Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%–347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.
Natural killer (NK) cells harbor efficient cytotoxicity against tumor cells without causing life-threatening cytokine release syndrome (CRS) or graft-versus-host disease (GvHD). When compared to chimeric antigen receptor (CAR) technology, Antibody-Cell Conjugation (ACC) technology has been developed to provide an efficient platform to arm immune cells with cancer-targeting antibodies to recognize and attack cancer cells. Recently, we established an endogenous CD16-expressing oNK cell line (oNK) with a favorable expression pattern of NK activation/inhibitory receptors. In this study, we applied ACC platform to conjugate oNK with trastuzumab and an anti-human epidermal growth factor receptor 2 (HER2) antibody. Trastuzumab-conjugated oNK, ACE-oNK-HER2, executed in vitro and in vivo cytotoxicity against HER2-expressing cancer cells and showed enhanced T cell-recruiting capability and secretion of IFNγ. The irradiated and cryopreserved ACE-oNK-HER2, designated as ACE1702, retained superior HER2-specific in vitro and in vivo potency with no tumorigenic potential. In conclusion, this study provides the evidence to support the potential clinical application of ACE1702 as a novel off-the-shelf NK cell therapy against HER2-expressing solid tumors.
Introduction Autologous or allogeneic natural killer (NK) cells possess efficient cytotoxicity against tumor cells without severe side effects such as CRS or graft-versus-host disease (GvHD). In addition to chimeric antigen receptor (CAR) strategy, antibody-cell conjugates (ACCTM) platform provides more efficient way to arm NK cells with binding specificity and enhanced potency against target cells. In this work, we develop a NK cell therapy product ACE1702, a novel NK cell line oNK conjugated with Trastuzumab, and assess its potency against HER2+ solid tumors. Methods oNK cells, a fluorescence-activated cell sorting (FACS) isolated CD16+ population sorted from NK-92 cells, was covalently conjugated with monoclonal antibody Trastuzumab after sublethal irradiation by our patented antibody-cell conjugates (ACCTM) platform to become our cryopreserved final product ACE1702 compliant with current good manufacturing practice (cGMP). Characterization of ACE1702 was analyzed by flow cytometry. Function of ACE1702 was validated by real-time xCELLigence analyzer in vitro. Efficacy of intraperitoneally (ip.) delivered ACE1702 was evaluated in tumor-bearing female immune compromised NSG mice. Tumorigenic potential of non-irradiated oNK and ACE1702 was evaluated in subcutaneous implantation female BALB/c nude mice model. Results We demonstrated that newly established CD16+ oNK cells harbor elevated basal cytotoxicity against target cells in the absence of antibodies without tumorigenic potential. The Trastuzumab-armed oNK cells, ACE1702, exerted human epidermal growth factor 2 (HER2) binding specificity and enhanced cytotoxicity against various types of cancer cells with grade 1 (MCF-7), grade 2 (OVCAR-3) and grade 3 (SK-OV-3) HER2 expression compared to control oNK cells in vitro. ACE1702 also displayed similar potency against Herceptin-resistant human breast cancer cell line BT-474 clone 5 and parental BT-474 cells in vitro. Furthermore, in vivo results in human ovarian cancer cell line SK-OV-3-bearing xenograft mouse model supported the in vitro observations. Compared to other immunotherapeutic strategies such as antibody-drug conjugate, allogeneic NK and chimeric antigen receptor (CAR) modification, ACCTM platform underscores its power in compatibility with diverse antibodies, ease for mass production compliant with good manufacturing practice and affordability to patients. Conclusion Here we introduced a novel Trastuzumab-modified oNK cell product with enhanced specificity against myriad types of HER2+ cancers even in the presence of Herceptin resistance, and demonstrated the non-tumorigenic potential of non-irradiated oNK cells and ACE1702. Citation Format: Hao-Kang H. Li, Tai-Sheng E. Wu, Ching-Wen S. Hsiao, Sen-Han S. Yang, Chia-Yun S. Lee, Yan-Liang J. Lin, Zih-Fei Z. Cheng, Yu-Bei Cheng, Yan-Da D. Lai, Hsiu-Ping C. Yang, Sai-Wen S. Tang, Wei-Lun S. Lo, Janet Pan, Shih-Chia S. Hsiao. ACE1702: A potent and off-the-shelf oNK cell therapy product [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2169.
BackgroundAutologous or allogeneic natural killer (NK) cells possess efficient cytotoxicity against tumor cells without severe side effects such as CRS or graft-versus-host disease (GvHD). In addition to chimeric antigen receptor (CAR) strategy, antibody-cell conjugates (ACC) platform provides more efficient way to arm NK cells with binding specificity and enhanced potency against target cells. In this work, we develop a NK cell therapy product ACE1702, a novel NK cell line oNK conjugated with trastuzumab, and assess its potency against HER2+ solid tumors.Methods oNK cells were covalently conjugated with monoclonal antibody Trastuzumab after sublethal irradiation by our patented antibody-cell conjugates (ACC) platform to become our cryopreserved final product ACE1702 compliant with current good manufacturing practice (cGMP). Function of ACE1702 was validated by real-time xCELLigence analyzer and MTT assay in vitro. Efficacy of intraperitoneally (ip.) delivered ACE1702 was evaluated in tumor-bearing female immune compromised NSG mice. Characterization of ACE1702 was analyzed by flow cytometry.ResultsWe demonstrated that the trastuzumab-armed oNK cells, ACE1702, exerted human epidermal growth factor 2 (HER2) binding specificity and enhanced cytotoxicity against various types of cancer cells with different grade of HER2 expressions compared to control oNK cells in vitro. In vivo results in human ovarian cancer cell line SK-OV-3-bearing xenograft mouse model further supported the in vitro observations. Of note, ACE1702 also displayed a better cytotoxicity against HER2+ cancer cells than trastuzumab and its derived antibody-drug conjugate. ACE1702 also remained cytotoxicity against cancer cells in the suppressive tumor microenvironment. Characterization revealed a preferential expression of NK activation receptors, and conjugation of trastuzumab with cell membrane proteins responsible for NK activity capacitated ACE1702 with enhanced cytotoxicity. These results underscore the potency of ACE1702 in eradication of cancer cells.ConclusionsHere we introduced a novel trastuzumab-modified oNK cell product with enhanced specificity against myriad types of HER2+ cancers. Selective conjugation of trastuzumab with membrane proteins contributing to NK activation conferred ACE1702 with enhanced cytotoxicity even in the suppressive tumor microenvironment.AcknowledgementsNoneTrial RegistrationNoneEthics ApprovalThe animal study was conducted according to protocols approved by the Institutional Animal Care and Use Committee of Muragenics.ConsentNone
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