Chimeric Antigen Receptor-modified T Research Articles

Co-infusion of CAR T cells with aAPCs expressing chemokines and costimulatory ligands enhances the anti-tumor efficacy in mice

Chimeric antigen receptor-modified T (CAR-T) cell therapy has shown curable efficacy for treating hematological malignancies, while in solid tumors, the immunosuppressive microenvironment causes poor activation, expansion and survival of CAR-T cells, accounting mainly for the unsatisfactory efficacy. The artificial antigen-presenting cells (aAPCs) have been used for ex vivo expansion and manufacturing of CAR-T cells. Here, we constructed a K562 cell-based aAPCs expressing human epithelial cell adhesion molecule (EpCAM), chemokines (CCL19 and CCL21) and co-stimulatory molecular ligands (CD80 and 4-1BBL). Our data demonstrated that the novel aAPCs enhanced the expansion, and increased the immune memory phenotype and cytotoxicity of CAR-T cells recognizing EpCAM, in vitro. Of note, co-infusion CAR-T and aAPC enhances the infiltration of CAR-T cells in solid tumors, which has certain potential for the treatment of solid tumors Moreover, IL-2-9-21, a cytokine cocktail, prevents CAR-T cells from entering the state of exhaustion prematurely after continuous antigen engagement and boosts the anti-tumor activity of CAR-T cells co-infused with aAPCs. These data provide a new strategy to enhance the therapeutic potential of CAR-T cell therapy for the treatment of solid tumors.

IL13Rα2-targeted third-generation CAR-T cells with CD28 transmembrane domain mediate the best anti-glioblastoma efficacy.

Chimeric antigen receptor (CAR)-modified T (CAR-T) cell therapy has been proven to be a powerful tool for the treatment of cancer, however, the limits are obvious, especially for solid tumors. Therefore, constantly optimizing the structure of CAR to improve its therapeutic effect is necessary. In this study, we generated three different third-generation CARs targeting IL13Rα2, with the same scFv, but different transmembrane domains (TMDs) from CD4, CD8 or CD28 (IL13-CD4TM-28.BB.ζ, IL13-CD8TM-28.BB.ζ and IL13-CD28TM-28.BB.ζ). CARs were transduced into primary T cells using retroviruses. The anti-GBM efficacy of CAR-T cells was monitored by flow cytometry and real-time cell analysis (RTCA) in vitro and examined in two xenograft mouse models. The differentially expressed genes related to different anti-GBM activity were screened by high throughput RNA sequencing. We observed that T cells transduced with these three CARs have similar anti-tumor activity when co-cultured with U373 cells which expressed higher IL13Rα2 but exhibited different anti-tumor activity when co-cultured with U251 cells that expressed lower IL13Rα2. All the three groups of CAR-T cells can be activated by U373 cells, but only IL13-CD28TM-28.BB.ζ CAR-T cells could be activated and expressed increased IFN-γ after co-culturing with U251 cells. IL13-CD28TM-28.BB.ζ CAR-T cells exhibited the best anti-tumor activity in xenograft mouse models which can infiltrate into the tumors. The superior anti-tumor efficacy of IL13-CD28TM-28.BB.ζ CAR-T cells was partially owing to differentially expressed extracellular assembly, extracellular matrix, cell migration and adhesion-related genes which contribute to the lower activation threshold, increased cell proliferation, and elevated migration capacity.

Convection-enhanced delivery of nanoencapsulated gene locoregionally yielding ErbB2/Her2-specific CAR-macrophages for brainstem glioma immunotherapy

Locoregional delivery of chimeric antigen receptor (CAR)-modified T (CAR-T) cells has emerged as a promising strategy for brain tumors. However, the complicated ex vivo cell manufacturing procedures and the rapid progression of the disease have limited its broader applications. Macrophages (MΦs) exhibit unique effector functions and a high degree of infiltration within the solid tumor microenvironment (TME), especially in the brain, where MΦs function as structural support, and the main immune effector cells of the CNS represent 5–12% of brain cells. Here, we report a synthetic universal DNA nanocarrier for in situ genetic editing of intratumoral MΦs with an ErbB2-specific CAR to direct their phagocytic activity towards tumors and subsequently initiate a locoregional antitumor immune response. Specifically, we demonstrated that when delivered locoregionally, the RP-182 peptide, located in the shell of a nanoparticle, targeted MΦs and reprogrammed M2-like tumor-associated macrophages (TAMs) to an antitumor M1-like phenotype. Subsequently, the CAR gene-laden DNA nanocomplex can be used to introduce ErbB2-targeted CAR, and the generated CAR-MΦs then act as “living” cures, thereby serially clearing the invasive tumor cells. Our work demonstrates a practical antitumor immunotherapy for brainstem gliomas (BSGs) that may be broadly applicable for patients suffering from other ErbB2-positive solid malignancies.

The Impact of Chimeric Antigen Receptor T Cell Quality to Clinical Safety and Efficacy in Non-Hodgkin Lymphomas

Background: Immunotherapy has become increasingly important in modern cancer treatments. It has become evident that chimeric antigen receptor-modified T (CAR-T) cells may be a promising treatment for hematological malignancies. Malignancies affecting B cells have been successfully treated using CAR-T cell therapies. Many studies have explored the influence of various factors on the efficacy and survival of CAR-T therapy from different aspects, including the patient's disease state, general condition, tumor burden, and even the dose of CAR-T cells infused. Few researchers have explored the effect of CAR-T cell quality on disease treatment impact on remission, survival, and prognosis. This study combines the essential condition of patients and quality data of CAR-T cells, such as transduction efficiency, killing efficiency, and the ratio of CD4+ and CD8+ subsets, to analyze CAR-T's safety and clinical efficacy in the treatment of non-Hodgkin's lymphoma in our center. Methods: This was a retrospective, single-center, investigator-initiated study. A total of 179 patients with NHL were included. Patients' baseline data were obtained from electronic medical records and telephone follow-ups. Laboratories provided data on the quality of patients' CAR-T cells. We conducted a multivariate analysis to determine the independent factors that influence treatment response and patient prognosis. Results: In our study, the median age of 179 NHL patients was 52 (range 17-78) years, and 51 (28.5%) were ≥60 years old. Before CAR-T treatment, 22 (12.3%) patients remained in complete remission (CR), and 83 (46.4%) patients were in progressive disease (PD). Among the available data, 105 (61.4%) patients had CAR-T cell killing efficiency (KE) <30%, while 66 (38.6%) patients had killing efficiency (KE) ≥30%. There were 101 (58%) patients with CAR-T cell transduction efficiency (TE) <40% and 73 (42%) with transduction efficiency (TE) ≥40%, respectively. CD4+/CD8+ ratio of CAR-T cells <1 in 32 (18.4%) patients and CD4+/CD8+ ratio of CAR-T cells ≥1 in 142 (81.6%) patients. Overall, the median event-free survival (PFS) was 10.6 months (95% CI: 4.064-17.136months), and the median overall survival has not yet been reached. The 6-month OS was 80.2%, and the 1-year OS was 70.6%. 2-year 0S is about 59.8%. In addition, we divided patients into different subgroups according to the TE and KE of CAR-T cells, and the results of multivariate logistic regression analysis were as follows. The subgroup with TE<40% had a higher complete remission rate than the subgroup with TE≥40%. And the subgroup with KE≥30% had a higher rate of complete remission than the subgroup with KE<30%, with p-values equal to 0.007 and 0.022, respectively. There was statistical significance, so TE and KE independently influenced efficacy factors. Multivariate cox regression analysis showed that the subgroup of CAR-T CD4+/CD8+<1 had better overall survival than CAR-T CD4+/CD8+≥1, p=0.039 (95% CI: 0.114-0.947). Therefore, the ratio of CAR-T CD4+/CD8+ was an independent factor for OS, while the subgroups of transduction efficiency and killing efficiency did not show statistical significance. Conclusion: In general, CAR-T therapy has shown significant efficacy in NHL, and the quality of CAR-T cells plays a vital role in treatment. Screening suitable CAR-T cells in future treatment will Make the treatment effect more satisfactory. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anti-CD 19 CAR-T Cell Therapy with Tisagenlecleucel for Ritcher Syndrome

About 2-10 percent of patients with chronic lymphocytic leukemia (CLL) will transform into an aggressive large cell lymphoma, called Ritcher syndrome (RS). The transformation is to DLBCL in 90-95% of cases and Hodgkin lymphoma in the remaining 5-10%. Current treatment strategies that typically mirror DLBCL have not significantly improved the historically dismal outcomes of RS. Chimeric antigen receptor-modified T (CAR-T) cells have revolutionized the treatment of B-cell malignancies and are currently approved for relapsed/refractory DLBCL. The impact of CAR-T cell therapy in the management of RS is limited by the small number of affected patients and published reports. We report the outcomes in 2 patients with RS treated with tisagenlecleucel (Tisa-cel) at our institution. Patient 1. This is a 75-year-old lady diagnosed with CLL at the age of 64, Rai stage 1, trisomy 12, mutated IGVH. She was on observation until she became symptomatic 5 yrs. after diagnosis and had a biopsy which showed CLL with RS, fluorescent in-situ hybridization (FISH) negative for C-MYC, BCL2 and BCL6. She was treated with RCHOP x 6 cycles with complete remission (CR) on PET. She received high dose chemotherapy with R-BEAM followed by autologous stem cell transplant, followed by 4 cycles of maintenance brentuximab given every 3 weeks for her CD30 positive large B cell lymphoma. About 5 yrs. later, surveillance PET showed hypermetabolic cervical lymph nodes and excisional biopsies showed CLL and DLBCL, non-germinal center type, concerning for relapsed RS. She was referred to us for CAR-T cell therapy evaluation. Bone marrow biopsy was negative for marrow involvement, minimal residual disease (MRD) by ClonoSEO was positive. She received lymphodepleting chemotherapy with fludarabine and cyclophosphamide followed by CAR-T cell infusion with tisa-cel 5x10^8 anti CD19 CAR-T cells on 4/18/22. Treatment was complicated by grade 1-2 cytokine release syndrome (CRS) (treated with tocilizumab and dexamethasone) requiring brief hospitalization. Day 90 PET showed CMR and bone marrow biopsy was negative for disease, MRD negative. She continues to be in CR 100 days post CAR-T cell therapy. Patient 2. A 63 years-old lady originally diagnosed with CLL in 2016 at the age of 57, with 17p deletion, 13q deletion and IgHV unmutated. She was started on ibrutinib shortly after due to a short lymphocyte doubling time. Three years later (in 2020), she developed worsening lymphadenopathy while on ibrutinib, underwent excisional biopsy and this showed RS with TP53 mutation, 17p deletion, gain of 13q, BCL2 positive. Venetoclax was added to ibrutinib and later, 2 cycles of Obinutuzumab were also given. Subsequent PET 7 months after showed disease progression with a lugano score of 5. She received 6 cycles of RCHOP in addition to venetoclax/ibrutinib but end of treatment PET showed disease progression. She was then referred to us for CAR-T cell evaluation. She received lymphodepleting chemotherapy with fludarabine and cyclophosphamide followed by CAR-T cell infusion with tis-cel 3.7 x 10^8 cells anti CD19 CAR-T cells on 6/7/21. This was complicated by grade 2 CRS that was treated with tocilizumab. Day 30 PET showed PR. Ibrutinib was resumed and the patient received radiation to right cervical lymph nodes. Day 100 PET reaffirmed PR and she subsequently underwent FluCyTBI RIC followed by haploidentical alloSCT from her daughter on 09/29/21. On day 180 post-transplant, the patient remained in CR by PET and bone marrow biopsy. She continues to be in CR now 1 year since her CAR-T cell infusion. Conclusion CAR-T cell therapy with Tisagenlecleucel shows promising activity in patients with RS. It is an evolving treatment strategy and more follow up is needed to assess the durability of the responses observed in these patients.

EP08.01-099 Activity of aPD1-MSLN-CART Cells against Metastatic Lung Cancer in a Phase 1 Trial

Mesothelin (MSLN) has been found to be highly expressive in multiple solid tumors including lung cancer. We engineered autocrine PD-1 nano antibodies MSLN CAR-T cells (aPD1-MSLN-CART), targeting MSLN positive tumor cells by chimeric antigen receptor-modified T (CAR-T) cells as well as secretion of PD1 antibodies, playing dual antitumor immune mechanism.

The tumor microenvironment of hepatocellular carcinoma and its targeting strategy by CAR-T cell immunotherapy.

Hepatocellular carcinoma (HCC) is the major subtype of liver cancer, which ranks sixth in cancer incidence and third in mortality. Although great strides have been made in novel therapy for HCC, such as immunotherapy, the prognosis remains less than satisfactory. Increasing evidence demonstrates that the tumor immune microenvironment (TME) exerts a significant role in the evolution of HCC and has a non-negligible impact on the efficacy of HCC treatment. In the past two decades, the success in hematological malignancies made by chimeric antigen receptor-modified T (CAR-T) cell therapy leveraging it holds great promise for cancer treatment. However, in the face of a hostile TME in solid tumors like HCC, the efficacy of CAR-T cells will be greatly compromised. Here, we provide an overview of TME features in HCC, discuss recent advances and challenges of CAR-T immunotherapy in HCC.

CAR-T cell therapy for lung cancer: Potential and perspective.

Lung cancer is the highest incidence and mortality of all cancers around the world. In the present immunotherapy era, an increasing number of immunotherapeutic agents including monoclonal antibody‐targeted drugs have been used in the clinical treatment of malignancy, but it still has many limitations. Chimeric antigen receptor‐modified T (CAR‐T) cells, a novel adoptive immunotherapy strategy, have not only been used successfully against hematological tumors, but have also opened up new avenues for immunotherapy of solid tumors, including lung cancer. However, targeting lung cancer‐specific antigens using engineered CAR‐T cells is complicated by the lack of proper tumor‐specific antigens, an immunosuppressive tumor microenvironment, a low level of CAR‐T cell infiltration into tumor tissues, along with off‐target effect, etc. Simultaneously, the clinical application of CAR‐T cells remains limited because of many challenges such as tumor lysis syndrome, neurotoxicity syndrome, and cytokine release syndrome. In this review, we outline the basic structure and generation characteristic of CAR‐T cells and summarize the common tumor‐associated antigens in clinical trials of CAR‐T cell therapy for lung cancer, and point out the current challenges and new strategies, aiming to provide new ideas and approaches for the pre‐clinical experiments and clinical trials of CAR‐T cell therapy in lung cancer.

The progress of research on chimeric antigen receptor NK cells

Chimeric antigen receptor-modified T (CAR-T) cells have brought a major breakthrough in tumor immunotherapy. As research continues to develop, CAR-T therapies have shown certain limitations, such as graft-versus-host reaction and the long time required to generate CAR-T cells. Natural killer (NK) cells, as the first line of defense against pathogens and cancer cells, belong to the innate immune system and have the advantage of being able to replace T cells, and can be used to prepare engineered chimeric antigen receptor-modified NK (CAR-NK) cells. The biological characteristics of CAR-NK cells, their sources, and the latest research progress and prospects for tumor treatment in China and worldwide are presented in this review.

Progress in the Application of Immune Checkpoint Inhibitor-Based Immunotherapy for Targeting Different Types of Colorectal Cancer.

Colorectal cancer (CRC), a common malignant disease, has the second highest mortality rate among all cancer types. Due to the diversity and heterogeneity of CRC, few effective treatment strategies have been developed in recent years, except for surgical resection. As immunotherapy has become a revolutionary treatment after surgery, along with chemoradiotherapy and targeted therapy, numerous basic research studies and clinical trials have been conducted on CRC. Therefore, immune checkpoint inhibitor (ICI) therapy has become the main anti-CRC immunotherapy method used at present. With the rapid development of biotechnology and cell research, an increasing number of monotherapy or combination therapy strategies using ICIs for CRC have been designed in recent years. Methods to classify and review ICI strategies for different types of CRC to better guide treatment are continuously investigated. However, the identification of why the ICIs would be more effective in targeting particular subtypes of CRC such as high microsatellite instability (MSI-H) is more important because of the different immune backgrounds in patients. This review intends to classify different subtypes of CRC and summarizes the basic and clinical studies on ICIs for each subtype of CRC currently available. In addition, we also attempt to briefly discuss the progress in immunotherapy methods other than ICI therapy, such as chemoimmunotherapy strategy, chimeric antigen receptor-modified T (CAR-T) cells, or immunotherapy based on oncolytic viruses. Finally, we provide a perspective on the development of immunotherapy in the treatment of CRC and attempt to propose a new systematic classification of CRC based on immunological strategies, which may improve guidance for the selection of immunotherapy strategies for different subtypes of CRC in the future.

Chimeric Antigen Receptor T Cell Targeting to CD19 and CD22 Combined with Anti-PD-1 Antibody Induce High Response in Patients with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

Objective: Despite the remarkable success of chimeric antigen receptor modified T (CAR-T) cell therapy for refractory or relapsed B cell non-Hodgkin lymphoma (R/R B-NHL), high rates of treatment failure and relapse after CAR-T cell therapy are considerable obstacles to overcome. Preclinical models have demonstrated that anti-PD-1 antibody is an attractive option following CAR-T therapy to reverse T cell exhaustion. Thus, we investigated their combination in R/R B-NHL.Methods: We performed a prospective, single-arm study of CAR-T cell combined with anti-PD-1 antibody treatment in R/R B-NHL (NCT04539444). Anti-PD-1 antibody was administrated on day 1 after patients received sequential infusion of anti-CD19 and anti-CD22 second-generation CAR-T cells, and the efficacy and safety of the combination treatment were evaluated.Results: From August 1, 2020 to June 30, 2021, a total of 11 patients were enrolled and completed at least 3 months follow-up. The median follow-up time is 5.8 months. Overall response was achieved in 9 of 11 patients (81.8%), and the complete response (CR) was achieved in 8 of 11 patients (72.7%). All 8 patients achieving CR still sustained remission at the last follow-up. The progression-free survival (PFS) and overall survival (OS) rates at 6 months were 80.8% and 100.0%, respectively. Cytokine release syndrome (CRS) occurred in only 4 patients (all were grade 1), and no neurotoxicity were observed.Conclusion: This study suggests that CAR-T cells combined with anti-PD-1 antibody elicit a safe and durable response in R/R B-NHL.Keywords: chimeric antigen receptor modified T cell, anti-PD-1 antibody, CD19/CD22, refractory or relapsed B cell non-Hodgkin lymphoma DisclosuresNo relevant conflicts of interest to declare. OffLabel Disclosure:We use the T cells were transduced with a lentivirus encoding the CD19-4-1BB-CD3 z and CD22-4-1BB-CD3 ztransgene to produce CAR-T cells. The main purpose of our study is to improve the response rate in patients with R/R B-NHL.

Primary mediastinal large B cell lymphoma.

Primary mediastinal large B cell lymphoma (PMBCL) is an aggressive large B cell lymphoma originating in the mediastinum, that mainly expresses B cell surface molecules, such as CD19, CD20, CD22, andCD79a. Clinically, they are characterized by rapidly increasing anterior mediastinal masses, which can cause compression of the surrounding tissues. The diagnosis of PMBCL mainly depends on the pathological features, imaging examination and clinical features. Currently, the most commonly used therapeutic regimens are R‐CHOP and R‐EPOCH. Radiotherapy is beneficial in some patients, but it can also lead to long‐term toxicity. The research and development of novel therapies are ongoing, and some studies have achieved encouraging results, including those conducted on chimeric antigen receptor‐modified T (CAR‐T) cell therapy and anti‐PD‐1 drugs. However, randomized controlled trials with larger sample sizes are still needed. Positron emission tomography‐computed tomography (PET‐CT) is mainly used to assess the curative effect after treatment and to guide the subsequent treatment strategy.

Cardiac Toxicity Associated with Cancer Immunotherapy and Biological Drugs.

Simple SummaryImmunotherapy is increasingly being used to treat solid tumors and lymphoproliferative diseases. The main classes of drugs are: HER-2-targeted therapies, CTLA-blockers, PD/PDL-1 inhibitors, CAR-T therapy. All these drugs are associated with meaningful cardiac toxicity, ranging from a transient decline of left ventricular function with complete reversibility to myocarditis with a high fatality rate.Cancer immunotherapy significantly contributed to an improvement in the prognosis of cancer patients. Immunotherapy, including human epidermal growth factor receptor 2 (HER2)-targeted therapies, immune checkpoint inhibitors (ICI), and chimeric antigen receptor-modified T (CAR-T), share the characteristic to exploit the capabilities of the immune system to kill cancerous cells. Trastuzumab is a monoclonal antibody against HER2 that prevents HER2-mediated signaling; it is administered mainly in HER2-positive cancers, such as breast, colorectal, biliary tract, and non-small-cell lung cancers. Immune checkpoint inhibitors (ICI) inhibit the binding of CTLA-4 or PD-1 to PDL-1, allowing T cells to kill cancerous cells. ICI can be used in melanomas, non-small-cell lung cancer, urothelial, and head and neck cancer. There are two main types of T-cell transfer therapy: tumor-infiltrating lymphocytes (or TIL) therapy and chimeric antigen receptor-modified T (CAR-T) cell therapy, mainly applied for B-cell lymphoma and leukemia and mantle-cell lymphoma. HER2-targeted therapies, mainly trastuzumab, are associated with left ventricular dysfunction, usually reversible and rarely life-threatening. PD/PDL-1 inhibitors can cause myocarditis, rare but potentially fulminant and associated with a high fatality rate. CAR-T therapy is associated with several cardiac toxic effects, mainly in the context of a systemic adverse effect, the cytokines release syndrome.

Infectious complications following chimeric antigen receptor T-cell therapy for a hematologic malignancy within 28 days

目的探讨血液肿瘤患者在回输嵌合抗原受体T细胞（CAR-T细胞）后28 d内感染的发生率、临床和微生物学特征及危险因素。方法回顾性分析2016年1月至2020年12月在华中科技大学同济医学院附属协和医院血液科成功接受CAR-T细胞输注的170例血液肿瘤患者基线资料以及回输后28 d内发生感染的临床与病原菌特征。使用泊松回归评估基线特征与感染密度的相关性，单因素和多因素Cox比例风险回归模型评估CAR-T细胞回输后感染的高危因素。结果170例患者中急性淋巴细胞白血病（ALL）72例、非霍奇金淋巴瘤（NHL）56例、多发性骨髓瘤（MM）42例，99例患者在28 d内共发生119次感染，累积感染发生率为（58.2±3.8）％；其中细菌感染98次（45.9％），总感染密度为2.01，首次感染发生的中位时间为回输后12（1～18）d。多因素泊松回归显示ALL患者、难治性疾病、既往30 d内感染病史、回输前ANC<0.5×109/L、前期治疗方案≥4种的患者在28 d内与感染密度有更高的相关性；多因素Cox回归显示≥3级细胞因子释放综合征（CRS）是发生感染的独立危险因素（HR＝3.74，95％CI 1.29～5.72，P<0.001）。结论感染是血液病患者CAR-T治疗后常见的并发症之一，不同肿瘤的感染发生率与类型有所差异，但均以细菌感染为主。ALL患者、难治性疾病、既往30天内感染病史、回输前ANC<0.5×109/L、前期治疗方案≥4种与感染密度相关；3级以上CRS是感染发生的独立危险因素。中国临床试验注册中心：ChiCTR-OIC-17011180、ChiCTR1800018143

Testicular involvement of acute lymphoblastic leukemia in children and adolescents: Diagnosis, biology, and management.

Acute lymphoblastic leukemia (ALL) in children and adolescents can involve the testes at diagnosis or upon relapse. The testes were long considered pharmacologic sanctuary sites, presumably because of the blood-testis barrier, which prevents the entry of large-molecular-weight compounds into the seminiferous tubule. Patients with testicular involvement were historically treated with testicular irradiation or orchiectomy. With the advent of contemporary intensive chemotherapy, including high-dose methotrexate, vincristine/glucocorticoid pulses, and cyclophosphamide, testicular leukemia present at diagnosis can be eradicated, with the risk of testicular relapse being 2% or lower. However, the management of testicular leukemia is not well described in the recent literature and remains relevant in low- and middle-income countries where testicular relapse is still experienced. Chemotherapy can effectively treat late, isolated testicular B-cell ALL relapses without the need for irradiation or orchiectomy in patients with an early response and thereby preserve testicular function. For refractory or early-relapse testicular leukemia, newer treatment approaches such as chimeric antigen receptor-modified T (CAR-T) cell therapy are under investigation. The control of testicular relapse with CAR-T cells and their penetration of the blood-testis barrier have been reported. The outcome of pediatric ALL has been improved remarkably by controlling the disease in the bone marrow, central nervous system, and testes, and such success should be extended globally. LAY SUMMARY: Acute lymphoblastic leukemia (ALL) in children and adolescents can involve the testes at diagnosis or upon relapse. Modern intensive chemotherapy has largely eradicated testicular relapse in high-income countries. Consequently, most current clinicians are not familiar with how to manage it if it does occur, and testicular relapse continues to be a significant problem in low- and middle-income countries that have not had access to modern intensive chemotherapy. The authors review the historical progress made in eradicating testicular ALL and use the lessons learned to make recommendations for treatment.

Antibodies against vaccine-preventable infections after CAR-T cell therapy for B cell malignancies.

BACKGROUNDLittle is known about pathogen-specific humoral immunity after chimeric antigen receptor–modified T (CAR-T) cell therapy for B cell malignancies.METHODSWe conducted a prospective cross-sectional study of CD19-targeted or B cell maturation antigen–targeted (BCMA-targeted) CAR-T cell therapy recipients at least 6 months posttreatment and in remission. We measured pathogen-specific IgG against 12 vaccine-preventable infections and the number of viral and bacterial epitopes to which IgG was detected (“epitope hits”) using a serological profiling assay. The primary outcome was the proportion of participants with IgG levels above a threshold correlated with seroprotection for vaccine-preventable infections.RESULTSWe enrolled 65 children and adults a median of 20 months after CD19- (n = 54) or BCMA- (n = 11) CAR-T cell therapy. Among 30 adults without IgG replacement therapy (IGRT) in the prior 16 weeks, 27 (90%) had hypogammaglobulinemia. These individuals had seroprotection to a median of 67% (IQR, 59%–73%) of tested infections. Proportions of participants with seroprotection per pathogen were comparable to population-based studies, but most individuals lacked seroprotection to specific pathogens. Compared with CD19-CAR-T cell recipients, BCMA-CAR-T cell recipients were half as likely to have seroprotection (prevalence ratio, 0.47; 95% CI, 0.18–1.25) and had fewer pathogen-specific epitope hits (mean difference, –90 epitope hits; 95% CI, –157 to –22).CONCLUSIONSeroprotection for vaccine-preventable infections in adult CD19-CAR-T cell recipients was comparable to the general population. BCMA-CAR-T cell recipients had fewer pathogen-specific antibodies. Deficits in both groups support the need for vaccine and immunoglobulin replacement therapy studies.FUNDINGSwiss National Science Foundation (Early Postdoc Mobility grant P2BSP3_188162), NIH/National Cancer Institute (NIH/NCI) (U01CA247548 and P01CA018029), NIH/NCI Cancer Center Support Grants (P30CA0087-48 and P30CA015704-44), American Society for Transplantation and Cellular Therapy, and Juno Therapeutics/BMS.

Secretion of bispecific protein of anti-PD-1 fused with TGF-β trap enhances antitumor efficacy of CAR-T cell therapy

Despite the remarkable success of chimeric antigen receptor-modified T (CAR-T) cell therapy for blood malignancies, the clinical efficacy of this novel therapy in solid tumor treatment is largely limited by the immunosuppressive tumor microenvironment (TME). For instance, immune checkpoints (e.g., programmed cell death protein 1 [PD-1]/programmed death ligand 1 [PD-L1]) in TME play an important role in inhibiting T cell proliferation and functions. Transforming growth factor β (TGF)-β secreted by cancer cells in TME induces regulatory T cells (Tregs) and inhibits cytotoxic T cells. To overcome the inhibitory effect of immune checkpoints, we have previously engineered CAR-T cells to secrete anti-PD-1 to block the PD-1/PD-L1 pathway activity, a step demonstrating superior antitumor efficacy compared with conventional CAR-T cells. In this study, we engineered CAR-T cells that secrete bispecific trap protein co-targeting PD-1 and TGF-β, with the aim of further improving antitumor immunity. Compared with conventional CAR-T cells and anti-PD-1-secreting CAR-T cells, data from in vitro and in vivo experiments showed that CAR-T cells with trap protein secretion further attenuated inhibitory T cell signaling, enhanced T cell persistence and expansion, and improved effector function and resistance to exhaustion. In the xenograft mouse model, CAR-T cells with trap protein secretion exhibited significantly enhanced antitumor immunity and efficacy. With these observations, we demonstrate the potential of trap protein self-secreting CAR-T cells as a potent therapy for solid tumors.

A phase I clinical trial of PSMA-directed/TGFβ-insensitive CAR-T cells in metastatic castration-resistant prostate cancer.

125 Background: Prostate specific membrane antigen (PSMA) is a highly expressed tumor-associated antigen potentially amenable to chimeric antigen receptor-modified T (CAR-T) cell therapy for castration-resistant prostate cancer (CRPC). However, a primary challenge to the success of CAR-T therapy in CRPC is the immunosuppressive microenvironment, characterized by high levels of TGFβ. The immunosuppressive functions of TGFβ can be inhibited in T cells using a dominant negative TGFβ receptor (TGFβRdn), thereby enhancing antitumor immunity. Methods: We conducted a first-in-human phase 1 clinical trial to evaluate the feasibility, safety and preliminary efficacy of PSMA-directed/TGFβ-insensitive CAR-T cells (CART-PSMA-TGFβRdn) in patients with metastatic CRPC (NCT03089203). In a 3+3 dose-escalation design, patients received a single dose of 1-3 x 107/m2 (Cohort 1) or 1-3 x 108/m2 (Cohort 2) CART-PSMA-TGFβRdn cells without lymphodepleting (LD) chemotherapy. In Cohort 3, one patient received 1-3 x 108/m2 CART-PSMA-TGFβRdn cells following a LD chemotherapy regimen of cyclophosphamide and fludarabine (Cy/Flu). In Cohort -3, three patients received 1-3 x 107/m2 CART-PSMA-TGFβRdn cells following Cy/Flu. Patients underwent metastatic tumor biopsies at baseline and on day 10 following treatment. Quantitative PCR of CART-PSMA-TGFβRdn DNA was performed at serial timepoints to evaluate for CAR-T expansion and persistence in peripheral blood and trafficking to target tissues. Multiplex cytokine analysis assessed CART-PSMA-TGFβRdn bioactivity. Results: Ten patients received CART-PSMA-TGFβRdn therapy across dose-level cohorts. All CART-PSMA-TGFβRdn infusion products met target transduction efficiency. Evaluation of CAR-T cellular kinetics demonstrated dose-dependent peripheral blood T cell expansion, as well as tumor tissue trafficking in post-treatment tumor biopsies. At Cohort 2 and above, 5 of 7 treated patients developed grade ≥2 cytokine release syndrome (CRS). Marked increases in inflammatory cytokines (IL-6, IL-15, IL-2, IFNγ) correlated with high-grade CRS events. One grade 5 adverse event (sepsis) occurred in Cohort 3. PSA decline was observed in 6 of 10 patients (median decline -33.2%, range -11.6% to -98.3%), and PSA30 response occurred in 4 of 10 patients (including one patient achieving PSA &lt; 0.1 ng/mL). Conclusions: Adoptive cellular therapy with CART-PSMA-TGFβRdn is safe and feasible in patients with metastatic CRPC. A dose-dependent and lymphodepletion chemotherapy-dependent relationship was observed with CART-PSMA-TGFβRdn cell expansion, cytokine expression, CRS, and anti-tumor effect. Correlative cell trafficking and paired tumor Nanostring analyses will be presented. Future clinical investigations seek to enhance anti-tumor efficacy, while optimizing the therapeutic window. Clinical trial information: NCT03089203.

Culturing adequate CAR-T cells from less peripheral blood to treat B-cell malignancies.

Objective:Chimeric antigen receptor-modified T (CAR-T) cells have shown impressive results against relapsed/refractory B cell malignancies. However, the traditional manufacture of CAR-T cells requires leukapheresis to isolate large amounts of peripheral blood T cells, thus making some patients ineligible for the procedure.Methods:We developed a simple method for CAR-T cell preparation requiring small volumes of peripheral blood. First, CD3+ T cells isolated from 50 mL peripheral blood from patients (B-cell malignancies) were stimulated with immobilized anti-CD3/RetroNectin in 6-well plates and then transduced with CAR-expressing lentiviral vector. After 4 d, the T cells were transferred to culture bags for large-scale CAR-T cell expansion. In vitro and animal experiments were performed to evaluate the activity of the manufactured CAR-T cells. Finally, 29 patients with B-cell acute lymphoblastic leukemia (B-ALL) and 9 patients with B-cell lymphoma were treated with the CAR-T cells.Results:The CAR-T cells were expanded to 1–3 × 108 cells in 8–10 d and successfully killed B cell-derived malignant tumor cells in vitro and in vivo. For patients with B-ALL, the complete remission rate was 93% 1 month after CAR-T cell infusion; after 12 months, the overall survival (OS) and leukemia-free survival rates were 69% and 31%, respectively. For patients with lymphoma, the objective response rate (including complete and partial remission) was 78% 2 months after CAR-T cell infusion, and after 12 months, the OS and progression-free survival rates were 71% and 43%, respectively. Cytokine-release syndrome (CRS) occurred in 65.51% and 55.56% of patients with B-ALL and B-cell lymphoma, respectively; severe CRS developed in 20.69% of patients with B-ALL and in no patients with lymphoma.Conclusions:Our novel method can generate sufficient numbers of CAR-T cells for clinical use from 50–100 mL peripheral blood, thus providing an alternative means of CAR-T cell generation for patients ineligible for leukapheresis.

Efficacy and Safety of CAR-Modified T Cell Therapy in Patients with Relapsed or Refractory Multiple Myeloma: A Meta-Analysis of Prospective Clinical Trials.

Background: In recent years, chimeric antigen receptor-modified T (CAR-T) cell therapy for B-cell leukemia and lymphoma has shown high clinical efficacy. Similar CAR-T clinical trials have also been carried out in patients with refractory/relapsed multiple myeloma (RRMM). However, no systematic review has evaluated the efficacy and safety of CAR-T cell therapy in RRMM. The purpose of this study was to fill this literature gap. Methods: Eligible studies were searched in PUBMED, EMBASE, the Cochrane Central Register of Controlled Trials (CENTRAL), CNKI, and WanFang from data inception to December 2019. For efficacy assessment, the overall response rate (ORR), minimal residual disease (MRD) negativity rate, strict complete response (sCR), complete response (CR), very good partial response (VGPR), and partial response (PR) were calculated. The incidence of any grade cytokine release syndrome (CRS) and grade ≥3 adverse events (AEs) were calculated for safety analysis. The effect estimates were then pooled using an inverse variance method. Results: Overall, 27 studies involving 497 patients were included in this meta-analysis. The pooled ORR and MRD negativity rate were 89% (95% Cl: 83–94%) and 81% (95% Cl: 67–91%), respectively. The pooled sCR, CR, VGPR, and PR were 14% (95% Cl: 5–27%), 13% (95% Cl: 4–26%), 23% (95% Cl: 14–33%), and 15% (95% Cl: 10–21%), respectively. Subgroup analyses of ORR by age, proportion of previous autologous stem cell transplantation (ASCT), and target selection of CAR-T cells revealed that age ≤ 55 years (≤55 years vs. > 55 years, p = 0.0081), prior ASCT ≤70% (≤70% vs. > 70%, p = 0.035), and bispecific CAR-T cells (dual B-cell maturation antigen (BCMA)/BCMA + CD19 vs specific BCMA, p = 0.0329) associated with higher ORR in patients. Subgroup analyses of remission depth by target selection suggested that more patients achieved a better response than VGPR with dual BCMA/BCMA + CD19 CAR-T cells compared to specific BCMA targeting (p = 0.0061). In terms of safety, the pooled incidence of any grade and grade ≥ 3 CRS was 76% (95% CL: 63–87%) and 11% (95% CL: 6–17%). The most common grade ≥ 3 AEs were hematologic toxic effects. Conclusion: In heavily treated patients, CAR-T therapy associates with promising responses and tolerable AEs, as well as CRS in RRMM. However, additional information regarding the durability of CAR-T cell therapy, as well as further randomized controlled trials, is needed.