Chimeric antigen receptor (CAR)-T cell-based immunotherapy of malignant disease relies on the specificity and association constant of single-chain variable fragments (scFvs). The latter are synthesized from parent antibodies by fusing their light (V_L) and heavy (V_H)-chain variable domains into a single chain using a flexible linker peptide. The fusion of V_L and V_H domains can distort their relative orientation, thereby compromising specificity and association constant of scFv, and reducing the lytic efficacy of CAR-T cells. Here, we circumvent the complications of domains’ fusion by designing scFv mutants that stabilize interaction between scFv and its target, thereby rescuing scFv efficacy. We employ an iterative approach, based on structural modeling and mutagenesis driven by computational protein design. To demonstrate the power of this approach, we use the scFv derived from an antibody specific to a human leukocyte antigen A2 (HLA-A2)-HER2-derived peptide complex. Whereas the parental antibody is highly specific to its target, the scFv showed reduced specificity. Using our approach, we design mutations into scFvs that restore specificity of the original antibody.

基于嵌合抗原受体（CAR）-T细胞的恶性肿瘤免疫疗法依赖于单链可变片段（scFvs）的特异性和缔合常数。后者由母体抗体通过使用柔性接头肽将其轻链（V _L）和重链（V _H）可变域融合到单链中而合成。V _L和V _H的融合结构域可能扭曲其相对方向，从而损害scFv的特异性和缔合常数，并降低CAR-T细胞的裂解效率。在这里，我们通过设计稳定scFv及其靶标之间相互作用的scFv突变体来避开域融合的复杂性，从而挽救scFv的效力。我们采用一种迭代方法，该方法基于结构化模型和由计算蛋白设计驱动的诱变。为了证明这种方法的功能，我们使用了对人白细胞抗原A2（HLA-A2）-HER2衍生的肽复合物具有特异性的抗体衍生的scFv。亲本抗体对其靶标具有高度特异性，而scFv显示出降低的特异性。使用我们的方法，我们将突变设计为可恢复原始抗体特异性的scFv。