Nanobody-based CAR T cell

Nanobody-based CAR T cells that target the tumor microenvironment inhibit the growth of solid tumors in immunocompetent mice

靶向肿瘤微环境的纳米体car t细胞抑制免疫活性小鼠实体瘤的生长

Chimeric antigen receptor (CAR) T cell therapy has been successful in clinical trials against hematological cancers, but has experienced challenges in the treatment of solid tumors. One of the main difficulties lies in a paucity of tumor-specific targets that can serve as CAR recognition domains. We therefore focused on developing VHH-based, single-domain antibody (nanobody) CAR T cells that target aspects of the tumor microenvironment conserved across multiple cancer types. Many solid tumors evade immune recognition through expression of checkpoint molecules, such as PD-L1, that down-regulate the immune response. We therefore targeted CAR T cells to the tumor microenvironment via the checkpoint inhibitor PD-L1 and observed a reduction in tumor growth, resulting in improved survival. CAR T cells that target the tumor stroma and vasculature through the EIIIB+ fibronectin splice variant, which is expressed by multiple tumor types and on neovasculature, are likewise effective in delaying tumor growth. VHH-based CAR T cells can thus function as antitumor agents for multiple targets in syngeneic, immunocompetent animal models. Our results demonstrate the flexibility of VHH-based CAR T cells and the potential of CAR T cells to target the tumor microenvironment and treat solid tumors.

嵌合抗原受体（CAR）T细胞治疗在血液肿瘤的临床试验中取得了成功，但在实体肿瘤的治疗中也遇到了挑战。其中一个主要的困难在于缺乏可作为car识别域的肿瘤特异性

靶点。因此，我们致力于开发基于VHH的单域抗体（纳米体）car t细胞，该细胞针对多种癌症类型中保存的肿瘤微环境的各个方面。许多实体肿瘤通过检测点分子（如pd-l1）的表达而逃避免疫识别，后者下调免疫反应。因此，我们通过检测点抑制剂pd-l1将car t细胞定位于肿瘤微环境，观察到肿瘤生长减少，从而提高生存率。通过EIIIB+纤维连接蛋白剪接变异体（通过多种肿瘤类型和新血管系统表达）靶向肿瘤基质和血管的car t细胞同样有效地延缓肿瘤生长。因此，基于VHH的Car T细胞可以作为同系物免疫活性动物模型中多个靶点的抗肿瘤药物。我们的研究结果表明，VHH基CAR T细胞的灵活性和CAR T细胞对肿瘤微环境和治疗实体肿瘤的潜力。

Cancers can avoid eradication by evading, and sometimes actively suppressing, the immune system, although they are often initially recognizable by immune cells. The rapidly evolving field of immunotherapy targets cancers by harnessing the power of the immune system. A key player in that approach is the chimeric antigen receptor (CAR) T cell (1–3). CAR T cells are T cells into which a recombinant receptor has been introduced to redirect their specificity toward an antigen of choice. Such receptors comprise an extracellular module that recognizes antigen independent of MHC restriction, in combination with cytoplasmic signaling domains. The antigen recognition module of CAR T cells is usually a single-chain variable fragment (scFv), linked to a costimulatory domain and a cytoplasmic activation domain, such as the CD3ζ or FcRγ intracellular signaling domain (4–6). The scFvs are composed of a heavy-chain variable fragment connected to a light-chain variable fragment by a flexible linker. They are typically reformatted from a full-length Ig, with the linker optimized to preserve heavy- and light-chain variable region pairing. However, scFvs do not always fold efficiently and can be prone to aggregation (7, 8). In contrast, the variable regions

of heavy-chain−only antibodies (VHHs or nanobodies) are small, stable, camelid-derived single-domain antibody fragments with affinities comparable to traditional scFvs (9, 10). VHHs are generally less immunogenic than murine scFvs and, owing to their small size, can access epitopes different from those seen by scFvs (11–13). VHHs could therefore serve as suitable antigen recognition domains in CAR T cells, and several potentially interesting VHHs (14–16) have been tested. Unlike scFvs, VHHs do not require the additional folding and assembly steps that come with V-region pairing. They allow surface display without the requirement for extensive linker optimization or other types of reformatting. The ability to switch out various VHH-based recognition domains yields a highly modular platform, accessible without having to reformat each new conventional antibody into an scFv.

癌症可以通过逃避，有时是积极抑制免疫系统来避免根除，尽管它们最初常常被免疫细胞识别。免疫疗法迅速发展的领域通过利用免疫系统的力量来针对癌症。其中一个关键因素是嵌合抗原受体（car）T细胞（1-3）。car t细胞是一种T细胞，在其中引入一种重组受体，以将其特异性导向所选抗原。这种受体包括一个细胞外模块，该模块与细胞质信号域结合，识别不受MHC限制的抗原。car t细胞的抗原识别模块通常是单链可变片段（scfv），与共刺激域和胞质激活域相连，如cd3ζ或fcrγ胞内信号域（4-6）。单链抗体由一个重链变量片段组成，该重链变量片段通过一个灵活的链接器连接到一个轻链变量片段。它们通常是从一个全长IG重新格式化，与链接器优化，以保持重链和轻链可变区域配对。然而，单链抗体并不总是能有效折叠，容易聚集（7，8）。相反，纯重链抗体（VHH或纳米体）的可变区域是小的、稳定的、由骆驼衍生的单域抗体片段，其亲和力与传统单链抗体（9，10）相当。VHH通常比鼠单链抗体免疫原性低，而且由于其体积小，可以访问不同于单链抗体（11-13）的表位。因此，VHH可以作为CART细胞中合适的抗原识别

域，并且已经测试了几种可能有趣的VHH（14-16）。与scfv不同，vhs不需要额外的折叠和组装步骤，这些步骤与v区域配对一起提供。它们允许表面显示，而无需进行广泛的链接器优化或其他类型的重新格式化。切换各种基于VHH的识别域的能力产生了一个高度模块化的平台，无需将每个新的常规抗体重新格式化为scfv即可访问。

Significance Despite its success in treating hematological cancers, chimeric antigen receptor (CAR) T cell therapy does not so easily eliminate solid tumors. Solid tumors generally develop in a highly immunosuppressive environment and are difficult to target, mostly due to a lack of tumor-specific antigen expression, but other factors contribute as well. This study develops a strategy to target multiple solid tumor types through markers in their microenvironment. The use of single-domain antibody (VHH)- based chimeric antigen receptor (CAR) T cells that recognize these markers circumvents the need for tumor-specific targets. VHH-based CAR T cells that target the tumor microenvironment through immune checkpoint receptors or through stroma and ECM markers are effective against solid tumors in syngeneic, immunocompetent animal models.

尽管嵌合抗原受体（car）T细胞疗法在治疗血液肿瘤方面取得了成功，但它并不能轻易地消除实体肿瘤。实体瘤通常在高度免疫抑制的环境中发展，很难定位，主要是由于缺乏肿瘤特异性抗原表达，但其他因素也有作用。本研究发展了一种策略，通过标记物在其微环境中定位多种实体肿瘤类型。单域抗体（VHH）为基础的嵌合抗原受体（CAR）T细胞识别这些标记物的使用，避免了对肿瘤特异性靶点的需要。在同基因、免疫活性动物模型中，通过免疫检查点受体或基质和ECM标记物靶向肿瘤微环境的基于VHH的Car T细胞对实体瘤有效。

CAR T cell therapies have proven clinically effective exclusively in hematological cancers. CD19-specific CAR T cells have shown success in treating a number of B cell leukemias and lymphomas, as B cell depletion is comparatively well tolerated (17, 18). However, not all tumors have highly specific biomarkers or antigens that are shared by dispensable cell types such as B cells, especially in the case of solid tumors. Antigens such as ErbB2, PSMA, and B7-H3 are considered possible CAR targets for solid tumors, but expression at low levels elsewhere may compromise such applications (19–21). Indeed, an ErbB2- targeted CAR T cell designed to treat metastatic colon cancer proved lethal in a patient, most likely due to off-tumor targeting of healthy lung epithelial cells (19). Off-tumor effects can include widespread cytokine release, which can lead to organ failure (19–21).

Car T细胞疗法已证明仅在血液学癌症中有临床疗效。CD19特异性Car T细胞在治疗一些B细胞白血病和淋巴瘤方面显示了成功，因为B细胞的衰竭相对来说是耐受良好的（17，18）。然而，并非所有的肿瘤都具有高度特异性的生物标记物或抗原，这些标记物或抗原可由B细胞等可有可无的细胞类型共享，特别是在实体肿瘤中。诸如erbb2、psma和b7-h3等抗原被认为是实体肿瘤的可能靶点，但是在其他地方低水平的表达可能会损害这种应用（19-21）。事实上，设计用于治疗转移性结肠癌的erbbb2靶向car t细胞在患者身上被证明是致命的，很可能是由于健康肺上皮细胞的非肿瘤靶向作用（19）。非肿瘤效应包括广泛的细胞因子释放，这可能导致器官衰竭（19-21）。

Current CAR T cell therapies target the tumor directly, as in the case of CD19 or mesothelin-specific CAR T cells. However, solid tumors rarely display unique antigenic markers, and exploitation of neoantigens would require their surface expression, as well as the production of immunoglobulins or VHHs that recognize

them, to generate appropriately specific CARs. To delay the growth of solid tumors, it may be helpful to compromise their microenvironment. Moreover, the microenvironments of many solid tumors share characteristics, for example, the expression of inhibitory molecules such as PD-L1 (22, 23). Using VHHs as recognition domains, we therefore explored PD-L1− specific CAR T cells to target the tumor microenvironment. PD-L1 is widely expressed on tumor cells, as well as on the infiltrating myeloid cells and lymphocytes. A CAR that recognizes PD-L1 should relieve immune inhibition and at the same time allow CAR T cell activation in the tumor microenvironment. PDL1−targeted CAR T cells might thus reprogram the tumor microenvironment, dampening immunosuppressive signals and promoting inflammation. To test this concept, we used the fully syngeneic B16 melanoma model, as well as a PD-L1−overexpressing B16 melanoma model and a colon adenocarcinoma cell line, MC38, in immunocompetent mice. Our results show a significant delay in tumor growth and improved survival by treatment with anti−PD-L1 CAR T cells.

目前的Car T细胞疗法直接针对肿瘤，如CD19或间皮素特异性Car T细胞。然而，实体肿瘤很少显示独特的抗原标记，新抗原的开发将需要它们的表面表达，以及产生识别它们的免疫球蛋白或VHH，以产生适当的特异性CAR。为了延缓实体瘤的生长，可能有助于破坏其微环境。此外，许多实体肿瘤的微环境都具有相同的特征，例如抑制分子如pd-l1（22，23）的表达。因此，我们利用VHHS作为识别域，探索了pd-l1特异性car t细胞，以定位肿瘤微环境。PD-L1广泛表达于肿瘤细胞，也表达于浸润的骨髓细胞和淋巴细胞。一辆能够识别pd-l1的CAR应该能够减轻免疫抑制，同时允许在肿瘤微环境中激活CART细胞。因此，pdl1靶向的car t细胞可能重新编程肿瘤微环境，抑制免疫抑制信号并促进炎症。为了验证这一概念，我们在免疫功能小鼠中使用了完全同源的B16黑色素瘤

模型，以及pd-l1-过度表达的B16黑色素瘤模型和结肠腺癌细胞系MC38。我们的结果显示，抗-PD-L1 car T细胞治疗可显著延缓肿瘤生长，提高生存率。

The reliance of solid tumors on extracellular matrix (ECM) and on neovasculature for nutrient supply affords yet another possible target for CAR T cells, as tumor ECM and newly formed blood vessels display unique antigens not commonly found in healthy adults (24, 25). As an extension of the concept that targeting PD-L1 in the tumor microenvironment may prove beneficial, we generated CAR T cells using a VHH that recognizes EIIIB, a splice variant of fibronectin strongly expressed in both the tumor ECM and the neovasculature (24, 26). These CAR T cells also reduce the rate of tumor growth in the B16 melanoma model. Attacking the tumor stroma and/or the neovasculature may not only help to establish a local inflammatory response that benefits subsequent immune recognition in a vaccinal manner, but it may also enhance access to the tumor for otherwise impermeant drugs in difficult to treat cancers. Many solid tumors depend on stromal ECM and neovasculature for survival, and, therefore, EIIIB serves as an easily generalizable target that is not limited to a specific tumor type. In this study, we establish VHH-based CAR T cells as a versatile, modular system to target various compartments of the solid tumor microenvironment.

实体瘤对细胞外基质（ECM）和新生血管对营养素供应的依赖性为Car T细胞提供了另一个可能的靶点，因为肿瘤ECM和新形成的血管显示出健康成人中不常见的独特抗原（24，25）。作为在肿瘤微环境中靶向pd-l1可能被证明是有益的概念的延伸，我们使用识别EIIIB的VHH生成了car t细胞，EIIIB是一种纤维连接蛋白的剪接变体，在肿瘤ECM和新血管系统中都有强烈表达（24，26）。在B16黑色素瘤模型中，这些car t细胞也降

低了肿瘤的生长速度。攻击肿瘤间质和/或新血管系统不仅有助于建立局部炎症反应，有助于随后的免疫识别以疫苗的方式，但它也可能增强获得肿瘤的途径，因为其他不必要的药物难以治疗癌症。许多实体瘤依靠基质ECM和新血管系统生存，因此，EIIIB是一个容易概括的靶点，不局限于特定的肿瘤类型。在本研究中，我们建立了以VHH为基础的CAR T细胞作为一个多功能、模块化的系统，以针对实体肿瘤微环境的不同区域。

Results

VHH-Based CAR T Cells Expressed with Retention of Antigen Specificity. The VHH-based CAR T cells generated in this study follow the principal design of scFv-based CAR T cells, where the VHH replaces the scFv as the recognition module. For the construction of these CARs, we used VHHs specific for GFP [referred to as “enhancer” or “Enh” (15)], for PD-L1 (B3 or A12), and for the EIIIB splice variant of fibronectin (NJB2) (14, 27–29). For most experiments, we used 1B7, a VHH that recognizes a Toxoplasma gondii kinase, as a negative (nonspecific) control (16). The lentiviral vector backbone is derived from murine stem cell virus and encodes the CAR construct in addition to an internal ribosomal entry site (IRES)-driven green fluorescent protein (GFP) or mCherry cassette to gauge transduction efficiency (Fig. 1A). Before transduction, T cells obtained from spleen were activated with plate-bound anti-mouse CD28 and anti-mouse CD3 (Fig. 1B). By gating on those cells that were successfully transduced [GFP or mCherry-positive, typically 40 to 80% transduced (SI Appendix, Fig. S1)], we assessed CAR expression and functionality by binding of suitably labeled CAR ligands. Immunoblots with Enh CAR lysate developed with an anti-Enh serum show a polypeptide of ∼40 kDa, the expected size of the Enh CAR (Fig. 1C). The

Enh CAR, when transduced into T cells, retained the ability to bind GFP, as evident from a FACS-based assay (Fig. 1D). We likewise showed that the anti−PD-L1 CAR, based on the A12 VHH, recognized a recombinant PD-L1−Fc fusion, as detected by fluorescently labeled anti-mouse IgG (Fig. 1E). In all cases, binding of antigen to CAR T cells was blocked by inclusion of a molar excess of the corresponding free VHH as competitor, indicating specificity of ligand binding. We conclude that VHHs are readily displayed as CAR recognition modules with full retention of antigen-binding specificity.

结果

以VHH为基础的Car T细胞表达，保留抗原特异性。本研究中产生的基于VHH的CART细胞遵循基于SCFV的CART细胞的主要设计，其中VHH取代SCFV作为识别模块。对于这些CAR的构造，我们使用了gfp（称为“增强剂”或“enh”（15））、pd-l1（b3或a12）和纤维连接蛋白（njb2）的eiiib剪接变体（14，27–29）的特异性VHH。在大多数实验中，我们使用1B7作为阴性（非特异性）对照。慢病毒载体骨架来源于小鼠干细胞病毒，除了内部核糖体进入位点（IRES）驱动的绿色荧光蛋白（GFP）或McHerry盒外，还编码CAR结构，以测量转导效率（图1a）。转导前，用平板结合的抗鼠CD28和抗鼠CD3激活脾脏T细胞（图1b）。通过对成功转导的细胞进行门控[gfp或mcherry阳性，通常40%到80%转导（si-附录，图s1）]，我们通过结合适当标记的car配体来评估car的表达和功能。用抗-EnH血清开发的带有Enh的CAR裂解物的免疫印迹显示了40 kDa的多肽，EnH小车的预期尺寸（图1C）。当转化为T细胞时，enh-car保留了结合gfp的能力，从基于facs的分析中可以明显看出（图1d）。我们同样表明，基于A12 VHH的抗-PD-L1抗体识别了一种重组的PD-L1−FC融合，如荧光标记的抗小鼠IgG所检测到的（图1e）。在所有情况下，抗原与Car T细胞的结合都被相应的游离VHH的摩尔过量

作为竞争对手所阻断，表明配体结合的特异性。我们得出结论，VHH很容易显示为具有抗原结合特异性的car识别模块。

In Vitro Activity of CAR T Cells: Cytokine Production and Cytotoxicity. Having shown the binding specificity of VHH-based CARs, we next determined the functional properties of VHH-based CAR T cells. Upon incubation of GFP-specific CAR T cells with platebound GFP, we observed an increase in IL-2 and IFNγ production in the culture supernatants (Fig. 2 A and B). Even though GFP in solution is a monomer, the plate-bound configuration allows multivalent engagement and ensures activation. Cytotoxicity of the PD-L1−targeted A12 CAR T cells was assessed on B16 melanoma cells, which express PD-L1. The A12 CAR T cells killed the B16 melanoma in a dose-dependent manner (Fig. 2C). IFNγ production from the CAR T cells in response to exposure to the B16 melanoma likewise increased at higher E:T ratios (Fig. 2D). PD-L1 is overexpressed on a number of different tumor types. We showed that the A12 PD-L1−targeted CAR can elicit cytotoxicity against several different cancer cell lines that express PD-L1, including C3.43 (Fig. 2 E and F), an HPV16- transformed cell line, and MC38 (Fig. 2 G and H), a colon adenocarcinoma, suggesting applicability across a spectrum of cancers. Cytotoxicity and IFNγ secretion again occurred in a dose-dependent manner. Both cytotoxicity (Fig. 2I) and IFNγ production (Fig. 2J) were blocked by inclusion of the corresponding soluble blocking VHH (B3), thus occluding other possible docking sites on the B16 melanoma for the CAR T cells to engage. We therefore conclude that cytotoxicity of the CAR T cells was specific for the target ligand.

Car T细胞的体外活性：细胞因子的产生和细胞毒性。在证明了基于VHH的CAR的结合特异性后，我们接下来确定了基于VHH的CART细胞的功能特性。在用血小板结合的gfp培养gfp特异性car t细胞后，我们观察到培养上清液中IL-2和IFNγ的产生增加（图2 a和b）。尽管溶液中的gfp是单体，但板结合结构允许多价结合并确保活化。在表达pd-l1的B16黑色素瘤细胞上评估pd-l1靶向a12 car t细胞的细胞毒性。a12 car t细胞以剂量依赖性方式杀死了b16黑色素瘤（图2c）。在暴露于B16黑色素瘤后，Car T细胞产生的IFNγ在较高的E:T比率下同样增加（图2d）。PD-L1在许多不同的肿瘤类型上过度表达。我们发现，A12 pd-l1靶向car可引起对几种表达pd-l1的不同癌细胞株的细胞毒性，包括c3.43（图2 e和f）、一种hpv16转化的细胞株和一种结肠癌MC38（图2 g和h），这表明其适用于各种癌症。细胞毒性和干扰素γ分泌再次发生，呈剂量依赖性。细胞毒性（图2i）和IFNγ的产生（图2j）都被相应的可溶性阻断VHH（B3）所阻断，从而阻断了B16黑色素瘤上其他可能的对接位点，使Car T细胞结合。因此我们得出结论，CAR T细胞的细胞毒性对靶配体是特异的。

Anti−PD-L1 CAR T Cells Are Generated More Effectively in a PDL1−Deficient Background. The design of CARs that recognize antigens expressed differentially on tumors versus normal cells poses a complication if the antigen is also expressed endogenously on the very same T cells programmed to display those CARs. This is the case for PD-L1, a possibly attractive target of solid tumors but expressed also at low levels on antigen-experienced T cells. We observed constitutively elevated IFNγ production when PD-L1− specific CARs were introduced into wild-type (WT), PD-L1− proficient T cells (Fig. 3A). Follicular T helper cells engage the PD-1/PD-L1 axis for proper function in the germinal center reaction (23). Consequently, “self”-activation of PD-L1−specific CAR T cells before they experience their targets could be problematic. Indeed, in the course of development of A12 CAR T

cells, these cells showed enhanced expression of exhaustion markers such as PD1, TIM3, and LAG-3 (Fig. 3B), presumably due to chronic activation by PD-L1 engagement either in cis or in trans. Through introduction of PD-L1 CARs into PD-L1−deficient, activated T cells, such premature activation was avoided. PD-L1−/− anti−PDL1 CAR T cells also persisted better in vivo. PD-L1−/− A12 PD-L1 CAR T cells or WT A12 PD-L1 CAR T cells were introduced into RAG−/− mice bearing B16 tumors, using 1B7 CAR T cells as controls (Fig. 3C). After 14 d of culture, spleens, tumors, and tumor-draining lymph nodes were harvested and probed for the presence of GFP+ CAR T cells. We saw that the A12 CAR CD4 and, to a lesser degree, CD8 T cells generated in a PD-L1−/− background expanded more in spleen and lymph nodes than A12 CAR T cells obtained from WT mice (Fig. 3 D–F). We also saw increased infiltration of PD-L1−/− CD8+ CAR T cells in the tumor compared with WT A12 CAR T cells or 1B7 (nonspecific) CAR T cells (Fig. 3 E and F). We conclude that persistent antigen recognition in the course of CAR T cell generation compromises activity and persistence of WT A12 CAR T cells in vivo. Interestingly, upon injection of varying amounts of A12 PD-L1−/− CAR T cells into WT hosts, we did not notice significant changes in the level of endogenous WT T cells, but did notice a decrease in CD45+CD11b+ cells in the spleen (SI Appendix, Fig. S2). This suggests that the level of PD-L1 expression, as well as the number of CAR T cells introduced, may determine whether cell killing occurs, or whether these T cells become exhausted.

在缺乏pd l1的背景下，抗-pd-l1 car t细胞的产生更为有效。如果抗原也在计划显示这些细胞的非常相同的T细胞内表达，那么识别肿瘤与正常细胞上不同表达抗原的CARS的设计就会带来并发症。这就是PD-L1的情况，它可能是实体肿瘤的一个有吸引力的靶点，

但在抗原经历的T细胞中也表达低水平。我们观察到当pd-l1-特异性CAR引入野生型（wt）、pd-l1-熟练T细胞（图3a）时，干扰素γ产生的组成性增加。滤泡T辅助细胞与pd-1/pd-l1轴结合，在生发中心反应中发挥适当的功能（23）。因此，在PD-L1-特异性CART细胞遇到目标之前，“自我激活”可能存在问题。事实上，在a12-car t细胞的发育过程中，这些细胞显示了衰竭标记物（如pd1、tim3和lag-3）的增强表达（图3b），这可能是由于pd-l1在顺式或反式中的慢性激活。通过将pd-l1CAR引入pd-l1缺陷、激活的T细胞，避免了这种过早激活。pd-l1−/−抗-pd l1 car T细胞在体内也保持较好。将pd-l1−/−a12 pd-l1 car t细胞或wt a12 pd-l1 car t细胞引入到患有B16肿瘤的RAG−/−小鼠中，使用1b7 car t细胞作为对照（图3c）。培养14天后，收集脾脏、肿瘤和肿瘤引流淋巴结，并检查是否存在GFP+Car T细胞。我们发现，在pd-l1−/−背景下产生的a12-car CD4和CD8 T细胞在脾脏和淋巴结中比从wt小鼠获得的a12-car T细胞扩张更多（图3 d-f）。我们还发现与wt a12 car t细胞或1b7（非特异性）car t细胞相比，肿瘤中pd-l1−/−cd8+car t细胞的浸润增加（图3 e和f）。我们的结论是，在T细胞生成过程中持续的抗原识别会影响体内wt a12 T细胞的活性和持续性。有趣的是，在将不同数量的A12 PD-L1−/−Car T细胞注入wt宿主后，我们没有注意到内源性wt T细胞水平的显著变化，但注意到脾脏中CD45+CD11b+细胞的减少（SI附录，图S2）。这表明pd-l1的表达水平，以及引入的car t细胞的数量，可能决定是否发生细胞杀伤，或者这些t细胞是否耗尽。

In Vivo Application of Anti−PD-L1 CAR T Cells Slows Growth of Solid Tumors. Since PD-L1 is up-regulated on several cancer types, we determined whether A12 CAR treatment would affect growth of various tumor models known to overexpress PD-L1. The first model we tested was the highly aggressive B16 melanoma (Fig. 4A). C57BL/6 PD-L1−/− mice were inoculated with both WT B16 cells and B16 cells transfected to overexpress PD-L1 under the control of a CMV

promoter (SI Appendix, Fig. S3). PD-L1−/− anti PD-L1 CAR T cells were injected into tumor-bearing mice once a week, for a total of three injections (9 × 106 to 14 × 106 cells per injection), using 1B7 CAR T cells as negative controls. Transduction rates of both CAR T cells were around 40% (SI Appendix, Fig. S1). TA-99, an anti-TRP1 monoclonal antibody that recognizes an antigen highly expressed on (a subset of) melanomas (30), was used in combination with CAR T cell treatment to enhance immune infiltration and delay tumor growth to allow the CAR T cells sufficient time to exert an effect. This aggressive melanoma model more accurately recapitulates human disease compared with standard NOD scid gamma (NSG) models, as the tumors are syngeneic and develop in the presence of a fully intact immune system, but with an ineffective immune response directed against the tumor. Mice treated with the A12 CAR T cells showed a statistically significant decrease in tumor growth rate and an increase in survival in both the B16 WT tumor model (P < 0.0001) and the PD-L1 overexpressing B16 model (P = 0.02) (Fig. 4 B–G). These experiments not only provide a system for studying CAR T cells in a syngeneic

immunocompetent

host

but

also

avoid

immune-depleting

chemotherapy. We next tested A12 CAR T cell efficacy in the syngeneic MC38 model, in fully immunocompetent C57BL/6 mice (Fig. 4 H–J). Mice were inoculated with tumors and were left untreated or treated with either the A12 CAR T cells or the nonspecific 1B7 CAR T cells once a week, starting on day 5, for a total of three injections of 1 × 107 to 1.6 × 107 cells per injection. A12 CAR T cell treatment increased survival (P = 0.003), as well as decreasing tumor growth compared with either no treatment or untargeted treatment. Low levels of immunogenicity against the A12 CAR were seen in a few mice, but no visible side effects developed upon repeated administration. Immunogenicity did not adversely affect survival

(SI Appendix, Fig. S4). Compared with mice actively immunized with VHHs, the levels of immunogenicity upon repeated CAR T cell injections are much lower (27, 28). A PD-L1−targeted VHH CAR T cell thus provides a significant survival benefit in several different tumor models. Immune checkpoints such as PD-L1 may serve as viable targets for CAR T cell therapy.

体内应用抗-pd-l1 car t细胞可减缓实体瘤的生长。由于pd-l1在几种癌症类型上上调，我们确定a12-car治疗是否会影响已知过度表达pd-l1的各种肿瘤模型的生长。我们测试的第一个模型是高度侵袭性的B16黑色素瘤（图4a）。将c57bl/6 pd-l1−/−小鼠接种wt-b16细胞和b16细胞，在CMV启动子的控制下将其过度表达pd-l1（SI附录，图S3）。pd-l1−/−抗pd-l1 car t细胞每周注射一次到荷瘤小鼠体内，共注射三次（每次注射9×106到14×106细胞），使用1b7 car t细胞作为阴性对照。两种细胞的转导率都在40%左右（见附录，图S1）。Ta-99是一种识别黑色素瘤（30）高度表达抗原的抗Trp1单克隆抗体，与Car T细胞治疗结合使用，以增强免疫渗透和延迟肿瘤生长，使Car T细胞有足够的时间发挥作用。与标准nod-scid-gamma（nsg）模型相比，这种侵袭性黑色素瘤模型更准确地再现了人类疾病，因为肿瘤是同基因的，在完全完整的免疫系统存在下发展，但针对肿瘤的免疫反应无效。用a12 car t细胞治疗的小鼠在b16 wt肿瘤模型（p<0.0001）和pd-l1过度表达b16模型（p=0.02）中均显示肿瘤生长率显著降低，生存率显著提高（图4 b-g）。这些实验不仅为研究同基因免疫活性宿主中的car t细胞提供了一个系统，而且避免了免疫耗竭的化疗。接下来，我们在具有完全免疫活性的C57BL/6小鼠的同基因MC38模型中测试了A12 CAR T细胞的功效（图4 H-J）。给小鼠接种肿瘤，从第5天开始，每周对其进行一次未经治疗或用A12 car t细胞或非特异性1b7 car t细胞治疗，每次注射共3次，每次注射1×107至1.6×107细胞。与未治疗或未靶向治疗相比，a12 car t细胞治疗可提高生存率（p=0.003），并降低肿瘤生长。少数小鼠对A12抗体免疫原性低，但重复给药后无明显副作用。免疫原性并未对存活率产生不利影响（SI附录，

图S4）。与用VHH主动免疫的小鼠相比，反复注射Car T细胞的免疫原性水平要低得多（27，28）。因此，在几种不同的肿瘤模型中，pd-l1靶向的VHH Car T细胞具有显著的生存益处。免疫检测点，如pd-l1，可能是CAR T细胞治疗的可行靶点。

Exhaustion of CAR T Cells Due to Persistent Activation Is Overcome by PD-L1 Blockade in Culture. Chronic PD-L1 exposure in the course of generating A12 CAR T cells decreases their persistence and proliferation. We reasoned that this phenomenon could be prevented by blocking PD-L1 exposure during culture. To prevent chronic activation of the A12 CAR T cells in culture, WT anti−PD-L1 CAR T cells were generated in the continuous presence of VHH B3, a high-affinity anti−PD-L1 VHH that blocks A12 binding of PD-L1 (27, 28). Indeed, blocking PD-L1 exposure in the course of CAR T cell generation decreases expression of exhaustion markers such as LAG3, TIM3, and PD-1 (Fig. 5A). We generated CAR T cells in either the WT or the PD-L1−/−background cultured with VHH B3 PD-L1 to prevent activation. We then introduced these A12 CAR T cells into WT C57BL/6 mice bearing a B16 tumor. After 2 wk, we harvested the spleens to determine persistence of CAR T cells. CD4 and, to a lesser extent, CD8 A12 CAR T cells generated in the presence of a PD-L1–blocking VHH expand more effectively in vivo than those generated in its absence (Fig. 5B). However, PD-L1−/− CAR T cells still proliferate more effectively. We next asked if decreasing the exhaustion level of these CAR T cells in the course of their production would improve an antitumor response in vivo. Mice inoculated with B16 tumors were treated with PDL1−targeted CAR T cells generated in the WT background, but cultured in the presence of excess soluble anti−PD-L1 VHH to prevent chronic activation (Fig. 5C). Since the in vitro data showed inferiority, we did not test the PD-L1−targeted CARs

generated in the WT background without PD-L1 blocking. We observed a delay in B16 tumor growth in mice that received WT anti−PD-L1 CAR T cells generated in the presence of the PDL1–blocking VHH (P = 0.04) (Fig. 5 D and E), showing that the prevention of early activation in culture is a viable means of allowing a PD-L1−targeted CAR T cell to be deployed in a patient setting.

在培养过程中，pd-l1阻滞剂可以克服由于持续活化导致的car t细胞衰竭。在产生A12 car T细胞的过程中长期暴露于pd-l1会降低其持久性和增殖。我们的理由是，在培养过程中通过阻断pd-l1的暴露可以预防这种现象。为了防止培养中a12 car t细胞的慢性活化，在持续存在VHH B3的情况下产生wt抗-pd-l1 car t细胞，这种高亲和力的抗-pd-l1 VHH阻止pd-l1的a12结合（27，28）。事实上，阻断T细胞生成过程中的pd-l1暴露会降低诸如lag3、tim3和pd-1等衰竭标记物的表达（图5a）。我们在wt或pd-l1−/−背景中产生car t细胞，用VHH B3 pd-l1培养以防止活化。然后，我们将这些a12-car T细胞导入wt c57bl/6小鼠体内，小鼠有一个b16肿瘤。2周后，我们采集脾脏来测定T细胞的持久性。CD4和，在较小程度上，在存在pd-l1-阻断VHH的情况下产生的CD8 A12 car T细胞在体内比在其缺失情况下产生的细胞更有效地扩张（图5b）。然而，pd-l1−/−car T细胞仍能更有效地增殖。接下来，我们问，在这些CART细胞的生产过程中，减少耗尽水平是否会提高体内的抗肿瘤反应。用wt背景下产生的pd l1-靶向car t细胞处理接种了B16肿瘤的小鼠，但在过量可溶性抗-pd-l1 vhh存在下培养以防止慢性活化（图5c）。由于体外数据显示出低劣性，我们没有在没有pd-l1阻断的情况下测试wt背景下产生的pd-l1靶向cars。我们观察到在存在p d l1-阻断VHH（p=0.04）的情况下，接受wt抗-p d-l1 car t细胞的小鼠中的b16肿瘤生长延迟（图5 d和e），表明预防培养中的早期激活是允许将p d-l1-靶向car t细胞部署到患者环境中的可行方法。

Anti-EIIIB Fibronectin-Targeted CAR T Cells Slow B16 Melanoma Growth in

Vivo. EIIIB is an alternatively spliced domain of fibronectin strongly expressed in tumors and during angiogenesis, but not in most normal tissues (26). We targeted CAR T cells specifically to the tumor microenvironment (stromal ECM and neovasculature) through recognition of the fibronectin EIIIB+ splice variant. We used VHH NJB2, which targets EIIIB (29), to generate B2 CAR T cells, and transduction rates of the B2 CAR were around 80% (SI Appendix, Fig. S1). We determined display of the B2 CAR by flow cytometry, using recombinant EIIIB-GST as the ligand and probing with rabbit anti-GST and fluorescently labeled anti-rabbit (Fig. 6A). Coculture of B2 CAR T cells with aortic endothelial cell lines that either contain or lack the EIIIB domain (gift from R.O.H.) confirm their specificity and cytotoxicity in vitro (Fig. 6B). Immunohistochemistry (IHC) and PET in survival or delay in tumor growth when tumor-bearing mice lacking adaptive immunity were treated with the B2 CAR T cells, despite efficient expansion of the B2 CAR T cells (SI Appendix, Fig. S7), indicating that the CAR treatment synergizes with the endogenous adaptive immune system to show efficacy in these immunocompetent tumor models. To further test the degree of efficacy of B2 CAR T cells in tumors with lower expression levels of EIIIB, we also investigated the MC38 colon carcinoma model. From immunohistological examination of excised MC38 tumors, we observed only low levels of EIIIB expression compared with the levels on B16 tumors (SI Appendix, Fig. S8). Mice inoculated with MC38 tumors and treated with B2 CAR T cells showed minimal effects on survival or tumor growth (Fig. 6F). We therefore suggest that the poor efficacy of B2 CAR T cells in the MC38 model is likely due to this lower expression level of EIIIB and that, for the B2 CAR T cells to be effective, a minimum level of EIIIB expression is required. These results show that targeting CAR T cells selectively to tumor ECM and

neovasculature can be very effective in suppressing tumor growth. We conclude that we can apply VHHs to generate CAR T cells that are effective in vivo against targets in the tumor microenvironment in fully immunocompetent mice.

抗EIIIB纤维连接蛋白靶向的Car T细胞在体内减缓了黑色素瘤的生长。EIIIB是纤维连接蛋白的一个选择性剪接域，在肿瘤和血管生成过程中强烈表达，但在大多数正常组织中不表达（26）。我们通过识别纤维连接蛋白EIIIB+剪接变异体，将Car T细胞特异性定位于肿瘤微环境（基质ECM和新血管系统）。我们使用靶向EIIIB（29）的VHH NJB2产生b2-car T细胞，b2-car的转导率约为80%（参见附录，图S1）。我们通过流式细胞术测定b2-car的显示，以重组EIIIb-GST为配体，用兔抗GST和荧光标记的兔抗GST进行探测（图6a）。b2-car-t细胞与主动脉内皮细胞系共培养，其中含有或不含有eiiib结构域（来自R.O.H.的礼物），证实了它们在体外的特异性和细胞毒性（图6b）。免疫组化（IHC）和PET在缺乏适应性免疫的荷瘤小鼠存活或肿瘤生长延迟中的作用，尽管b2-car-t细胞有效率的扩增（si-appendix，图S7），表明car治疗与内源性适应性免疫系统协同，以显示这些免疫系统的疗效。免疫适应肿瘤模型。为了进一步验证b2-car-t细胞在低表达EIIIb的肿瘤中的作用程度，我们还研究了MC38结肠癌模型。通过对切除的MC38肿瘤的免疫组织学检查，我们发现与B16肿瘤相比，EIIIB表达水平较低（SI附录，图S8）。接种MC38肿瘤并用b2-car t细胞治疗的小鼠对生存或肿瘤生长的影响最小（图6f）。因此，我们认为，在MC38模型中，b2-car T细胞的低效性可能是由于EIIIb的低表达水平，并且，为了使b2-car T细胞有效，需要最低水平的EIIIb表达。这些结果表明，选择性靶向肿瘤ECM和新生血管对抑制肿瘤生长是非常有效的。我们的结论是，我们可以应用VHH来产生在体内对完全免疫功能小鼠肿瘤微环境中的靶点有效的car t细胞。

Treatment with Anti-EIIIB Fibronectin-Targeted CAR T Cells Leads to Tumor Immune Infiltration and Necrosis. To more closely analyze the mechanisms of B2

CAR treatment, we performed IHC on tumors excised while undergoing treatment. WT C57BL/6 mice were inoculated with B16 tumors, and mice were either treated with B2 CAR T cells or left untreated. At day 16, when there was a significant difference in tumor sizes between the treated and control group (Fig. 7 A and B), tumors were excised, fixed, and subjected to IHC. Tumor samples were then stained with secondary only (control) or for EIIIB, CD31, CD3, CD4, and CD8 to determine how ECM, vasculature, and immune cell populations were affected by the B2 CAR T cell treatment. The structure of the untreated tumors appeared healthy and intact, while the treated tumors showed clear signs of disruption. In the untreated samples, we saw expression of EIIIB in the tumor stroma and capsule, as well as around the vasculature, as indicated by its partial colocalization with CD31 (Fig. 7C). Expression of EIIIB in the tumor stroma appeared heterogeneous. In contrast, two of the three smaller treated tumors were highly necrotic, as indicated by the lack of healthy nuclear staining and disintegration of the matrix (Fig. 7D). Furthermore, these two treated samples showed decreased levels of CD31-positive vasculature compared with controls. Since B2 CAR T cells are targeted to EIIIB, which is expressed in tumor stroma and on neovasculature, the necrotic nature and lack of CD31 expression in the treated samples is perhaps to be expected. The third treated tumor was slightly larger (Fig. 7B) and was heterogeneous, showing a mixture of live, healthy tumor and necrotic, damaged tissue (Fig. 7E, Top). The healthy tumor regions expressed EIIIB and showed heavy T cell infiltration throughout the tissue, compared with untreated tumors. CD31 staining of this heterogeneous tumor indicated the presence of intact vasculature in the healthy sections with immune cell infiltration, while the necrotic regions displayed a lack of vasculature with less T cell infiltration (Fig. 7E, Bottom). Averaging across all

tumors, those treated with B2 CAR T cells had elevated levels of immune cells (Fig. 7F). The heterogeneous treated tumor showed many more infiltrating immune cells in those regions that were still alive (Fig. 7G). A reasonable interpretation is that the B2 CAR T cells infiltrate the tumors and possibly also recruit additional immune cells. These data further corroborate the ability of B2 CAR T cells to infiltrate and damage EIIIBexpressing tumors. Tumors rely on support and nutrients delivered by their stroma and vasculature, and, by compromising these interactions, the B2 CAR T cells markedly delay tumor growth.

抗EIIIB纤维连接蛋白靶向Car T细胞治疗可导致肿瘤免疫浸润和坏死。为了更深入地分析b2-car治疗的机制，我们对治疗过程中切除的肿瘤进行了IHC。wt c57bl/6小鼠接种了B16肿瘤，小鼠要么用b2-car t细胞治疗，要么不治疗。在第16天，当治疗组和对照组的肿瘤大小有显著差异时（图7a和b），肿瘤被切除、固定并接受IHC治疗。然后用二级纯（对照）或EIIIB、CD31、CD3、CD4和CD8对肿瘤样本进行染色，以确定b2-car-t细胞治疗对细胞外基质、血管系统和免疫细胞群的影响。未经治疗的肿瘤结构看起来健康完整，而治疗后的肿瘤显示出明显的破坏迹象。在未经治疗的样本中，我们发现肿瘤基质和包膜以及血管系统周围的EIIIB表达，如其与CD31的部分共定位所示（图7c）。EIIIB在肿瘤基质中的表达呈现异质性。相比之下，三个较小的治疗肿瘤中有两个是高度坏死的，这表明缺乏健康的核染色和基质的解体（图7d）。此外，与对照组相比，这两个处理过的样本显示CD31阳性血管系统的水平降低。由于b2-car-t细胞的靶向是在肿瘤基质和新生血管中表达的eiiib，因此治疗样本中的坏死性质和缺乏CD31表达可能是可以预料的。第三个治疗的肿瘤稍大（图7b），并且是异质的，显示了活的、健康的肿瘤和坏死的、受损的组织的混合物（图7e，顶部）。与未经治疗的肿瘤相比，健康的肿瘤区域表达EIIIB并在整个组织中显示出大量T细胞浸润。这种异质性肿瘤的CD31染色显示免疫细胞浸润的健康切片中存在完整的血管系统，而坏死区域显示缺乏血管系统，T细胞浸润较少（图7e，

底部）。在所有肿瘤中，b2-car-t细胞治疗的患者的免疫细胞水平均升高（图7f）。异种治疗的肿瘤显示在那些仍然存活的区域有更多的免疫细胞浸润（图7g）。一个合理的解释是，b2-car-t细胞浸润肿瘤，并可能招募更多的免疫细胞。这些数据进一步证实了b2-car-t细胞浸润和损伤eiiibexpressing肿瘤的能力。肿瘤依赖于基质和血管系统提供的支持和营养，并且，通过破坏这些相互作用，b2-car-t细胞明显延迟肿瘤生长。

Discussion

Although CAR T cells have shown success in treating several types of hematological cancers, their deployment will require further refinement for an attack on solid tumors. Limitations in biomarker availability, insufficient delivery of CAR T cells, and an increased immunosuppressive environment within the tumor may account for poor CAR T cell performance in the treatment of solid tumors (31). Physical barriers, such as a dense ECM that encapsulates the tumor, or properties of the vasculature that preclude adhesion and diapedesis of CAR T cells could likewise compromise their efficacy (31). Indeed, many solid tumors suppress immunity through expression of checkpoint proteins such as PD-L1, which engage corresponding inhibitory receptors on T cells (23). PD-L1 has not been exploited as a target for CAR T cells in vivo.

讨论

尽管Car T细胞在治疗几种血液学癌症方面已显示出成功，但它们的部署将需要进一步完善，以应对实体肿瘤的攻击。生物标记物可用性的限制、Car T细胞的输送不足以及肿瘤内免疫抑制环境的增加可能导致实体肿瘤治疗中Car T细胞表现不佳（31）。物理屏

障，如包裹肿瘤的致密ECM，或阻止Car T细胞粘附和滞育的血管系统特性，同样会损害其疗效（31）。事实上，许多实体肿瘤通过检测点蛋白（如pd-l1）的表达抑制免疫，pd-l1与T细胞上相应的抑制受体结合（23）。pd-l1在体内还没有被开发成CAR T细胞的靶点。

Establishing a more inflammatory local environment might be beneficial to overcoming

immune

suppression.

Monoclonal

antibodies

that

inhibit

development of the tumor vasculature by targeting VEGF, or cytokine therapies such as provision of IL2 or IL-12, can increase inflammation in the tumor for more effective immune control (27, 32–34). Cytokine release by activated CAR T cells might help establish the requisite local conditions, in addition to exerting their cytolytic effects. We therefore generated CAR T cells that either target the checkpoint protein PD-L1 or the tumor stromal ECM and neovasculature through EIIIB, a fibronectin splice variant strongly expressed in both murine and human tumors, both recognized by NJB2 VHH (25, 29). A major difficulty in developing CAR T cells for solid tumor treatment is the lack of targetable antigens. Most antigens proposed as CAR T cell targets to treat solid tumors are exclusive to a specific cancer type, and limited information on cancer-specific antigens for the vast majority of solid tumors puts many tumors out of reach for CAR T cell therapy (35). By targeting markers in the tumor microenvironment that are expressed in a variety of tumors, the CAR T cells described here show versatility for several different tumor models. They have the potential to target other cancers that lack identified tumor-specific antigens. PD-L1 is overexpressed on a majority of tumors and on immune cells within the tumor microenvironment (36). EIIIB is expressed in the neovasculature and tumor stroma of a range of tumor subtypes (25). The EIIIB-targeted VHH has already been tested against a panel of multiorgan human

tissue metastasis biopsies and reacts with a diverse set of tumor samples, further demonstrating the possible broad applicability of the B2 CAR T cells (29). We optimized the production of VHH-based CAR T cells and verified their function in vitro and in vivo by direct ligandbinding assays, cytotoxicity, cytokine production, and inhibition of tumor growth. VHH-based CAR T cells that recognize PD-L1 show ligand-specific cytotoxicity and are effective in highly aggressive, syngeneic tumor models in immunocompetent mice without prior immunodepletion. As long as the immune system contributes to eradication of solid tumors, as in the case of melanoma, lymphodepletion may have significant deleterious effects. We suggest that the mode of action for these PD-L1 targeted CAR T cells is at least twofold. First, anti−PD-L1 CAR T cells exert direct cytotoxicity and produce cytokines. Second, binding of a CAR to the relevant checkpoint molecules should block their interaction with natural ligands on host T cells, resulting in less immune suppression and exhaustion. In vitro, PD-L1−targeted CAR T cells show cytotoxicity against several types of solid tumors, including B16 melanoma, MC38 colon adenocarcinoma, and C3.43 HPV-transformed cell lines. In vivo, PD-L1−targeted CAR T cells significantly inhibit growth of B16 and MC38 tumors and provide a survival benefit.

建立更具炎性的局部环境可能有助于克服免疫抑制。通过靶向血管内皮生长因子或细胞因子疗法（如提供IL2或IL-12）抑制肿瘤血管系统发育的单克隆抗体可增加肿瘤炎症，从而实现更有效的免疫控制（27，32–34）。活化的car t细胞释放细胞因子除了发挥其细胞溶解作用外，还可能有助于建立必要的局部条件。因此，我们通过EIIIB产生了靶向检测点蛋白pd-l1或肿瘤间质ECM和新血管系统的car t细胞，这是一种在小鼠和人类肿瘤中都强烈表达的纤连蛋白剪接变体，这两种细胞都被NJB2-VHH（25，29）识别。发展用

于实体肿瘤治疗的car t细胞的一个主要困难是缺乏靶向抗原。大多数被提议作为治疗实体肿瘤的CAR T细胞靶点的抗原仅限于特定的癌症类型，而对于绝大多数实体肿瘤而言，关于癌症特异性抗原的有限信息使许多肿瘤无法进行CAR T细胞治疗（35）。通过在肿瘤微环境中定位各种肿瘤中表达的标记物，本文所描述的Car T细胞显示了几种不同肿瘤模型的多功能性。它们有可能针对其他缺乏肿瘤特异性抗原的癌症。PD-L1在大多数肿瘤和肿瘤微环境中的免疫细胞上过表达（36）。EIIIB在一系列肿瘤亚型（25）的新生血管和肿瘤基质中表达。EIIIB靶向的VHH已经在一组多器官人体组织转移活检中进行了测试，并与一组不同的肿瘤样本发生反应，进一步证明了B2 car t细胞（29）可能具有广泛的适用性。我们通过直接配体结合试验、细胞毒性、细胞因子产生和抑制肿瘤生长，优化了VHH-car T细胞的产生，并在体外和体内验证了其功能。识别pd-l1的基于VHH的Car T细胞表现出配体特异性细胞毒性，在免疫活性小鼠的高侵袭性同基因肿瘤模型中有效，而无免疫耗竭。只要免疫系统有助于根除实体瘤，就像黑色素瘤一样，淋巴耗竭可能具有显著的有害作用。我们建议这些pd-l1靶向的car t细胞的作用模式至少是两倍。首先，抗-pd-l1 CAR T细胞具有直接的细胞毒性并产生细胞因子。第二，一辆CAR与相关的检查点分子的结合应该阻止它们与宿主T细胞上的天然配体的相互作用，从而减少免疫抑制和疲劳。在体外，pd-l1靶向的car t细胞对几种类型的实体瘤具有细胞毒性，包括B16黑色素瘤、MC38结肠癌和C3.43 hpv转化的细胞系。在体内，pd-l1靶向的car t细胞显著抑制了b16和mc38肿瘤的生长，并提供了生存益处。

The production of anti−PD-L1 CAR T cells is complicated by the fact that WT T cells express low, endogenous levels of PDL1. Anti−PD-L1 CAR T cells generated in the WT background therefore constantly experience low levels of antigen exposure. This leads to some degree of T cell exhaustion and impairs function, in vivo persistence, and proliferation of the CAR T cells. This phenomenon is not unique to the PD-L1 target, as several desirable tumor antigens are also expressed

at low levels elsewhere in the tumor, because, with the exception of neoantigens, very few truly tumor-specific antigens exist. We found two ways to overcome this hurdle. First, mice treated with anti− PD-L1 CAR T cells generated in a PD-L1−deficient background showed a delay in tumor growth, indicating that these VHH-based CAR T cells are indeed effective in tumor treatment. Second, by generating anti−PD-L1 CAR T cells in the continuous presence of a saturating dose of an anti−PD-L1 VHH in solution, engagement of the PD-1/PD-L1 axis is blocked, and the resulting CAR T cells retain efficacy in vivo. Genetic ablation of PD-L1 using CRISPR-Cas9 in the course of CAR generation would likewise be possible, but involves genetic modifications in addition to provision of the CAR construct (37). We therefore preferred provision of the CAR ectodomain in soluble form in the course of generating anti−PD-L1 CAR T cells. In our experiments, we saw no obvious untoward effects upon transfer of these CAR T cells at our injection levels. We noticed a decrease in CD11b+ cells, which were highly PD-L1−positive, but did not see significant changes in other immune populations. Generation of the PD-L1−targeted CAR T cells in a WT background did not result in fratricide, possibly due to sequestering of the PD-L1 ligand by PD1 on the T cell surface in cis, as reported for antigen presenting cells (APCs) (38), or an insufficient level of PD-L1 expression to induce killing.

抗-pd-l1 car t细胞的产生由于wt t细胞表达低的内源性pd l1而变得复杂。因此，在wt背景下产生的抗-pd-l1 car T细胞经常经历低水平的抗原暴露。这会导致某种程度的T细胞衰竭，并损害功能、体内持久性和Car T细胞的增殖。这种现象不是PD-L1靶的独特现象，因为除了新抗原外，肿瘤其他部位的一些理想肿瘤抗原也表达的很低，因为真正存在的肿瘤特异性抗原很少。我们找到了两种克服这一障碍的方法。首先，在pd-l1缺乏

的背景下用抗-pd-l1 car t细胞治疗的小鼠显示肿瘤生长延迟，这表明这些基于VHH的car t细胞在肿瘤治疗中确实有效。第二，通过在溶液中持续存在抗-pd-l1 VHH饱和剂量的情况下产生抗-pd-l1 car T细胞，阻断pd-1/pd-l1轴的接合，并且由此产生的car T细胞在体内保持有效性。在CAR制造过程中使用CRISPR-CAS9对pd-l1进行基因消融也是可能的，但除了提供CAR构造外，还涉及基因修饰（37）。因此，在产生抗-pd-l1 Car T细胞的过程中，我们更倾向于以可溶性形式提供Car胞外域。在我们的实验中，我们没有看到在我们的注射水平上对这些CART细胞的转移有明显的不良影响。我们注意到CD11b+细胞减少，这是高度pd-l1-阳性，但没有看到其他免疫人群的显著变化。在wt背景下产生pd-l1靶向的car t细胞不会导致自相残杀，可能是由于pd1将pd-l1配体固定在顺式细胞的t细胞表面，如抗原呈递细胞（apcs）（38）报告的，或pd-l1表达水平不足以诱导杀伤。

Targeting the tumor ECM or neovasculature in the tumor microenvironment rather than the tumor directly may serve as another method to target multiple tumor types. Since most solid tumors require angiogenesis to provide nutrients for survival, targeting stromal and neoangiogenic markers may be a viable strategy (39). Indeed, an EIIIB+ fibronectin CAR (B2 CAR) T cell targeted to tumor ECM and the neovasculature inhibited growth of the aggressive B16 melanoma in an immunocompetent mouse. B16 tumors are strongly positive for EIIIB as assessed by IHC. B2 CAR T cell-treated B16 tumors are largely necrotic and show vascular and stromal damage, delaying tumor growth, as fewer nutrients can be delivered to support tumor growth. Treated tumor tissue that is not already necrotic shows immune cell infiltration, suggesting that B2 CAR T cells and possibly other endogenous immune cells localize to damaged tumor ECM and vasculature. In contrast, the MC38 tumor, which showed less expression of the EIIIB fibronectin

splice variant, failed to respond to treatment with anti-EIIIB CAR T cells. Even though solid tumors may share a need for ECM and angiogenesis, not all tumors display the FN EIIIB variant equally. It may be possible to identify other vascular and stromal markers that might serve a similar purpose. These B2 CAR T cell models further highlight the importance of using syngeneic animal models for CAR T cell treatment. When RAG−/− mice inoculated with B16 were treated with B2 CAR T cells, the survival benefit was lost, highlighting the importance of the endogenous immune system in synergizing with CAR treatment. Unlike the B2 CAR T cell treatment, when the A12 CAR T cell treatment was tested in RAG−/− mice, we noticed a survival benefit. As PD-L1 is expressed by the actual tumor cells, unlike EIIIB, a survival benefit would be expected. However, with the EIIIBtargeted CAR T cell treatment, it may be possible that compromising the matrix allows for greater immune infiltration and buildup of an endogenous immune response to antigens directly on the tumor itself, explaining why treatment was effective in immunocompetent mice but not in immunodeficient mice.

以肿瘤微环境中的肿瘤细胞外基质或新生血管为靶点，而不是直接以肿瘤为靶点，可以作为另一种针对多种肿瘤类型的方法。由于大多数实体肿瘤需要血管生成来为生存提供营养，靶向基质和新血管生成标记物可能是一种可行的策略（39）。事实上，以肿瘤ECM和新血管系统为靶点的EIIIB+纤维连接蛋白-Car（b2-Car）T细胞抑制免疫功能小鼠侵袭性黑色素瘤B16的生长。根据IHC的评估，B16肿瘤对EIIIB呈强阳性。b2-car-t细胞治疗的b16肿瘤大部分坏死，显示血管和基质损伤，延迟肿瘤生长，因为可以提供较少的营养以支持肿瘤生长。尚未坏死的治疗肿瘤组织显示免疫细胞浸润，提示b2-car-t细胞和其他可能的内源性免疫细胞定位于受损的肿瘤ECM和血管系统。相比之下，MC38肿瘤显示出较少的EIIIB纤维连接蛋白剪接变异体的表达，对抗EIIIB Car T细胞的治疗没有反应。

尽管实体瘤可能共享一个ECM和血管生成的需要，并不是所有的肿瘤显示FN-EIIIB的变异相同。可能有可能识别其他血管和基质标记物，这些标记物可能有类似的用途。这些b2-car t细胞模型进一步强调了使用同基因动物模型治疗car t细胞的重要性。当用b2-car t细胞处理接种了b16的RAG−/−小鼠时，存活率降低，这突出了内源性免疫系统与car治疗协同作用的重要性。与b2 car t细胞治疗不同，当a12 car t细胞治疗在RAG−/−小鼠中进行试验时，我们注意到了生存益处。由于pd-l1是由实际的肿瘤细胞表达的，与eiiib不同，预期会有生存益处。然而，通过靶向Car T细胞治疗，破坏基质可能允许更大的免疫渗透，并直接在肿瘤本身上建立对抗原的内源性免疫反应，这解释了为什么治疗对免疫活性小鼠有效，而对免疫缺陷小鼠无效。

Targeting the tumor neovasculature and tumor stroma with EIIIBtargeted CAR T cells may not only compromise the blood supply of the tumor, it might also serve as a means for improving tumor accessibility for small-molecule drugs and other therapies that can be used in combination with the CAR T cells, even if only transiently. Much like therapies that combine different checkpoint-blocking antibodies, the most likely route forward for solid tumors lies in combinations of CAR T cells with antibodies, radiation, or small-molecule drugs. From our experiments with both the PD-L1–targeted and EIIIB-targeted CAR T cells, we conclude that the VHHbased CAR approach is highly modular and broadly applicable to various tumors. Once a VHH of the appropriate specificity has been identified, it can be slotted into the CAR backbone for expression without the need for modification and optimization of linkers that connect VH and VL, which are an integral part of scFv-based CAR T cells. A platform for producing VHH-based CAR T cells expands the range of syngeneic tumors targetable by CAR T cells in a fully immunocompetent murine model. VHHs are appealing as antigen recognition

domains for CAR T cells, as they are easily expressible and have no obvious stability concerns (9, 11, 40–42).

以肿瘤新血管和肿瘤基质为靶向的靶向car t细胞不仅可能损害肿瘤的血液供应，而且可能是改善小分子药物和其他可与car t细胞结合使用的治疗方法的一种手段，即使只是短暂的。就像结合不同的检查点阻断抗体的治疗方法一样，实体肿瘤最有可能的前进路线是将Car T细胞与抗体、辐射或小分子药物结合在一起。通过我们对pd-l1靶向和eiiib靶向car t细胞的实验，我们得出结论：vhHBased car方法是高度模块化的，广泛适用于各种肿瘤。一旦识别出具有适当特异性的VHH，就可以将其插入到CAR主干中进行表达，而无需修改和优化连接VH和VL的链接器，后者是基于SCFV的CAR T细胞的一个组成部分。在一个完全免疫的小鼠模型中，一个产生基于VHH的car t细胞的平台扩大了car t细胞可靶向的同基因肿瘤的范围。VHH作为T细胞的抗原识别域很有吸引力，因为它们很容易表达并且不存在明显的稳定性问题（9，11，40–42）。

Immunodeficient mouse models are still largely the most commonly used models in CAR T cell research (43–45). They are beneficial in that human tumor models and CAR T cells can be studied, but also suffer from a number of drawbacks. Without the presence of intact innate and adaptive immunity, these animal models do not accurately depict the potential of immune suppression that may occur in the clinic. The use of immunocompetent mice as a tumor model has the added benefit of endogenous immunity and more accurately depicts clinical effects and recapitulates the degree of efficacy. Development of therapies that do not require immune depletion would seem further desirable, as endogenous antitumor immunity plays a large role in tumor surveillance (46). Compared with xenograft models, immunocompetent models also allow for better assessment of

the safety profile of treatment. The results from these models demonstrate feasibility and efficacy of CAR T cells that target the tumor microenvironment against aggressive solid tumors in a fully immunocompetent system. Our models show generalizability across multiple tumor types. Future efforts should be directed at incorporation of combination therapies, including checkpoint blockade and cytokine therapies to further improve treatment of solid tumors.

免疫缺陷小鼠模型仍然是CART细胞研究中最常用的模型（43-45）。它们有利于人体肿瘤模型和CART细胞的研究，但也有一些缺点。如果没有完整的先天免疫和适应性免疫，这些动物模型不能准确描述可能在临床上发生的免疫抑制的可能性。使用免疫活性小鼠作为肿瘤模型具有内源性免疫的额外好处，更准确地描述了临床效果并概括了疗效的程度。由于内源性抗肿瘤免疫在肿瘤监测中起着重要作用（46），因此开发不需要免疫耗竭的疗法似乎更为可取。与异种移植模型相比，免疫活性模型还可以更好地评估治疗的安全性。这些模型的结果表明，在完全免疫系统中，以肿瘤微环境为靶点的CAR T细胞对抗侵袭性实体肿瘤的可行性和有效性。我们的模型显示了多种肿瘤类型的普遍性。今后的工作应集中在联合治疗上，包括检查点阻断和细胞因子治疗，以进一步改善实体肿瘤的治疗。

Materials and Methods CAR T cells were generated through retroviral infection of primary murine T cells. In vitro assays were performed using Cell Titer Glo (Promega) and IFNγ and IL-2 ELISAs (BD). All animal procedures performed were in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of Boston Children’s Hospital (IACUC Protocol 16-12- 3328). A detailed description of the materials and methods used in this study is provided in SI Appendix, Supplementary Materials and Methods.

材料和方法通过原代小鼠T细胞的逆转录病毒感染产生细胞。采用细胞滴度GLO（promega）、IFNγ和IL-2 Elisas（bd）进行体外检测。所有动物程序均按照机构指南进行，并经波士顿儿童医院机构动物护理和使用委员会（IACUC协议16-12-3328）批准。本研究所用材料和方法的详细说明见SI附录“补充材料和方法”。

ACKNOWLEDGMENTS. We thank the H. Lodish laboratory for providing the XZ vector for CAR transduction. We thank M. Maus for helpful discussion in designing the CAR constructs. We thank D. Wittrup for providing the TA99 antibody. We thank S. Alamo for providing recombinant PD-L1−Fc. We thank S. Kolifrath for assistance in animal models. We thank the Koch histology core facility for assistance in immunohistological staining. This work was supported by the Lustgarten Foundation Grant 80939. Y.J.X. is supported by the National Science Foundation Graduate Research Fellowship. M.D. was supported by the National Institutes of Health Mentored Clinical Scientist Development Award 1K08DK114563-01 and the American Gastroenterological Association Research Scholars Award. N.J. was supported by the Mazumdar-Shaw International Oncology Fellowship. R.O.H. is supported by Howard Hughes Medical Institute Department of Defense (HHMI DOD) Innovator Award W81XWH-14-1-0240 and, in part, by Core Grant National Institutes of Health/National Cancer Institute (NIH/NCI) P30-CA14051 to the Koch Institute.

致谢。我们感谢H.Lodish实验室为CAR转导提供了XZ载体。我们感谢M.Maus在设计CAR结构方面的帮助性讨论。感谢D.Wittrup提供TA99抗体。感谢S.Alamo提供重组pd-l1−fc。感谢S.Kolifrath为动物模型提供帮助。我们感谢科赫组织学中心协助免疫组织染色。这项工作得到了LuestGARTN基金会80939的支持。Y.J.X.由国家科学基金

会研究生奖学金资助。医学博士由美国国立卫生研究院指导临床科学家发展奖1K08DK114563-01和美国胃肠学会研究学者奖资助。N.J.得到了马祖姆达肖国际肿瘤研究所的支持。R.O.H.得到了霍华德休斯医学研究所国防部（HHMI DoD）创新者奖W81XWH-14-1-0240的支持，部分还得到了国家卫生研究所/国家癌症研究所（NIH/NCI）P30-CA14051的核心拨款。

Fig. 1. VHH-based CAR T cells expressed with retention of antigen specificity. (A) Retroviral construct of VHH-based CAR T cells and their introduction into mouse T cells. (B) Production process for generation of CAR T cells. (C) Immunoblot on T cells transduced with Enh CAR construct. Lysates from transduced and untransduced T cells were blotted against using antiEnh serum generated from immunization of mice with the Enh VHH. Polypeptides corresponding to the Enh CAR and soluble Enh were seen. (D) Schematic of assay to test for Enh CAR display. T cells were transduced with Enh CAR and probed for

binding to GFP by flow cytometry. Nonspecific binding was measured by incubation with an irrelevant protein, TIM3−Fc fusion, probed for with an anti-mouse IgG conjugated to APC. (E) T cells were transduced with A12 CAR targeted to PD-L1. Successful display is probed by binding to recombinant PD-L1−Fc fusion and detected by an antimouse IgG conjugated to APC.

图1。以VHH为基础的Car T细胞表达，保留抗原特异性。（a）逆转录病毒构建基于VHH的CAR T细胞并将其导入小鼠T细胞。（b）生产汽车t电池的生产工艺。（c）用enh-car结构转导的T细胞免疫印迹。用EnH-VHH免疫小鼠产生的抗EnEH血清，对转导的和未转导的T细胞裂解物进行了印迹。发现与enh-car和可溶性enh相对应的多肽。（d）用于测试Enh汽车显示器的分析示意图。用Enh-Car转导T细胞，流式细胞仪检测T细胞与gfp的结合。非特异性结合是通过与一种无关的蛋白（tim3-fc融合）孵育来测量的，用一种与apc结合的抗鼠IgG进行探索。（e）以pd-l1为靶点，用A12-car转导T细胞。通过结合重组pd-l1−fc融合来探索成功的显示，并通过与apc结合的抗鼠IgG检测。

Fig. 2. In vitro activity of CAR T cells: cytokine production and cytotoxicity. T

cells were transduced with Enh CAR. (A) IL-2 and (B) IFNγ levels in the supernatant of CAR T cells cultured for 24 h with GFP or an irrelevant protein (TIM3−Fc). (C–J) T cells were transduced with A12 CAR targeted to PD-L1. (C and D) A12 CAR T cells recognized and killed B16 tumors. Coculture of anti− PD-L1 A12 CAR and a nonspecific control 1B7, recognizing a T. gondii calcium-dependent protein kinase, with B16 cells. Cells were cultured for 48 h at various effector:target (E:T) ratios. (C) A Cell Titer Glo assay was performed to measure cytotoxicity. (D) Supernatants were collected and IFNγ levels were measured. (E and F) A12 PD-L1−targeted cells were also effective in killing C3.43 HPV-transformed cancer cell lines. C3.43 cells were cultured with A12 CAR T cells at various E:T ratios. (E) C3.43 killing was measured by Cell Titer Glo, and (F) CAR activation was measured by IFNγ secretion. (G and H) A12 CAR T cells were cytotoxic against MC38 colon adenocarcinoma cells. A12 CAR T cells were cocultured with MC38 cells at various E:T ratios, and (G) MC38 killing and (H) A12 CAR T cell activation and cytokine secretion were measured. (I and J) Blocking experiments were performed using the B16 coculture setup. Cytotoxicity assay mixtures were incubated with varying concentrations soluble A12 VHH, B3 VHH, or an irrelevant 96G3M VHH (14). B3 binds PD-L1 with higher affinity than does A12. Higher levels of target antigen blockade lead to (I) better B16 survival and (J) less IFNγ secretion, indicating specificity. ****P ≤ 0.0001

图2。汽车T细胞的体外活性：细胞因子的产生和细胞毒性。用Enh-Car转导T细胞。（a）用gfp或无关蛋白（tim3-fc）培养24小时的car t细胞上清液中的IL-2和（b）ifnγ水平。（c-j）T细胞被靶向pd-l1的a12-car转导。（c和d）A12 car T细胞识别并杀死了B16个肿瘤。抗-pd-l1 a12-car和非特异性对照1b7的共培养，识别一种t.gondii

钙依赖性蛋白激酶，与B16细胞。细胞以不同的效应器：靶（E:T）比率培养48小时。（c）进行细胞滴度GLO测定以测量细胞毒性。（d）收集上清液，测量IFNγ水平。（e和f）a12 pd-l1-靶向细胞也能有效杀死C3.43 hpv转化的癌细胞株。用不同E:T比的A12-Car T细胞培养C3.43细胞。（e）用细胞滴度Glo测定C3.43的杀灭率，用干扰素γ分泌测定（f）car的激活率。（g和h）A12 car T细胞对MC38结肠癌细胞具有细胞毒性。在不同的E:T比率下，将a12-car t细胞与mc38细胞共培养，并测定（g）mc38杀伤和（h）a12-car t细胞活化和细胞因子分泌。（i和j）采用B16共培养装置进行阻断实验。用不同浓度的可溶性A12 VHH、B3 VHH或不相关的96G3M VHH（14）培养细胞毒性分析混合物。B3结合pd-l1的亲和力高于a12。较高水平的靶抗原阻断导致（i）较好的B16存活率和（j）较低的干扰素γ分泌，表明特异性。****P≤0.0001

Fig. 3. Anti−PD-L1 CAR T cells are generated more effectively in a PD-L1− deficient background. (A) WT T cells were transduced with the A12 CAR and cultured with B16 cells for 48 h. Supernatants from the B16 coculture experiments were probed for levels of IFNγ. (B) A12 PD-L1−targeted CAR T cells were generated in WT T cells and PD-L1 KO T cells. A12 CAR T cells generated in WT T cells showed increased levels of PD1, TIM3, and LAG3 expression. (C) RAG−/− mice were injected with B16 tumors s.c., and, 2 d later, A12 CAR T cells generated in WT T cells and PD-L1KO T cells were adoptively transferred. On day 15, tumors, spleens, and lymph nodes were harvested to determine the relative numbers of persisting CAR T cells. (D) Splenocytes were analyzed for the presence of GFP-labeled A12 CAR T cells. More CD4 and CD8 CAR T cells made in the PD-L1 KO background persisted. (E) Greater levels of CD4 CAR T cells made in the PD-L1 KO background were found in the spleen and draining lymph node (for 1B7 PD-L1−/− vs. A12 PDL1−/−: spleen CD4, P = 0.0014; spleen CD8, P = 0.0023; LN CD4, P < 0.0001; LN CD8, P = 0.0757; tumor CD4, P = 0.0238; tumor CD8, P = 0.0162; for A12 PDL1−/− vs. A12 WT: LN CD4, P = 0.0007). (F) More CD8 CAR T cells made in the PD-L1 KO background were present in the spleen, draining lymph nodes, and

tumor. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

图3。抗-pd-l1的car t细胞在pd-l1缺乏的背景下更有效地产生。（a）将wt t细胞用A12载体转导，用B16细胞培养48小时，同时检测b16共培养实验的上清液中IFNγ的水平。（b）在wt t细胞和pd-l1 ko t细胞中产生A12 pd-l1靶向car t细胞。wt t细胞中产生的a12 car t细胞显示pd1、tim3和lag3表达水平增加。（c）向RAG−/−小鼠注射B16肿瘤s.c.，2 d后，采用转移wt T细胞和pd-l1ko T细胞中产生的A12 car T细胞。第15天，收集肿瘤、脾脏和淋巴结，以确定持续存在的car t细胞的相对数量。（d）分析脾细胞是否存在GFP标记的A12 car T细胞。在pd-l1 ko背景下，更多的cd4和cd8 car T细胞持续存在。（e）在脾脏和引流淋巴结（1b7 pd-l1−/−vs.A12 pd l1−/−：脾脏cd4，p=0.0014；脾脏cd8，p=0.0023；ln cd4，p<0.0001；ln cd8，p=0.0757；肿瘤cd4，p=0.0238；肿瘤cd8，p=0.0162；A12 pd l1−/−vs.A12 wt:ln cd4，p=0.0007）中发现较高水平的cd4 car t细胞。（f）在pd-l1 ko背景下，脾脏、引流淋巴结和肿瘤中出现更多的CD8-car T细胞。*p≤0.05，**p≤0.01，***p≤0.001，***p≤0.0001。

Fig. 4. In vivo application of anti−PD-L1 CAR T cells slows growth of solid tumors. (A) PD-L1 KO mice were inoculated with B16 tumor cells. On days 2, 7, and 14, mice were treated with A12 CAR T cells (n = 10) or 1B7-irrelevant CAR T cells (n = 5) or left untreated (n = 5). All mice were given an anti-TRP1 antibody, TA99, in combination with CAR T cell treatment. (B) Kaplan−Meier curves showing survival of each treatment condition (P < 0.0001, Mantel− Cox log-rank test). (C) The average tumor area with SEM and (D) individual tumor area of each mouse was measured. Treatment with the A12 CAR T cells delayed tumor growth (none/A12 P = 0.0296, 1B7/A12 P = 0.04). (E) PD-L1 KO mice were inoculated with B16 tumor cells engineered to express high levels of PD-L1 under the control of a CMV promoter (n = 5). (F) Kaplan− Meier curve showing survival of each group (P =

0.0233, Mantel−Cox log rank). Mice treated with A12 CAR T cells showed improved survival. (G) Average tumor area (none/A12 P = 0.0029, 1B7/A12 P = 0.0422, unpaired t test with Bonferroni correction) and individual tumor area for each group were measured. SEM is shown. (H) PD-L1 KO mice were inoculated with MC38 colon adenocarcinoma. Mice were either left untreated (n = 5), treated with irrelevant CAR T cells (n = 5), or treated with PD-L1−targeted CAR T cells (n = 8). (I) Survival was measured and plotted on a Kaplan−Meier curve, showing that A12 CAR treatment improved survival (P = 0.003). (J) The tumor area average for each group was monitored (none/A12 P = 0.003, 1B7/ A12 P = 0.009, unpaired t test with Bonferroni correction)

图4。体内应用抗-pd-l1 car t细胞可减缓实体瘤的生长。（a）将pd-l1 ko小鼠接种B16肿瘤细胞。第2天、第7天和第14天，小鼠接受A12 car t细胞（n=10）或1b7无关car t细胞（n=5）治疗或不治疗（n=5）。所有小鼠均给予抗trp1抗体ta99，并结合car t细胞治疗。（b）Kaplan−Meier曲线显示每个治疗条件的生存率（p<0.0001，Mantel−Cox对数秩检验）。（c）用扫描电镜和（d）测量每只小鼠的平均肿瘤面积。用A12 Car T细胞治疗延迟肿瘤生长（无/A12 p=0.0296，1B7/A12 p=0.04）。（e）在CMV启动子的控制下（n=5），用设计用于表达高水平PD-L1的B16肿瘤细胞接种PD-L1 ko小鼠。（f）显示各组生存率的kaplan−meier曲线（p=0.0233，mantel−cox对数秩）。用A12 car t细胞治疗的小鼠存活率提高。（g）测量各组的平均肿瘤面积（none/a12 p=0.0029，1b7/a12 p=0.0422，未配对t检验和Bonferroni校正）和单个肿瘤面积。SEM显示。（h）PD-L1-KO小鼠接种MC38结肠癌。小鼠要么未经治疗（n=5），用不相关的car t细胞治疗（n=5），要么用pd-l1-靶向car t细胞治疗（n=8）。（i）测量存活率并绘制卡普兰-迈耶曲线，显示a12-car治疗可改善存活率（p=0.003）。（j）监测各组的肿瘤面积平均值（无/A12 p=0.003，1B7/A12 p=0.009，未配对t检验，用Bonferroni校正）。

Fig. 5. Exhaustion of CAR T cells due to persistent activation overcome by PD-L1 blockade in culture. (A) Chronic antigen exposure and exhaustion of A12 CAR T cells made in a WT background can be blocked by incubation in the course of culture with soluble anti−PD-L1 VHH to mask endogenous PDL1. A12 CAR T cells were generated in the presence of soluble B3 VHH, which binds PD-L1 with higher affinity than A12. Expression of common exhaustion markers was analyzed using flow cytometry. (B) WT mice were inoculated with B16 tumors, and A12 CAR T cells made in a WT background with and without inclusion of soluble B3 were introduced and compared with A12 CAR T cells made in the PD-L1 KO background. A12 CAR T cells made in a WT background in the presence of soluble B3 showed better persistence than A12 CAR T cells made without inclusion of B3 (CD4: A12 WT vs. A12 WT+ B3, P = 0.0283; CD8: A12 WT vs. A12 WT+ B3, P = 0.1346). (C)

Mice were inoculated with B16 overexpressing PD-L1 tumors on day 0, and either A12 or B3 CAR T cells generated in the presence of soluble B3 were introduced on days 3, 10, and 17 (n = 5). (D) Kaplan−Meier curve showing survival of each group. Mice treated with the A12 and B3 CAR T cells showed a slight increase in survival (P = 0.0058, Mantel−Cox log-rank test). (E) Individual tumor area for each group was measured. The A12 CAR T cells generated in the presence of soluble B3 slightly delayed tumor growth (P = 0.0483). SEM is shown. *P ≤ 0.05, **P ≤ 0.01.

图5。在培养过程中，由于pd-l1阻滞剂持续激活而导致的car t细胞衰竭。（a）在wt背景下制造的A12 car T细胞的慢性抗原暴露和耗尽可通过在培养过程中与可溶性抗-pd-l1 VHH孵育来阻断，以掩盖内源性pd l1。a12-car T细胞在可溶性b3-vhh存在下产生，与pd-l1结合的亲和力高于a12。用流式细胞仪分析了常见疲劳标志物的表达。（b）wt小鼠接种了B16肿瘤，并在wt背景下制备了含有和不含有可溶性B3的A12 car T细胞，并与pd-l1 ko背景下制备的A12 car T细胞进行了比较。在含有可溶性B3的wt背景下制备的a12 car t细胞比不含B3的a12 car t细胞表现出更好的持久性（CD4:a12 wt vs.a12 wt+b3，p=0.0283；CD8:a12 wt vs.a12 wt+b3，p=0.1346）。（c）在第0天给小鼠接种过表达pd-l1肿瘤的b16，在第3、10和17天引入在可溶性b3存在下产生的a12或b3 car t细胞（n=5）。（d）显示各组存活率的卡普兰-迈耶曲线。用a12和b3 car t细胞处理的小鼠存活率略有增加（p=0.0058，mantel-cox对数秩检验）。（e）测量各组的单个肿瘤面积。在可溶性B3存在下产生的a12 car T细胞略微延迟肿瘤生长（p=0.0483）。SEM显示。*P≤0.05，**P≤0.01。

Fig. 6. Anti-EIIIB fibronectin-targeted CAR T cells slow B16 melanoma growth in vivo. (A) T cells were transduced with the EIIIB-specific B2 CAR construct, and transduction efficiency was monitored by mCherry expression. Cells were then incubated with recombinant EIIIB-GST and probed with rabbit anti-GST and anti-rabbit A647 to determine ligand binding. (B) B2 CAR T cells show cytotoxicity in response to ligand recognition. B2 CAR T cells were cocultured with aortic endothelial cells (AEC) that either express the EIIIB fibronectin domain (AEC FN+/+) or lack it (AEC FN−/−). (C) Mice were inoculated with B16 tumors on day 0, and B2 CAR T cells (n = 10) were introduced on days 4, 15, and 20. (D) Tumor area was

measured for individual mice. Kaplan−Meier curve showing survival of each group (P = 0.0001, Mantel−Cox log-rank test with the Bonferroni correction for multiple comparisons). Mice treated with the B2 CAR T cells showed improved survival. SEM is shown. (E) RAG−/− mice were inoculated with B16 tumors and treated with B2 (n = 4) or 1B7 CAR T cells (n = 3) on day 4. RAG−/− mice treated with B2 CAR T cells do not show improved survival increase or delayed tumor growth. SEM is shown. (F) MC38 expresses lower levels of EIIIB (SI Appendix, Fig. S8). MC38 survival curves (P = 0.1895, ns, Mantel−Cox log-rank test) and MC38 individual tumor areas were not significantly affected by treatment with B2 CAR T cells (n = 7). ***P ≤ 0.001, ****P ≤ 0.0001; ns, nonsignificant.

图6。抗EIIIB纤维连接蛋白靶向的Car T细胞在体内减缓了黑色素瘤的生长。（a）用EIIIB特异性b2-car结构转导T细胞，用McHerry表达监测其转导效率。然后用重组EIIIB-GST培养细胞，用兔抗GST和兔抗A647探针检测配体结合。（b）b2-car T细胞对配体识别有细胞毒性反应。b2-car t细胞与主动脉内皮细胞（aec）共培养，后者表达eiiib纤维连接蛋白结构域（aec-fn+/+）或缺乏该结构域（aec-fn−/-）。（c）小鼠于第0天接种B16肿瘤，于第4、15和20天导入b2-car T细胞（n=10）。（d）测量单个小鼠的肿瘤面积。Kaplan−Meier曲线显示各组的生存率（p=0.0001，Mantel−Cox对数秩检验，采用Bonferroni校正进行多次比较）。经b2-car-t细胞处理的小鼠存活率提高。SEM显示。（e）RAG−/−小鼠在第4天接种了B16肿瘤，并用B2（n=4）或1B7 Car T细胞（n=3）治疗。用b2-car t细胞治疗的RAG−/−小鼠没有显示生存率提高或肿瘤生长延迟。SEM显示。（f）MC38表示更低水平的EIIIB（SI附录，图S8）。b2-car t细胞治疗对MC38生存曲线（p=0.1895，ns，mantel-cox对数秩检验）和MC38个体肿瘤区域没有显著影响（n=7）。***P≤0.001，***P≤0.0001；ns，无显著性。

Fig. 7. Treatment with anti-EIIIB fibronectin-targeted CAR T cells leads to tumor immune infiltration and necrosis. (A) WT mice were inoculated with tumors on day 0 and either left untreated (N = 2) or treated with B2 CAR T cells (n = 3) twice, on days 4 and 11. On day 16, tumors were harvested, fixed, and embedded for IHC and stained for EIIIB, CD31, CD3, CD4, and CD8. (B) The tumor area average measurements and values for individual mice are plotted. (C) Tumor samples were stained with PBS and secondary only (control), NJB2 VHH, anti-CD31, anti-CD3, anti-CD4, and anti-CD8. One representative image is shown. A 20× magnification of the edge (E), capsular region (Top) of the tumor is shown. A similar

magnification of a core (C) (Bottom) regions of the tumor is shown. EIIIB is present in the tumor capsule, tumor stroma, and surrounding the tumor vasculature, as inferred from colocalization with CD31 staining. In untreated samples, tumors appeared healthy and live, with intact matrix throughout the tissue. Little T cell and immune infiltration was apparent. (D) Necrotic B2 CAR T cell-treated tumors. Two of the three smaller treated tumors were highly necrotic, with a disintegrated matrix. CD31 staining shows a lack of tumor vasculature with little immune infiltration. (E) One treated tumor appeared to be heterogeneous and showed both (Bottom) necrotic [dead (D)] and (Top) live (L) sectors. The live tissue showed CD31 staining and was heavily infiltrated by CD3-, CD4-, and CD8-positive cells. (F) The number of CD3-, CD4-, and CD8- positive cells was quantified for both treated and untreated tumors. (G) The number of CD3-, CD4-, and CD8-positive cells was quantified for both the live and dead sections of the treated and untreated tumors

图7。抗EIIIB纤维连接蛋白靶向Car T细胞治疗可导致肿瘤免疫浸润和坏死。（a）在第0天给wt小鼠接种肿瘤，第4天和第11天两次不治疗（n=2）或用b2 car t细胞（n=3）治疗。在第16天，对肿瘤进行采集、固定和植入以进行IHC，并对EIIIB、CD31、CD3、CD4和CD8进行染色。（b）绘制单个小鼠的肿瘤面积平均测量值和值。（c）肿瘤标本用PBS和二级纯（对照）、NJB2 VHH、抗CD31、抗CD3、抗CD4和抗CD8染色。显示了一幅具有代表性的图像。显示肿瘤边缘（E）、包膜区（顶部）的20倍放大。肿瘤核心（c）（底部）区域的放大率相似。EIIIB存在于肿瘤包膜、肿瘤基质和肿瘤血管周围，由CD31染色的共定位推断。在未经治疗的样本中，肿瘤看起来健康而活跃，整个组织的基质完整。T细胞数量少，免疫浸润明显。（d）坏死性b2 car t细胞治疗肿瘤。三个较小的治疗肿瘤中，有两个是高度坏死的，基质已崩解。CD31染色显示肿瘤血管系统缺失，免疫浸润少。（e）一个治疗肿瘤似乎是异质的，显示（底部）坏死[死亡（d）]和（顶部）

活（l）部分。活组织呈CD31染色，CD3、CD4和CD8阳性细胞大量浸润。（f）对治疗和未治疗肿瘤的CD3、CD4和CD8阳性细胞的数量进行量化。（g）对治疗和未治疗肿瘤的活段和死亡段的CD3、CD4和CD8阳性细胞的数量进行量化。