摘要

背景：

系统性红斑狼疮（Systemic Lupus erythematosus, SLE）是一种经典的自身免疫性疾病，其发病机制复杂，遗传易感性及异常的表观遗传学修饰共同导致免疫系统紊乱，患者的临床表现具有高度异质性。尽管传统药物如糖皮质激素和免疫抑制剂在一定程度上提高了患者生存率，但仍然存在部分患者不应答、病情反复发作等问题，且长期使用会导致感染、代谢紊乱等副作用的发生。靶向免疫疗法（如单克隆抗体Belimumab、Anifrolumab等）通过阻断B细胞激活因子（B cell activating factor belonging to the TNF family，BAFF）、干扰素受体等靶点部分缓解病情，但仍存在应答率有限、停药复发及感染风险升高等问题，因此亟需进一步探索新的治疗靶点以提高治疗效果。

CD132（Common gamma chain，γ-chain，γc），又称IL2RG，是IL-2（Interleukin-2，IL-2）、IL-4、IL-7、IL-9、IL-15和IL-21一共6种γ-chain家族细胞因子受体的共同亚基，广泛表达于T细胞、B细胞、NK细胞等免疫细胞。研究发现SLE患者血清中IL-7、IL-15、IL-21等γ-chain因子显著上调，促进T、B细胞过度活化及自身抗体产生，而IL-2则通过激活调节性T细胞（Regulatory T cell，Treg）发挥保护作用。因此，差异性阻断CD132（即在阻断IL-7、IL-15和IL-21等促炎细胞因子下游信号通路的同时保留IL-2信号传导）可能削弱效应性T细胞和B细胞的功能，同时维持IL-2对Treg的激活作用，从而促进免疫平衡的恢复。但是目前尚无差异性阻断CD132的抗体，研发该抗体有望为SLE治疗提供新药物。

目的：

探究CD132在SLE疾病发生发展中扮演的角色，研发出差异性阻断CD132的单克隆抗体，进一步揭示差异性阻断CD132治疗SLE的疗效及机制，为治疗SLE等自身免疫病提供全新选择。

方法：

1. 检测SLE患者CD132的表达水平，分析其与疾病活动度的相关性；磁珠分选人T细胞和B细胞，体外培养探究阻断CD132对T细胞及B细胞功能活性等影响。

2. 通过免疫小鼠、构建噬菌体文库、高通量筛选以及阻断活性筛选等实验技术，构建出差异性阻断CD132的单克隆嵌合体。

3. 嵌合抗体人源化，并利用FcRn人源化（FcRn^hu/hu）小鼠对抗CD132抗体的药代动力学（Pharmacokinetics, PK）进行评价。

4. 对CD132人源化（CD132^hu/hu）小鼠进行抗CD132抗体注射，评价抗CD132抗体对小鼠血细胞的影响，并在此基础上对小鼠进行血蓝蛋白（Keyhole Limpet Hemocyanin，KLH）免疫造模，评估抗CD132抗体对KLH免疫小鼠的作用。

5. 选择两种常用的SLE动物模型，姥鲛烷（Pristane）诱导SLE样动物模型和用SLE患者血细胞构建移植物抗宿主病（Graft-versus-host disease，cGVHD）模型）小鼠进行抗CD132抗体皮下注射，评价抗CD132抗体对SLE的治疗效果。

6. 选择CD132人源化小鼠（CD132^hu/hu）和BAFF及BAFFR双人源化（BAFF^hu/hu/BAFFR^hu/hu）小鼠进行pristane造模，分别注射抗CD132抗体和Belimumab，对比抗CD132抗体和Belimumab的治疗效果。

结果：

1. CD132在SLE患者多种淋巴细胞中表达增高，与SLE疾病活动指数（Systemic lupus erythematosus disease activity index，SLEDAI）评分呈正相关。抗CD132抗体阻断人T细胞分泌多种促炎因子，同时抑制抗体分泌。

2. 成功开发出一款靶向CD132的差异性阻断嵌合抗体2D4。该抗体在有效抑制IL-21等促炎细胞因子信号的同时，最大限度减少了对IL-2下游信号通路的干扰。

3. 嵌合抗体2D4人源化得到终分子h2D4H4K12，该分子在保留了与2D4相似阻断活性同时，降低了嵌合体的免疫原性，并在FcRn^hu/hu小鼠中表现出优异的药代动力学特性。

4. h2D4H4K12能够显著减少小鼠循环T、B、NK细胞的数量，且对效应淋巴细胞的抑制作用强于幼稚淋巴细胞，对Treg细胞的抑制强度远弱于对标分子（由Regeneron公司开发的全阻断CD132抗体REGN7257）；同时降低小鼠对KLH的免疫反应，降低记忆B细胞、浆母细胞、抗体分泌细胞（Antibody secreting cells，ASCs）等细胞比例，抑制抗体分泌。

5. h2D4H4K12能够缓解两种常用的SLE模型鼠疾病表型，显著降低尿蛋白/肌酐比、循环自身抗体和促炎因子水平。h2D4H4K12同时还能降低淋巴结和脾脏中T、B细胞比例。

6. h2D4H4K12通过同时抑制T细胞和B细胞，在治疗狼疮性肾炎方面明显优于Belimumab。

结论：

本研究首次发现CD132可以作为SLE治疗新靶点，并成功开发出人源化单抗 h2D4H4K12，其通过选择性阻断IL-21、IL-15等γ-chain细胞因子信号（保留IL-2活性），显著抑制T、B细胞过度活化及自身抗体分泌。在两种SLE样小鼠模型中，h2D4H4K12均展现出显著治疗效果，而在pristane诱导的SLE样小鼠模型中，h2D4H4K12展现出较Belimumab更强的疗效，尤其在减轻肾脏损伤方面表现突出。该新药的研发有望为SLE临床治疗提供新的选择。

Abstract

Background:

Systemic Lupus Erythematosus (SLE) is a classical autoimmune disease characterized by complex pathogenesis involving genetic predisposition, environmental triggers, and widespread immune dysregulation, with high clinical heterogeneity. Although conventional therapies such as glucocorticoids and immunosuppressants have improved patient survival to some extent, a subset of patients still face challenges including inadequate efficacy, disease relapse, and long-term side effects such as infections and metabolic disorders. Targeted immunotherapies (e.g., monoclonal antibodies like Belimumab and Anifrolumab) partially alleviate symptoms by blocking BAFF, interferon receptors, and other targets, yet limitations persist, including suboptimal response rates, relapse upon discontinuation, and increased infection risks. Thus, there is an urgent need to explore novel therapeutic targets to enhance treatment outcomes.

CD132 (Common gamma chain, γc or IL2RG), a shared subunit of six γ-chain family cytokine receptors (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21), is widely expressed on immune cells such as T, B, and NK cells. Studies have shown that γ-chain cytokines, including IL-7, IL-15, and IL-21, are markedly upregulated in SLE patients, promoting hyperactivation of T/B cells and autoantibody production, while IL-2 exerts protective effects by activating regulatory T cells (Tregs). Therefore, differentially blocking CD132 (specifically inhibiting downstream pathways of proinflammatory cytokines (IL-7, IL-15, IL-21) while preserving IL-2-mediated signaling) may simultaneously suppress signaling of multiple pathogenic cytokines, attenuate effector T/B cell functions, and preserve IL-2-mediated Treg activation, thereby restoring immune balance. However, no CD132-targeting antibodies currently exist, and their development may offer a novel therapeutic strategy for SLE.

Objective:

This study aims to investigate the role of CD132 in SLE pathogenesis, develop a differentially blocking monoclonal antibody targeting CD132, elucidate its therapeutic efficacy and mechanisms in SLE, and provide a novel approach for treating SLE and other autoimmune diseases.

Methods:

1. Assess CD132 expression levels in SLE patients and correlate with disease activity. Isolate human T and B cells via magnetic sorting to evaluate the effects of CD132 blockade on cellular function in vitro.

2. Generate a differentially blocking CD132-targeting chimeric antibody through mouse immunization, phage display library construction, high-throughput screening, and functional validation.

3. Humanize the chimeric antibody and evaluate its pharmacokinetics in FcRn-humanized (FcRn^hu/hu) mice.

4. Administer the anti CD132 antibody to CD132-humanized (CD132^hu/hu) mice to assess its effects on blood cells. Immunize mice with Keyhole Limpet Hemocyanin (KLH) to evaluate the antibody’s impact on KLH-induced immune responses.

5. Test the therapeutic efficacy of the anti CD132 antibody in two SLE-related murine models: pristane-induced SLE-like disease and a graft-versus-host disease (GVHD) model constructed using blood cells from SLE patients.

6. Compare the therapeutic effects of the anti CD132 antibody and Belimumab in pristane-induced SLE models using CD132^hu/hu mice and BAFF/BAFFR-double-humanized (BAFF^hu/hu/BAFFR^hu/hu) mice.

Results:

1. CD132 expression was elevated in multiple lymphocyte subsets of SLE patients and positively correlated with SLEDAI scores. Anti-CD132 antibody blocked the secretion of proinflammatory cytokines by human T cells and inhibited antibody production.

2. A chimeric antibody targeting CD132 with selective blocking activity (h2D4H4K12) was successfully developed. 2D4 effectively inhibited signaling from proinflammatory cytokines like IL-21 while minimally interfering with IL-2 downstream pathways.

3. The humanized version of the chimeric antibody (h2D4H4K12) retained blocking activity comparable to the original chimeric antibody, exhibited reduced immunogenicity, and demonstrated superior pharmacokinetic properties in FcRn^hu/hu mice.

4. h2D4H4K12 significantly reduced circulating T, B, and NK cell counts, with stronger inhibitory effects on effector lymphocytes than naïve lymphocytes and markedly weaker suppression of Tregs. It also dampened KLH-induced immune responses by reducing memory B cells, plasmablasts, antibody-secreting cells (ASCs), and antibody secretion.

5. h2D4H4K12 alleviated disease phenotypes in both SLE-related murine models, significantly lowering urine protein/creatinine ratios, circulating autoantibodies, and proinflammatory cytokine levels. It also reduced T and B cell populations in lymph nodes and spleens.

6. h2D4H4K12 demonstrated superior efficacy over Belimumab in treating lupus nephritis by simultaneously inhibiting both T and B cells.

Conclusions:

This study identifies CD132 as a novel therapeutic target for SLE and develops h2D4H4K12, a humanized monoclonal antibody that selectively blocks IL-21 and IL-15 signaling while preserving IL-2 activity. h2D4H4K12 effectively suppresses pathogenic T/B cell hyperactivation and autoantibody production, demonstrating significant therapeutic efficacy in two SLE-like murine models. Notably, it outperforms Belimumab in pristane-induced SLE-like mice, particularly in mitigating renal injury. This novel agent provides a differentiated targeting strategy for SLE treatment.