similar patterns of signal transducer and activator of transcription 1 (STAT-1), -2, -3, and -5 phosphorylation and nearly identical patterns of gene expression when analyzed in two distinct cell types by microarray analysis. Interestingly, the IL-29 receptor is preferentially expressed on primary hepatocytes within normal liver and pegylated forms of IL-29 and IFN-alpha induced equivalent 2'5' oligoadenylate synthetase (OAS) and MX1 gene expression in this cell type. Pegylated IL-29 also produced a significant reduction in human hepatitis B and hepatitis C viral load in vitro and reduced the cytopathic effect caused by the fully replicating flavivirus, West Nile virus. In conclusion, IL-29 and IFN-alpha stimulate identical antiviral responses despite their utilization of different receptors. This fact, combined with significant receptor expression in hepatitis virus-infected livers, suggests that IL-29 may have therapeutic value against chronic viral hepatitis in human patients.

IL-21 enhances tumor-specific CTL induction by anti-DR5 antibody therapy.

Tumor cell apoptosis is the basis of many cancer therapies, and tumor-specific T cells are the principal effectors of successful anti-tumor immunotherapies. In this study, we show that induction of tumor cell apoptosis by agonistic mAb against DR5, combined with delayed IL-21 treatment, suppressed tumor growth and pre-established tumor metastases. Synergistic effects of the combination were observed in several tumor models where the target tumor was sensitive to DR5-mediated apoptosis. IL-21 promoted tumor-specific CTL activity and enhanced memory responses to tumor rechallenge. These results indicate that a rational combination of Ab-based therapy that causes tumor cell apoptosis and a cytokine that promotes T cell memory is a useful new strategy for cancer immunotherapy.

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.

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.