Characteristics of premanufacture CD8+T cells determine CAR-T efficacy in patients with diffuse large B-cell lymphoma.

Although chimeric antigen receptor (CAR) T cells have become an important treatment option for patients with relapsed/refractory B-cell malignancies, more than 60% of patients with diffuse large B-cell lymphoma (DLBCL) treated with CAR-T cell therapies fail to achieve a durable response. To reveal changes in CAR-T cell therapy and identify response biomarkers, we conducted a retrospective analysis of pre-manufacture source T cells and CAR-T cell products and their association with outcome in 58 patients with r/rDLBCL who received tandem CD19/CD20 CAR-T cell therapy. We performed bulk RNA-Seq, single-cell RNA-Seq, and paired T cell receptor sequencing on CAR-T cell products and pre-manufacture T cells from DLBCL patients. We note that a CD8+ stem cell-like memory T cell population with a higher proportion and enhanced activating capacity of the CAR-T cell products was key to achieving durable clinical response. By analysing autologously-derived, pre-manufacture T cells, our data suggest that heterogeneity in the cellular and molecular features of pre-manufacture T cells contribute to the variation in efficacy after CAR-T cell therapy in DLBCL. The differences in anti-tumour efficacy of CAR-T cells among patients with different clinical outcomes appear to be due to the loss of CCR7 gene expression, coupled with increased expression of activation- and inhibitor-related genes in the CD8+ naïve-T cell populations among the apheresis T cells from patients with a poor molecular response. These findings significantly advance our understanding of the underlying molecular determinants of pre-manufacture T cell function.

CD58 loss in tumor cells confers functional impairment of CAR T cells.

Chimeric antigen receptor (CAR) T-cell therapy has achieved significant success in treating a variety of hematologic malignancies, but resistance to this treatment in some patients limited its wider application. Using an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) screening, we identified and validated loss of CD58 conferred immune evasion from CAR T cells in vitro and in vivo. CD58 is a ligand of the T-cell costimulatory molecule CD2, and CD58 mutation or downregulated expression is common in hematological tumors. We found that disruption of CD58 in tumor cells induced the formation of suboptimal immunological synapse (IS) with CAR T cells, which conferred functional impairment of CAR T cells, including the attenuation of cell expansion, degranulation, cytokine secretion, and cytotoxicity. In summary, we describe a potential mechanism of tumor-intrinsic resistance to CAR T-cell therapy and suggest that this mechanism may be leveraged for developing therapeutic strategies to overcome resistance to CAR T-cell therapy in B-cell malignancies.

Identification of NOXA as a pivotal regulator of resistance to CAR T-cell therapy in B-cell malignancies.

Despite the remarkable success of chimeric antigen receptor (CAR) T-cell therapy for treating hematologic malignancies, resistance and recurrence still occur, while the markers or mechanisms underlying this resistance remain poorly understood. Here, via an unbiased genome-wide CRISPR/Cas9 screening, we identified loss of NOXA, a B-cell lymphoma 2 (BCL2) family protein in B-cell malignancies, as a pivotal regulator of resistance to CAR T-cell therapy by impairing apoptosis of tumor cells both in vitro and in vivo. Notably, low NOXA expression in tumor samples was correlated with worse survival in a tandem CD19/20 CAR T clinical trial in relapsed/refractory B-cell lymphoma. In contrast, pharmacological augmentation of NOXA expression by histone deacetylase (HDAC) inhibitors dramatically sensitized cancer cells to CAR T cell-mediated clearance in vitro and in vivo. Our work revealed the essentiality of NOXA in resistance to CAR T-cell therapy and suggested NOXA as a predictive marker for response and survival in patients receiving CAR T-cell transfusions. Pharmacological targeting of NOXA might provide an innovative therapeutic strategy to enhance CAR T-cell therapy.

Long-term activity of tandem CD19/CD20 CAR therapy in refractory/relapsed B-cell lymphoma: a single-arm, phase 1-2 trial.

Increasing the remission rate and reducing the recurrence rate can improve the clinical efficacy of chimeric antigen receptor (CAR) T cell therapy in recurrent/refractory non-Hodgkin lymphoma (r/rNHL). In this open-label, single-arm phase I/II trial, 87 patients with r/rNHL, including 58 patients with aggressive diffuse large B-cell lymphoma and 24 with high tumour burden, received an infusion at doses of 0.5 × 106-8 × 106 TanCAR7 T cells per kilogram of body weight after conditioning chemotherapy. The best overall response rate was 78% (95% confidence interval [CI], 68-86); response rates were consistent across prognostic subgroups. The median follow-up was 27.7 months. The median progression-free survival was 27.6 months (95% CI, 11 to not reached). Cytokine release syndrome (CRS) occurred in 61 patients (70%) with 60% of cases being grade 1 or 2 and 10% being grade 3 or greater. Grade 3 CAR T cell-related encephalopathy syndrome (CRES) occurred in 2 patients (2%). Two patients died from treatment-associated severe pulmonary infection, and one died from CRS-related pulmonary injury between 1 and 3 months post infusion. Long-term remissions were observed following the use of TanCAR7 T cells in r/rNHL with a safety profile that included CRS but few cases of CRES.

Mutant B2M-HLA-E and B2M-HLA-G fusion proteins protects universal chimeric antigen receptor-modified T cells from allogeneic NK cell-mediated lysis.

Recent studies have indicated the antitumor activity and reduced allogeneic response of universal chimeric antigen receptor-modified T (UCAR T) cells lacking endogenous T cell receptors and beta-2 microglobulin (B2M) generated using gene-editing technologies. However, these cells are vulnerable to lysis by allogeneic natural killer (NK) cells due to their lack of human leukocyte antigen (HLA) class I molecule expression. Here, constitutive expression of mutant B2M-HLA-E (mBE) and B2M-HLA-G (mBG) fusion proteins in anti-CD19 UCAR T (UCAR T-19) cells was conducted to protect against allogeneic NK cell-mediated lysis. The ability of cells expressing mBE or mBG to resist NK cell-mediated lysis was observed in gene-edited Jurkat CAR19 cells. UCAR T-19 cells constitutively expressing the mBE and mBG fusion proteins were manufactured and showed effective and specific anti-tumor activity. Constitutive expression of the mBE and mBG fusion proteins in UCAR T-19 cells prevented allogeneic NK cell-mediated lysis. In addition, these cells were not recognizable by allogeneic T cells. Additional experiments, including those in animal models and clinical trials, are required to evaluate the safety and efficacy of UCAR T-19 cells that constitutively express mBE and mBG.

Low-dose decitabine priming endows CAR T cells with enhanced and persistent antitumour potential via epigenetic reprogramming.

Insufficient eradication capacity and dysfunction are common occurrences in T cells that characterize cancer immunotherapy failure. De novo DNA methylation promotes T cell exhaustion, whereas methylation inhibition enhances T cell rejuvenation in vivo. Decitabine, a DNA methyltransferase inhibitor approved for clinical use, may provide a means of modifying exhaustion-associated DNA methylation programmes. Herein, anti-tumour activities, cytokine production, and proliferation are enhanced in decitabine-treated chimeric antigen receptor T (dCAR T) cells both in vitro and in vivo. Additionally, dCAR T cells can eradicate bulky tumours at a low-dose and establish effective recall responses upon tumour rechallenge. Antigen-expressing tumour cells trigger higher expression levels of memory-, proliferation- and cytokine production-associated genes in dCAR T cells. Tumour-infiltrating dCAR T cells retain a relatively high expression of memory-related genes and low expression of exhaustion-related genes in vivo. In vitro administration of decitabine may represent an option for the generation of CAR T cells with improved anti-tumour properties.

Clinical development of CAR T cell therapy in China: 2020 update.

Chimeric antigen receptor (CAR) T-cell therapy has achieved significant success in the treatment of hematological malignancies. In recent years, fast-growing CAR T clinical trials have actively explored their potential application scenarios. According to the data from the clinicaltrials.gov website, China became the country with the most registered CAR T trials in September 2017. As of June 30, 2020, the number of registered CAR T trials in China has reached 357. In addition, as many as 150 other CAR T trials have been registered on ChiCTR. Although CAR T therapy is flourishing in China, there are still some problems that cannot be ignored. In this review, we aim to systematically summarize the clinical practice of CAR T-cell therapy in China. This review will provide an informative reference for colleagues in the field, and a better understanding of the history and current situation will help us more reasonably conduct research and promote cooperation.

Efficacy and biomarker analysis of CD133-directed CAR T cells in advanced hepatocellular carcinoma: a single-arm, open-label, phase II trial.

Expressed by cancer stem cells of various epithelial cell origins and hepatocellular carcinoma (HCC), CD133 is an attractive therapeutic target for HCC. The marker CD133 is highly expressed in endothelial progenitor cells (EPC). EPCs circulate in increased numbers in the peripheral blood of patients with highly vascularized HCC and contribute to angiogenesis and neovascularization. This phase II study investigated CD133-directed chimeric antigen receptor (CAR) T (CART-133) cells in adults with HCC. Patients with histologically confirmed and measurable advanced HCC and adequate hematologic, hepatic, and renal functions received CART-133 cell infusions. The primary endpoints were safety in phase I and progression-free survival (PFS) and overall survival (OS) in phase II. Other endpoints included biomarkers for CART-133 T cell therapy. Between June 1, 2015, and September 1, 2017, this study enrolled 21 patients who subsequently received CART-133 T cells across phases I and II. The median OS was 12 months (95% CI, 9.3-15.3 months) and the median PFS was 6.8 months (95% CI, 4.3-8.4 months). Of 21 evaluable patients, 1 had a partial response, 14 had stable disease for 2 to 16.3 months, and 6 progressed after T-cell infusion. The most common high-grade adverse event was hyperbilirubinemia. Outcome was correlated with the baseline levels of vascular endothelial growth factor (VEGF), soluble VEGF receptor 2 (sVEGFR2), stromal cell-derived factor (SDF)-1, and EPC counts. Changes in EPC counts, VEGF, SDF-1, sVEGFR2, and interferon (IFN)-γ after cell infusion were associated with survival. In patients with previously treated advanced HCC, CART-133 cell therapy demonstrates promising antitumor activity and a manageable safety profile. We identified early changes in circulating molecules as potential biomarkers of response to CART-133 cells. The predictive value of these proangiogenic and inflammatory factors as potential biomarkers of CART-133 cell therapy in HCC will be explored in prospective trials. This study is registered at ClinicalTrials.gov (NCT02541370).

Optimized tandem CD19/CD20 CAR-engineered T cells in refractory/relapsed B-cell lymphoma.

Chimeric antigen receptor (CAR) T cells targeting CD19 have achieved breakthroughs in the treatment of hematological malignancies, such as relapsed/refractory non-Hodgkin lymphoma (r/rNHL); however, high rates of treatment failure and recurrence after CAR T-cell therapy are considerable obstacles to overcome. In this study, we designed a series of tandem CARs (TanCARs) and found that TanCAR7 T cells showed dual antigen targeting of CD19 and CD20, as well as formed superior and stable immunological synapse (IS) structures, which may be related to their robust antitumor activity. In an open-label single-arm phase 1/2a trial (NCT03097770), we enrolled 33 patients with r/rNHL; 28 patients received an infusion after conditioning chemotherapy. The primary objective was to evaluate the safety and tolerability of TanCAR7 T cells. Efficacy, progression-free survival, and overall survival were evaluated as secondary objectives. Cytokine release syndrome occurred in 14 patients (50%): 36% had grade 1 or 2 and 14% had grade 3. No cases of CAR T-cell-related encephalopathy syndrome (CRES) of grade 3 or higher were confirmed in any patient. One patient died from a treatment-associated severe pulmonary infection. The overall response rate was 79% (95% confidence interval [CI], 60-92%), and the complete response rate was 71%. The progression-free survival rate at 12 months was 64% (95% CI, 43-79%). In this study, TanCAR7 T cells elicited a potent and durable antitumor response, but not grade 3 or higher CRES, in patients with r/rNHL.

Anti-EGFR chimeric antigen receptor-modified T cells in metastatic pancreatic carcinoma: A phase I clinical trial.

The current clinical outcome for patients with metastatic pancreatic carcinoma (PC) remains poor. Epidermal growth factor receptor (EGFR) is detectable in PC, suggesting that EGFR is a rational target in PC. We conducted a phase I clinical trial to evaluate the safety and efficacy of autologous anti-EGFR chimeric antigen receptor-modified T (CAR T-EGFR) cells in patients with metastatic PC. The expression levels of EGFR on tumor cells detected by immunohistochemistry were required to be more than 50%. Sixteen patients were enrolled and received one to three cycles of the CAR T-EGFR cell infusion within 6 months (median dose of CAR T cells: 3.48 × 106/kg; range, 1.31 to 8.9 × 106/kg) after the conditioning regimen with 100 to 200 mg/m2 nab-paclitaxel and 15 to 35 mg/kg cyclophosphamide. Grade ≥3 adverse events included fever/fatigue, nausea/vomiting, mucosal/cutaneous toxicities, pleural effusion and pulmonary interstitial exudation and were reversible. Of 14 evaluable patients, four achieved partial response for 2-4 months, and eight had stable disease for 2-4 months. The median progression-free survival was 3 months (range, 4-months) from the first cycle of CAR T-EGFR cell treatment, and the median overall survival of all 14 evaluable patients was 4.9 months (range, 2.9-30 months). Decreased EGFR expression on tumor cells was observed in patients who achieved stable disease with shrinkage of metastatic lesions in the liver, and enrichment of central memory T cells in infused cells improved the clinical response. In conclusion, the treatment with CAR T-EGFR cells is safe and effective in patients with metastatic PC. This trial was registered at www.clinicaltrials.gov (identifier no: NCT01869166).

Correction to: Bispecific CAR-T cells targeting both CD19 and CD22 for therapy of adults with relapsed or refractory B cell acute lymphoblastic leukemia.

An amendment to this paper has been published and can be accessed via the original article.

Bispecific CAR-T cells targeting both CD19 and CD22 for therapy of adults with relapsed or refractory B cell acute lymphoblastic leukemia.

BACKGROUND: Despite the impressive complete remission (CR) induced by CD19 CAR-T cell therapy in B-ALL, the high rate of complete responses is sometimes limited by the emergence of CD19-negative leukemia. Bispecific CAR-modified T cells targeting both CD19 and CD22 may overcome the limitation of CD19-negative relapse. METHODS: We here report the design of a bispecific CAR simultaneous targeting of CD19 and CD22. We performed a phase 1 trial of bispecific CAR T cell therapy in patients with relapsed/refractory precursor B-ALL at a dose that ranged from 1.7 × 106 to 3 × 106 CAR T cells per kilogram of body weight. RESULTS: We demonstrate bispecific CD19/CD22 CAR T cells could trigger robust cytolytic activity against target cells. MRD-negative CR was achieved in 6 out of 6 enrolled patients. Autologous CD19/CD22 CAR T cells proliferated in vivo and were detected in the blood, bone marrow, and cerebrospinal fluid. No neurotoxicity occurred in any of the 6 patients treated. Of note, one patient had a relapse with blast cells that no longer expressed CD19 and exhibited diminished CD22 site density approximately 5 months after treatment. CONCLUSION: In brief, autologous CD19/CD22 CAR T cell therapy is feasible and safe and mediates potent anti-leukemic activity in patients with relapsed/refractory B-ALL. Furthermore, the emergence of target antigen loss and expression downregulation highlights the critical need to anticipate antigen escape. Our study demonstrates the reliability of bispecific CD19/CD22 CAR T cell therapy in inducing remission in adult patients with relapsed/refractory B-ALL. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03185494.

PD-1 silencing impairs the anti-tumor function of chimeric antigen receptor modified T cells by inhibiting proliferation activity.

BACKGROUND: Blocking programmed death-1 (PD-1) is considered to be a promising strategy to improve T cell function, and this is being explored in many ongoing clinical trials. In fact, our knowledge about PD-1 is primarily based on the results of short-term experiments or observations, but how long-lasting PD-1 blockade can affect T cell function remains unclear. METHODS: We planned to use shRNA-based gene knockdown technology to mimic long-lasting PD-1 blockade. We constructed PD-1 steadily blocked chimeric antigen receptor modified T (CAR-T) cells, and with these cells we can clearly study the effects of PD-1 knockdown on T cell function. The anti-tumor function, proliferation ability and differentiation status of PD-1 silenced CAR-T cells were studied by in vitro and animal experiments. RESULTS: According to short-term in vitro results, it was reconfirmed that the resistance to programmed death-ligand 1 (PD-L1)-mediated immunosuppression could be enhanced by PD-1 blockade. However, better anti-tumor function was not presented by PD-1 blocked CAR-T cells in vitro or in vivo experiments. It was found that PD-1 knockdownmight impair the anti-tumor potential of CAR-T cells because it inhibited T cells' proliferation activity. In addition, we observed that PD-1 blockade would accelerate T cells' early differentiation and prevent effector T cells from differentiating into effect memory T cells, and this might be the reason for the limited proliferation of PD-1 silenced CAR-T cells. CONCLUSION: These results suggest that PD-1 might play an important role in maintaining the proper proliferation and differentiation of T cells, and PD-1 silencing would impair T cells' anti-tumor function by inhibiting their proliferation activity.

Haploidentical CD19/CD22 bispecific CAR-T cells induced MRD-negative remission in a patient with relapsed and refractory adult B-ALL after haploidentical hematopoietic stem cell transplantation.

BACKGROUND: Chimeric antigen receptor T (CAR-T) cell therapy simultaneously against CD19 and CD22 is an attractive strategy to address the antigen escape relapse after CD19-directed CAR-T cell therapies. However, the potential of optimizing the durability of remission by this approach in patients with B cell acute lymphoblastic leukemia (B-ALL) remains a critical unanswered question so far. CASE PRESENTATION: We treated an adult patient with relapsed and refractory B-ALL after haploidentical hematopoietic stem cell transplantation (HSCT) by administering haploidentical CAR-T cells targeting both CD19 and CD22 following preparative lymphodepleting chemotherapy. This patient has remained in minimal residual disease-negative remission for more than 14 months and has been tapered off graft versus host disease prophylaxis. CONCLUSIONS: CAR simultaneously targeting CD19 and CD22 has the potential of inducing long-term remission in patients with B-ALL.

Restoration of CD3+CD56+ cell level improves skin lesions in severe psoriasis: A pilot clinical study of adoptive immunotherapy for patients with psoriasis using autologous cytokine-induced killer cells.

Psoriasis is a chronic inflammatory skin disorder mediated by the cells and molecules of both the innate and adaptive immune systems. Autologous cytokine-induced killer (CIK) cell infusion is considered an effective and safe cancer treatment and is licensed for this use in China. Accumulated evidence indicating that CD3+CD56+ cells are significantly decreased in psoriatic patients prompted us to investigate if the restoration of CD3+CD56+ cells may be beneficial for psoriatic patients. We designed a clinical trial for psoriasis treatment that involved CIK cell infusion because CIK cells include a large amount of CD3+CD56+ T cells (NCT01894373 at www.clinicaltrials.gov). Six patients with severe psoriasis were initially enrolled, and four of them exhibited markedly lower levels of CD3+CD56+ cells in their peripheral blood (PB) relative to healthy donors. CIK cell infusion-associated toxicity was not observed in any infusion. The percentage of CD3+CD56+ cells in the PB markedly increased and the psoriasis area and severity index (PASI) synchronously decreased in four patients with lower CD3+CD56+ cell contents, and two of them obtained a more than 4-month PASI75 after completing a four-cycle treatment. However, a decrease in the CD3+CD56+ cells was observed concomitantly with disease recurrence after short-term amelioration. In contrast, no obvious improvement was observed in the two patients with nearly normal CD3+CD56+ cells in the PB before treatment. These observations suggest that the normalization of the CD3+CD56+ cell level may improve the skin lesions of severe psoriasis and warrant further clinical trials for severe psoriasis using repeated CIK adoptive immunotherapy.

CD133-directed CAR T cells for advanced metastasis malignancies: A phase I trial.

Expressed by cancer stem cells of various epithelial cell origins, CD133 is an attractive therapeutic target for cancers. Autologous chimeric antigen receptor-modified T-cell directed CD133 (CART-133) was first tested in this trial. The anti-tumor specificity and the postulated toxicities of CART-133 were first assessed. Then, we conducted a phase I clinical study in which patients with advanced and CD133-positive tumors received CART-133 cell-infusion. We enrolled 23 patients (14 with hepatocellular carcinoma [HCC], 7 with pancreatic carcinomas, and 2 with colorectal carcinomas). The 8 initially enrolled patients with HCC were treated by a CART-133 cell dose escalation scheme (0.05-2 × 106/kg). The higher CAR-copy numbers and its reverse relationship with the count of CD133+ cells in peripheral blood led to the determination of an acceptable cell dose is 0.5-2 × 106/kg and reinfusion cycle in 23 patients. The primary toxicity is a decrease in hemoglobin/platelet (≤ grade 3) that is self-recovered within 1 week. Of 23 patients, three achieved partial remission, and 14 achieved stable disease. The 3-month disease control rate was 65.2%, and the median progression-free survival was 5 months. Repeated cell infusions seemed to provide a longer period of disease stability, especially in patients who achieved tumor reduction after the first cell-infusion. 21 out of 23 patients had not developed detectable de novo lesions during this term. Analysis of biopsied tissues by immunohistochemistry showed CD133+ cells were eliminated after CART-133 infusions. This trial showed the feasibility, controllable toxicities, and effective activity of CART-133 transfer for treating patients with CD133-postive and late-stage metastasis malignancies.

Excessive activated T-cell proliferation after anti-CD19 CAR T-cell therapy.

Excessive activated T-cell proliferation was observed in vivo in one patient after an anti-CD19-chimeric antigen receptor (CAR) T-cell infusion. The patient, who had chemotherapy refractory and CD19+ diffuse large B-cell lymphoma (DLBCL), received an anti-CD19 CAR T-cell infusion following conditioning chemotherapy (fludarabine/cyclophosphamide). The lymphocyte count in the peripheral blood (PB) increased to 77 × 109/L on day 13 post infusion, and the proportion of CD8+ actived T cells was 93.06% of the lymphocytes. Then, the patient suffered from fever and hypoxaemia. Significant increases in serum cytokine, lactate dehydrogenase, aspartate aminotransferase (AST), alanine transaminase (ALT), and glutamic-oxalacetic transaminase (γ-GT) levels were observed. A high-throughput sequencing analysis for T-cell receptors (TCRs) and whole-genome sequencing were used to explore the mechanisms underlying this excessive T-cell proliferation. TCR diversity was demonstrated, but no special gene mutation was found. The patient was found to be infected with the John Cunningham polyomavirus (JCV). It cannot be ruled out the bystander activation pathway induced by JCV infections related the excessive activated T-cell proliferation. Although the clinical and laboratory data do not fully explain the reason for excessive T-cell proliferation after the anti-CD19 CAR T-cell infusion, the risk of this type of toxicity should be emphasized. This study was registered at www.clinicaltrials.gov as NCT01864889.