T Cell Receptor-Engaging Monoclonal Antibodies Mobilize the Anti-Tumor Functions of Invariant Natural Killer T Cells

Abstract

Invariant natural killer T cells (iNKTs) are innate-type T lymphocytes that directly kill tumor cells or tumor-growth promoting immunosuppressive cells such astumor-associated macrophages. Additionally, iNKTs robustly transactivate the antitumor functions of T, B, natural killer, and dendritic cells as well as reinvigorate exhausted immune cells in the tumor microenvironment. As such, iNKTs make excellent candidates for inclusion in anti-cancer cellular therapies. However, to capitalize on the potential benefits of iNKT cell-based approaches, it is imperative that we develop new and clinically viable strategies to enhance their antitumor function. To that end, two novel monoclonal antibodies (mAbs) that selectively bind to the human (NKTT320) or murine (NKT14m) invariant T cell receptor have been recently developed and characterized. Studies using purified human iNKTs (in vitro) and a model of non-human primate (in vivo) reveal that NKTT320 promotes swift, vigorous and sustained iNKT cell activation that is accompanied by robust production of inflammatory mediators and bystander immune cell activation. Furthermore, NKTT320 augments expression of cytotoxic markers and human iNKT cell degranulation. Similarly, NKT14m prompts dramatic murine iNKT cell activation and functional response both in vitro and in vivo. However, antitumor efficacy of a single dose of NKT14m injection in tumor-bearing mice is limited and tumor-model dependent. In contrast, combination treatment of NKT14m with either low dose interleukin (IL)-12 or the chemotherapeutic agent, cyclophosphamide results in a superior antitumor response in vivo. This is evident by activation of both iNKTs and other immune cells, prolonged survival of the tumor-challenged mice, and long-lasting immunity. Collectively, these recent studies justify further development of anti-iTCR mAbs that can be used alone or in conjunction with immunomodulatory agents to enhance iNKT cell antitumor immunity against various cancers.

FIG. 1:

Invariant NKTs utilize various mechanisms to exert cytotoxicity against tumor cells. Invariant NKTs can indirectly kill tumor targets by transactivating cytotoxic effector cells such as the CD8+T and NK cells. When antigen presenting cells (such as DCs) present a tumor-derived glycolipid to iNKTs, it leads to their activation, IFN-γ production and CD40 stimulation. This iNKT cell-derived IFNγ induces DCs to produce IL-12, which further augments IFNγ secretion by iNKT and NK cells, and activates CD8+ T cell- and NK cell-dependent killing of tumor cells. Alternatively, iNKTs recognize glycolipid antigens presented by CD1d on tumor cells and mount direct cytotoxicity via exocytosis of lytic molecules such as perforin/granzyme or via interaction of death receptors such as Fas-FasL. Immunosuppressive cells such as tumor-associated macrophages (TAMs) and/or IL-10 producing neutrophils promote tumor growth. Invariant NKTs can limit tumor cells by either directly killing TAMs or by alleviating the suppressive effect of neutrophils via CD40-CD40L interactions. TAA: tumor-associated antigen.

FIG. 2:

NKTT320 promotes human iNKT cell activation and immunomodulatory functions. (A) NKTT320 binds specifically to the invariant TCR of human iNKTs and induces rapid and robust iNKT cell activation as well as Th1/Th2 cytokine and chemokine production in vitro, which in turn invigorates bystander immune cells. Additionally, NKTT320-stimulated iNKTs express enhanced levels of granzyme B and exhibit increased degranulation. Thus, NKTT320 can potentially facilitate both direct and indirect human iNKT cell cytotoxicity against tumor targets. (B) The effects of NKTT320 in vivo have been characterized in Mauritian-origin cynomolgus macaques (MCM). In vivo injection of NKTT320 in MCM leads to rapid iNKT cell activation, increased polyfunctionality, and robust production of several cytokines and chemokines. This subsequently allows for the recruitment and activation of both the innate and adaptive immune cells, sustained iNKT cell activation (no anergy), and upregulation of several genes that modulate activation and inhibition of inflammatory host responses.

FIG. 3:

Antitumor effects of NKT14m in vivo. (A) The antitumor efficacy of a single dose of NKT14m in vivo yields moderate (B-cell lymphoma) to no (T-cell lymphoma) antitumor efficacy but is associated with an increase in IFN-γ-producing iNKTs. (B) An improved antitumor response is observed in tumor-challenged (B-cell lymphoma) mice after a second dose of NKT14m injection. Retreatment with NKT14m results in enhanced IFN-γ production and no iNKT cell anergy. (C) Administration of NKT14m with low dose IL12 augments iNKT cell IFN-γ production in vivo, transactivation of other immune cells and a superior suppression of T-cell lymphoma growth (as compared to NKT14m or IL-12 alone). (D) Combination of a single dose of NKT14m with cyclophosphamide (Cy) induces a potent antitumor response, IFN-γ production by iNKT and CD4+T cells and improved survival of B-cell lymphoma-challenged mice. Re-injection of tumor cells in these animals results in long-lasting immunity in vivo.