Cytokine-induced killer (ClK) cells:from basic research to clinical translation

The accumulation of basic researches and clinical studies related to cytokine-induced killer(CIK) cells has confirmed their safety and feasibility in treating malignant diseases.This review summarizes the available published literature related to the biological characteristics and clinical applications of CIK cells in recent years.A number of clinical trials with CIK cells have been implemented during the progressive phases of cancer,presenting potential widespread applications of CIK cells for the future.Furthermore,this review briefly compares clinical applications of CIK cells with those of other adoptive immunotherapeutic cells.However,at present,there are no uniform criteria or large-scale preparations of CIK cells.The overall clinical response is difficult to evaluate because of the use of autologous CIK cells.Based on these observations,several suggestions regarding uniform criteria and universal sources for CIK cell preparations and the use of CIK cells either combined with chemotherapy or alone as a primary strategy are briefly proposed in this review.Large-scale,controlled,grouped,and multi-center clinical trials on CIK cell-based immunotherapy should be conducted under strict supervision.These interventions might help to improve future clinical applications and increase the clinical curative effects of CIK cells for a broad range of malignancies in the future.

Reciprocal activation of antigen-presenting cells and CAR T cells triggers a widespread endogenous anti-tumor immune response through sustained high-level IFNγproduction

Adoptive cell transfer(ACT)using chimeric antigen receptor(CAR)modified T cells and T cell receptor(TCR)engineered T cells has shown therapeutic efficacy in cancer treatment1,2.CAR T cells are widely applicable to tumor patients because of their ability to directly identify tumor cells in an MHCindependent manner.

Chimeric antigen receptor-modified T cells for the immunotherapy of patients with EGFR-expressing advanced relapsed/refractory non-small cell lung cancer

The successes achieved by chimeric antigen receptor-modified T （CAR-T） cells in hematological malignancies raised the pos- sibility of their use in non-small lung cancer （NSCLC）. In this phase I clinical study （NCT01869166）, patients with epidermal growth factor receptor （EGFR）-positive （〉50% expression）, relapsed/refractory NSCLC received escalating doses of EGFR-targeted CAR-T cell infusions. The EGFR-targeted CAR-T cells were generated from peripheral blood after a 10 to 13-day in vitro expansion. Serum cytokines in peripheral blood and copy numbers of CAR-EGFR transgene in peripheral blood and in tissue biopsy were monitored periodically. Clinical responses were evaluated with RECISTI.1 and im- mune-related response criteria, and adverse events were graded with CTCAE 4.0. The EGFR-targeted CAR-T cell infusions were well-tolerated without severe toxicity. Of 11 evaluable patients, two patients obtained partial response and five had stable disease for two to eight months. The median dose of transfused CAR＋ T cells was 0.97x 10^7 cells kg J （interquar- tile range （IQR）, 0.45 to 1.09x 10^7 cells kg 1）. Pathological eradication of EGFR positive tumor cells after EGFR-targeted CAR-T cell treatment can be observed in tumor biopsies, along with the CAR-EGFR gene detected in tumor-infiltrating T cells in all four biopsied patients. The EGFR-targeted CAR-T cell therapy is safe and feasible for EGFR-positive advanced re- lapsed/refractory NSCLC.

Phase I study of chimeric antigen receptor modified T cells in treating HER2-positive advanced biliary tract cancers and pancreatic cancers

This phase I clinical trial （NCT01935843） is to evaluate the safety, feasibility, and activity of chimeric antigen receptor-engineered T cell （CART） immunotherapy tar- geting human epidermal growth factor receptor 2 （HER2） in patients with advanced biliary tract cancers （BTCs） and pancreatic cancers （PCs）. Eligible patients with HER2-positive （〉50%） BTCs and PCs were enrolled in the trial. Well cultured CART-HER2 cells were infused following the conditioning treatment composed of nab- paclitaxel （100-200 mg/m2） and cyclophosphamide （15-35 mglkg）. CAR transgene copy number in the peripheral blood was serially measured to monitor the expansion and persistence of CART-HER2 cells in vivo. Eleven enrolled patients received 1 to 2-cycle CART- HER2 cell infusion （median CAR＋ T cell 2.1× 10^6/kg）. The conditioning treatment resulted in mild-to-moderate fatigue, nausea/vomiting, myalgialarthralgia, and lym- phopenia. Except one grade-3 acute febrile syndrome and one abnormal elevation of transaminase （〉9 ULN）, adverse events related to the infusion of CART-HER2 cells were mild-to-moderate. Post-infusion toxicities included one case of reversible severe upper gastroin- testinal hemorrhage which occurred in a patient with gastric antrum invaded by metastasis 11 days after the CART-HER2 cell infusion, and 2 cases of grade 1-2delayed fever, accompanied by the release of C-reactive protein and interleukin-6. All patients were evaluable for assessment of clinical response, among which 1 obtained a 4.5-months partial response and 5 achieved stable disease. The median progression free survival was 4.8 months （range, 1.5-8.3 months）. Finally, data from this study demonstrated the safety and feasibility of CART-HER2 immunotherapy, and showed encourag- ing signals of clinical activity.

Current status and perspectives of chimeric antigen receptor modified T cells for cancer treatment

Chimeric antigen receptor （CAR） is a recombinant immunoreceptor combining an antibody-derived target- ing fragment with signaling domains capable of acti- vating cells, which endows T cells with the ability to recognize tumor-associated surface antigens indepen- dent of the expression of major histocompatibiiity complex （MHC） molecules. Recent early-phase clinical trials of CAR-modified T （CAR-T） cells for relapsed or refractory B cell malignancies have demonstrated promising results （that is, anti-CD19 CAR-T in B cell acute lymphoblastic leukemia （B-ALL））. Given this suc- cess, broadening the clinical experience of CAR-T cell therapy beyond hematological malignancies has been actively investigated. Here we discuss the basic design of CAR and review the clinical results from the studies of CAR-T cells in B cell leukemia and lymphoma, and several solid tumors. We additionally discuss the major challenges in the further development and strategies for increasing anti-tumor activity and safety, as well as for successful commercial translation.

Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro and xenotransplanted tumors in vivo

Human epidermal growth factor receptor 2 （HER2） pro- teins are overexpressed in a high proportion of gastric cancer （GC） cases and affect the maintenance of cancer stem cell （CSC） subpopulations, which are used as tar- gets for the clinical treatment of patients with HER2- positive GC. Despite improvements in survival, numer- ous HER2-positive patients fail treatment with trastuzu- mab, highlighting the need for more effective therapies. In this study, we generated a novel type of genetically modified human T cells, expressing a chimeric antigen receptor （CAR）, and targeting the GC cell antigen HER2, which harbors the CD137 and CD3/; moieties. Our findings show that the expanded CART cells, expressing an increased central memory phenotype, were activated by the specific recognition of HER2 antigens in an MHC-in- dependent manner, and effectively killed patient-derived HER2-positive GC cells. In HER2-positive xenograft tumors, CART cells exhibited considerably enhanced tumor inhibition ability, long-term survival, and homing totargets, compared with those of non-transduced T cells. The sphere-forming ability and in vivo tumorigenicity of patient-derived gastric cancer stem-like cells, expressing HER2 and the CD44 protein, were also inhibited. Our results support the future development and clinical application of this adoptive immunotherapy in patients with HER2-positive advanced GC.

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.

Targeting cancer stem cells by using chimeric antigen receptor-modified T cells： a potential and curable approach for cancer treatment

Cancer stem cells （CSCs）, a subpopulation of tumor cells, have self-renewal and multi-lineage differentiation abilities that play an important role in cancer initiation, mainte- nance, and metastasis. An accumulation of evidence indicates that CSCs can cause conventional therapy fail- ure and cancer recurrence because of their treatment resistance and self-regeneration characteristics. There- fore, approaches that specifically and efficiently eliminate CSCs to achieve a durable clinical response are urgently needed. Currently, treatments with chimeric antigen receptor-modified T （CART） cells have shown successful clinical outcomes in patients with hematologic malignan- cies, and their safety and feasibility in solid tumors was confirmed. In this review, we will discuss in detail the possibility that CART cells inhibit CSCs by specifically targeting their cell surface markers, which will ultimately improve the clinical response for patients with various types of cancer. A number of viewpoints were summarized to promote the application of CSC-targeted CART cells in clinical cancer treatment. This review covers the key aspects of CSC-targeted CART cells against cancers in accordance with the premise of the model, from bench to bedside and back to bench.

Efficiency and side effects of anti-CD38 CAR T cells in an adult patient with relapsed B-ALL after failure of bi-specific CD19/CD22 CAR T cell treatment

(CAR)T cells and immune checkpoint blockade therapies has shown remarkable outcomes in the field of cancer treatment.1,2 In particular,great success has been achieved in B cell acute lymphocytic leukemia(B-ALL)treated with anti-CD19 CAR T cells.2 However,the loss,mutation,and reduced expression of CD19 are crucial causes for the failure of CAR T cell treatment in leukemia patients.3,4 CD38,a 45-kDa type II glycoprotein,is detectable in BALL,and several studies have indicated the efficacy and safety of CD38 monoclonal antibodies in clinical applications,suggesting that CD38 represents a suitable therapeutic target for the treatment of B-ALL.5,6 In addition,we developed a CAR against CD38,and our preclinical study indicated that anti-CD38 CAR T(CAR T-38)cells could specifically lyse CD38-positive tumor cells.7 Herein,we present the anti-leukemia ability of CAR T-38 cells in an adult relapsed B-ALL patient after failure of bi-specific CD19/CD22 CAR T cell treatment,and this administration of CAR T-38 cells was accompanied by the occurrence of target-mediated toxicities and severe and uncontrollable cytokine release syndrome(CRS).

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.

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.

An analytical biomarker for treatment of patients with recurrent B-ALL after remission induced by infusion of anti-CD19 chimeric antigen receptor T(CAR-T) cells

Anti-CD19 chimeric antigen receptor-modified T(CAR-T-19) cells have emerged as a powerful targeted immunotherapy for B-cell lineage acute lymphoblastic leukemia with a remarkable clinical response in recent trials. Nonetheless, few data are available on the subsequent clinical monitoring and treatment of the patients, especially those with disease recurrence after CAR-T-19 cell infusion. Here, we analyzed three patients who survived after our phase I clinical trial and who were studied by means of biomarkers reflecting persistence of CAR-T-19 cells in vivo and predictive factors directing further treatment. One patient achieved 9-week sustained complete remission and subsequently received an allogeneic hematopoietic stem cell transplant. Another patient who showed relapse after 20 weeks without detectable leukemia in the cerebrospinal fluid after CAR-T-19 cell treatment was able to achieve a morphological remission under the influence of stand-alone low-dose chemotherapeutic agents. The third patient gradually developed extensive extramedullary involvement in tissues with scarce immune-cell infiltration during a long period of hematopoietic remission after CAR-T-19 cell therapy. Long-term and discontinuous increases in serum cytokines(mainly interleukin 6 and C-reactive protein) were identified in two patients(Nos. 1 and 6) even though only a low copy number of CAR molecules could be detected in their peripheral blood. This finding was suggestive of persistent functional activity of CAR-T-19 cells. Combined analyses of laboratory biomarkers with their clinical manifestations before and after salvage treatment showed that the persistent immunosurveillance mediated by CAR-T-19 cells would inevitably potentiate the leukemia-killing effectiveness of subsequent chemotherapy in patients who showed relapse after CAR-T-19-induced remission.