Department of Biology, University of Erlangen-Nuremberg, Erlangen, Germany.
MAbs. 2011 Jan-Feb;3(1):21-30. doi: 10.4161/mabs.3.1.14057. Epub 2011 Jan 1.
A single-chain triplebody (sctb) 33-ds16-ds19 comprising two distal single-chain Fv fragments (scFvs) specific for the lymphoid antigen CD19 and the myeloid antigen CD33 flanking a central scFv specific for CD16, which is the low affinity Fc-receptor (FcγRIII) present on natural killer cells and macrophages, was produced and its properties were investigated. CD33 and CD19 in combination are present on acute leukemiablasts with mixed lineage phenotype, but not on normal human hematopoietic cells. For comparison, two bispecific scFvs (bsscFvs), ds19-ds16 and 33-ds16, with monovalent binding to CD19 and CD33, respectively, were also studied. The sctb 33-ds16-ds19 specifically interacted with all 3 antigens. On the antigen double-positive cell line BV-173, the sctb bound with 2-fold greater avidity than bsscFv ds19-ds16 (KD = 21 vs. 42 nM) and with 1.4-fold greater avidity than bsscFv 33-ds16 (KD = 29 nM). All 3 fusion proteins had similar affinity for CD16 and sufficient thermic stability in human serum. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, the sctb promoted lysis of BV-173 cells at 23-fold lower concentrations than bsscFv ds19-ds16 and at 1.4-fold lower concentrations than bsscFv 33-ds16. The sctb also mediated potent ADCC of the antigen double-positive mixed lineage leukemia cell line SEM, and the half-maximal concentration EC50 for BV-173 cells was 7 pM. Therefore, CD19 and CD33 are present on the surface of these leukemic cell lines such that they can be connected by a single sctb molecule, permitting the recruitment of NK cells via CD16 and tumor cell lysis.
一种由两个针对淋巴细胞抗原 CD19 和髓样抗原 CD33 的远端单链 Fv 片段（scFv）组成的单链三聚体（sctb）33-ds16-ds19，夹在一个针对 CD16 的中央 scFv 之间，CD16 是自然杀伤细胞和巨噬细胞上的低亲和力 Fc 受体（FcγRIII），该 sctb 被生产出来并研究了其性质。CD33 和 CD19 同时存在于具有混合谱系表型的急性白血病blasts 中，但不存在于正常的人类造血细胞中。为了比较，还研究了两种单价结合 CD19 和 CD33 的双特异性 scFv（bsscFv）ds19-ds16 和 33-ds16。sctb 33-ds16-ds19 特异性地与所有 3 种抗原相互作用。在抗原双阳性细胞系 BV-173 上，sctb 的结合亲和力比 bsscFv ds19-ds16 高 2 倍（KD = 21 对 42 nM），比 bsscFv 33-ds16 高 1.4 倍（KD = 29 nM）。所有 3 种融合蛋白对 CD16 的亲和力相似，在人血清中有足够的热稳定性。在以人单核细胞为效应物的抗体依赖性细胞毒性（ADCC）反应中，sctb 促进 BV-173 细胞的裂解，浓度比 bsscFv ds19-ds16 低 23 倍，比 bsscFv 33-ds16 低 1.4 倍。sctb 还介导抗原双阳性混合谱系白血病细胞系 SEM 的有效 ADCC，BV-173 细胞的半最大浓度 EC50 为 7 pM。因此，CD19 和 CD33 存在于这些白血病细胞系的表面，使得它们可以通过单个 sctb 分子连接，通过 CD16 募集 NK 细胞并裂解肿瘤细胞。
