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Cancer immunotherapy using a bispecific NK receptor fusion protein that engages both T cells and tumor cells.

ABSTRACT: T-cell immunotherapy is a promising strategy to treat cancer, but its efficacy, complexity, and costs may pose challenges. In this study, we report the results of an investigation of a new approach to selectively activate a T-cell attack against tumor cells. The immunotherapeutic approach we developed utilizes a bifunctional fusion protein that binds tumor cells through NK (natural killer)-activating receptor NKG2D and that recruits and stimulates T cells through an anti-CD3 single-chain variable fragment (scFv-NKG2D). In vitro, this scFv-NKG2D fusion protein engaged both T cells and tumor cells, stimulating T cells to produce IFN-?, and cytotoxicity against NKG2D ligand-positive tumor cells. In vivo, expression of scFv-NKG2D by NKG2D ligand-positive tumor cells reduced tumor burden and, in some cases, led to tumor-free survival. Administration of scFv-NKG2D in vivo also promoted survival in a murine lymphoma model. Tumor-free mice were resistant to rechallenge with cognate tumor cells, suggesting that a host-specific immunologic memory response had been generated. Host adaptive immunity including ?? T cells was required for scFv-NKG2D-mediated therapeutic efficacy. ScFv-NKG2D also inhibited the growth of NKG2D ligand-negative B16F10 tumors, reduced the percentage of myeloid-derived suppressor cells and regulatory T cells, and increased the infiltration of T cells, suggesting that scFv-NKG2D may target these immune suppressive cells. Together, these results establish scFv-NKG2D as a promising biological fusion protein to induce effective antitumor immunity.

SUBMITTER: Zhang T

PROVIDER: S-EPMC3095211 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Cancer immunotherapy using a bispecific NK receptor fusion protein that engages both T cells and tumor cells.

Zhang Tong T Sentman Charles L CL

Cancer research 20110131 6

T-cell immunotherapy is a promising strategy to treat cancer, but its efficacy, complexity, and costs may pose challenges. In this study, we report the results of an investigation of a new approach to selectively activate a T-cell attack against tumor cells. The immunotherapeutic approach we developed utilizes a bifunctional fusion protein that binds tumor cells through NK (natural killer)-activating receptor NKG2D and that recruits and stimulates T cells through an anti-CD3 single-chain variabl ...[more]

PMID: 21282338

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Project description:BackgroundThe cytotoxicity of NK cells is largely dependent on IgG Fc receptor CD16a, which mediates antibody-dependent cell-mediated cytotoxicity (ADCC). The high-affinity and non-cleavable CD16 (hnCD16) is developed and demonstrated a multi-tumor killing potential. However, the hnCD16 receptor activates a single CD16 signal and provides limited tumor suppression. How to exploit the properties of hnCD16 and incorporate NK cell-specific activation domains is a promising development direction to further improve the anti-tumor activity of NK cells.MethodsTo expand the applications of hnCD16-mediated ADCC for NK cell-based immunotherapy in cancer, we designed the hnCD16 Fusion Receptor (FR) constructs with the ectodomain of hnCD16 fused with NK cell-specific activating domains in the cytoplasm. FR constructs were transduced into CD16-negative NK cell line and human iPSC-derived NK (iNK) cells and effective FR constructs were screened. The up-regulation of immune activation- and cytokine-releasing-related pathways in FR-transduced NK cells was screened and validated by RNA sequencing and multiplex cytokines release assay, respectively. The tumor-killing efficiency was tested in vitro and in vivo via co-culture with tumor cell lines and xenograft mice-bearing human B-cell lymphoma, respectively.ResultsWe screened the most effective combination to kill B cell lymphoma, which was fused with the ectodomain of hnCD16a, NK-specific co-stimulators (2B4 and DAP10) and CD3ζ in cytoplasmic domains. The screened construct showed excellent cytotoxicity effects and sharp multiple cytokines releasing both in the NK cell line and iNK cells. The transcriptomic analysis and validation assays of hnCD16- and hnCD16FR-transduced NK cells showed that hnCD16FR transduction remodeled immune-related transcriptome in NK cells, where significant upregulation of genes related to cytotoxicity, high cytokines releasing, induced tumor cell apoptosis, and ADCC in comparison with hnCD16 transduction were highlighted. In vivo xenograft studies demonstrated that a single low-dose regimen of engineered hnCD16FR iPSC-derived NK cells co-administered with anti-CD20 mAb treatment mediated potent activity and significantly improved survival.ConclusionWe developed a novel hnCD16FR construct that exhibits more potent cytotoxicity than reported hnCD16, which is a promising approach to treat malignancies with improved ADCC properties. We also offer a rationale for NK activation domains that remodel immune response to enhance CD16 signaling in NK cells.

Project description:IntroductionThe Fc region of monoclonal antibodies (mAbs) interacts with the CD16a receptor on natural killer (NK) cells with "low affinity" and "low selectivity". This low affinity/selectivity interaction results in not only suboptimal anticancer activity but also induction of adverse effects. CD16a on NK cells binds to the antibody-coated cells, leading to antibody-dependent cell-mediated cytotoxicity (ADCC). Recent clinical data have shown that the increased binding affinity between mAb Fc region and CD16a receptor is responsible for significantly improved therapeutic outcomes. Therefore, the objective of this study was to develop a bispecific killer cell engager (BiKE) with high affinity and specificity/selectivity toward CD16a receptor for NK cell-based cancer immunotherapy.MethodsTo engineer BiKE, a llama was immunized, then high binding anti-CD16a and anti-HER2 VHH clones were isolated using phage display. ELISA, flow cytometry, and biolayer interferometry (BLI) data showed that the isolated anti-CD16a VHH has high affinity (sub-nanomolar) toward CD16a antigen without cross-reactivity with CD16b-NA1 on neutrophils or CD32b on B cells. Similarly, the data showed that the isolated anti-HER2 VHH has high affinity/specificity toward HER2 antigen. Using a semi-flexible linker, anti-HER2 VHH was recombinantly fused with anti-CD16a VHH to create BiKE:HER2/CD16a. Then, the ability of BiKE:HER2/CD16a to activate NK cells to release cytokines and kill HER2+ cancer cells was measured. As effector cells, both high-affinity haNK92 (CD16+, V176) and low-affinity laNK92 (CD16+, F176) cells were used.Results and discussionThe data showed that the engineered BiKE:HER2/CD16a activates haNK92 and laNK92 cells to release cytokines much greater than best-in-class mAbs in the clinic. The cytotoxicity data also showed that the developed BiKE induces higher ADCC to both ovarian and breast cancer cells in comparison to Trazimera™ (trastuzumab). According to the BLI data, BiKE:HER2/CD16 recognizes a different epitope on CD16a antigen than IgG-based mAbs; thus, it provides the opportunity for not only monotherapy but also combination therapy with other antibody drugs such as checkpoint inhibitors and antibody-drug conjugates. Taken together, the data demonstrate the creation of a novel BiKE with high affinity and specificity toward CD16a on NK cells with the potential to elicit a superior therapeutic response in patients with HER2+ cancer than existing anti-HER2 mAbs.

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Cancer immunotherapy using a bispecific NK receptor fusion protein that engages both T cells and tumor cells.

Publications

Cancer immunotherapy using a bispecific NK receptor fusion protein that engages both T cells and tumor cells.

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