Feigelson Sara W, Dadosh Tali, Levi Nehora, Sapoznikov Anita, Weinstein-Marom Hadas, Blokon-Kogan Dayana, Avraham Yahel, Unger Tamar, Gross Gideon, Dahan Rony, Alon Ronen
Department of Immunology and Regenerative Biology, Weizmann Institute of Science, Rehovot, Israel.
Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel.
Front Immunol. 2025 Feb 10;16:1398757. doi: 10.3389/fimmu.2025.1398757. eCollection 2025.
Targeting cytotoxic T lymphocytes (CTLs), as chimeric antigen T cells (CAR-T), T cell receptor-engineered (TCR)-T cells or adoptive cell transfer of tumor infiltrating T cells (TILs) to solid tumors is a major therapeutic challenge. We describe a new strategy to confer these lymphocytes with adhesiveness to surface proteins enriched in the tumor microenvironment. This approach is based on decorating CTLs with monoclonal antibodies (mAbs) specific to any surface protein of interest within the stroma and the extracelullar matrix of solid tumors. For efficient mAb decoration, we have introduced a mAb binding Fc receptor (FcR) scaffold, FcγRIIB1 (CD32B1), which we found to be enriched on B lymphocyte microvilli (MV). This isoform contains an inhibitory ITIM motif within a cytoplasmic tail anchored to the cortical cytoskeleton. We thus generated a non-signaling CD32B1 mutant lacking the ITIM motif (termed ITIM-less CD32B1, or ILCD32B1) and successfully expressed it in human T cells which normally do not express this FcR. The ILCD32B1 expressing lymphocytes bound multiple IgG1 mAbs whose Fc domain was engineered with a 5-residue substitution to reach a nM range of Fc-FcγCR dissociation constants. The mAb decorated ILCD32B1 expressing T cells could readily adhere to a surface-bound cognate antigen. To broaden the utility of this scaffold, we have also generated a new fusion protein in which the entire Fc binding domain was truncated (tILCD32B1) and replaced with a monomeric streptavidin variant, mSA2, via a CD8 hinge. The molecule, termed mSA2-CD8h-tILCD32B1, was also successfully expressed in T cells, readily and stably bound biotinylated IgG mAbs and once decorated with the biotin labeled mAbs, conferred the T cells with high adhesiveness to multiple surface-coated antigens. mSA2-CD8h-tILCD32B1 expressing human T cells decorated ex vivo with a biotin-labeled mAb retained the antibody for hours after accumulation inside breast tumors implanted in immunodeficient recipient mice. Our results collectively suggest that a non-signaling CD32B1 can be used as a versatile scaffold for mAb decoration of T cells. Our mAb decoration approach can confer new cell adhesive reactivities to improve tumor CTL (CAR-T and TIL) accumulation and retention inside solid tumors.
将细胞毒性T淋巴细胞（CTL），如嵌合抗原T细胞（CAR-T）、工程化T细胞受体（TCR）-T细胞或肿瘤浸润T细胞（TIL）过继性细胞转移用于实体瘤治疗是一项重大的治疗挑战。我们描述了一种新策略，使这些淋巴细胞能够黏附于肿瘤微环境中富集的表面蛋白。该方法基于用针对实体瘤基质和细胞外基质中任何感兴趣表面蛋白的单克隆抗体（mAb）修饰CTL。为了实现高效的mAb修饰，我们引入了一种mAb结合Fc受体（FcR）支架，即FcγRIIB1（CD32B1），我们发现它在B淋巴细胞微绒毛（MV）上富集。该异构体在锚定到皮质细胞骨架的细胞质尾部含有一个抑制性免疫受体酪氨酸抑制基序（ITIM）。因此，我们生成了一种缺乏ITIM基序的无信号CD32B1突变体（称为无ITIM的CD32B1，或ILCD32B1），并成功地在通常不表达这种FcR的人T细胞中表达。表达ILCD32B1的淋巴细胞结合了多种IgG1 mAb，其Fc结构域经过5个残基的替换工程改造，以达到纳摩尔范围的Fc-FcγCR解离常数。用mAb修饰的表达ILCD32B1的T细胞能够很容易地黏附到表面结合的同源抗原上。为了拓宽这种支架的应用范围，我们还生成了一种新的融合蛋白，其中整个Fc结合结构域被截短（tILCD32B1），并通过CD8铰链替换为单体链霉亲和素变体mSA2。这种称为mSA2-CD8h-tILCD32B1的分子也成功地在T细胞中表达，能够快速稳定地结合生物素化的IgG mAb，一旦用生物素标记的mAb修饰，就能使T细胞对多种表面包被抗原具有高黏附性。用生物素标记的mAb在体外修饰的表达mSA2-CD8h-tILCD32B1的人T细胞，在植入免疫缺陷受体小鼠体内的乳腺肿瘤中积聚后，能将抗体保留数小时。我们的结果共同表明，无信号的CD32B1可以用作T细胞mAb修饰的通用支架。我们的mAb修饰方法可以赋予新的细胞黏附反应性，以改善肿瘤CTL（CAR-T和TIL）在实体瘤内的积聚和滞留。
