Combining the best of two worlds: highly flexible chimeric antigen receptor adaptor molecules (CAR-adaptors) for the recruitment of chimeric antigen receptor T cells

Chimeric antigen receptor (CAR)-engineered T cells have a proven efficacy for the treatment of refractory hematological B cell malignancies. While often accompanied by side effects, CAR-T technology is getting more mature and will become an important treatment option for various tumor indications. In this review, we summarize emerging approaches that aim to further evolve CAR-T cell therapy based on combinations of so-called universal or modular CAR-(modCAR-)T cells, and their respective adaptor molecules (CAR-adaptors), which mediate the crosslinking between target and effector cells. The activity of such modCAR-T cells is entirely dependent on binding of the respective CAR-adaptor to both a tumor antigen and to the CAR-expressing T cell. Contrary to conventional CAR-T cells, where the immunological synapse is established by direct interaction of CAR and membrane-bound target, modCAR-T cells provide a highly flexible and customizable development of the CAR-T cell concept and offer an additional possibility to control T cell activity.     

Optimization of manufacturing conditions for chimeric antigen receptor T cells to favor cells with a central memory phenotype

BackgroundChimeric antigen receptor (CAR)-T cells are genetically engineered to recognize tumor-associated antigens and have potent cytolytic activity against tumors. Adoptive therapy with CAR-T cells has been highly successful in B-cell leukemia and lymphoma. However, in solid tumor settings, CAR-T cells face a particularly hostile tumor microenvironment where multiple immune suppressive factors serve to thwart the anti-cancer immune response. Clinical trials of solid tumor antigen-targeted CAR-T cells have shown limited efficacy, and issues for current CAR-T cell therapies include failures of expansion and persistence, tumor entry, deletion and functional exhaustion.MethodsWe compared our standard protocol for CAR-T cell manufacturing, currently used to generate CAR-T cells for a phase 1 clinical trial, with two alternative approaches for T-cell activation and expansion. The resulting cultures were analyzed using multicolor flow cytometry, cytokine bead array and xCELLigence cytotoxicity assays.ResultsWe have found that by changing the method of activation we can promote generation of CAR-T cells with enhanced CD62L and CCR7 expression, increased interleukin (IL)-2 production and retention of cytolytic activity, albeit with slower kinetics.DiscussionWe propose that these phenotypic characteristics are consistent with a central memory phenotype that will better enable CAR-T cell survival and persistence after activation in vivo, and we aim to test this in a continuation of our current phase 1 clinical trial of CAR-T cells in patients with advanced melanoma.     

Targeted BRAF Inhibition Impacts Survival in Melanoma Patients with High Levels of Wnt/β-Catenin Signaling

Unprecedented clinical responses have been reported in advanced stage metastatic melanoma patients treated with targeted inhibitors of constitutively activated mutant BRAF, which is present in approximately half of all melanomas. We and others have previously observed an association of elevated nuclear β-catenin with improved survival in molecularly-unselected melanoma patients. This study sought to determine whether levels of Wnt/β-catenin signaling in melanoma tumors prior to treatment might predict patient responses to BRAF inhibitors (BRAFi). We performed automated quantification of β-catenin immunohistochemical expression in pretreatment BRAF-mutant tumors from 32 BRAFi-treated melanoma patients. Unexpectedly, patients with higher nuclear β-catenin in their tumors did not exhibit the survival advantage previously observed in molecularly-unselected melanoma patients who did not receive BRAFi. In cultured melanoma cells treated with long-term BRAFi, activation of Wnt/β-catenin signaling is markedly inhibited, coinciding with a loss of the enhancement of BRAFi-induced apoptosis by WNT3A observed in BRAFi-naïve cells. Together, these observations suggest that long-term treatment with BRAFi can impact the interaction between BRAF/MAPK and Wnt/β-catenin signaling to affect patient outcomes. Studies with larger patient cohorts are required to determine whether nuclear β-catenin expression correlates with clinical responses to BRAFi and to specific mechanisms of acquired resistance to BRAFi. Understanding these pathway interactions will be necessary to facilitate efforts to individualize therapies for melanoma patients.     

稳定表达CD19-FLUC-GFP的CT26细胞系的构建及鉴定

实体瘤缺乏明确的嵌合抗原受体T细胞(chimeric antigen receptor T-cell, CAR-T)治疗靶点。因此,通过慢病毒将已经明确的靶点分子CD19带入实体瘤细胞系,研究CD19 CAR-T细胞对其的杀伤,能够为CAR-T细胞针对实体瘤的治疗提供潜在的支撑。本研究利用三质粒慢病毒系统构建了稳定表达CD19、萤火虫荧光素酶(firefly luciferase, FLUC)和绿色荧光蛋白(green fluorescent protein, GFP)的结肠癌CT26细胞系CT26-CD19-FLUC-GFP。该细胞系与CT26细胞系的生长活性一致。通过流式细胞术检测不同代次CT26-CD19-FLUC-GFP细胞,证实了CT26-CD19-FLUC-GFP细胞连续传代至第5、10、22代后CD19及GFP的稳定表达。进一步证实,连续传代至第22代的CT26-CD19-FLUC-GFP细胞中的CD19 mRNA及FLUC表达水平显著高于对照组CT26细胞。与T细胞相比,CD19 CAR-T细胞能够显著杀伤CT26-CD19-FLUC-GFP细胞及MC38-CD19细胞。CT26-CD19-FLUC-GFP细胞腹腔植入小鼠体内1周后,通过活体成像仪可以检测到腹腔区域的FLUC表达。上述结果表明,成功构建了稳定表达CD19-FLUC-GFP的CT26细胞系,且该细胞系能够被CD19 CAR-T细胞特异性杀伤。     

共表达IL-15和CCL19的EGFRvⅢ CAR-T细胞的构建和功能探究

本研究分析了共表达白细胞介素-15 (interleukin-15, IL-15)和趋化因子配体19 (C-C chemokine ligand 19, CCL19)的EGFRvⅢ CAR-T细胞的功能特性及其体外特异性杀伤效果，旨在优化CAR-T细胞多项功能，提高靶向EGFRvⅢ 的CAR-T细胞对胶质母细胞瘤(glioblastoma, GBM)的治疗效果。通过基因工程技术获得重组慢病毒质粒，转染293T细胞获得慢病毒并感染T细胞获得靶向EGFRvⅢ的第四代CAR-T细胞(EGFRvⅢ-IL-15-CCL19 CAR-T)。利用流式细胞仪、细胞计数仪、趋化小室、凋亡试剂盒等检测了第四代和第二代CAR-T细胞(EGFRvⅢ CAR-T)的CAR分子表达率、增殖、趋化能力、体外特异性杀伤能力及抗凋亡能力等。结果表明，与EGFRvⅢ CAR-T细胞相比，EGFRvⅢ-IL-15-CCL19 CAR-T细胞能成功分泌IL-15和CCL19，具有更强的体外增殖能力、趋化能力以及抗凋亡能力(P值均<0.05)，而体外特异性杀伤能力无显著差异。因此，靶向EGFRvⅢ且同时分泌IL-15和CCL19的CAR-T细胞有望提高胶质母细胞瘤的治疗效果，为临床试验提供一定的参考依据。     

Impact of Exogenous Galectin-9 on Human T Cells: CONTRIBUTION OF THE T CELL RECEPTOR COMPLEX TO ANTIGEN-INDEPENDENT ACTIVATION BUT NOT TO APOPTOSIS INDUCTION*

Galectin-9 (gal-9) is a multifunctional β-galactoside-binding lectin, frequently released in the extracellular medium, where it acts as a pleiotropic immune modulator. Despite its overall immunosuppressive effects, a recent study has reported bimodal action of gal-9 on human resting blood T cells with apoptosis occurring in the majority of them, followed by a wave of activation and expansion of Th1 cells in the surviving population. Our knowledge of the signaling events triggered by exogenous gal-9 in T cells remains limited. One of these events is cytosolic calcium (Ca²⁺) release reported in some murine and human T cells. The aim of this study was to investigate the contribution of Ca²⁺ mobilization to apoptotic and nonapoptotic effects of exogenous gal-9 in human T cells. We found that the T cell receptor (TCR)-CD3 complex and the Lck kinase were required for Ca²⁺ mobilization but not for apoptosis induction in Jurkat cells. These data were confirmed in human CD4⁺ T cells from peripheral blood as follows: a specific Lck chemical inhibitor abrogated Ca²⁺ mobilization but not apoptosis induction. Moreover, Lck activity was also required for the production of Th1-type cytokines, i.e. interleukin-2 and interferon-γ, which resulted from gal-9 stimulation in peripheral CD4⁺ T cells. These findings indicate that gal-9 acts on T cells by two distinct pathways as follows: one mimicking antigen-specific activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis that is independent of this complex.     

Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells

Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis, showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use.     

Interleukin-1α Activity in Necrotic Endothelial Cells Is Controlled by Caspase-1 Cleavage of Interleukin-1 Receptor-2: IMPLICATIONS FOR ALLOGRAFT REJECTION*

Inflammation is a key instigator of the immune responses that drive atherosclerosis and allograft rejection. IL-1α, a powerful cytokine that activates both innate and adaptive immunity, induces vessel inflammation after release from necrotic vascular smooth muscle cells (VSMCs). Similarly, IL-1α released from endothelial cells (ECs) damaged during transplant drives allograft rejection. However, IL-1α requires cleavage for full cytokine activity, and what controls cleavage in necrotic ECs is currently unknown. We find that ECs have very low levels of IL-1α activity upon necrosis. However, TNFα or IL-1 induces significant levels of active IL-1α in EC necrotic lysates without alteration in protein levels. Increased activity requires cleavage of IL-1α by calpain to the more active mature form. Immunofluorescence and proximity ligation assays show that IL-1α associates with interleukin-1 receptor-2, and this association is decreased by TNFα or IL-1 and requires caspase activity. Thus, TNFα or IL-1 treatment of ECs leads to caspase proteolytic activity that cleaves interleukin-1 receptor-2, allowing IL-1α dissociation and subsequent processing by calpain. Importantly, ECs could be primed by IL-1α from adjacent damaged VSMCs, and necrotic ECs could activate neighboring normal ECs and VSMCs, causing them to release inflammatory cytokines and up-regulate adhesion molecules, thus amplifying inflammation. These data unravel the molecular mechanisms and interplay between damaged ECs and VSMCs that lead to activation of IL-1α and, thus, initiation of adaptive responses that cause graft rejection.     

SPSB1, a Novel Negative Regulator of the Transforming Growth Factor-β Signaling Pathway Targeting the Type II Receptor

Appropriate cellular signaling is essential to control cell proliferation, differentiation, and cell death. Aberrant signaling can have devastating consequences and lead to disease states, including cancer. The transforming growth factor-β (TGF-β) signaling pathway is a prominent signaling pathway that has been tightly regulated in normal cells, whereas its deregulation strongly correlates with the progression of human cancers. The regulation of the TGF-β signaling pathway involves a variety of physiological regulators. Many of these molecules act to alter the activity of Smad proteins. In contrast, the number of molecules known to affect the TGF-β signaling pathway at the receptor level is relatively low, and there are no known direct modulators for the TGF-β type II receptor (TβRII). Here we identify SPSB1 (a Spry domain-containing Socs box protein) as a novel regulator of the TGF-β signaling pathway. SPSB1 negatively regulates the TGF-β signaling pathway through its interaction with both endogenous and overexpressed TβRII (and not TβRI) via its Spry domain. As such, TβRII and SPSB1 co-localize on the cell membrane. SPSB1 maintains TβRII at a low level by enhancing the ubiquitination levels and degradation rates of TβRII through its Socs box. More importantly, silencing SPSB1 by siRNA results in enhanced TGF-β signaling and migration and invasion of tumor cells.     

Guanine Nucleotide Exchange Factor αPIX Leads to Activation of the Rac 1 GTPase/Glycogen Phosphorylase Pathway in Interleukin (IL)-2-stimulated T Cells

Recently, we have reported that the active form of Rac 1 GTPase binds to the glycogen phosphorylase muscle isoform (PYGM) and modulates its enzymatic activity leading to T cell proliferation. In the lymphoid system, Rac 1 and in general other small GTPases of the Rho family participate in the signaling cascades that are activated after engagement of the T cell antigen receptor. However, little is known about the IL-2-dependent Rac 1 activator molecules. For the first time, a signaling pathway leading to the activation of Rac 1/PYGM in response to IL-2-stimulated T cell proliferation is described. More specifically, αPIX, a known guanine nucleotide exchange factor for the small GTPases of the Rho family, preferentially Rac 1, mediates PYGM activation in Kit 225 T cells stimulated with IL-2. Using directed mutagenesis, phosphorylation of αPIX Rho-GEF serines 225 and 488 is required for activation of the Rac 1/PYGM pathway. IL-2-stimulated serine phosphorylation was corroborated in Kit 225 T cells cultures. A parallel pharmacological and genetic approach identified PKCθ as the serine/threonine kinase responsible for αPIX serine phosphorylation. The phosphorylated state of αPIX was required to activate first Rac 1 and subsequently PYGM. These results demonstrate that the IL-2 receptor activation, among other early events, leads to activation of PKCθ. To activate Rac 1 and consequently PYGM, PKCθ phosphorylates αPIX in T cells. The biological significance of this PKCθ/αPIX/Rac 1 GTPase/PYGM signaling pathway seems to be the control of different cellular responses such as migration and proliferation.     

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell–bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.     

Genetic engineering of T cells with chimeric antigen receptors for hematological malignancy immunotherapy

The host immune system plays an instrumental role in the surveillance and elimination of tumors by recognizing and destroying cancer cells. In recent decades, studies have mainly focused on adoptive immunotherapy using engineered T cells for the treatment of malignant diseases. Through gene engraftment of the patient’s own T cells with chimeric antigen receptor (CAR), they can recognize tumor specific antigens effectively and eradicate selectively targeted cells in an MHC-independent fashion. To date, CAR-T cell therapy has shown great clinical utility in patients with B-cell leukemias. Owing to different CAR designs and tumor complex microenvironments, genetically redirected T cells may generate diverse biological properties and thereby impact their long-term clinical performance and outcome. Meanwhile some unexpected toxicities that result from CAR-T cell application have been examined and limited the curative effects. Diverse important parameters are closely related with adoptively transferred cell behaviors, including CAR-T cells homing, CAR constitutive signaling, T cell differentiation and exhaustion. Thus, understanding CARs molecular design to improve infused cell efficacy and safety is crucial to clinicians and patients who are considering this novel cancer therapeutics. In this review, the developments in CAR-T cell therapy and the limitations and perspectives in optimizing this technology towards clinical application are discussed.     

Clinical trials of CAR-T cells in China

Novel immunotherapeutic agents targeting tumor-site microenvironment are revolutionizing cancer therapy. Chimeric antigen receptor (CAR)-engineered T cells are widely studied for cancer immunotherapy. CD19-specific CAR-T cells, tisagenlecleucel, have been recently approved for clinical application. Ongoing clinical trials are testing CAR designs directed at novel targets involved in hematological and solid malignancies. In addition to trials of single-target CAR-T cells, simultaneous and sequential CAR-T cells are being studied for clinical applications. Multi-target CAR-engineered T cells are also entering clinical trials. T cell receptor-engineered CAR-T and universal CAR-T cells represent new frontiers in CAR-T cell development. In this study, we analyzed the characteristics of CAR constructs and registered clinical trials of CAR-T cells in China and provided a quick glimpse of the landscape of CAR-T studies in China.     

T-cell Receptor (TCR)-Peptide Specificity Overrides Affinity-enhancing TCR-Major Histocompatibility Complex Interactions

αβ T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short “foreign” peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery.     

Transient Combination Therapy Targeting the Immune Synapse Abrogates T Cell Responses and Prolongs Allograft Survival in Mice

T cells play a major role in allograft rejection, which occurs after T cell activation by the engagement of several functional molecules to form an immune synapse with alloantigen presenting cells. In this study, the immune synapse was targeted using mAbs directed to the TCR beta-chain (TCRβ) and lymphocyte function-associated antigen−1 (LFA1) to induce long-term allograft survival. Evaluation of antigen-specific T cell responses was performed by adoptively transferring CFSE labeled transgenic OT-II cells into wild-type mice and providing OVA peptide by intravenous injection. Graft survival studies were performed in mice by transplanting BALB/c ear skins onto the flanks of C57BL/6 recipients. The anti-TCRβ plus anti-LFA1 mAb combination (but not either mAb alone) abrogated antigen-specific T cell responses invitro and invivo. Transient combination therapy with these agents resulted in significantly prolonged skin allograft survival in mice (51±10 days; p<0.01) when compared to treatment with either anti-TCRβ mAb (24±5 days) or anti-LFA1 mAb (19±3 days) alone or no treatment (10±1 days). When lymphoid tissues from these mice were analyzed at different times post-transplant, only those receiving the combination of anti-TCRβ and anti-LFA1 mAbs demonstrated long-lasting reductions in total T cell numbers, cellular and humoral anti-donor responses, and expression of CD3 on the surface of T cells. These results demonstrate that transient anti-TCRβ and anti-LFA1 mAb combination therapy abrogates antigen-reactive T cell responses with long-lasting effects that significantly prolong allograft survival.     

Targeting CD276 by CAR-T cells induces regression of esophagus squamous cell carcinoma in xenograft mouse models

Esophageal cancer, including esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), has a poor prognosis and limited therapeutic options. Chimeric antigen receptor (CAR)-T cells represent a potential ESCC treatment. In this study, we examined CD276 expression in healthy and esophageal tumor tissues and explored the tumoricidal potential of CD276-targeting CAR-T cells in ESCC. CD276 was strongly and homogenously expressed in ESCC and EAC tumor lesions but mildly in healthy tissues, representing a good target for CAR-T cell therapy. We generated CD276-directed CAR-T cells with a humanized antigen-recognizing domain and CD28 or 4–1BB co-stimulation. CD276-specific CAR-T cells efficiently killed ESCC tumor cells in an antigen-dependent manner both in vitro and in vivo. In patient-derived xenograft models, CAR-T cells induced tumor regression and extended mouse survival. In addition, CAR-T cells generated from patient T cells demonstrated potent cytotoxicity against autologous tumor cells. Our study indicates that CD276 is an attractive target for ESCC therapy, and CD276-targeting CAR-T cells are worth testing in ESCC clinical trials.     

Potential of chimeric antigen receptor (CAR)‐redirected immune cells in breast cancer therapies: Recent advances

Despite substantial developments in conventional treatments such as surgery, chemotherapy, radiotherapy, endocrine therapy, and molecular‐targeted therapy, breast cancer remains the leading cause of cancer mortality in women. Currently, chimeric antigen receptor (CAR)–redirected immune cell therapy has emerged as an innovative immunotherapeutic approach to ameliorate survival rates of breast cancer patients by eliciting cytotoxic activity against cognate tumour‐associated antigens expressing tumour cells. As a crucial component of adaptive immunity, T cells and NK cells, as the central innate immune cells, are two types of pivotal candidates for CAR engineering in treating solid malignancies. However, the biological distinctions between NK cells‐ and T cells lead to differences in cancer immunotherapy outcomes. Likewise, optimal breast cancer removal via CAR‐redirected immune cells requires detecting safe target antigens, improving CAR structure for ideal immune cell functions, promoting CAR‐redirected immune cells filtration to the tumour microenvironment (TME), and increasing the ability of these engineered cells to persist and retain within the immunosuppressive TME. This review provides a concise overview of breast cancer pathogenesis and its hostile TME. We focus on the CAR‐T and CAR‐NK cells and discuss their significant differences. Finally, we deliver a summary based on recent advancements in the therapeutic capability of CAR‐T and CAR‐NK cells in treating breast cancer.     

Adoptive therapy with CAR redirected T cells for hematological malignancies

The survival of patients with hematological malignancies has been significantly improved due to the development of new therapeutic agents. However, relapse remains a major matter for concern. Recently, T cells engineered with chimeric antigen receptor(CAR) were reported to show unprecedented responses in a range of hematological malignancies. The persistence of the CAR-T cell can last for years and tends toward long-term antitumor memory by which relapses can be effectively prevented. The primary side effects that appear in most clinical trials are cytokine release syndrome and neurotoxicity. However, these symptoms can be treated and reversed. In this review, we describe CAR structure and function and summarize recent advances in CAR-T cell therapy in hematological malignancies.     

靶向GPC3的第四代嵌合抗原受体T细胞 (分泌IL-7和CCL19) 的构建以及功能

基于嵌合抗原受体修饰的T细胞(CAR-T)的过继免疫疗法已被证明是治疗恶性肿瘤最有希望的策略之一,但是目前其在实体瘤中的应用依然有限。研究表明磷脂酰肌醇蛋白聚糖3 (GPC3)对肝细胞癌来说是一种有意义的诊断、治疗和预后生物标志物,且已有利用第二代/第三代GPC3靶向的CAR-T细胞治疗肝细胞癌的研究报道。为了进一步提高治疗效果,构建同时表达GPC3 CAR、人源IL-7和CCL19细胞因子的第四代慢病毒载体,转染293T细胞包装慢病毒并感染人T淋巴细胞制备靶向GPC3的第四代CAR-T细胞(GPC3-BBZ-7×19)。利用细胞计数、趋化小室、荧光素酶生物发光法以及流式细胞术等比较其与第二代GPC3 CAR-T细胞(GPC3-BBZ)在增殖、迁移、杀伤以及亚型分布方面的区别,评估GPC3-BBZ-7×19 CAR-T细胞对免疫缺陷小鼠体内GPC3阳性的肝细胞癌腹腔移植瘤生长的作用。结果表明与GPC3-BBZ CAR-T细胞相比,GPC3-BBZ-7×19 CAR-T细胞具备更强的增殖能力、趋化能力以及记忆干细胞(Stem memory T cell,Tscm)组成比(P值均0.05),而在体外特异性杀伤GPC3阳性的肝细胞癌细胞以及细胞因子分泌水平方面无显著差异。此外,GPC3-BBZ-7×19 CAR-T细胞能够明显抑制免疫缺陷小鼠体内的GPC3阳性肝细胞癌移植瘤模型。因此靶向GPC3的第四代CAR-T细胞(分泌IL-7和CCL19)有望比第二代GPC3靶向的CAR-T细胞更持久有效地对抗肿瘤,并产生针对肿瘤的记忆功能,为以后的临床试验提供了临床前研究基础。     

Attenuated T Cell Responses to a High-Potency Ligand In Vivo

αβ T cell receptor (TCR) recognition of foreign peptides bound to major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells is a key event in the initiation of adaptive cellular immunity. In vitro, high-affinity binding and/or long-lived interactions between TCRs and pMHC correlate with high-potency T cell activation. However, less is known about the influence of TCR/pMHC interaction parameters on T cell responses in vivo. We studied the influence of TCR/pMHC binding characteristics on in vivo T cell immunity by tracking CD4⁺ T cell activation, effector, and memory responses to immunization with peptides exhibiting a range of TCR/pMHC half-lives and in vitro T cell activation potencies. Contrary to predictions from in vitro studies, we found that optimal in vivo T cell responses occur to ligands with intermediate TCR/pMHC half-lives. The diminished in vivo responses we observed to the ligand exhibiting the longest TCR/pMHC half-life were associated with attenuation of intracellular signaling, expansion, and function over a broad range of time points. Our results reveal a level of control over T cell activation in vivo not recapitulated in in vitro assays and highlight the importance of considering in vivo efficacy of TCR ligands as part of vaccine design.