为了利用T细胞的强大的肿瘤杀伤能力来治疗CD19(+)恶性肿瘤,我们构建了AFM11,一种仅由Fv结构域组成的人源化四价双特异性CD19/CD3串联双抗体(TandAb).该分子表现出良好的可制造性和稳定性。AFM11具有2个针对CD3的结合位点和2个针对CD19的结合位点,CD19是一种从早期B细胞发育通过分化为浆细胞表达的抗原,并且是CD20作为开发治疗性抗体以治疗B细胞恶性肿瘤的靶标的有吸引力的替代品。AFM11与靶向相同抗原的串联scFv双特异性T细胞衔接剂(BiTE)分子的结合和细胞毒性的比较表明,AFM11引起更有效的体外B细胞裂解。尽管TandAb对CD3具有很高的亲和力,但它介导了对CD19(+)细胞的连续杀伤,几乎不依赖于效应器:靶标比率的效力或功效,与BiTE不同。TandAb相对于BiTE的优势在较低的效应子:靶比率下最显著。AFM11介导的严格的靶依赖性T细胞活化由CD25和CD69诱导证明,扩散,和细胞因子释放,尽管二价CD3参与。在NOD/SCID异种移植模型中,AFM11在体内诱导Raji肿瘤的剂量依赖性生长抑制,和放射性标记的TandAb表现出对肿瘤的优异定位,但不对正常组织。小鼠静脉给药后,半衰期范围为18.4至22.9小时。在人类离体B细胞慢性淋巴细胞白血病研究中,AFM11在自体环境中表现出实质性的细胞毒性活性。因此,AFM11可以代表具有有利的安全性风险特征和预期的给药方案的用于治疗CD19(+)恶性肿瘤的有希望的治疗剂。
To harness the potent tumor-killing capacity of T cells for the treatment of CD19(+) malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19(+) cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly target-dependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19(+) malignancies with an advantageous safety risk profile and anticipated dosing regimen.