Directing CAR T cells towards the tumor vasculature for the treatment of solid tumors

For successful application of chimeric antigen receptor (CAR) T cell therapy in solid tumors, major hurdles have to be overcome. CAR T cells have to cross the vascular barrier, which is hampered by the anergic state of the tumor vasculature, characterized by suppressed levels of leukocyte adhesion molecules on the endothelium. Additional immunosuppressive mechanisms in the solid tumor microenvironment can affect infiltration, activity and persistence of CAR T cells. Redirecting CAR T cells towards the tumor vasculature poses a possible solution, as molecular targets of tumor endothelial cells can be directly engaged from within the blood.In this review, we discuss recent advances in CAR T cell therapy against solid tumors, with a focus on targeting the tumor vasculature. Furthermore, we discuss opportunities to overcome challenges and barriers through engineering of CAR T cells to enhance trafficking, safety and efficacy.     

The progress and perspectives of CAR-T cell therapy

CAR-T cell therapy has already achieved world-renowned clinical effects in the treatment of hematological malignancies. Due to the tumor heterogeneity, immunosuppressive microenvironment, and other factors, CAR-T cell therapy has still not shown obvious clinical efficacy in clinical treatment of solid tumors. However, great progress has been made in the preparation of CAR-T cells in recent years, including T cells redirected for universal cytokine mediated killing, universal CAR -T cells, non-viral vector CAR-T cells, SynNotch technology, SUPRA CAR technology, regulated CAR-T cells, and bi-specific CAR-T cells, etc. Future research and development of CAR-T cell therapy will be focused on these following aspects: the combined application of CAR-T cells with different targets, known as "Cocktail CAR-T cells", is expected to increase efficiency toward solid tumors; based on systemic biology/synthetic biology theories, CAR-T cells are likely to be transformed to robot or intelligent system by introducing sensors, logic gates, and logic circuits. This article mainly comments on research progress and perspectives on CAR-T cell therapy in solid tumor treatment.     

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.     

Chimeric antigen receptor T cells in solid tumors: a war against the tumor microenvironment

Chimeric antigen receptor(CAR) T cell is a novel approach, which utilizes anti-tumor immunity for cancer treatment. As compared to the traditional cell-mediated immunity, CAR-T possesses the improved specificity of tumor antigens and independent cytotoxicity from major histocompatibility complex molecules through a monoclonal antibody in addition to the Tcell receptor. CAR-T cell has proven its effectiveness, primarily in hematological malignancies, specifically where the CD19 CAR-T cells were used to treat B-cell acute lymphoblastic leukemia and B-cell lymphomas. Nevertheless, there is little progress in the treatment of solid tumors despite the fact that many CAR agents have been created to target tumor antigens such as CEA,EGFR/EGFRvIII, GD2, HER2, MSLN, MUC1, and other antigens. The main obstruction against the progress of research in solid tumors is the tumor microenvironment, in which several elements, such as poor locating ability, immunosuppressive cells,cytokines, chemokines, immunosuppressive checkpoints, inhibitory metabolic factors, tumor antigen loss, and antigen heterogeneity, could affect the potency of CAR-T cells. To overcome these hurdles, researchers have reconstructed the CAR-T cells in various ways. The purpose of this review is to summarize the current research in this field, analyze the mechanisms of the major barriers mentioned above, outline the main solutions, and discuss the outlook of this novel immunotherapeutic modality.     

嵌合抗原受体T细胞介绍及抗肿瘤临床应用

过继性细胞免疫治疗（adoptive cellular immunotherapy,ACI）是目前较为有效的恶性肿瘤的治疗方法之一。随着技术的日趋成熟,已在多种实体瘤和血液肿瘤的t临床治疗中取得较好疗效。其中,嵌合抗原受体（chimeric antigen receptor,CAR）T细胞技术是近年来发展非常迅速的一种细胞治疗技术。通过基因改造技术,效应T细胞的靶向性、杀伤活性和持久性均较常规应用的免疫细胞高,并可克服肿瘤局部免疫抑制微环境和打破宿主免疫耐受状态。目前,CAR的信号域已从第一代的单一信号分子发展为包含CD28、4—1BB等共刺激分子的多信号结构域（第二、三代）,临床应用广泛。但是,该技术也存在脱靶效应、插入突变等临床应用风险。该文将就CAR—T细胞技术在恶性肿瘤免疫治疗中的应用及可能存在的问题作一综述。     

Hurdles of CAR-T cell-based cancer immunotherapy directed against solid tumors

Recent reports on the impressive efficacy of chimeric antigen receptor (CAR)-modified T cells against hematologic malignancies have inspired oncologists to extend these efforts for the treatment of solid tumors. Clinical trials of CAR-T-based cancer immunotherapy for solid tumors showed that the efficacies are not as remarkable as in the case of hematologic malignancies. There are several challenges that researchers must face when treating solid cancers with CAR-T cells, these include choosing an ideal target, promoting efficient trafficking and infiltration, overcoming the immunosuppressive microenvironment, and avoiding associated toxicity. In this review, we discuss the obstacles imposed by solid tumors on CAR-T cell-based immunotherapy and strategies adopted to improve the therapeutic potential of this approach. Continued investigations are necessary to improve therapeutic outcomes and decrease the adverse effects of CAR-T cell therapy in patients with solid malignancies in the future.     

Inducible secretion of IL-21 augments anti-tumor activity of piggyBac-manufactured chimeric antigen receptor T cells

BackgroundThe efficiency of chimeric antigen receptor (CAR) T-cell-based therapies depends on a sufficient expansion of CAR T cells in vivo and can be weakened by intra-tumoral suppression of CAR T cell functions, leading to a failure of therapy. For example, certain B-cell malignancies such as chronic lymphocytic leukemia are weakly sensitive to treatment with CAR T cells. Co-expression of proinflamatory cytokines such as IL-12 and IL-18 by CAR T cells have been shown to enhance their antitumor function. We similarly engineered CAR T cell to co-express IL-21 and studied the effects of IL-21 on CAR T cells specific to CD19 and prostate-specific membrane antigens using an in vitro co-culture model and NSG mice transplanted with B-cell tumors.ResultsIL-21 enhanced the expansion of CAR T cells after antigenic stimulation, reduced the level of apoptosis of CAR T cells during co-culture with tumor cells and prevented differentiation of CAR T cells toward late memory phenotypes. In addition, induced secretion of IL-21 by CAR T cells promoted tumor infiltration by CD19-specific CAR (CAR19) T cells in NSG mice, resulting in reduced tumor growth. By co-culturing CAR19 T cells with bone-marrow fragments infiltrated with CLL cells we demonstrate that IL-21 reduces the immunosupressive activity of CLL cells against CAR19 T cells.ConclusionsCAR19 T cells armed with IL-21 exhibited enhanced antitumor functions. IL-21 promoted their proliferation and cytotoxicity against chronic lymphocytic leukemia (CLL). The results suggest that arming CAR T cells with IL-21 could boost the effectiveness of CAR T-mediated therapies.     

Resistance against anti-CD19 and anti-BCMA CAR T cells: Recent advances and coping strategies

Chimeric antigen receptor T (CAR T) cell therapy is a new treatment paradigm that has revolutionized the treatment of CD19-positive B cell malignancies and BCMA-positive plasma cell malignancies. The response rates are highly impressive in comparison to historical cohorts, but the responses are not durable. The most recent results from pivotal trials show that current CAR T cell products fail to demonstrate optimal long-term disease control. Resistance to CAR T cells is related to CAR structure, T cell factors, tumor factors and the immunosuppressive microenvironment. Novel strategies are needed following failure with CAR T cell treatment. In this review, we discuss the resistance mechanisms to CAR T cell treatment according to disease and the emerging strategies to overcome resistance.     

Expression and prognostic relevance of PRAME in primary osteosarcoma

The preferentially expressed antigen of melanoma (PRAME), a cancer-testis antigen with unknown function, is expressed in many human malignancies and is considered an attractive potential target for tumor immunotherapy. However, studies of its expression and function in osteosarcoma have rarely been reported. In this study, we found that PRAME is expressed in five osteosarcoma cell lines and in more than 70% of osteosarcoma patient specimens. In addition, an immunohistochemical analysis showed that high PRAME expression was associated with poor prognosis and lung metastasis. Furthermore, PRAME siRNA knockdown significantly suppressed the proliferation, colony formation, and G1 cell cycle arrest in U-2OS cells. Our results suggest that PRAME plays an important role in cell proliferation and disease progression in osteosarcoma. However, the detail mechanisms of PRAME function in osteosarcoma require further investigation.     

Label-free in vitro assays predict the potency of anti-disialoganglioside chimeric antigen receptor T-cell products

Background aimsChimeric antigen receptor (CAR) T cells have demonstrated remarkable efficacy against hematological malignancies; however, they have not experienced the same success against solid tumors such as glioblastoma (GBM). There is a growing need for high-throughput functional screening platforms to measure CAR T-cell potency against solid tumor cells.MethodsWe used real-time, label-free cellular impedance sensing to evaluate the potency of anti-disialoganglioside (GD2) targeting CAR T-cell products against GD2+ patient-derived GBM stem cells over a period of 2 days and 7 days in vitro. We compared CAR T products using two different modes of gene transfer: retroviral transduction and virus-free CRISPR-editing. Endpoint flow cytometry, cytokine analysis and metabolomics data were acquired and integrated to create a predictive model of CAR T-cell potency.ResultsResults indicated faster cytolysis by virus-free CRISPR-edited CAR T cells compared with retrovirally transduced CAR T cells, accompanied by increased inflammatory cytokine release, CD8+ CAR T-cell presence in co-culture conditions and CAR T-cell infiltration into three-dimensional GBM spheroids. Computational modeling identified increased tumor necrosis factor α concentrations with decreased glutamine, lactate and formate as being most predictive of short-term (2 days) and long-term (7 days) CAR T cell potency against GBM stem cells.ConclusionsThese studies establish impedance sensing as a high-throughput, label-free assay for preclinical potency testing of CAR T cells against solid tumors.     

CAR T-cell therapy: Balance of efficacy and safety

Early results from clinical trials of autologous chimeric antigen receptor (CAR)-expressing T cells for the therapy of B-cell malignancies have encouraged extending the potency of this therapy to other cancers. However, the success of using CAR T-cells to treat patients with solid tumors has been limited. In this review, we summarize current knowledge on the design and applications of CARs for the targeted therapy of cancer. We describe existing issues that limit the widespread application of CAR T cells and discuss the optimization steps needed to further improve safety and efficacy of this therapeutic platform.     

CAR19/22 T cell cocktail therapy for B-ALL relapsed after allogeneic hematopoietic stem cell transplantation

B cell acute lymphocytic leukemia (B-ALL) patients who have relapsed after hematopoietic stem cell transplantation (HSCT) have a poor prognosis, and there is currently no standard approach available. Chimeric antigen receptor (CAR)-T cells induce high rates of initial response and long-term remission among patients with B-cell malignancies, especially B-ALL. Meanwhile, sequential infusion of CAR19/22 T cells has been proven to be effective at preventing tumor immune escape. In the present study, we retrospectively analyzed 23 B-ALL patients who relapsed after allogeneic (allo)-HSCT and underwent sequential infusion of CAR19/22 T cells, including nine donor-derived and 14 recipient-derived, in our center from July 2016 to July 2020, to evaluate the safety and efficacy of the cocktail of two single-specific CAR-T cells in B-ALL patients relapsed after transplantation. Except for one patient refusing evaluation, the remaining 22 patients achieved minimal residual disease (MRD)-negative complete remission within 30 days after CAR-T infusion. Most toxicities were slight and reversible. The estimated 12-month progression-free survival (PFS) rate was 59.2% (95% confidence interval [CI], 35.9% to 76.5%), and the estimated 12-month overall survival (OS) rate was 67.4% (95% CI, 43.2% to 83.1%). Only two patients had CD19-negative recurrence. In addition, early recurrence after transplantation, graft-versus-host disease (GVHD) and severe infection after CAR-T infusion were poor prognostic factors. Our results indicate that sequential infusion of CAR19/22 T cells is safe and effective for relapsed ALL patients after HSCT. This trial was registered at www.chictr.org.cn as #ChiCTR-OPN-16008526.     

CAR-NK治疗肿瘤的研究与临床进展

肿瘤细胞免疫疗法近年来的发展颇为瞩目,嵌合抗原受体T（CAR-T）的临床研究显示其对血液系统肿瘤具有良好的治疗效果。自然杀伤细胞（NK）是人体固有免疫的一类重要细胞,其不同于T细胞的非特异性识靶及杀伤机制吸引科学家将工程CAR-T技术沿袭并用于嵌合抗原受体NK（CAR-NK）改造。目前,无论在体外细胞模型还是小鼠动物模型中,CAR-?NK均显示出良好的肿瘤杀伤效果。最新的临床研究显示,CAR-NK细胞对血液系统肿瘤有良好的治疗效果,但治疗实体瘤效果尚待验证。与CAR-T细胞疗法一样,CAR-NK也有问题亟需解决,但是NK细胞作为效应细胞,其自身优点预示CAR-NK细胞在实体瘤治疗方面拥有良好的发展前景。     

Micro-hydrogel injectables that deliver effective CAR-T immunotherapy against 3D solid tumor spheroids

Chimeric antigen receptor (CAR-) T cells are revolutionizing cancer treatment, as a direct result of their clinical impact on the treatment of hematological malignancies. However for solid tumors, CAR-T cell therapeutic efficacy remains limited, primarily due to the complex immunosuppressive tumor microenvironment, inefficient access to tumor cells and poor persistence of the killer cells. In this in vitro study, an injectable, gelatin-based micro-hydrogel system that can encapsulate and deliver effective CAR-T therapy is investigated. CAR-T cells targeting TAG-72, encapsulated in these microgels possessed high viability (> 87%) after 7 days, equivalent to those grown under normal expansion conditions, with retention of the T cell phenotype and functionality. Microgel recovered CAR-T cells demonstrated potent on-target cytotoxicity against human ovarian cancer in vitro and on three-dimensional tumor spheroids, by completely eliminating tumor cells. The gelatin-based micro-hydrogels have the potential to serve as carrier systems to augment CAR-T immunotherapeutic treatment of solid tumors.     

嵌合抗原受体T细胞治疗的现状与展望

肿瘤严重威胁着人类健康,当前肿瘤传统的治疗方法有手术治疗、化疗、放疗和靶向药物治疗等。近年来,肿瘤免疫治疗,尤其是嵌合抗原受体（chimeric antigen receptor,CAR） T细胞免疫疗法在基础研究与临床应用中蓬勃发展,并在治疗血液系统恶性肿瘤方面取得了巨大成功。然而,大量研究显示,细胞免疫治疗后可出现不同程度的毒副反应,且部分患者缓解后再次复发。因此,了解细胞治疗面临的挑战与局限性,寻找解决的办法,对继续发挥细胞免疫疗法的潜能具有重要意义。本文就免疫细胞的CAR结构、病毒载体的选择、细胞治疗面临的挑战及前景进行综述。     

MSLN (Mesothelin), ANTXR1 (TEM8), and MUC3A are the potent antigenic targets for CAR T cell therapy of gastric adenocarcinoma

Gastric adenocarcinoma is usually diagnosed in late stages, necessitating the use of different therapeutic modalities. Currently, antibody-based therapies have also been approved through with limited clinical efficacy. Reinforcing antibody-based immunotherapy by using chimeric antigen receptor (CAR) T cells may enhance the approach. However, the cells can cause severe on-target and off-tumor toxicities owing to their higher sensitivity to low-level antigen expressions. To address the need for safe and reliable targets, we made a bioinformatics pipeline by which we screened overexpressed genes in the disease for off-tumor sites in many normal tissues. Our inspection showed that MSLN (Mesothelin), ANTXR1 (TEM8), and MUC3A are the probable targets of CAR T cell therapy in gastric adenocarcinoma. The proposed antigenic targets might respond to the need to simultaneously target multiple antigens in a tumor matrix to prevent resistance.     

Targeting the tumor and its associated stroma: One and one can make three in adoptive T cell therapy of solid tumors

Adoptive T cell therapy (ACT) has become a promising immunotherapeutic option for cancer patients. The proof for ACT therapeutic efficacy was first obtained with allogenic T cells and then reproduced with T cells isolated from patients’ tumor samples (i.e. tumor-infiltrating lymphocytes). It is now clear that specificity of ACT products can be educated by genetically engineering T cells with classical T Cell Receptors (TCR) or chimeric antigen receptors (CAR). To date a poor accessibility of the tumor mass and a hostile microenvironment, influenced by genetic and epigenetic instability, mainly limit ACT therapeutic efficacy in the case of solid tumors. Available data indicate that these hurdles might be overcome by combinatorial therapeutic strategies targeting the tumor and its associated stroma. Here we review some of the available dual targeting strategies focusing on given combination of TCR/CAR-redirected T cell products and their association with drugs targeting the tumor-vessel and/or epigenetic modifiers, with the ability to sensitize tumors to T cell recognition. Existing data have proven synergistic effects in combined settings (one and one can indeed make three) and suggest that further benefit might be achieved by additional combinatorial therapeutic approaches (could one + one + one make ten?) in ACT of solid tumor.     

Role of Chimeric Antigen Receptor T Cell Therapy in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most common primary malignant cancer of brain, which is extremely aggressive and carries a dreadful prognosis. Current treatment protocol runs around radiotherapy, surgical resection, and temozolomide with median overall survival of around 12–15 months. Due to its heterogeneity and mutational load, immunotherapy with chimeric antigen receptor (CAR) T cell therapy can be a promising treatment option for recurrent glioblastoma. Initial phase 1 studies have shown that this therapy is safe without dose-limiting side effects and it also has a better clinical outcome. Therefore, CAR T cell therapy can be a great future tool in our armamentarium to treat advanced GBM. In this article, we have explained the structure, mechanism of action, and rationale of CAR T cell therapy in GBM; we also discussed various antigenic targets and clinical outcome of initial studies of this novel therapy.     

Secretion of human soluble programmed cell death protein 1 by chimeric antigen receptor-modified T cells enhances anti-tumor efficacy

Background aimsChimeric antigen receptor (CAR) T cells have achieved favorable responses in patients with hematologic malignancies, but the outcome has been far from satisfactory in the treatment of tumors with high expression of immunosuppressive molecules. To overcome this limitation, we modified CAR T cells to secrete types of human soluble programmed cell death protein 1 (PD-1) called sPD-1 CAR T cells.MethodsTo compare the effector function between second (conventional second-generation CAR targeting CD19) and sPD-1 CAR T cells, we measured cytotoxicity, cytokine secretion and activation markers incubated with or without tumor cells expressing CD19 and/or programmed cell death ligand 1 (PD-L1). Furthermore, the anti-tumor efficacy of second and sPD-1 CAR T cells was determined using an NSG mouse model bearing NALM-6-PD-L1. Finally, the underlying mechanism was investigated by metabolic parameters and RNA sequencing analysis of different CAR T cells.ResultsCompared with second CAR T cells, sPD-1 CAR T cells enhanced killing efficiency toward CD19⁺PD-L1⁺ tumor cells in vitro. Furthermore, sPD-1 CAR T cells reduced the tumor burden and prolonged overall survival of the NSG (NOD-SCID-IL2rg) mice bearing NALM-6-PD-L1. To explore the effect of soluble PD-1 on CAR T cells, we found that sPD-1 CAR T cells exhibited higher levels of activation and ameliorative profiles of differentiation, exhaustion, glycolysis and apoptosis.ConclusionsWith constitutive soluble PD-1 secretion, sPD-1 CAR T cells have tended to eradicate tumors with a high expression of PD-L1 more effectively than second CAR T cells. This may be due to soluble PD-1 enhancing apoptosis resistance, aerobic metabolism and a more “stem” differentiation of CAR T cells. Overall, our study presents a feasible strategy to increase the efficacy of CAR T cells.