Selective targeting of GARP-LTGFb axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8+ T cell anti-tumor immunity. Selective targeting of GARP-LTGFb axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8+ T cell anti-tumor immunity

Immune checkpoint blockade (ICB) has revolutionized cancer immunotherapy. However, the majority of cancer patients fail to respond clinically. One potential reason is the accumulation of transforming growth factor-beta (TGFb) in the tumor microenvironment (TME). TGFb drives cancer immune evasion in part by inducing regulatory T cells (Tregs) and limiting CD8+ T cell function. Glycoprotein-A repetitions predominant (GARP) is a cell surface docking receptor for activating latent TGF1, TGFb2 and TGFb3, with its expression restricted in effector Tregs, cancer cells as well as platelets. Herein we demonstrate that GARP overexpression in human cancers correlates with unfavorable TME and poor clinical response to ICB, raising a possibility of GARP blockade to improve cancer immunotherapy. However, it is unclear if targeting GARP in the TME is feasible without affecting platelets. We report an anti-human GARP antibody (named PIIO-1) specific for its ligand-interacting domain, effectively blocking activation of latent TGFb1. PIIO-1 lacks recognition of GARP-TGF complex on platelets. Using human GARP knock-in mice, we demonstrate that PIIO-1 does not cause thrombocytopenia, and is preferentially distributed in the TME and effective for treating both GARP+ and GARP- cancers, alone or in combination with anti-PD-1 antibody. PIIO-1 treatment reduces canonical TGFb signaling in tumor-infiltrating immune cells, prevents T cell exhaustion, and enhances CD8+ T cell migration into the TME in a CXCR3-dependent manner. Collectively, we conclude that GARP contributes to multiple aspects of immune resistance in cancer; anti-GARP antibody PIIO-1 is an efficacious and safe strategy to overcome ICB resistance, therefore warranting clinical development. Overall design: 1x10^5 MB-49 Bladder Cancer cells were injected s.c. on the right flank of hLrrc32KI mice. PIIO-1 (200 ug/mouse, i.p.) were delivered on day 6 and 9 for 2 doses. Tumors were collected on day 10. Single cell suspension and RNA isolation were prepared, and then subjected to bulk RNA sequencing.

免疫检查点阻断（Immune checkpoint blockade, ICB）彻底革新了癌症免疫治疗。然而绝大多数癌症患者无法产生临床应答，其中一个潜在原因是肿瘤微环境（tumor microenvironment, TME）中转化生长因子-β（transforming growth factor-beta, TGFβ）的蓄积。TGFβ可通过诱导调节性T细胞（regulatory T cells, Tregs）、抑制CD8+ T细胞功能，部分介导癌症免疫逃逸。糖蛋白A重复序列主要蛋白（Glycoprotein-A repetitions predominant, GARP）是一种可激活潜伏型TGFβ1、TGFβ2及TGFβ3的细胞表面停靠受体，其表达局限于效应性Tregs、癌细胞及血小板中。本研究证实，人类癌症中GARP的过表达与不良肿瘤微环境及ICB临床应答不佳相关，这提示阻断GARP或可改善癌症免疫治疗。然而目前尚不明确，在肿瘤微环境中靶向GARP而不影响血小板功能是否可行。本研究报道一种靶向人GARP配体结合结构域的抗人GARP抗体（命名为PIIO-1），可有效阻断潜伏型TGFβ1的激活；PIIO-1不会识别血小板表面的GARP-TGFβ复合物。通过人源GARP敲入小鼠模型，本研究证实PIIO-1不会引发血小板减少症，且可优先富集于肿瘤微环境中，无论单独使用还是与抗PD-1抗体联合使用，均可有效治疗GARP阳性及GARP阴性癌症。PIIO-1治疗可降低肿瘤浸润免疫细胞中的经典TGFβ信号通路活性，抑制T细胞耗竭，并以CXCR3依赖的方式增强CD8+ T细胞向肿瘤微环境的迁移。综上，本研究认为GARP参与介导癌症免疫抵抗的多个环节；抗GARP抗体PIIO-1是一种可有效且安全地克服ICB耐药的策略，因此具备临床开发价值。整体实验设计：向hLrrc32KI小鼠右侧胁腹皮下注射1×10^5个MB-49膀胱癌细胞。分别于第6天和第9天给予PIIO-1（200 μg/只，腹腔注射（i.p.）），共给药2次。于第10天收集肿瘤组织，制备单细胞悬液并提取RNA，随后进行批量RNA测序。