Das Gargi, Ptacek Jakub, Havlinova Barbora, Nedvedova Jana, Barinka Cyril, Novakova Zora
Laboratory of Structural Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Prumyslova 595, 252 50 Vestec, Czech Republic.
Department of Cell Biology, Faculty of Science, Charles University, 128 00 Prague, Czech Republic.
ACS Pharmacol Transl Sci. 2023 Oct 6;6(11):1703-1714. doi: 10.1021/acsptsci.3c00159. eCollection 2023 Nov 10.
Prostate cancer (PCa) tops the list of cancer-related deaths in men worldwide. Prostate-specific membrane antigen (PSMA) is currently the most prominent PCa biomarker, as its expression levels are robustly enhanced in advanced stages of PCa. As such, PSMA targeting is highly efficient in PCa imaging as well as therapy. For the latter, PSMA-positive tumors can be targeted directly by using small molecules or macromolecules with cytotoxic payloads or indirectly by engaging the immune system of the host. Here we describe the engineering, expression, purification, and biological characterization of bispecific T-cell engagers (BiTEs) that enable targeting PSMA-positive tumor cells by host T lymphocytes. To this end, we designed the 5D3-αCD3 BiTE as a fusion of single-chain fragments of PSMA-specific 5D3 and anti-CD3 antibodies. Detailed characterization of BiTE was performed by a combination of size-exclusion chromatography, differential scanning fluorimetry, and flow cytometry. Expressed in insect cells, BiTE was purified in monodisperse form and retained thermal stability of both functional parts and nanomolar affinity to respective antigens. 5D3-αCD3's efficiency and specificity were further evaluated using PCa-derived cell lines together with peripheral blood mononuclear cells isolated from human blood. Our data revealed that T-cells engaged via 5D3-αCD3 can efficiently eliminate tumor cells already at an 8 pM BiTE concentration in a highly specific manner. Overall, the data presented here demonstrate that the 5D3-αCD3 BiTE is a candidate molecule of high potential for further development of immunotherapeutic modalities for PCa treatment.
前列腺癌（PCa）是全球男性癌症相关死亡的首要原因。前列腺特异性膜抗原（PSMA）是目前最突出的PCa生物标志物，因为其表达水平在PCa晚期显著增强。因此，靶向PSMA在PCa成像和治疗中都非常有效。对于后者，PSMA阳性肿瘤可以通过使用携带细胞毒性载荷的小分子或大分子直接靶向，或者通过激活宿主免疫系统间接靶向。在这里，我们描述了双特异性T细胞衔接器（BiTEs）的工程设计、表达、纯化和生物学特性，该衔接器能够使宿主T淋巴细胞靶向PSMA阳性肿瘤细胞。为此，我们设计了5D3-αCD3 BiTE，它是PSMA特异性5D3单链片段和抗CD3抗体的融合体。通过尺寸排阻色谱、差示扫描荧光法和流式细胞术相结合的方法对BiTE进行了详细表征。BiTE在昆虫细胞中表达后，以单分散形式纯化，并保留了两个功能部分的热稳定性以及对各自抗原的纳摩尔亲和力。使用PCa衍生的细胞系以及从人血中分离的外周血单核细胞进一步评估了5D3-αCD3的效率和特异性。我们的数据显示，通过5D3-αCD3衔接的T细胞能够在8 pM的BiTE浓度下以高度特异性的方式有效消除肿瘤细胞。总体而言，本文提供的数据表明，5D3-αCD3 BiTE是一种具有很高潜力的候选分子，可用于进一步开发PCa治疗的免疫治疗方法。
