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Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor activity.

ABSTRACT: Antibody-dependent cellular cytotoxicity (ADCC) is a key effector mechanism of natural killer (NK) cells that is mediated by therapeutic monoclonal antibodies (mAbs). This process is facilitated by the Fc receptor CD16a on human NK cells. CD16a appears to be the only activating receptor on NK cells that is cleaved by the metalloprotease a disintegrin and metalloproteinase-17 upon stimulation. We previously demonstrated that a point mutation of CD16a prevents this activation-induced surface cleavage. This noncleavable CD16a variant is now further modified to include the high-affinity noncleavable variant of CD16a (hnCD16) and was engineered into human induced pluripotent stem cells (iPSCs) to create a renewable source for human induced pluripotent stem cell-derived NK (hnCD16-iNK) cells. Compared with unmodified iNK cells and peripheral blood-derived NK (PB-NK) cells, hnCD16-iNK cells proved to be highly resistant to activation-induced cleavage of CD16a. We found that hnCD16-iNK cells were functionally mature and exhibited enhanced ADCC against multiple tumor targets. In vivo xenograft studies using a human B-cell lymphoma demonstrated that treatment with hnCD16-iNK cells and anti-CD20 mAb led to significantly improved regression of B-cell lymphoma compared with treatment utilizing anti-CD20 mAb with PB-NK cells or unmodified iNK cells. hnCD16-iNK cells, combined with anti-HER2 mAb, also mediated improved survival in an ovarian cancer xenograft model. Together, these findings show that hnCD16-iNK cells combined with mAbs are highly effective against hematologic malignancies and solid tumors that are typically resistant to NK cell-mediated killing, demonstrating the feasibility of producing a standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies that are otherwise refractory.

SUBMITTER: Zhu H

PROVIDER: S-EPMC7005364 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:Erectile dysfunction (ED) is an important kind of postoperative complication of pelvic surgery that affects patients' quality of life. Transplantation of mesenchymal stem cells (MSC) has been found to alleviate ED caused by cavernous nerve injury (CNI) in rats. However, little is known about whether induced pluripotent stem cell-derived mesenchymal stem cells (iMSC) have a therapeutic effect on CNI ED. We established an ED model on rats and evaluated the effect of iMSC on it. Methods: Eight-week-old male Sprague-Dawley rats were assigned to four groups and received following operation: sham operation (sham group); bilateral CNI and phosphate-buffered saline (PBS) injections (PBS group); bilateral CNI and adipose-derived mesenchymal stem cells transplantation (adMSC group); or bilateral CNI and iMSC injection (iMSC group). After therapy, the cavernous nerve was stimulated by electricity and the intracavernous pressure (IAP)/mean arterial blood pressure (MAP) was measured. The endothelial and smooth muscle tissue in the penis was assessed histologically with Masson's trichrome stain. Immunofluorescence/immunohistochemical stains were applied for the detection of nNOS, vWF, eNOS, SMA, Desmin, S100?, and caspase-3. Nude rats CNI ED model was established for the evaluation of iMSC longevity and differentiation capacity. The paracrine factors were assessed by real-time PCR. Results: Transplantation of iMSC significantly restored the IAP/MAP in this CNI ED model and showed long-term effects. It could rescue the expression of vWF, eNOS, SMA, and Desmin, which indicated the alleviation of endothelial and smooth muscle tissues of the penis. iMSC therapy also could increase the expression of nNOS in the cavernosum and S100? in the major pelvic ganglia (MPG) which contributed to the erectile function. Moreover, the level of BAX and caspase-3 were reduced and Bcl-2 was increased, which indicated the anti-apoptosis effects of iMSC. The iMSC showed little transdifferentiation and exerted their function by activating the secretome of the host. Conclusion: Transplantation of iMSC significantly improved ED induced by CNI. The iMSC may exert their effects via paracrine factors and may be a promising therapeutic candidate for treating CNI ED in the future.

Project description:Somatic cell nuclear transfer (SCNT) has been proved capable of reprogramming various differentiated somatic cells into pluripotent stem cells. Recently, induced pluripotent stem cells (iPS) have been successfully derived from mouse and human somatic cells by the over-expression of a combination of transcription factors. However, the molecular mechanisms underlying the reprogramming mediated by either the SCNT or iPS approach are poorly understood. Increasing evidence indicates that many tumor pathways play roles in the derivation of iPS cells. Embryonal carcinoma (EC) cells have the characteristics of both stem cells and cancer cells and thus they might be the better candidates for elucidating the details of the reprogramming process. Although previous studies indicate that EC cells cannot be reprogrammed into real pluripotent stem cells, the reasons for this remain unclear. Here, nuclei from mouse EC cells (P19) were transplanted into enucleated oocytes and pluripotent stem cells (P19 NTES cells) were subsequently established. Interestingly, P19 NTES cells prolonged the development of tetraploid aggregated embryos compared to EC cells alone. More importantly, we found that the expression recovery of the imprinted H19 gene was dependent on the methylation state in the differential methylation region (DMR). The induction of Nanog expression, however, was independent of the promoter region DNA methylation state in P19 NTES cells. A whole-genome transcriptome analysis further demonstrated that P19 NTES cells were indeed the intermediates between P19 cells and ES cells and many interesting genes were uncovered that may be responsible for the failed reprogramming of P19 cells. To our knowledge, for the first time, we linked incomplete reprogramming to the improved pluripotency of EC cell-derived pluripotent stem cells. The candidate genes we discovered may be useful not only for understanding the mechanisms of reprogramming, but also for deciphering the transition between tumorigenesis and pluripotency.

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Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor activity.

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