Tutt A, Stevenson G T, Glennie M J
Tenovus Research Laboratory, General Hospital, Southampton, United Kingdom.
J Immunol. 1991 Jul 1;147(1):60-9.
To investigate whether the retargeting of resting CTL can benefit from cooperative signaling between the TCR/CD3 complex and various accessory molecules, such as CD2, CD4, CD5, and CD8, we have constructed a series of trispecific F(ab')3 derivatives. Each derivative was designed to engage effector T lymphocytes with two Fab' arms, and tumor cells with a single Fab' arm. They were constructed by selective coupling of three mAb Fab' fragments, primarily via their hinge-region sulfhydryl groups, using the cross-linker o-phenylenedimaleimide. En route to the production of trispecific F(ab')3 antibodies a range of bispecific F(ab')2 derivatives was first prepared which could bind simultaneously to two different receptor molecules on T cells. Bispecific derivatives containing specificities for (CD2 (T11(1)) x CD3), (CD3 x CD4), (CD3 x CD8) or two epitopes on CD2, ((T11(1) x (T11(3)), all yielded two to three times the uptake of [3H]thymidine with fresh PBMC to that seen with intact IgG from anti-CD3 (OKT3). The exception to these findings was a bispecific F(ab')2 derivative with specificities for (CD3 x CD5) which caused slightly less proliferation than the control reagent, OKT3 IgG. When these bispecific antibodies were converted into trispecific antibodies (TsAb) by the addition of a Fab' from anti-CD37 they were then all able to retarget resting, unprimed, T cells from fresh PBMC for lysis of CD37+ tumor cells. However, the cytotoxic activity of these reagents fell into two distinct groups: group one, containing (anti-CD3 x anti-CD4 x anti-CD37), (anti-CD3 x anti-CD5 x anti-CD37), and (anti-CD3 x anti-CD8 x anti-CD37), gave minimal lysis and behaved in a similar way to the BsAb, (anti-CD3 x anti-CD37), i.e., no evidence of cooperative signaling for lysis; and group two, containing (anti-T11(1) x anti-CD3 x anti-CD37) and (anti-T11(1) x anti-T11(3) x anti-CD37), which were highly cytotoxic and gave up to 80% specific 51Cr-release. The failure of group one TsAb, in particular (anti-CD3 x anti-CD8 x anti-CD37) which should recruit CD8+ CTL, to give efficient lysis despite having anti-T cell arms that were mitogenic as a bispecific antibody, indicates that the cooperative signaling for proliferation is probably distinct from the signal(s) provided by group two TsAb that activate for both proliferation and lysis.(ABSTRACT TRUNCATED AT 400 WORDS)
为了研究静息细胞毒性T淋巴细胞（CTL）的重定向是否能受益于T细胞受体/CD3复合物与各种辅助分子（如CD2、CD4、CD5和CD8）之间的协同信号传导，我们构建了一系列三特异性F(ab')3衍生物。每个衍生物设计为由两条Fab'臂结合效应T淋巴细胞，一条Fab'臂结合肿瘤细胞。它们通过使用交联剂邻苯二甲酰亚胺，主要通过其铰链区巯基选择性偶联三个单克隆抗体Fab'片段构建而成。在制备三特异性F(ab')3抗体的过程中，首先制备了一系列双特异性F(ab')2衍生物，它们可以同时结合T细胞上的两种不同受体分子。含有针对（CD2（T11(1)）×CD3）、（CD3×CD4）、（CD3×CD8）或CD2上两个表位（（T11(1)×（T11(3)））特异性的双特异性衍生物，与新鲜外周血单个核细胞（PBMC）一起培养时，[3H]胸腺嘧啶核苷的摄取量均是抗CD3（OKT3）完整IgG的两到三倍。这些结果的例外是一种针对（CD3×CD5）特异性的双特异性F(ab')2衍生物，其诱导的增殖略低于对照试剂OKT3 IgG。当通过添加抗CD37的Fab'将这些双特异性抗体转化为三特异性抗体（TsAb）时，它们都能够重定向新鲜PBMC中静息、未致敏的T细胞，以裂解CD37+肿瘤细胞。然而，这些试剂的细胞毒性活性分为两个不同的组：第一组，包含（抗CD3×抗CD4×抗CD37）、（抗CD3×抗CD5×抗CD37）和（抗CD3×抗CD8×抗CD37），裂解作用最小，其行为方式与双特异性抗体（抗CD3×抗CD37）相似，即没有协同信号传导促进裂解的证据；第二组包含（抗T11(1)×抗CD3×抗CD37）和（抗T11(1)×抗T11(3)×抗CD37），具有高度细胞毒性，特异性51Cr释放高达80%。第一组TsAb，特别是应该募集CD8+CTL的（抗CD3×抗CD8×抗CD37），尽管其抗T细胞臂作为双特异性抗体具有促有丝分裂作用，但仍不能有效裂解，这表明增殖的协同信号传导可能与第二组TsAb提供的激活增殖和裂解的信号不同。（摘要截断于400字）
