NKG2D-bispecific enhances NK and CD8+ T cell antitumor immunity

Abstract

Background: Cancer immunotherapy approaches that elicit immune cell responses, including T and NK cells, have revolutionized the field of oncology. However, immunosuppressive mechanisms restrain immune cell activation within solid tumors so additional strategies to augment activity are required.

Methods: We identified the co-stimulatory receptor NKG2D as a target based on its expression on a large proportion of CD8+ tumor infiltrating lymphocytes (TILs) from breast cancer patient samples. Human and murine surrogate NKG2D co-stimulatory receptor-bispecifics (CRB) that bind NKG2D on NK and CD8+ T cells as well as HER2 on breast cancer cells (HER2-CRB) were developed as a proof of concept for targeting this signaling axis in vitro and in vivo.

Results: HER2-CRB enhanced NK cell activation and cytokine production when co-cultured with HER2 expressing breast cancer cell lines. HER2-CRB when combined with a T cell-dependent-bispecific (TDB) antibody that synthetically activates T cells by crosslinking CD3 to HER2 (HER2-TDB), enhanced T cell cytotoxicity, cytokine production and in vivo antitumor activity. A mouse surrogate HER2-CRB (mHER2-CRB) improved in vivo efficacy of HER2-TDB and augmented NK as well as T cell activation, cytokine production and effector CD8+ T cell differentiation.

Conclusion: We demonstrate that targeting NKG2D with bispecific antibodies (BsAbs) is an effective approach to augment NK and CD8+ T cell antitumor immune responses. Given the large number of ongoing clinical trials leveraging NK and T cells for cancer immunotherapy, NKG2D-bispecifics have broad combinatorial potential.

Keywords: Bispecifics; CD8+ T cells; NK cells; NKG2D; Synthetic immunity.

Fig. 1

NKG2D is expressed on CD8 + T cells from human breast cancer samples and is associated with better prognosis in atezolizumab-treated TNBC patients. Percentage of co-stimulatory receptor positive A CD8 + , B FoxP3- CD4 + and C FoxP3 + CD4 + T cells from human-dissociated breast cancer tumor cells. D Kaplan–Meier analysis of Overall Survival (OS, left) and Progression-free Survival (PFS, right) among patients with TNBC treated with placebo + nanoparticle albumin-bound (Nab)-paclitaxel (top) or with Atezolizumab + Nab-Paclitaxel (bottom) stratified by high and low KLRK1 (encoding NKG2D) expression normalized to a CD8 signature shown above. (E) Frequency of NKG2D + CD4 + and CD8 + T cells (top) and corresponding NKG2D mean fluorescence intensities (MFI, bottom) from matched PBMCs and human-dissociated HER2 + breast cancer DTCs. Mann–Whitney test used for statistics; *, p ≤ 0.05; **p, ≤ 0.01

Fig. 2

HER2-CRB induces NK cell immunological synapse formation and signaling. A Diagram of HER2-CRB, NIST-CRB and HER2/NIST molecules. B Flow cytometry-based conjugate quantification following 10 min incubation of primary NK cells conjugated with BT474 cells in the absence or presence of 1 or 10 µg/mL HER2-CRB. Results obtained from two independent healthy donors. Representative dot plot (left) and quantified data (right). C Representative images of cell trace violet (CTV)-labeled primary NK cells conjugated with CMTMR-labeled BT474 cells in the presence of NIST- or HER2-CRB. Quantified area depicting immunological synapse used to quantify phosphotyrosine (pTyr) and phalloidin signals. D Quantification of actin and pTyr intensities by confocal imaging of 30 individual interactions after 10 min incubation of conjugation of primary NK cells with BT474 cells in the presence of NIST- or HER2-CRB. Similar results were obtained from another independent healthy donor. E Western blot of phosphorylated ERK, total ERK and loading control Hsp60 after 5 and 15 min incubation of NK cells, HER2-coated beads and either HER2-CRB or NIST-CRB. Mann–Whitney test used for statistics; ****, p ≤ 0.0001

Fig. 3

HER2-CRB elicits NK cell cytotoxicity. A Percentage of killing normalized to NIST-CRB for NK92.6DF5 cells co-cultured with HER2 + HCC2218 cells for three days in the presence of titrated NIST-l or HER2-CRB. B Fold-change of BT474 cell killing normalized to time 0 h. BT474 cells were co-cultured with IL-15 prestimulated primary NK cells with an E:T ratio of 1:1 in the absence or presence of HER2-CRB. Data derived from three independent donors. C NK cell secretion of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) as well as chemokines CXC chemokine motif ligand 10 (CXCL10), macrophage inflammatory protein-1α (MIP-1α) and MIP-1β from experiment depicted in (B) on day 4. Three independent donors illustrated. D Fold-change of BT474 cell killing normalized by time 0 h. BT474 cells were co-cultured with primary NK cells at an E:T ratio of 1:1 in the presence of HER2-CRB, negative controls or trastuzumab with conditioned media obtained from 24 h co-culture of PBMCs and BT474 cells in the presence of HER2-4D5-TDB or NIST-TDB. Three independent donors are represented for donor conditioned media mix cultured with NK cells from 3 separate donors (9 total samples). E Area under the curve quantification from (D). Mann–Whitney test was used for statistical significance; *p, ≤ 0.05

Fig. 4

HER2-CRB enhances HER2-TDB-mediated T cell activation and cytotoxic activity. A Fold-change of luciferase activity normalized to untreated Jurkat-NKG2D + NFAT-Luc cells. Jurkat cells were co-cultured with BT474 cells for 6 h with either HER2.2C4-TDB or HER2-TDB or NIST-TDB in combination with either HER2-CRB or controls. B BT474 target cell killing normalized to time 0 h. BT474 cells were co-cultured for over five days with primary CD8 + T cells at an E:T ratio of 1:1 in the presence of 10 ng/ml of HER2-2C4-TDB alone or in combination with titrated HER2-CRB. Representative of four independent donors. C Cytokine quantification of CD8 + T cell and BT474 cell co-culture at an E:T of 1:1 in the presence of 10 ng/ml of HER2.2C4-TDB alone or in combination with HER2-CRB after 96 h. D Differential gene expression analysis from bulk RNA-seq on CD8 + T cells co-cultured for 24 h with BT474 cells in the presence of 10 ng/mL of HER2.2C4-TDB alone or in combination with HER2-CRB represented as a volcano plot. Blue dots represent Log(fold-change) of  ≤ -0.7 and red dots depict Log(fold-change) ≥ 0.7, p ≤ 0.05. Black points are below stated thresholds. E GSEA data from Hallmark (top) and Reactome (bottom) gene sets from results in (D). F GSEA from immunologic gene sets (C7) from data in (D)

Fig. 5

HER2-CRB improves HER2-TDB in vivo activity. A JimT-1 tumor volume growth over-time in NSG mice (left) with or without adoptive transfer of human PBMCs treated with HER2-TDB alone, HER2-CRB alone or in combination (combo). Individual mouse tumor volumes are represented in right panel. Slash represents a mouse death due to health conditions. (B, left) Change in tumor volumes of the Fo5 tumor model. Mice were treated with mHER2-TDB, mHER2-CRB or in combination (combo). Vehicle treatment was used as a negative control. (Right) Individual tumor volumes at day 4 and day 7 from. Mann–Whitney test used for statistical analysis; *p, ≤ 0.05; **, p ≤ 0.01

Fig. 6

HER2-CRB promotes NK cell and CD8 + T cell effector function in vivo. Fo5 tumor-bearing mice were treated with NIST- or mHER2-TDB with NIST- or mHER2-CRB. Tumors were harvested 4 days following treatment for analysis. A Frequency of Ki67 + CD8 + T and NK cells. B Percent Granzyme B + and CD107a/b + CD8 + T and NK cells. C Percentages of IFN-γ + and TNF-α + CD8 + T and NK cells. D Differential gene expression analysis of bulk RNA-seq from sorted CD8 + TILs. Blue dots represent Log(fold-change) of  ≤ − 0.7 and red dots depict Log(fold-change) ≥ 0.7, p ≤ 0.05. Black points are below stated thresholds. (E) GSEA data from Hallmark (top) and Reactome (bottom) gene sets from results in (D). F GSEA from immunologic gene sets (C7) from data in (D). Mann–Whitney test used for statistical analysis; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001