造血细胞移植（HCT）的结果证明，免疫细胞能够消除白血病细胞。然而，诱导治疗失败的患者由于其微小残留病 (MRD) 状态，将无法从 HCT 中受益。因此，我们的目标是开发一种免疫调节剂，通过一种新型融合蛋白在白血病细胞存在的情况下激活免疫效应细胞来减少 MRD，该融合蛋白嵌合两种临床耐受的生物制剂：CD33 抗体和 IL15Ra/IL15 复合物 (CD33xIL15)。一组具有不同配置的 CD33xIL15 融合蛋白构建体，并鉴定了具有最佳体外 AML 结合、T 细胞激活和 NK 细胞增强能力的构建体。然后，我们使用 89Zr-immunoPET 成像评估了 AML 异种移植模型中最有利的 CD33xIL15 构建体的生物分布和体内肿瘤保留。体外生物分布研究用于确认构建体的药代动力学。生成的两种融合蛋白 CD33xIL15 (N72D) 和 CD33xIL15wt 表现出最佳的体外行为，并在体内进行了进一步评估。这些研究表明，CD33xIL15wt 候选物能够在肿瘤中保留与其亲代 CD33 抗体 Lintuzumab 一样长的时间（120 小时时为 13.9±±3.1%ID/g vs 18.6±±1.1%ID/g）。这项工作表明CD33xIL15 融合蛋白能够靶向白血病细胞并在体外刺激局部 T 细胞，并集中在 AML 异种移植物的肿瘤中。它还强调了 89Zr-immunoPET 在指导肿瘤靶向抗体-细胞因子融合蛋白的开发和选择方面的重要性。© 2024。作者获得 Springer-Verlag GmbH 德国（Springer Nature 旗下公司）的独家许可。

Immune cells are capable of eliminating leukemic cells, as evidenced by outcomes in hematopoietic cell transplantation (HCT). However, patients who fail induction therapy will not benefit from HCT due to their minimal residual disease (MRD) status. Thus, we aimed to develop an immunomodulatory agent to reduce MRD by activating immune effector cells in the presence of leukaemia cells via a novel fusion protein that chimerises two clinically tolerated biologics: a CD33 antibody and the IL15Ra/IL15 complex (CD33xIL15).We generated a set of CD33xIL15 fusion protein constructs with varying configurations and identified those with the best in vitro AML-binding, T cell activation, and NK cell potentiation. Using 89Zr-immunoPET imaging we then evaluated the biodistribution and in vivo tumour retention of the most favourable CD33xIL15 constructs in an AML xenograft model. Ex vivo biodistribution studies were used to confirm the pharmacokinetics of the constructs.Two of the generated fusion proteins, CD33xIL15 (N72D) and CD33xIL15wt, demonstrated optimal in vitro behaviour and were further evaluated in vivo. These studies revealed that the CD33xIL15wt candidate was capable of being retained in the tumour for as long as its parental CD33 antibody, Lintuzumab (13.9 ± 3.1%ID/g vs 18.6 ± 1.1%ID/g at 120 h).This work demonstrates that CD33xIL15 fusion proteins are capable of targeting leukemic cells and stimulating local T cells in vitro and of concentrating in the tumour in AML xenografts. It also highlights the importance of 89Zr-immunoPET to guide the development and selection of tumour-targeted antibody-cytokine fusion proteins.© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.