Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-Stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

CD28 is a key T cell costimulatory receptor implicated
in antitumor
immunity and immune-related disorders, yet no small molecule modulators
of CD28 have reached clinical development. Here, we report the discovery
and characterization of small molecule CD28 antagonists identified
through affinity selection-mass spectrometry (AS-MS). Subsequent catalog-based
structure–activity relationship (SAR) optimization led to the
identification of validated hits, 5MS-5 and 19MS-5, which exhibit
direct CD28 binding and potent inhibition of CD28-B7 interactions
in cellular reporter assays. Pharmacokinetic profiling demonstrated
favorable solubility, metabolic stability, permeability, and oral
exposure in vivo. Functionally, both compounds suppressed cytokine
production in primary human T cells cocultured with tumor spheroids
and human epithelial tissues, validating their ability to inhibit
CD28-driven immune activation in physiologically relevant models.
These findings establish 5MS-5 and 19MS-5 as promising CD28 inhibitors
and provide a foundation for developing orally bioavailable immunomodulators
targeting T cell costimulation.

分化簇28（CD28）是关键的T细胞共刺激受体，与抗肿瘤免疫及免疫相关疾病密切相关，但目前尚无CD28的小分子调节剂（small molecule modulators）进入临床研发阶段。本文报道了通过亲和选择-质谱（affinity selection-mass spectrometry, AS-MS）技术筛选得到的CD28小分子拮抗剂的发现与表征过程。后续基于化合物库的构效关系（structure–activity relationship, SAR）优化，成功鉴定出经过验证的命中化合物5MS-5与19MS-5，二者可直接结合CD28，并在细胞报告试验中强效抑制CD28与B7的相互作用。药代动力学表征显示，这两种化合物在体内具备良好的溶解性、代谢稳定性、膜通透性及口服生物利用度。功能实验表明，在与肿瘤球体（tumor spheroids）及人上皮组织共培养的原代人T细胞中，两种化合物均可抑制细胞因子生成，证实其在生理相关模型中可阻断CD28介导的免疫激活。本研究确立了5MS-5与19MS-5作为极具潜力的CD28抑制剂的价值，为开发靶向T细胞共刺激通路的口服可生物利用免疫调节剂奠定了坚实基础。