Abstract：
Objective: To construct a novel dual-specific antibody targeting human CD123 (CD123 DuAb) and study its effects in acute myeloid leukemia (AML) . Methods: Based on the variable region of the CD123 monoclonal antibody independently developed at our institution, the CD123 DuAb expression plasmid was constructed by molecular cloning and transfected into ExpiCHO-S cells to prepare the antibody protein. Through a series of in vitro experiments, its activation and proliferation effect on T cells, as well as the effect of promoting T-cell killing of AML cells, were verified. Results: ① A novel CD123 DuAb plasmid targeting CD123 was successfully constructed and expressed in the Expi-CHO eukaryotic system. ②The CD123 DuAb could bind both CD3 on T cells and CD123 on CD123(+) tumor cells. ③When T cells were co-cultured with MV4-11 cells with addition of the CD123 DuAb at a concentration of 1 nmol/L, the positive expression rates of CD69 and CD25 on T cells were 68.0% and 44.3%, respectively, which were significantly higher than those of the control group (P<0.05). ④Co-culture with CD123 DuAb at 1 nmol/L promoted T-cell proliferation, and the absolute T-cell count increased from 5×10(5)/ml to 3.2×10(6)/ml on day 9, and CFSE fluorescence intensity decreased significantly. ⑤ With the increase in CD123 DuAb concentration in the culture system, T-cell exhaustion and apoptosis increased. When the CD123 DuAb was added at a concentration of 1 nmol/L to the culture system, the proportion of CD8(+) PD-1(+) LAG-3(+) T cells was 10.90%, and the proportion of propidium iodide (PI) (-) Annexin Ⅴ(+) T cells and PI(+) Annexin Ⅴ(+) T cells was 18.27% and 11.43%, respectively, which were significantly higher than those in the control group (P<0.05). ⑥ The CD123 DuAb significantly activated T cells, and the activation intensity was positively correlated with its concentration. The expression rate of CD107a on T cells reached 16.05% with 1 nmol/L CD123 DuAb, which was significantly higher than that of the control group (P<0.05). ⑦The CD123 DuAb promoted cytokine secretion by T cells at a concentration of 1 nmol/L, and the concentration of IFN-γ and TNF-α in the supernatant of the co-culture system reached 193.8 pg/ml and 169.8 pg/ml, respectively, which was significantly higher than that of the control group (P<0.05). ⑧When CD123 DuAb was added at a concentration of 1 nmol/L to the co-culture system of T cells and CD123(+) tumor cells, the killing intensity of T cells significantly increased, and the residual rates of CD123(+) MV4-11 cells, CD123(+) Molm13 cells, and CD123(+) THP-1 cells were 7.4%, 6.7%, and 14.6% on day 3, respectively, which were significantly lower than those in the control group (P<0.05) . Conclusion: In this study, a novel CD123 DuAb was constructed and expressed. In vitro experiments verified that the DuAb binds to CD123(+) tumor cells and T cells simultaneously, promotes T-cell activation and proliferation, and facilitates their anti-leukemia effect, which provides a basis for further clinical research.
目的： 构建一种新的靶向CD123抗原的双特异性抗体（CD123 DuAb），研究CD123DuAb在急性髓系白血病（AML）治疗中的作用。 方法： 以自主研发的CD123单克隆抗体可变区为基础，利用分子克隆技术，构建CD123 DuAb表达质粒，转染ExpiCHO-S细胞，表达该双功能抗体。通过功能实验，验证CD123 DuAb在T细胞活化及增殖中的作用，及其促进T细胞对AML细胞的杀伤作用。 结果： ①构建了CD123 DuAb表达质粒，并通过Expi-CHO真核系统表达。②CD123 DuAb可以分别与T细胞上的CD3位点及CD123阳性肿瘤细胞上的CD123位点结合。③将1 nmol/L CD123 DuAb加入T细胞与MV4-11细胞共培养体系中，T细胞中CD69阳性表达率为68.0%，CD25阳性表达率为44.3%，均显著高于对照组（P值均<0.05）。④在CD123 DuAb浓度为1 nmol/L的条件下培养T细胞，可促进T细胞增殖，其绝对计数第1天为5×10(5)/ml，第9天扩增至3.2×10(6)/ml，且CFSE荧光强度显著降低。⑤CD123 DuAb能够显著激活T细胞，且激活强度与其浓度呈正相关，浓度为1 nmol/L时，T细胞CD107a表达率可达16.05%，较对照组显著升高（P<0.05）。⑥随培养体系中CD123 DuAb浓度的升高，T细胞耗竭和凋亡也随之增加，浓度为1 nmol/L时，T细胞CD8(+)PD-1(+)LAG-3(+)比例为10.90%，PI(-)Annexin Ⅴ(+)比例为18.27%，PI(+)Annexin Ⅴ(+)比例为11.43%，均较对照组显著升高（P<0.05）。⑦CD123 DuAb能够促进T细胞分泌细胞因子，浓度为1 nmol/L时，共培养体系上清中的IFN-γ和TNF-α的浓度可分别达到193.8 pg/ml和169.8 pg/ml，较对照组显著升高（P<0.05）。⑧将CD123 DuAb以1 nmol/L的浓度加入T细胞与CD123阳性肿瘤细胞共培养体系中，能显著增强T细胞对肿瘤细胞的杀伤作用，共培养3 d，CD123(+)MV4-11细胞、CD123(+)Molm13细胞和CD123(+)THP-1细胞的残留率分别为7.4%、6.7%和14.6%，较对照组显著降低（P<0.05）。 结论： 本研究构建及表达了一种靶向CD123的双特异性抗体，并通过体外实验验证其可以同时结合CD123阳性肿瘤细胞和T细胞，促进T细胞的活化和增殖，增强其抗白血病作用，为进一步临床研究提供了基础。.
目的： 构建一种新的靶向CD123抗原的双特异性抗体（CD123 DuAb），研究CD123DuAb在急性髓系白血病（AML）治疗中的作用。 方法： 以自主研发的CD123单克隆抗体可变区为基础，利用分子克隆技术，构建CD123 DuAb表达质粒，转染ExpiCHO-S细胞，表达该双功能抗体。通过功能实验，验证CD123 DuAb在T细胞活化及增殖中的作用，及其促进T细胞对AML细胞的杀伤作用。 结果： ①构建了CD123 DuAb表达质粒，并通过Expi-CHO真核系统表达。②CD123 DuAb可以分别与T细胞上的CD3位点及CD123阳性肿瘤细胞上的CD123位点结合。③将1 nmol/L CD123 DuAb加入T细胞与MV4-11细胞共培养体系中，T细胞中CD69阳性表达率为68.0%，CD25阳性表达率为44.3%，均显著高于对照组（P值均<0.05）。④在CD123 DuAb浓度为1 nmol/L的条件下培养T细胞，可促进T细胞增殖，其绝对计数第1天为5×10(5)/ml，第9天扩增至3.2×10(6)/ml，且CFSE荧光强度显著降低。⑤CD123 DuAb能够显著激活T细胞，且激活强度与其浓度呈正相关，浓度为1 nmol/L时，T细胞CD107a表达率可达16.05%，较对照组显著升高（P<0.05）。⑥随培养体系中CD123 DuAb浓度的升高，T细胞耗竭和凋亡也随之增加，浓度为1 nmol/L时，T细胞CD8(+)PD-1(+)LAG-3(+)比例为10.90%，PI(-)Annexin Ⅴ(+)比例为18.27%，PI(+)Annexin Ⅴ(+)比例为11.43%，均较对照组显著升高（P<0.05）。⑦CD123 DuAb能够促进T细胞分泌细胞因子，浓度为1 nmol/L时，共培养体系上清中的IFN-γ和TNF-α的浓度可分别达到193.8 pg/ml和169.8 pg/ml，较对照组显著升高（P<0.05）。⑧将CD123 DuAb以1 nmol/L的浓度加入T细胞与CD123阳性肿瘤细胞共培养体系中，能显著增强T细胞对肿瘤细胞的杀伤作用，共培养3 d，CD123(+)MV4-11细胞、CD123(+)Molm13细胞和CD123(+)THP-1细胞的残留率分别为7.4%、6.7%和14.6%，较对照组显著降低（P<0.05）。 结论： 本研究构建及表达了一种靶向CD123的双特异性抗体，并通过体外实验验证其可以同时结合CD123阳性肿瘤细胞和T细胞，促进T细胞的活化和增殖，增强其抗白血病作用，为进一步临床研究提供了基础。.
摘要：
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