Wenxin Xu

Efficacy and Mechanism-of-Action of a Novel Superagonist Interleukin-15: Interleukin-15 Receptor αSu/Fc Fusion Complex in Syngeneic Murine Models of Multiple Myeloma

ALT-803, a complex of an interleukin (IL)-15 superagonist mutant and a dimeric IL-15 receptor αSu/Fc fusion protein, was found to exhibit significantly stronger in vivo biologic activity on NK and T cells than IL-15. In this study, we show that a single dose of ALT-803, but not IL-15 alone, eliminated well-established 5T33P and MOPC-315P myeloma cells in the bone marrow of tumor-bearing mice. ALT-803 treatment also significantly prolonged survival of myeloma-bearing mice and provided resistance to rechallenge with the same tumor cells through a CD8(+) T-cell-dependent mechanism. ALT-803 treatment stimulated CD8(+) T cells to secrete large amounts of IFN-γ and promoted rapid expansion of CD8(+)CD44(high) memory T cells in vivo. These memory CD8(+) T cells exhibited ALT-803-mediated upregulation of NKG2D (KLRK1) but not PD-1 (PDCD1) or CD25 (IL2RA) on their cell surfaces. ALT-803-activated CD8(+) memory T cells also exhibited nonspecific cytotoxicity against myeloma and other tumor cells in vitro, whereas IFN-γ had no direct effect on myeloma cell growth. ALT-803 lost its antimyeloma activity in tumor-bearing IFN-γ knockout mice but retained the ability to promote CD8(+)CD44(high) memory T-cell proliferation, indicating that ALT-803-mediated stimulation of CD8(+)CD44(high) memory T cells is IFN-γ-independent. Thus, besides well-known IL-15 biologic functions in host immunity, this study shows that IL-15-based ALT-803 could activate CD8(+)CD44(high) memory T cells to acquire a unique innate-like phenotype and secrete IFN-γ for nonspecific tumor cell killing. This unique immunomodulatory property of ALT-803 strongly supports its clinical development as a novel immunotherapeutic agent against cancer and viral infections.

Novel Human Interleukin-15 Agonists

IL-15 is an immunostimulatory cytokine trans-presented with the IL-15 receptor α-chain to the shared IL-2/IL-15Rβ and common γ-chains displayed on the surface of T cells and NK cells. To further define the functionally important regions of this cytokine, activity and binding studies were conducted on human IL-15 muteins generated by site-directed mutagenesis. Amino acid substitutions of the asparagine residue at position 72, which is located at the end of helix C, were found to provide both partial agonist and superagonist activity, with various nonconservative substitutions providing enhanced activity. Particularly, the N72D substitution provided a 4–5-fold increase in biological activity of the IL-15 mutein compared with the native molecule based on proliferation assays with cells bearing human IL-15Rβ and common γ-chains. The IL-15N72D mutein exhibited superagonist activity through improved binding ability to the human IL-15Rβ-chain. However, the enhanced potency of IL-15N72D was not observed with cells expressing the mouse IL-15Rα-IL-15Rβ-γc complex, suggesting that this effect is specific to the human IL-15 receptor. The enhanced biological activity of IL-15N72D was associated with more intense phosphorylation of Jak1 and Stat5 and better anti-apoptotic activity compared with the wild-type IL-15. IL-15N72D superagonist activity was also preserved when linked to a single-chain TCR domain to generate a tumor-specific fusion protein. Thus, the human IL-15 superagonist muteins and fusions may create opportunities to construct more efficacious immunotherapeutic agents with clinical utility.

Comparison of the superagonist complex, ALT-803, to IL15 as cancer immunotherapeutics in animal models

IL15 stimulates T-cell and NK-cell responses, but not Tregs. The antitumor efficacy, biodistribution, and toxicity of an IL15-based superagonist, ALT-803, was examined in animal models and was superior, supporting its clinical development for advanced hematologic or solid tumors. IL15, a potent stimulant of CD8+ T cells and natural killer (NK) cells, is a promising cancer immunotherapeutic. ALT-803 is a complex of an IL15 superagonist mutant and a dimeric IL15 receptor αSu/Fc fusion protein that was found to exhibit enhanced biologic activity in vivo, with a substantially longer serum half-life than recombinant IL15. A single intravenous dose of ALT-803, but not IL15, eliminated well-established tumors and prolonged survival of mice bearing multiple myeloma. In this study, we extended these findings to demonstrate the superior antitumor activity of ALT-803 over IL15 in mice bearing subcutaneous B16F10 melanoma tumors and CT26 colon carcinoma metastases. Tissue biodistribution studies in mice also showed much greater retention of ALT-803 in the lymphoid organs compared with IL15, consistent with its highly potent immunostimulatory and antitumor activities in vivo. Weekly dosing with 1 mg/kg ALT-803 in C57BL/6 mice was well tolerated, yet capable of increasing peripheral blood lymphocyte, neutrophil, and monocyte counts by >8-fold. ALT-803 dose-dependent stimulation of immune cell infiltration into the lymphoid organs was also observed. Similarly, cynomolgus monkeys treated weekly with ALT-803 showed dose-dependent increases of peripheral blood lymphocyte counts, including NK, CD4+, and CD8+ memory T-cell subsets. In vitro studies demonstrated ALT-803–mediated stimulation of mouse and human immune cell proliferation and IFNγ production without inducing a broad-based release of other proinflammatory cytokines (i.e., cytokine storm). Based on these results, a weekly dosing regimen of ALT-803 has been implemented in multiple clinical studies to evaluate the dose required for effective immune cell stimulation in humans. Cancer Immunol Res; 4(1); 49–60. ©2015 AACR.

IL-15 superagonist/IL-15RαSushi-Fc fusion complex (IL-15SA/IL-15RαSu-Fc; ALT-803) markedly enhances specific subpopulations of NK and memory CD8+ T cells, and mediates potent anti-tumor activity against murine breast and colon carcinomas

Interleukin (IL)-15-N72D superagonist-complexed with IL-15RαSushi-Fc fusion protein (IL-15SA/IL-15RαSu-Fc; ALT-803) has been reported to exhibit significant anti-tumor activity in murine myeloma, rat bladder cancer, and murine glioblastoma models. In this study, we examined the immunomodulatory and anti-tumor effects of IL-15SA/IL-15RαSu-Fc in tumor-free and highly metastatic tumor-bearing mice. Here, IL-15SA/IL-15RαSu-Fc significantly expanded natural killer (NK) and CD8+ T cells. In examining NK cell subsets, the greatest significant increase was in highly cytotoxic and migrating (CD11b+, CD27hi; high effector) NK cells, leading to enhanced function on a per-cell basis. CD8+ T cell subset analysis determined that IL-15SA/IL-15RαSu-Fc significantly increased IL-15 responding memory (CD122+, CD44+) CD8+ T cells, in particular those having the innate (NKG2D+, PD1−) phenotype. In 4T1 breast tumor–bearing mice, IL-15SA/IL-15RαSu-Fc induced significant anti-tumor activity against spontaneous pulmonary metastases, depending on CD8+ T and NK cells, and resulting in prolonged survival. Similar anti-tumor activity was seen in the experimental pulmonary metastasis model of CT26 colon carcinoma cells, particularly when IL-15SA/IL-15RαSu-Fc was combined with a cocktail of checkpoint inhibitors, anti-CTLA-4 and anti-PD-L1. Altogether, these studies showed for the first time that IL-15SA/IL-15RαSu-Fc (1) promoted the development of high effector NK cells and CD8+ T cell responders of the innate phenotype, (2) enhanced function of NK cells, and (3) played a vital role in reducing tumor metastasis and ultimately survival, especially in combination with checkpoint inhibitors.

Novel antitumor mechanism-of-action of an IL-2 fusion protein mediated by tumor associated macrophage repolarization and innate-like CD8+ memory T cells

ALT-801, a fusion of interleukin-2 and a single-chain T cell receptor domain specific to a p53 peptide/HLA-A*0201 complex, has shown immune cell-mediated activity in human xenograft tumor SCID mouse models and in patients with metastatic malignancies. To further investigate the antitumor efficacy of this fusion protein in immunocompetent mice, we conducted mechanism-of-action studies in an orthotopic murine muscle invasive bladder cancer model. ALT-801 treatment (1.6 mg/kg i.v., day 7, 10, 14, 17 post-tumor instillation) significantly prolonged survival of C57BL/6 mice bearing orthotopic MB49luc bladder tumors when compared to equivalent treatment with either IL-2 (0.4 mg/kg) or PBS (8 mice/group; median survival: ALT-801, 48.5 days vs. IL-12, 36 days; vs.PBS, 33.5 days, p 0.05; PBS vs. ALT-801 treatment of tumor-bearing IFN-γ or IFN-γR KO mice, P > 0.05). Consistent with these results, IHC, adoptive cell transfer and expression array studies showed that ALT-801 treatment led to activation and proliferation of CD4+ and CD8+ T cells which migrate from the lymphoid tissues to the tumor site where they secrete IFN-γ. Twenty hours after ALT-801 administration, tumor associated-macrophages (TAM) in the bladder of tumor-bearing mice were also transiently repolarized from an M2 (tumor promoting) to an M1 (tumor killing) phenotype in an IFN-γ dependent manner. Additionally, in vivo ALT-801 treatment induced innate-like CD8+CD44high memory T cells to proliferate and upregulate NKG2D receptors. These cells may contribute to elevated splenocyte cytotoxic activity against mouse and human bladder tumor cells that was seen after ALT-801 administration. The results of these studies suggest that ALT-801 induces IFN-γ-dependent TAM repolarization and non-specific CD8+ memory effector T cells that promote robust and rapid antitumor activity in mice bearing orthotopic MB49luc bladder tumors. This novel immunostimulatory mechanism-of-action appears to be distinct from that of IL-2 or other T cell-based immunotherapeutics and is currently being assessed in bladder cancer patients under treatment with ALT-801.

Abstract 3245: IL-15 superagonist/IL-15RαSushi-Fc fusion complex (IL-15SA/IL-15RαSu-Fc; ALT-803) markedly enhances specific subpopulations of NK and memory CD8+ T cells, and mediates potent anti-tumor activity of murine breast and colon carcinomas

Interleukin (IL)-15-N72D superagonist-complexed with IL-15RαSushi-Fc fusion protein (IL-15SA/IL-15RαSu-Fc; ALT-803) has been reported to exhibit significant anti-tumor activity in murine myeloma, rat bladder cancer, and murine glioblastoma models. In this study, we examined the immunomodulatory and anti-tumor effects of IL-15SA/IL-15RαSu-Fc in tumor-free and highly metastatic tumor-bearing mice. Here, IL-15SA/IL-15RαSu-Fc significantly expanded NK and CD8+ T cells. In examining natural killer (NK) cell subsets, the greatest significant increase was in highly cytotoxic and migrating (CD11b+, CD27hi; high effector) NK cells, leading to enhanced function on a per-cell basis. CD8+ T cell subset analysis determined that IL-15SA/IL-15RαSu-Fc significantly increased IL-15 responding memory (CD122+, CD44+) CD8+ T cells, in particular those having the innate (NKG2D+, PD1-) phenotype. In 4T1 breast tumor-bearing mice, IL-15SA/IL-15RαSu-Fc induced significant anti-tumor activity against spontaneous pulmonary metastases, depending on CD8+ T and NK cells, and resulting in prolonged survival. Similar anti-tumor activity was seen in the experimental pulmonary metastasis model of CT26 colon carcinoma cells, particularly when IL-15SA/IL-15RαSu-Fc was combined with a cocktail of checkpoint inhibitors, anti-CTLA-4 and anti-PD-L1. Altogether, these studies showed for the first time that IL-15SA/IL-15RαSu-Fc (1) promoted the development of high effector NK cells, (2) enhanced function of NK cells, and (3) played a vital role in reducing tumor metastasis and ultimately survival, especially in combination with checkpoint inhibitors. Citation Format: Peter S. Kim, Anna R. Kwilas, Wenxin Xu, Sarah Alter, Emily K. Jeng, Hing C. Wong, Jeffrey Schlom, James W. Hodge. IL-15 superagonist/IL-15RαSushi-Fc fusion complex (IL-15SA/IL-15RαSu-Fc; ALT-803) markedly enhances specific subpopulations of NK and memory CD8+ T cells, and mediates potent anti-tumor activity of murine breast and colon carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3245.

Enhanced efficacy of ALT-803, an IL-15-based superagonist complex, in combination with immune checkpoint inhibitors in an orthotopic muscle invasive bladder tumor model in mice

Immune checkpoint therapy, which can target regulatory pathways of T cells to enhance antitumor immune responses, has led to important clinical advances and provides novel strategies for combination immunotherapies against cancer. ALT-803 is an IL-15 superagonist complex capable of stimulating T cell and NK cell responses without triggering Treg activity. ALT-803 exhibits improved pharmacokinetics and lymphoid organ biodistribution compared to native IL-15, allowing this complex to have more potent efficacy against various hematologic and solid tumor models. Antibodies that block checkpoint inhibitor interactions, such as PD-1/PD-L1 or CTLA-4, have shown clinical efficacy against both melanoma and lung cancer, and may also be active in other tumor types, including bladder cancer. Here, we report that the combination of immune checkpoint blockers with ALT-803 has potent anti-tumor activity in C57BL/6 mice bearing syngenic orthotopic MB49-luc bladder tumors. These tumor cells were found to express CTLA-4 ligands and PD-L1 on the cell surface; thus, blockade of both CTLA-4 and PD-1/PD-L1 pathways were evaluated. Treatment of mice bearing established MB49luc bladder tumors with ALT-803 monotherapy exhibited a statistically significant increase in survival compared to controls (P < 0.05). However, the combination of ALT-803 with anti-PD-L1 and anti-CTLA-4 Abs further prolonged survival compared to control or related monotherapies. This effect was also seen with combination therapy of ALT-803+anti-PD-1 and ALT-803+anti-PD-1/anti-CTLA-4 Abs. Additionally, mice that were cured of MB49-luc tumors by ALT-803 plus anti-PD-L1/anti-CTLA-4 Ab therapy were resistant from bladder tumor rechallenge without further drug treatment whereas age-matched treatment-naive mice developed tumors and die following MB49-luc tumor cell instillation. These results demonstrate that a short course of ALT-803 plus immune checkpoint inhibitor treatment not only has potent efficacy against established MB49luc tumor, but is also capable of inducing long-lasting protective immunologic memory against subsequent tumor cell rechallenge. Characterization of immune cell activity responsible for improved antitumor efficacy is underway. Overall, these results confirm that enhanced antitumor responses can be achieved by combining immune checkpoint blockers with the immunostimulatory agonist ALT-803, warranting evaluation of these strategies in the clinical setting.

Enhanced efficacy of IL-15-based ALT-803 superagonist complex in combination with immune checkpoint inhibitors in hematologic and metastatic mouse tumor models

Recent advances in achieving highly durable clinical responses via inhibition of immune checkpoint molecules have revolutionized the outlook for cancer immunotherapy. However, such responses are only observed in a minority of patients, suggesting that strategies to further augment antitumor immune activity may provide additional clinical benefit. ALT-803 is an IL-15 superagonist:IL-15Rα-Fc complex capable of stimulating T cell and NK cell responses without inducing Treg activity. ALT-803 has improved pharmacokinetics and biodistribution compared to native IL-15, allowing this complex to exhibit more potent efficacy than IL-15 in various hematologic and solid tumor models. Mechanism-of-action studies showed that ALT-803 is capable of inducing both innate effector-like and adaptive T cell antitumor activity, suggesting that the immunostimulatory effects of ALT-803 could compliment the activity of checkpoint inhibitors to block tumor immune evasion. To assess this, we utilized a murine 5T33P myeloma model in which ALT-803 was shown to be effective at decreasing tumor burden in the bone marrow and prolonging animal survival. Treatment of mice bearing established 5T33P tumors (5/group) with 5 mg/kg anti-PD-L1 mAb also prolonged survival compared to controls (P > 0.01), whereas treatment with 1.25 mg/kg anti-PD-L1 mAb or 10 mg/kg anti-CTLA-4 mAb had no significant antitumor effect (p < 0.05). Consistent with these results, 5T33P cells were found to express PD-L1 but not CD80/CD86. Treatment with sub-optimal 0.25 mg/kg anti-PD-L1 mAb or 50 µg/kg ALT-803 was ineffective at prolonging survival of 5T33P-bearing mice (median survival; PBS, 27 days; anti-PD-L1 mAb, 31 day; ALT-803, 28 days). However, combination therapy with sub-optimal anti-PD-L1 mAb plus ALT-803 extended median survival to 66 days (p < 0.05 vs. controls). These combination therapies were also evaluated in a metastatic murine CT26 colon carcinoma model that was previous used to show increased efficacy of IL-15+anti-PD-L1+anti-CTLA-4 mAb treatment. Notably, ALT-803+anti-PD-L1+anti-CTLA-4 mAb treatment provided greater survival benefit to mice with metastatic CT26 tumors than treatment with vehicle or ALT-803 alone (median survival: combined therapy, 53 days vs. PBS, 15 days; vs. ALT-803, 22.5 days; both p < 0.01) or the IL-15+anti-PD-L1+anti-CTLA-4 mAb combination (median survival: 18 days; p < 0.001). In all of these models, combination therapies of ALT-803 and anti-checkpoint Abs were well tolerated. Characterization of immune cell activity responsible for improved antitumor efficacy is underway. Overall, these results confirm that enhanced antitumor responses can be achieved by combining immune checkpoint blockers with ALT-803, warranting evaluation of these strategies in the clinical setting.