Immunogenicity of CAR T cells in cancer therapyCAR T 细胞在癌症治疗中的免疫原性

Key points 要点

1.Pre-existing and/or treatment-induced immunity to chimeric antigen receptor(CAR) constructs containing mouse-derived single-chain variable fragments are associated with treatment failure in certain patients and might limit the success of redosing strategies.1.某些患者对含有小鼠衍生单链可变片段的嵌合抗原受体（CAR）构建体存在预先免疫和/或治疗诱导免疫，这与治疗失败有关，并可能限制重新给药策略的成功。

2.The possible effects of immunogenicity on CAR T cell persistence and function are currently poorly understood.2.目前对免疫原性对 CAR T 细胞持久性和功能可能产生的影响了解甚少。

3.Novel technologies designed to enhance CAR T cell performance and/or the application of allogeneic CAR T cells might further amplify the likelihood of anti-CAR immune responses,thus necessitating strategies to overcome such risks.3.旨在提高 CAR T 细胞性能的新技术和/或异体 CAR T 细胞的应用可能会进一步扩大抗 CAR 免疫反应的可能性，因此需要制定策略来克服这些风险。

4.Various monitoring, mitigation and management approaches can be used to reduce the risk of anti-CAR immunity, although validated assays enabling adequate assessments of anti-CAR immune responses remain an unmet need.4.可以采用各种监测、缓解和管理方法来降低抗 CAR 免疫的风险，但目前仍未满足充分评估抗 CAR 免疫反应的有效检测方法的需求。

5.We advocate for the inclusion of CAR-associated immunogenicity analysis in both preclinical and clinical investigations of CAR T cell therapy.5.我们主张将 CAR 相关免疫原性分析纳入 CAR T 细胞疗法的临床前和临床研究中。

Mechanisms of action of anti-CaR immune responses. 抗 CAR 免疫反应的作用机制。

Acquired anti-chimeric antigen receptor (CAR) immune responses can be humoral or cellular. Cellular immunity probably arises from the processing and cross-presentation of foreign (mouse, viral or non-self human) sequences to the CAR molecule in the context of a major histocompatibility complex (MHC). CAR peptides from apoptotic or necrotic CAR T cells can be displayed by antigen-presenting cells and used to prime T cell responses in secondary lymphoid organs (panel a). CAR-specific CD8+ T cells or cytotoxic CD4+ T cells153 can eliminate CAR T cells that present fragments of the CAR molecule via MHC-mediated recognition. Humoral immunity can be primed through CAR proteins in apoptotic bodies presented by follicular dendritic cells to B cells. Supported by anti-CAR T cells, CAR-specific B cells can expand and then undergo class switching and plasma cell differentiation, producing different classes of immunoglobulins with distinct functions. Anti-CAR antibodies can potentially induce the death of CAR T cells via several mechanisms, including antibody-dependent cellular cytotoxicity, whereby interactions between CAR-bound antibodies and the Fc receptor (FcR) domains of innate immune cells, such as natural killer (NK) cells or macrophages, leads to cytotoxicity via the release of perforin and/or granzymes, or phagocytosis (panel b) and complement[1]dependent cytotoxicity owing to CAR-bound antibodies that activate the complement cascade, leading to the formation of membrane attack complexes and cell lysis (panel c). Following CAR engagement, anti-CAR IgE could also bind to the FcR of mast cells, thus promoting degranulation and CAR T cell death (panel d). Excessive release of multiple vasoactive mediators in mast cell granula could lead to systemic anaphylaxis, which was lethal in one patient获得性抗嵌合抗原受体（CAR）免疫反应可以是体液免疫反应，也可以是细胞免疫反应。细胞免疫可能源于外来（小鼠、病毒或非人类自身）序列在主要组织相容性复合体（MHC）的背景下对 CAR 分子的处理和交叉呈递。来自凋亡或坏死的 CAR T 细胞的 CAR 肽可由抗原递呈细胞显示，并用于激发次级淋巴器官中的 T 细胞反应（面板 a）。CAR 特异性 CD8+ T 细胞或细胞毒性 CD4+ T 细胞153 可通过 MHC 介导的识别消除呈现 CAR 分子片段的 CAR T 细胞。通过滤泡树突状细胞向 B 细胞展示的凋亡体中的 CAR 蛋白，可启动体液免疫。在抗 CAR T 细胞的支持下，CAR 特异性 B 细胞可以扩增，然后进行类别转换和浆细胞分化，产生具有不同功能的不同类别的免疫球蛋白。抗 CAR 抗体有可能通过多种机制诱导 CAR T 细胞死亡，包括抗体依赖性细胞毒性，即 CAR 结合抗体与先天性免疫细胞（如自然杀伤（NK）细胞或巨噬细胞）的 Fc 受体（FcR）结构域相互作用、通过释放穿孔素和/或颗粒酶或吞噬作用，产生细胞毒性（b 组）；由于 CAR 结合抗体激活补体级联，产生补体[1]依赖性细胞毒性，导致膜攻击复合物的形成和细胞裂解（c 组）。CAR 接合后，抗 CAR IgE 还可与肥大细胞的 FcR 结合，从而促进脱颗粒和 CAR T 细胞死亡（图示 d）。肥大细胞颗粒中多种血管活性介质的过度释放可导致全身性过敏性休克，其中一名患者因过敏性休克而死亡。

Differences in anti-CAR immune responses following the targeting of B cell versus solid-tumour antigens.以 B 细胞抗原和实体瘤抗原为靶点的抗 CAR 免疫反应的差异。

a | Chimeric antigen receptor (CAR) T cells targeting B cell malignancies induce aplasia of non-malignant B cells, thus reducing the potential for anti-CAR humoral immunity. b | CAR T cells directed to solid-tumour antigens (targets not expressed on B cells) might have an increased risk of inducing both cellular and humoral anti-CAR immunity.a | 以 B 细胞恶性肿瘤为靶点的嵌合抗原受体（CAR）T 细胞会诱导非恶性 B 细胞增生，从而降低抗 CAR 体液免疫的潜力。 b | 以实体瘤抗原（B 细胞不表达的靶点）为靶点的 CAR T 细胞可能会增加诱导细胞和体液抗 CAR 免疫的风险。

Engineering CAR T cells to reduce their inherent immunogenicity. 改造 CAR T 细胞，降低其固有的免疫原性。

Improving chimeric antigen receptor (CAR) design in order to limit inherent immunogenicity involves selecting tumour-binding moieties with the least immunogenic potential. Here, we show methods of replacing elements of single-chain variable fragments (scFvs) derived from mouse antibodies with human sequences or replacing the entire mouse-derived scFv with human-derived heavy-chain variable fragments or fully human scFvs. CARs with heavy-chain-only variable fragment tumour-recognition domains have the additional advantage of not requiring linkers, which are another possible source of immunogenicity. As an alternative to scFvs, the ligand of a tumour-associated receptor (or vice versa) can be attached to the constant regions of the CAR. Such constructs exploiting interactions between endogenous receptors and their ligands might enable tumour targeting with minimal risk of immunogenicity.改进嵌合抗原受体（CAR）设计以限制固有免疫原性涉及到选择免疫原性最小的肿瘤结合分子。在这里，我们展示了用人源序列替代小鼠抗体单链可变片段（scFv）元素或用人源重链可变片段或全人源scFv替代整个小鼠源scFv的方法。 具有纯重链可变片段肿瘤识别域的CAR具有不需要连接体的额外优势，而连接体是另一种可能的免疫原性来源。作为 scFvs 的替代品，肿瘤相关受体的配体（或反之亦然）可以连接到 CAR 的恒定区。这种利用内源性受体及其配体之间相互作用的构建物可能会在将免疫原性风险降至最低的情况下实现肿瘤靶向。

Monitoring, mitigation and management of anti-CAR immunity in the clinic. 临床中抗 CAR 免疫的监测、缓解和管理。

Mitigation of anti-CAR immunity through lymphodepletion regimens can reduce the numbers of circulating lymphoid cells as well as of antigen-presenting cells prior to CAR T cell infusion. Furthermore, cytoreductive treatments might reduce the numbers of certain immuno[1]suppressive cells in the tumour microenvironment, leading to tumour cell death and creating a pro-inflammatory milieu that promotes the antitumour efficacy of CAR T cells154. The monitoring of patients receiving CAR T cells should begin as soon as a patient is considered for therapy. Conventional cancer treatment regimens, including monoclonal antibodies of mouse origin155, can induce human anti-mouse antibodies (HAMAs) in patients12,13. Therefore, careful evaluation of pre[1]existing immunity is important for the avoidance of anaphylaxis after CAR T cell infusion17. Subsequently, monitoring in clinical trials should include surveillance for possible HAMAs and specific anti-CAR immune responses, including antibody and T cell analysis. The management of low-level anti-CAR immunity can be achieved using repeat lymphodepletion to reduce anti-CAR immunity prior to redosing with the same CAR T cell product. Humanized CAR T cell constructs could be considered in patients who progressed or relapsed after a previous infusion of CAR T cells containing mouse single-chain variable fragments (scFvs) or who required subsequent infusions to prevent disease progression. However, this approach could be associated with additional costs and/or treatment delays.通过淋巴清除疗法减轻抗CAR免疫可在CAR T细胞输注前减少循环淋巴细胞和抗原递呈细胞的数量。此外，细胞吞噬治疗可能会减少肿瘤微环境中某些免疫[1]抑制细胞的数量，导致肿瘤细胞死亡，并创造一种促进 CAR T 细胞抗肿瘤疗效的促炎环境154。一旦考虑对接受 CAR T 细胞治疗的患者进行监测，就应立即开始。传统的癌症治疗方案，包括小鼠单克隆抗体155 ，可诱导患者产生人类抗小鼠抗体（HAMAs）12,13。因此，要避免 CAR T 细胞输注后出现过敏性休克，就必须仔细评估前期已有的免疫力17。随后，临床试验中的监测应包括对可能的 HAMA 和特异性抗 CAR 免疫反应的监控，包括抗体和 T 细胞分析。低水平的抗 CAR 免疫可通过重复淋巴清扫进行管理，以在重新输注相同的 CAR T 细胞产品前降低抗 CAR 免疫。人源化 CAR T 细胞构建物可用于先前输注含有小鼠单链可变片段（scFvs）的 CAR T 细胞后病情恶化或复发的患者，或需要后续输注以防止病情恶化的患者。不过，这种方法可能会增加费用和/或延误治疗。

Assays designed to monitor anti-CAR immunity旨在监测抗 CAR 免疫的检测方法

Cellular immunity 细胞免疫

Cytotoxicity assays 细胞毒性试验

Cytotoxicity assays measure the killing of chimeric antigen receptor(CAR)-transduced target cells(including T cells and autologouslymphoid cell lines) by ex vivo expanded T cells and/or mononuclear cells from peripheral blood samples via stimulation with irradiated CAR-expressing targets. Such assays typically use 51Cr labelling for quantification of target cell death after co-culture for 4–6 hours.细胞毒性测定法测量的是体外扩增的 T 细胞和/或外周血样本中的单核细胞通过照射表达 CAR 的靶细胞，杀死嵌合抗原受体（CAR）转导的靶细胞（包括 T 细胞和自体类细胞系）的情况。此类检测通常使用 51Cr 标记，用于定量检测共培养 4-6 小时后靶细胞的死亡情况。

Degranulation assays 脱颗粒测定

Degranulation assays measure CD107a (also known as Lamp1) surface expression, which indicates recent secretion of cytolytic vesicles, on ex vivo expanded patient-derived T cells from peripheral blood samples co-cultured with irradiated CAR-expressing T cells using flow cytometry.脱颗粒测定法利用流式细胞术测量外周血样本中与辐照 CAR 表达 T 细胞共培养的体外扩增患者 T 细胞的 CD107a（又称 Lamp1）表面表达，这表明 T 细胞最近分泌了细胞溶解小泡。

In vitro proliferation assays体外增殖试验

In vitro proliferation assays measure the ex vivo proliferation of patient-derived T cells after stimulation with irradiated CAR-expressing target cells or CAR–peptide mixtures typically through 3 H-thymidine incorporation or flow cytometric assessment (dilution of a proliferation dye such as carboxyfluorescein diacetate succinimidyl ester or similar).体外增殖检测通常通过 3 H-胸苷掺入或流式细胞仪评估（稀释增殖染料，如羧基荧光素二乙酸琥珀酰亚胺酯或类似物），测量患者衍生 T 细胞在接受辐照表达 CAR 的靶细胞或 CAR 肽混合物刺激后的体内外增殖情况。

Enzyme-Linked Immunosorbent Spot (ELISpot) assays酶联免疫吸附点（ELISpot）测定

ELISpot assays detect IFNγ released by ex vivo expanded patient-derived CAR-specific T cells. These cells are typically derived from peripheral blood mononuclear cells followed by 2-week expansion in the presence of irradiated CAR-expressing T cells and IL-2. After co-culture,these cells are exposed to either irradiated CAR-expressing target cells or CAR-derived peptide pools. This approach has the advantage of enabling the identification of the distinct protein fragments recognized by anti-CAR T cells.ELISpot 检测法可检测体外扩增的患者 CAR 特异性 T 细胞释放的 IFNγ。这些细胞通常来源于外周血单核细胞，然后在经过辐照的 CAR 表达 T 细胞和 IL-2 存在下扩增 2 周。共培养后，这些细胞暴露于经过辐照的 CAR 表达靶细胞或 CAR 衍生肽池。这种方法的优点是可以识别抗 CAR T 细胞识别的不同蛋白质片段。

Intracellular cytokine staining and flow cytometry细胞内细胞因子染色和流式细胞术

Intracellular cytokine staining and flow cytometry involve the detection of IFNγ or other cytokine-producing T cells among peripheral blood mononuclear cells after stimulation with mouse-derived CAR sequence-spanning peptides (or potentially non-mouse CAR peptides). This approach uses intracellular staining with fluorochrome-labelled antibodies typically including but not limited to IFNγ, TNF, and/orIL-2 and/or extracellular staining of activation markers such as CD137 and CD40 ligand followed by flow cytometry for quantification purposes.细胞内细胞因子染色和流式细胞术是指用小鼠衍生的 CAR 序列跨肽（或潜在的非小鼠 CAR 肽）刺激外周血单核细胞后，检测其中的 IFNγ 或其他产生细胞因子的 T 细胞。这种方法使用通常包括但不限于IFNγ、TNF和/或IL-2的荧光标记抗体进行细胞内染色，和/或使用CD137和CD40配体等活化标记物进行细胞外染色，然后使用流式细胞术进行量化。

T cell receptor (TCR) repertoire analysisT 细胞受体 (TCR) 重排分析

TCR repertoire analysis involves either next-generation sequencing or TCR Vβ spectratyping using flow cytometry. This approach enables the identification of expanded T cell clones in vivo based on individual TCR complementarity defining regions or TCR Vβ usage. Such analysis can be used to detect infused clones if TCR repertoire analysisis conducted before infusion.TCR 基因库分析包括下一代测序或使用流式细胞仪进行 TCR Vβ 图谱分析。这种方法能根据单个 TCR 互补性定义区域或 TCR Vβ 使用情况识别体内扩增的 T 细胞克隆。如果在输注前进行 TCR 重排分析，这种分析可用于检测输注的克隆。

Humoral anti-CAR immunity体液抗 CAR 免疫

Enzyme-linked immunosorbent assays (ELISAs)酶联免疫吸附试验（ELISA）

ELISAs are standardized assays that enable the semi-quantitative analysis of human anti-mouse antibodies(HAMAs). To this end,patient serum is added to mouse immunoglobulin-coated wells.After washing,human immunoglobulins bound to the mouse immunoglobulins are visualized using secondary antibody staining with an anti-human IgG typically linked to a reporter enzyme (such as horseradish peroxidase). Subsequently, enzyme-mediated luminescence or colour changes in a solution are monitored for quantification. Standards run in the same assay enable the estimation of HAMA levels.ELISAs 是一种标准化检测方法，可对人抗鼠抗体（HAMAs）进行半定量分析。为此，将患者血清加入涂有小鼠免疫球蛋白的孔中，经洗涤后，使用通常与报告酶（如辣根过氧化物酶）相连的抗人 IgG 二抗染色，观察与小鼠免疫球蛋白结合的人免疫球蛋白。随后，监测溶液中酶介导的发光或颜色变化，以进行量化。在同一检测中运行的标准品可用于估算 HAMA 水平。

Cell-based flow cytometry基于细胞的流式细胞仪

Cell-based flow cytometry involves the incubation of CAR-expressing target cells(such as Jurkat T cells or Chinese hamster ovary (CHO) cells) with patient serum samples, followed by washing and secondary staining for anti-human IgG. Additional steps are required to remove theHAMA signal and thusidentify specific anti-CAR/idiotype antibodies unrelated to HAMA基于细胞的流式细胞术包括将表达 CAR 的靶细胞（如 Jurkat T 细胞或中国仓鼠卵巢 (CHO) 细胞）与患者血清样本孵育，然后进行洗涤和抗人 IgG 的二次染色。还需要额外的步骤来去除 HAMA 信号，从而鉴定出与 HAMA 无关的特异性抗 CAR/idiotype 抗体。

ELISA anti-drug antibody bridging assaysELISA 抗药抗体桥接试验

ELISA anti-drug antibody bridging assays use two soluble CAR proteins, one that binds to the vessel's surface while the second is linked to a reporter such as an alkaline phosphatase or horseradish peroxidase. This complex only becomes fixed during the wash stages in the presence of an anti-CAR antibody. Additional steps are needed to remove the HAMA signal. This approach does not enable the identification of specific conformational CAR epitopes, which would require anchoring of the entire CAR to a cell membraneELISA 抗药抗体桥接检测法使用两种可溶性 CAR 蛋白，其中一种与血管表面结合，另一种则与碱性磷酸酶或辣根过氧化物酶等报告物相连。只有在抗 CAR 抗体存在的洗涤阶段，这种复合物才会被固定。去除 HAMA 信号还需要额外的步骤。这种方法无法鉴定特定构象的 CAR 表位，因为这需要将整个 CAR 固定在细胞膜上。