Overcoming Resistance to Natural Killer Cell Based Immunotherapies for Solid Tumors

Abstract

Despite advances in the diagnostic and therapeutic modalities, the prognosis of several solid tumor malignancies remains poor. Different factors associated with solid tumors including a varied genetic signature, complex molecular signaling pathways, defective cross talk between the tumor cells and immune cells, hypoxic and immunosuppressive effects of tumor microenvironment result in a treatment resistant and metastatic phenotype. Over the past several years, immunotherapy has emerged as an attractive therapeutic option against multiple malignancies. The unique ability of natural killer (NK) cells to target cancer cells without antigen specificity makes them an ideal candidate for use against solid tumors. However, the outcomes of adoptive NK cell infusions into patients with solid tumors have been disappointing. Extensive studies have been done to investigate different strategies to improve the NK cell function, trafficking and tumor targeting. Use of cytokines and cytokine analogs has been well described and utilized to enhance the proliferation, stimulation and persistence of NK cells. Other techniques like blocking the human leukocyte antigen-killer cell receptors (KIR) interactions with anti-KIR monoclonal antibodies, preventing CD16 receptor shedding, increasing the expression of activating NK cell receptors like NKG2D, and use of immunocytokines and immune checkpoint inhibitors can enhance NK cell mediated cytotoxicity. Using genetically modified NK cells with chimeric antigen receptors and bispecific and trispecific NK cell engagers, NK cells can be effectively redirected to the tumor cells improving their cytotoxic potential. In this review, we have described these strategies and highlighted the need to further optimize these strategies to improve the clinical outcome of NK cell based immunotherapy against solid tumors.

Keywords: bispecific antibody; checkpoint inhibitors; chimeric antigen receptor; cytokines; immunotherapy; natural killer cell; solid tumor; tumor microenvironment.

Figure 1

Target recognition, tolerance, missing self. (A) NK cells recognize and kill their targets by an integrated balance of inhibitory and activating signals to discriminate between healthy cells (tolerance) vs. elimination of transformed or virally infected targets (killing). NK-cell tolerance depends on several MHC class I inhibitory signals (either classical, HLA-A, -B, or -C, or nonclassical, HLA-E) expressed by healthy cells that engage KIR or NKG2A with minimal activation signals resulting in tolerance. Malignant transformation or viral infection promotes target cell killing by downregulation of MHC class I expression and an upregulation of signals from activating NK-cell receptors. (B) Although in some cases, MHC downregulation is variable or incomplete, target cell killing can still occur by changing the balance with activating signals upregulated by stress-induced activating receptor ligands. (C) This balance between inhibition and activation can be uniquely manipulated in the hematopoietic transplant setting by selection of donors who will respond to apparent missing self HLA class I in the HLA-mismatched recipients. For example, reconstitution with a high frequency of donor KIR2DL1⁺ NK cells would not be inhibited in a HLA-C1 (C1⁺-HLA-C) recipient (KIR ligand mismatch). Here, NK-cell alloreactivity would kill the recipient's tumor. In contrast, when the same KIR2DL1⁺ NK cells reconstitute in an HLA-C2 recipient (C2⁺-HLA-C) (KIR ligand match), the recipient's tumor would be seen as having self HLA class I and would not provoke an alloreactive NK-cell response. Reproduced with permission from Cooley et al. (30).

Figure 2

Schema for NK cell manufacturing with artificial antigen-presenting cells. Artificial antigen-presenting cells were produced by genetic modification of K562 to express costimulatory molecules and membrane-bound cytokines. To expand NK cells ex vivo, unfractionated PBMC are stimulated weekly with irradiated PBMC, inducing rapid proliferation of NK cells and in some cases non-specific expansion of T cells. Contaminating T cells may be depleted, and the remaining purified NK cells may be stimulated weekly by the artificial antigen-presenting cells as needed to obtain sufficient numbers. Expanded NK cells may be used directly or cryopreserved for future use. Reproduced with permission from Denman et al. (49).

Figure 3

Chimeric antigen receptors (CARs). The first-generation CARs only have CD3 zeta signaling domain; the second-generation CARs include one CD28 or 4-1BB co-stimulatory components combined with CD3 zeta signaling domain; the third-generation CARs include two co-stimulatory domains; the fourth-generation CARs are designed with new elements including a controllable suicide gene like caspase 9 or loaded with IL-12 secretion. Reproduced with permission from: Barth et al. (39).

Figure 4

The interaction between NKG2D on NK cells and NKG2D ligands on tumor cells. In normal cells, NKG2D ligands express is very low. The functions of NK cells are balanced by the signals from the inhibitory and activating receptors. In humans, when normal cells are transformed into cancer cells. NKG2D ligands such as MICA/B and ULBP proteins, are often overexpressed. The engagement of NKG2D and NKG2D ligands overcomes inhibitory signals on NK cells, activates NK cells to release cytotoxic molecules such as perforin and granzyme, and trigger apoptosis of tumor cells.

Figure 5

Strategies to overcome NK resistance in solid tumors. To enhance targeting specificity, NK cells have been engineered to express CAR such as anti-HER2 CAR, anti-EGFR CAR, anti-GD2 CAR et al. to target a specific antigen on tumor cell surface. NK cells can be activated by cytokines and cytokine fusion proteins such as IL-2, IL-15, IL-12, IL-18, IL-21, ALT-803 (an IL-15 superagonist), NKTR-255 (a polymer-engineered IL-15 molecule), anti-GD2-IL2, and anti-GD2-RLI fusions et al. Bispecific antibodies are novel molecules where two antigens can be targeted at the same time by combining the specificities of two antibodies. Bispecific antibodies can enhance NK cells targeting and killing. Preventing CD16 shedding and expressing high affinity CD16 on NK cells combined with novel engineered humanized antibodies will enhance NK mediated ADCC. The inhibitory roles of checkpoint proteins PD-1, TIGIT, IL-1R8, and KIR on NK cells are well documented. Blocking PD-1, TIGIT, and KIR with specific antibodies or knocking down IL-1R8 in NK cells unleash human NK-cell effector function. The membrane-bound ligands such as MICA/B binding to NKG2D play an important role in NK cell activation and tumor immune surveillance. Therapeutic strategies have focused on enhancing NKG2D expression and signaling on NK cells and enhancing the level of membrane-bound NKG2D ligand on tumor cells; and eliminating soluble NKG2D ligands. To enhance NK homing and tumor infiltration, NK cells can be enhanced to express chemokine receptors such as CXCR3, CXCR2 to be attracted to tumor cells that secret CXCL10, CXCL1, CXCL8, or CCL5. TGF-β plays an immunosuppressive effect of on NK cells. The approaches to block TGF-β and inhibit TGF-β pathway including TGF-β neutralizing antibody, TGF-β receptor I kinase inhibitors, SMAD3-Silenced (Smad3knd) NK Cells, NK cells engineered with a dominant negative receptor II for TGF-β (DNRII), NK cells engineered to express a chimeric receptor with TGF-β type II receptor extracellular and transmembrane domains and the intracellular domain of NK cell-activating receptor NKG2D (RIIG2D).