In this project, we developed a novel fully humanized anti-EGFR/anti-CD3 bispecific antibody (αEGFR/αCD3 BsAb) and its application platform in armed-T cell therapy. Addition of BsAb into culture medium of human peripheral blood mononuclear cells (PBMCs) can efficiently differentiate, expand, and arm human CD3+ T cell to selectively kill EGFR+ colon cancer cells. This culture process does not need the addition of mouse anti-CD3 antibody OKT3 which has been widely used to ex vivo expand human CD3+ T cell for clinical T cell therapy, including armed-T cells and CAR-T cells. Our preliminary results demonstrated that the novel BsAb culture method can efficiently stimulate the differentiation of human CD3+ T cell (>90%) from human PBMCs and can maintain T cell proliferation more than 21 days, which is comparable to traditional OKT3 culture method. More importantly, during the culture process of the BsAb culture method, αEGFR/αCD3 BsAb can effectively arm to the surface of CD3+ T cells, transforming naïve T cells into EGFR specific T cells, thus dramatically enhance killing efficacy of T cells against EGFR+ colon cancer cells. At an E/T of 25:1, the mean cytotoxicity of BsAb-T cells increased by 60.6% over that seen in traditional OKT3-T cells. Moreover, to reduce the immunogenicity of αEGFR/αCD3 BsAb (anti-EGFR Fab comes from mouse/human chimerical antibody Erbitux; anti-CD3 scFv comes from mouse antibody OKT3), we have designed a fully humanized αEGFR/αCD3 BsAb (the degree of humanization: anti-EGFR VH/92.5%, VL/93.5%；anti-CD3 VH/94.1%, VL/92.5%). Based on these promising results, this 3-year project has the following goals: Aim 1. Optimizing the mixing ratio of T cells and BsAb to improve T cell proliferation and BsAb arming efficiency; Aim 2. Assessing the tumor targeting efficacy and the survival of BsAb-T cells in vivo; Aim 3. Determining the therapeutic index of BsAb-T cells against subcutaneous or orthotopic EGFR+colon tumors; Aim 4. Enhancing the counterwork ability of BsAb-T cell against immunosuppressive tumors by ex vivo modification of anti-PD-1 antibody; Aim 5. Development of fully humanized αEGFR/αCD3 BsAb to improve its safety in clinical. Successful development of this strategy is expected to provide valuable tools for differentiating, expanding, and arming human CD3+ T cell by one-step addition of BsAb into human PBMC culture medium, allowing enhanced specific targeting and therapeutic efficacy of human T cells for tumors in the clinic
|Effective start/end date||8/1/18 → 7/1/19|
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