Kaiping Han

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.

IL-15:IL-15 receptor alpha superagonist complex: High-level co-expression in recombinant mammalian cells, purification and characterization

IL-15, a promising cytokine for treating cancer and viral diseases, is presented in trans by the IL-15 receptor (IL-15R) alpha-chain to the IL-15Rβγc complex displayed on the surface of T cells and natural killer (NK) cells. We previously reported that an asparagine to aspartic acid substitution at amino acid 72 (N72D) of IL-15 provides a 4-5-fold increase in biological activity compared to the native molecule. In this report, we describe Chinese hamster ovary (CHO) cell expression of a soluble complex (IL-15 N72D:IL-15RαSu/Fc) consisting of the IL-15 N72D superagonist and a dimeric IL-15Rα sushi domain-IgG1 Fc fusion protein. A simple but readily scalable affinity and ion exchange chromatography method was developed to highly purify the complex having both IL-15 binding sites fully occupied. The immunostimulatory effects of this complex were confirmed using cell proliferation assays. Treatment of mice with a single intravenous dose of IL-15N72D:IL-15RαSu/Fc resulted in a significant increase in CD8+ T cells and NK cells that was not observed following IL-15 treatment. Pharmacokinetic analysis indicated that the complex has a 25-h half-life in mice which is considerably longer than <40-min half-life of IL-15. Thus, the enhanced activity of the IL-15N72D:IL-15RαSu/Fc complex is likely the result of the increased binding activity of IL-15N72D to IL-15Rβγc, optimized cytokine trans-presentation by the IL-15RαSu domain, the dimeric nature of the cytokine domain and its increased in vivo half-life compared to IL-15. These findings indicate that this IL-15 superagonist complex could serve as a superior immunostimulatory therapeutic agent.

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.

The IL-15-Based ALT-803 Complex Enhances FcγRIIIa-Triggered NK Cell Responses and in Vivo Clearance of B Cell Lymphomas

Purpose: Anti-CD20 monoclonal antibodies (mAb) are an important immunotherapy for B-cell lymphoma, and provide evidence that the immune system may be harnessed as an effective lymphoma treatment approach. ALT-803 is a superagonist IL-15 mutant and IL-15Rα–Fc fusion complex that activates the IL-15 receptor constitutively expressed on natural killer (NK) cells. We hypothesized that ALT-803 would enhance anti–CD20 mAb-directed NK-cell responses and antibody-dependent cellular cytotoxicity (ADCC). Experimental Design: We tested this hypothesis by adding ALT-803 immunostimulation to anti-CD20 mAb triggering of NK cells in vitro and in vivo. Cell lines and primary human lymphoma cells were utilized as targets for primary human NK cells. Two complementary in vivo mouse models were used, which included human NK-cell xenografts in NOD/SCID-γc−/− mice. Results: We demonstrate that short-term ALT-803 stimulation significantly increased degranulation, IFNγ production, and ADCC by human NK cells against B-cell lymphoma cell lines or primary follicular lymphoma cells. ALT-803 augmented cytotoxicity and the expression of granzyme B and perforin, providing one potential mechanism for this enhanced functionality. Moreover, in two distinct in vivo B-cell lymphoma models, the addition of ALT-803 to anti-CD20 mAb therapy resulted in significantly reduced tumor cell burden and increased survival. Long-term ALT-803 stimulation of human NK cells induced proliferation and NK-cell subset changes with preserved ADCC. Conclusions: ALT-803 represents a novel immunostimulatory drug that enhances NK-cell antilymphoma responses in vitro and in vivo, thereby supporting the clinical investigation of ALT-803 plus anti-CD20 mAbs in patients with indolent B-cell lymphoma. Clin Cancer Res; 22(3); 596–608. ©2015 AACR.

Targeting activity of a TCR/IL-2 fusion protein against established tumors

We have previously reported that a single-chain T cell receptor/IL-2 fusion protein (scTCR-IL2) exhibits potent targeted antitumor activity in nude mice bearing human tumor xenografts that display cognate peptide/HLA complexes. In this study, we further explore the mechanism of action of this molecule. We compared the biological activities of c264scTCR-IL2, a scTCR-IL2 protein recognizing the aa264–272 peptide of human p53, with that of MART-1scTCR-IL2, which recognizes the MART-1 melanoma antigen (aa27–35). In vitro studies showed that c264scTCR-IL2 and MART-1scTCR-IL2 were equivalent in their ability to bind cell-surface IL-2 receptors and stimulate NK cell responses. In mice, MART-1scTCR-IL2 was found to have a twofold longer serum half-life than c264scTCR-IL2. However, despite its shorter serum half-life, c264scTCR-IL2 showed significantly better antitumor activity than MART-1scTCR-IL2 against p53+/HLA-A2+ tumor xenografts. The more potent antitumor activity of c264scTCR-IL2 correlated with an enhanced capacity to promote NK cell infiltration into tumors. Similar differences in antigen-dependent tumor infiltration were observed with activated splenocytes pre-treated in vitro with c264scTCR-IL2 or MART-1scTCR-IL2 and then transferred into p53+/HLA-A2+ tumor bearing recipients. The data support a model where c264scTCR-IL2 activates immune cells to express IL-2 receptors. Following stable interactions with cell-surface IL-2 receptors, c264scTCR-IL2 fusion molecule enhances the trafficking of immune cells to tumors displaying target peptide/HLA complexes where the immune cells mediate antitumor effects. Thus, this type of fusion molecule could be used directly as a targeted immunotherapeutic or in adoptive cell transfer approaches to activate and improve the anti-cancer activities of immune cells by providing them with pre-selected antigen recognition capability.

A Novel Fusion of ALT-803 (Interleukin (IL)-15 Superagonist) with an Antibody Demonstrates Antigen-specific Antitumor Responses

IL-15 and its receptor α (IL-15Rα) are co-expressed on antigen-presenting cells, allowing transpresentation of IL-15 to immune cells bearing IL-2RβγC and stimulation of effector immune responses. We reported previously that the high-affinity interactions between an IL-15 superagonist (IL-15N72D) and the extracellular IL-15Rα sushi domain (IL-15RαSu) could be exploited to create a functional scaffold for the design of multivalent disease-targeted complexes. The IL-15N72D·IL-15RαSuFc complex, also known as ALT-803, is a multimeric complex constructed by fusing IL-15N72D·IL-15RαSu to the Fc domain of IgG1. ALT-803 is an IL-15 superagonist complex that has been developed as a potent antitumor immunotherapeutic agent and is in clinical trials. Here we describe the creation of a novel fusion molecule, 2B8T2M, using the ALT-803 scaffold fused to four single chains of the tumor-targeting monoclonal antibody rituximab. This molecule displays trispecific binding activity through its recognition of the CD20 molecule on tumor cells, stimulation via IL-2RβγC displayed on immune effector cells, and binding to Fcγ receptors on natural killer cells and macrophages. 2B8T2M activates natural killer cells to enhance antibody-dependent cellular cytotoxicity, mediates complement-dependent cytotoxicity, and induces apoptosis of B-lymphoma cells. Compared with rituximab, 2B8T2M exhibits significantly stronger antitumor activity in a xenograft SCID mouse model and depletes B cells in cynomolgus monkeys more efficiently. Thus, ALT-803 can be modified as a functional scaffold for creating multispecific, targeted IL-15-based immunotherapeutic agents and may serve as a novel platform to improve the antitumor activity and clinical efficacy of therapeutic antibodies.

Interleukin-15:Interleukin-15 receptor α scaffold for creation of multivalent targeted immune molecules

Human interleukin-15 (hIL-15) and its receptor α (hIL-15Rα) are co-expressed in antigen presenting cells allowing trans-presentation of the cytokine to immune effector cells. We exploited the high-affinity interactions between hIL-15 and the extracellular hIL-15Rα sushi domain (hIL-15RαSu) to create a functional scaffold for the design of multispecific fusion protein complexes. Using single-chain T cell receptors (scTCRs) as recognition domains linked to the IL-15:IL-15Rα scaffold, we generated both bivalent and bispecific complexes. In these fusions, the scTCR domains retain the antigen-binding activity and the hIL-15 domain exhibits receptor binding and biological activity. As expected, bivalent scTCR fusions exhibited improved antigen binding due to increased avidity, whereas fusions comprising two different scTCR domains were capable of binding two cognate peptide/MHC complexes. Bispecific molecules containing scTCR and scCD8αβ domains also exhibit enhanced binding to peptide/MHC complexes, demonstrating that the IL-15:IL-15Rα scaffold displays flexibility necessary to support multi-domain interactions with a given target. Surprisingly, functional heterodimeric molecules could be formed by co-expressing the TCR α and β chains separately as fusions to the hIL-15 and hIL-15RαSu domains. Together, these properties indicate that the hIL-15 and hIL-15RαSu domains can be used as versatile, functional scaffold for generating novel targeted immune molecules.

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.

Combination therapy of an IL-15 superagonist complex, ALT-803, and a tumor targeting monoclonal antibody promotes direct antitumor activity and protective vaccinal effect in a syngenic mouse melanoma model

Cytokine-based and antibody-targeted immunotherapies have both been important approaches in the treatment of malignant cancers. However, combinational therapies of cytokines and tumor-targeting antibodies remain to be further explored, especially in advanced solid tumors. In this study, C57BL/6 mice bearing established subcutaneous B16F10 melanoma were treated with mouse melanoma targeting anti-gp75 monoclonal antibody (mAb), TA99, combined with interleukin (IL)-15 based superagonist ALT-803. This soluble protein complex consists of an IL-15 superagonist mutant (IL-15N72D) associated with an IL-15 receptor α Sushi domain - human IgG1 Fc fusion protein. Compared to native IL-15, ALT-803 possesses superior in vivo biologic activity for stimulating NK and CD8+ memory T cells. The combined ALT-803+TA99 therapy significantly exceeded either monotherapy in inhibiting melanoma tumor growth (p < 0.001) and prolonging survival (p < 0.01) of B16F10 tumor-bearing mice. Through immune cell depletion studies and immunophenotyping of peripheral cells as well as tumor-infiltrating leukocyte subsets, we found that ALT-803 enhances TA99-mediated antitumor immunity through activation of NK cells and expansion of the CD8+CD44high memory T cell arm. In contrast, CD4+ T cells were shown to play more of a suppressive role in the therapeutic effect of ALT-803+TA99, possibly through involvement of regulatory T cells or ALT-803-mediated induction of PD-L1 on CD4+ T cells in the periphery and tumor microenvironment. Addition of anti-PD-L1 mAb to ALT-803+TA99 therapy resulted in a further increase in antitumor activity against subcutaneous B16F10 tumors. Furthermore, tumor-bearing mice that survived due to ALT-803+TA99 combination therapy exhibited long term antitumor memory against B16F10 tumor cell rechallenge. Immune-depletion studies revealed that this antitumor memory was associated with CD4+ T cells, CD8+ T cells and NK cells. Our findings suggest a therapeutic opportunity for ALT-803 in combination with tumor-targeting antibodies to simultaneously augment targeted antitumor activities of therapeutic antibodies and induce a long-term vaccinal effect which will provide durable responses in the treated host.