Influencing factors and solution strategies of chimeric antigen receptor T-cell therapy(CAR–T)cell immunotherapy

Chimeric antigen receptor T-cesll therapy(CAR–T)has achieved groundbreaking advancements in clinical application,ushering in a new era for innovative cancer treatment.However,the challenges associated with implementing this novel targeted cell therapy are increasingly significant.Particularly in the clinical management of solid tumors,obstacles such as the immunosuppressive effects of the tumor microenvironment,limited local tumor infiltration capability of CAR–T cells,heterogeneity of tumor targeting antigens,uncertainties surrounding CAR–T quality,control,and clinical adverse reactions have contributed to increased drug resistance and decreased compliance in tumor therapy.These factors have significantly impeded the widespread adoption and utilization of this therapeutic approach.In this paper,we comprehensively analyze recent preclinical and clinical reports on CAR–T therapy while summarizing crucial factors influencing its efficacy.Furthermore,we aim to identify existing solution strategies and explore their current research status.Through this review article,our objective is to broaden perspectives for further exploration into CAR–T therapy strategies and their clinical applications.

T cells expressing a LMP1-specific chimeric antigen receptor mediate antitumor effects against LMP1-positive nasopharyngeal carcinoma cells in vitro and in vivo

T cells modified with chimeric antigen receptor are an attractive strategy to treat Epstein-Barr virus（EBV） associated malignancies.The EBV latent membrane protein 1（LMP1） is a 66-KD integral membrane protein encoded by EBV that consists of transmembrane-spanning loops.Previously,we have identified a functional signal chain variable fragment（scFv） that specifically recognizes LMP1 through phage library screening.Here,we constructed a LMP1 specific chimeric antigen receptor containing anti-LMP1 scFv,the CD28 signalling domain,and the CD3ζchain（HELA/CAR）.We tested its functional ability to target LMP1 positive nasopharyngeal carcinoma cells.HELA/CAR cells were efficiently generated using lentivirus vector encoding the LMP1-specific chimeric antigen receptor to infect activated human CD3＋ T cells.The HELA/CAR T cells displayed LMP1 specific cytolytic action and produced IFN-γ and IL-2 in response to nasopharyngeal carcinoma cells overexpressing LMP1.To demonstrate in vivo anti-tumor activity,we tested the HELA/CAR T cells in a xenograft model using an LMP1 overexpressing tumor.Intratumoral injection of anti-LMP1 HELA/CAR-T cells significantly reduced tumor growth in vivo.These results show that targeting LMP1 using HELA/CAR cells could represent an alternative therapeutic approach for patients with EBV-positive cancers.

Adoptive immunotherapy for acute leukemia:New insights in chimeric antigen receptors

Relapses remain a major concern in acute leukemia. It is well known that leukemia stem cells(LSCs) hide in hematopoietic niches and escape to the immune system surveillance through the outgrowth of poorly immunogenic tumor-cell variants and the suppression of the active immune response. Despitethe introduction of new reagents and new therapeutic approaches, no treatment strategies have been able to definitively eradicate LSCs. However, recent adoptive immunotherapy in cancer is expected to revolutionize our way to fight against this disease, by redirecting the immune system in order to eliminate relapse issues. Initially described at the onset of the 90's, chimeric antigen receptors(CARs) are recombinant receptors transferred in various T cell subsets, providing specific antigens binding in a non-major histocompatibility complex restricted manner, and effective on a large variety of human leukocyte antigen-divers cell populations. Once transferred, engineered T cells act like an expanding "living drug" specifically targeting the tumor-associated antigen, and ensure long-term antitumor memory. Over the last decades, substantial improvements have been made in CARs design. CAR T cells have finally reached the clinical practice and first clinical trials have shown promising results. In acute lymphoblastic leukemia, high rate of complete and prolonged clinical responses have been observed after anti-CD19 CAR T cell therapy, with specific but manageable adverse events. In this review, our goal was to describe CAR structures and functions, and to summarize recent data regarding pre-clinical studies and clinical trials in acute leukemia.

Focused evaluation of the roles of macrophages in chimeric antigen receptor (CAR) T cell therapy associated cytokine release syndrome

Cytokine release syndrome(CRS)is a major obstacle to the widespread clinical application of chimeric antigen receptor(CAR)T cell therapies.CRS can also be induced by infections(such as SARS-CoV-2),drugs(such as therapeutic antibodies),and some autoimmune diseases.Myeloid-derived macrophages play key roles in the pathogenesis of CRS,and participate in the production and release of the core CRS cytokines,including interleukin(IL)-1,IL-6,and interferon-γ.In this review,we summarize the roles of macrophages in CRS and discuss new developments in macrophage activation and the related mechanisms of cytokine regulation in CRS.

c-Met-targeted chimeric antigen receptor T cells inhibit hepatocellular carcinoma cells in vitro and in vivo

c-Met is a hepatocyte growth factor receptor overexpressed in many tumors such as hepatocellular carcinoma(HCC).Therefore,c-Met may serve as a promising target for HCC immunotherapy.Modifying T cells to express c-Met-specific chimeric antigen receptor(CAR)is an attractive strategy in treating c-Met-positive HCC.This study aimed to systematically evaluate the inhibitory effects of 2^(nd)-and 3^(rd)-generation c-Met CAR-T cells on hepatocellular carcinoma(HCC)cells.Here,2^(nd)-and 3^(rd)-generation c-Met CARs containing an anti-c-Met singlechain variable fragment(scFv)as well as the CD28 signaling domain and CD3ζ(c-Met-28-3ζ),the CD137 signaling domain and CD3ζ(c-Met-137-3ζ),or the CD28 and CD137 signaling domains and CD3ζ(c-Met-28-137-3ζ)were constructed,and their abilities to target c-Met-positive HCC cells were evaluated in vitro and in vivo.All c-Met CARs were stably expressed on T cell membrane,and c-Met CAR-T cells aggregated around c-Met-positive HCC cells and specifically killed them in vitro.c-Met-28-137-3ζCAR-T cells secreted more interferon-gamma(IFN-γ)and interleukin 2(IL-2)than c-Met-28-3ζCAR-T cells and c-Met-137-3ζCAR-T cells.Compared with c-Met low-expressed cells,c-Met CAR-T cells secreted more cytokines when co-cultured with c-Met high-expressed cells.Moreover,c-Met-28-137-3ζCAR-T cells eradicated HCC more effectively in xenograft tumor models compared with the control groups.This study suggests that 3^(rd)-generation c-Met CAR-T cells are more effective in inhibiting c-Met-positive HCC cells than 2^(nd)-generation c-Met CAR-T cells,thereby providing a promising therapeutic intervention for c-Met-positive HCC.

Advancement of chimeric antigen receptor-natural killer cells targeting hepatocellular carcinoma

With the advance of genome engineering technology,chimeric antigen receptors(CARs)-based immunotherapy has become an emerging therapeutic strategy for tumors.Although initially designed for T cells in tumor immunotherapy,CARs have been exploited to modify the function of natural killer(NK)cells against a variety of tumors,including hepatocellular carcinoma(HCC).CAR-NK cells have the potential to sufficiently kill tumor antigen-expressing HCC cells,independent of major histocompatibility complex matching or prior priming.In this review,we summarize the recent advances in genetic engineering of CAR-NK cells against HCC and discuss the current challenges and prospects of CAR-NK cells as a revolutionary cellular immunotherapy against HCC.

Chimeric Antigen Receptors and Regulatory T Cells:The Potential for HLA-Specific Immunosuppression in Transplantation

Chimeric antigen receptors(CARs)are a breakthrough in genetic engineering that have revolutio nized the field of adoptive cellular therapy(ACT).Cells expressing these receptors are rerouted to a predefined target by the inclusion of an antigen-specific binding region within the synthetic CAR construct.The advantage of cells with programmed specificity has been demonstrated clinically in the field of oncology,and it is clear that such cells have greater accuracy,potency,and reduced off-target therapeutic effects compared with their unmodified counterparts.In contrast to conventional T cells(Tconvs),regulatory T cells(Tregs)play a major role in suppressing immune activation and regulating the host immune response.CAR expression within Tregs has been proposed as a therapy for autoimmune and inflammatory diseases,graft-versus-host disease(GVHD),and organ transplant rejectio n.In the latter,they hold immense potential as mediators of immune tolerance for recipients of allotransplants.However,current research into CAR-Treg engineering is extremely limited,and there is uncertainty regarding optimal design for therapeutic use.This review examines the rationale behind the development of CAR-Tregs,their significance for human transplantation,potential designs,safety considerations,and comparisons of CAR-Tregs in transplantation models to date.

Chidamide and sintilimab combination in diffuse large B-cell lymphoma progressing after chimeric antigen receptor T therapy

BACKGROUND Diffuse large B-cell lymphoma(DLBCL)is curable with first-line chemoimmunotherapy but patients with relapsed/refractory(R/R)DLBCL still face a poor prognosis.For patients with R/R DLBCL,the complete response rate to traditional next-line therapy is only 7%and the median overall survival is 6.3 mo.Recently,CD19-targeting chimeric antigen receptor T cells(CAR-T)have shown promise in clinical trials.However,approximately 50%of patients treated with CAR-T cells ultimately progress and few salvage therapies are effective.CASE SUMMARY Here,we report on 7 patients with R/R DLBCL whose disease progressed after CAR-T infusion.They received a PD-1 inhibitor(sintilimab)and a histone deacetylase inhibitor(chidamide).Five of the 7 patients tolerated the treatment without any serious adverse events.Two patients discontinued the treatment due to lung infection and rash.At the 20-mo follow-up,the median overall survival of these 7 patients was 6 mo.Of note,there were 2 complete response rates(CRs)and 2 partial response rates(PRs)during this novel therapy,with an overall response rate(ORR)of 57.1%,and one patient had a durable CR that lasted at least 20 mo.CONCLUSION In conclusion,chidamide combined with sintilimab may be a choice for DLBCL patients progressing after CD19-targeting CAR-T therapy.

Refractory lymphoma treated with chimeric antigen receptor T cells combined with programmed cell death-1 inhibitor:A case report

BACKGROUND Diffuse large B-cell lymphoma(DLBCL)is a common aggressive non-Hodgkin's lymphoma(NHL),accounting for 30%-40%of adult NHL.Primary testicular(PT)lymphoma is an uncommon extranodal disease representing approximately 1%-2%of lymphoma.Approximately 30%–40%of patients are refractory to frontline therapy or relapse after complete remission.Refractory DLBCL responds poorly to other lines of chemotherapy,and experiences short-term survival.CASE SUMMARY We present a 41-year-old male patient who was diagnosed with PT-DLBCL.Further disease progression was observed after multiline chemotherapy.Chimeric antigen receptor T cells(CAR-T)therapy salvaged the patient.Unfortunately,a new mass was observed in the right adrenal area after six months.The patient was administered programmed cell death protein-1(PD-1)inhibitor therapy and maintained progression-free survival at more than 17 mo of follow-up.CONCLUSION Our findings support the potential benefit of CAR-T combined with PD-1 inhibitor therapies in this type of relapsed and refractory PT-DLBCL.

Programmed cell death protein-1 inhibitor combined with chimeric antigen receptor T cells in the treatment of relapsed refractory non- Hodgkin lymphoma: A case report

BACKGROUND Chimeric antigen receptor T cell(CART)therapy has benefited many refractory lymphoma patients,but some patients experience poor effects.Previous studies have shown that programmed cell death protein-1(PD-1)inhibitors can improve and prolong the therapeutic effect of CAR-T cell treatment.CASE SUMMARY A 61-year-old male presented with 15-d history of diarrhea and lower-limb edema.A large mass was detected in the pelvis,and pathology indicated non-Hodgkin diffuse large B-cell lymphoma.After three cycles of the R-CHOP chemotherapeutic regimen,the patient showed three subcutaneous nodules under the left armpit and both sides of the cervical spine.Pathological examination of the nodules indicated DLBCL again.The patient was diagnosed with relapsed and refractory diffuse large B-cell lymphoma.We recommended CAR-T cell treatment.Before treatment,the patient’s T cell function and expression of immune detection points were tested.Expression of PD-1 was obviously increased(52.7%)on cluster of differentiation(CD)3+T cells.The PD-1 inhibitor(3 mg/kg)was infused prior to lymphodepleting chemotherapy with fludarabine and cyclophosphamide.CAR-CD19 T cells of 3×10^(6)/kg and CAR-CD22 T cells 1×10^(6)/kg were infused,respectively.The therapeutic effect was significant,and the deoxyribonucleic acid copy numbers of CAR-CD19 T cells and CAR-CD22 T cells were stable.Presently,the patient has been disease-free for more than 12 mo.CONCLUSION This case suggests that the combination of PD-1 inhibitors and CAR-T cellsimproved therapeutic efficacy in B-cell lymphoma.

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.

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.

ncreasing the safety and efficacy of chimeric antigen receptor T cell therapy

Chimeric antigen receptor （CAR） T cell therapy is a promising cancer treatment that has recently been undergoing rapid development. However, there are still some major challenges, including precise tumor targeting to avoid off-target or ＂on-target/off-tumor＂ toxicity, adequate T cell infiltration and migration to solid tumors and T cell proliferation and persistence across the physical and biochemical barriers of solid tumors. In this review, we focus on the primary challenges and strafegies to design safe and effective CAR T cells, including using novel cutting-edge technologies for CAR and vecfor designs to increase both the safety and efficacy, further T cell modification to overcome the tumorassociated immune suppression, and using gene editing technologies to generate universal CAR T cells. All these efforts promote the development and evolution of CAR T cell therapy and move toward our ultimate goal--curing cancer with high safety, high efficacy, and low cost.

Chimeric antigen receptor- and natural killer cell receptor-engineered innate killer cells in cancer immunotherapy

Chimeric antigen receptor(CAR)-engineered T-cell(CAR-T)therapy has demonstrated impressive therapeutic efficacy against hematological malignancies,but multiple challenges have hindered its application,particularly for the eradication of solid tumors.Innate killer cells(IKCs),particularly NK cells,NKT cells,andγδT cells,employ specific antigen-independent innate tumor recognition and cytotoxic mechanisms that simultaneously display high antitumor efficacy and prevent tumor escape caused by antigen loss or modulation.IKCs are associated with a low risk of developing GVHD,thus offering new opportunities for allogeneic“off-the-shelf”cellular therapeutic products.The unique innate features,wide tumor recognition range,and potent antitumor functions of IKCs make them potentially excellent candidates for cancer immunotherapy,particularly serving as platforms for CAR development.In this review,we first provide a brief summary of the challenges hampering CAR-T-cell therapy applications and then discuss the latest CAR-NK-cell research,covering the advantages,applications,and clinical translation of CAR-and NK-cell receptor(NKR)-engineered IKCs.Advances in synthetic biology and the development of novel genetic engineering techniques,such as gene-editing and cellular reprogramming,will enable the further optimization of IKC-based anticancer therapies.

Advancing chimeric antigen receptor T cel therapy with CRISPR/Cas9

The clustered regularly interspaced short palindromic repeats （CRISPR）/CRISPR-associated 9 （CRISPR/Casg） system, an RNA-guided DNA targeting technology, is triggering a revolution in the field of biology. CRISPR/ Cas9 has demonstrated great potential for genetic manipulation. In this review, we discuss the current development of CRISPR/Cas9 technologies for thera- peutic applications, especially chimeric antigen receptor （CAR） T cell-based adoptive immunotherapy. Different methods used to facilitate efficient CRISPR delivery and gene editing in T cells are compared. The potential of genetic manipulation using CRISPR/Cas9 system to generate universal CAR T cells and potent T cells that are resistant to exhaustion and inhibition is explored. We also address the safety concerns associated with the use of CRISPR/Cas9 gene editing and provide potential solutions and future directions of CRISPR application in the field of CAR T cell immunotherapy. As an integration-free gene insertion method, CRISPR/Cas9 holds great promise as an efficient gene knock-in platform. Given the tremendous progress that has been made in the past few years, we believe that the CRISPPJ Cas9 technology holds immense promise for advancing immunotherapy.

Current advances in chimeric antigen receptor T-cell therapy for refractory/relapsed multiple myeloma

Multiple myeloma(MM),considered an incurable hematological malignancy,is characterized by its clonal evolution of malignant plasma cells.Although the application of autologous stem cell transplantation(ASCT)and the introduction of novel agents such as immunomodulatory drugs(IMiDs)and proteasome inhibitors(PIs)have doubled the median overall survival to eight years,relapsed and refractory diseases are still frequent events in the course of MM.To achieve a durable and deep remission,immunotherapy modalities have been developed for relapsed/refractory multiple myeloma(RRMM).Among these approaches,chimeric antigen receptor(CAR)T-cell therapy is the most promising star,based on the results of previous success in B-cell neoplasms.In this immunotherapy,autologous T cells are engineered to express an artificial receptor which targets a tumor-associated antigen and initiates the T-cell killing procedure.Tisagenlecleucel and Axicabtagene,targeting the CD19 antigen,are the two pacesetters of CAR T-cell products.They were approved by the US Food and Drug Administration(FDA)in 2017 for the treatment of acute lymphocytic leukemia(ALL)and diffuse large B-cell lymphoma(DLBCL).Their development enabled unparalleled efficacy in combating hematopoietic neoplasms.In this review article,we summarize six promising candidate antigens in MM that can be targeted by CARs and discuss some noteworthy studies of the safety profile of current CAR T-cell therapy.

Efficacy and safety of chimeric antigen receptor-T cells in the treatment of B cell lymphoma:a systematic review and meta-analysis

Background:Conventional treatment has limited efficacy in relapsed/refractory B-cell lymphoma.Since chimeric antigen receptor T-cell(CAR-T)technology has shown high safety and results in high remission rates,we investigated its efficacy and safety in B-cell lymphoma treatment and analyzed potential affecting factors to provide evidence for therapeutic strategies and applications.Methods:We searched databases including PubMed,Embase,and Cochrane up to July 2019.Meta-analysis 1 was conducted to study the efficacy of CAR-T cell for treating B-cell lymphoma,measuring the response rate and complete remission rate as outcomes.Sub-group analysis was performed for age,pathological type,target antigen,co-stimulatory molecule,and conditioning chemotherapy.Meta-analysis 2 was undertaken on the safety of the treatment with the incidence rate of toxicity(cytokine-releasing syndrome[CRS],neurotoxicity)as an outcome.Results:Seventeen studies were included in the systematic review and meta-analysis.It was found that CAR-T cells had good therapeutic effects in the following cases:B-cell lymphoma(patients≥65 years old);diffuse large B-cell lymphoma pathological type;patients with treatment target antigen other than CD19;patients treated with co-stimulatory molecules other than CD28,including 4-1BB+CD28 or 4-1BB;and patients treated with cyclophosphamide/fludarabine pre-treatment protocol conditioning chemotherapy.Although the CRS and neurotoxicity incidences were high,most were reversible with minimal risk of death.Conclusion:CAR-T cell treatment is safe for clinical application;however,toxicity effects should be monitored.

Adoptive transfer of T cells transduced with a chimeric antigen receptor to treat relapsed or refractory acute leukemia: efficacy and feasibility of immunotherapy approaches

Treatment outcomes of acute leukemia(AL) have not improved over the past several decades and relapse rates remain high despite the availability of aggressive therapies. Conventional relapsed leukemia treatment includes second allogeneic hematopoietic stem cell transplantation(allo-HSCT) and donor lymphocyte infusion(DLI), which in most cases mediate, at best, a modest graft-versus-leukemia effect, although their clinical efficacy is still limited. Although allo-HSCT following myeloablative conditioning is a curative treatment option for younger patients with acute myeloid leukemia(AML) in a first complete remission(CR), allo-HSCT as a clinical treatment is usually limited because of treatment-related toxicity. The overall DLI remission rate is only 15%–42% and 2-year overall survival(OS) is approximately 15%–20%, with a high(40%–60%) incidence of DLI-related graft-versus-host disease(GVHD). Therefore, development of new, targeted treatment strategies for relapsed and refractory AL patients is ongoing. Adoptive transfer of T cells with genetically engineered chimeric antigen receptors(CARs) is an encouraging approach for treating hematological malignancies. These T cells are capable of selectively recognizing tumor-associated antigens and may overcome many limitations of conventional therapies, inducing remission in patients with chemotherapy-refractory or relapsed AL. In this review, we aimed to highlight the current understanding of this promising treatment modality, discussing its adverse effects and efficacy.

Clinical development of chimeric antigen receptor-T cell therapy for hematological malignancies

Cellular therapies have revolutionized the treatment of hematological malignancies since their conception and rapid development.Chimeric antigen receptor(CAR)-T cell therapy is the most widely applied cellular therapy.Since the Food and Drug Administration approved two CD19-CAR-T products for clinical treatment of relapsed/refractory acute lymphoblastic leukemia and diffuse large B cell lymphoma in 2017,five more CAR-T cell products were subsequently approved for treating multiple myeloma or B cell malignancies.Moreover,clinical trials of CAR-T cell therapy for treating other hematological malignancies are ongoing.Both China and the United States have contributed significantly to the development of clinical trials.However,CAR-T cell therapy has many limitations such as a high relapse rate,adverse side effects,and restricted availability.Various methods are being implemented in clinical trials to address these issues,some of which have demonstrated promising breakthroughs.This review summarizes developments in CAR-T cell trials and advances in CAR-T cell therapy.

Cancer stem cell-targeted chimeric antigen receptor(CAR)-T cell therapy: Challenges and prospects

Cancer stem cells(CSCs)with their self-renewal ability are accepted as cells which initiate tumors.CSCs are regarded as interesting targets for novel anticancer therapeutic agents because of their association with tumor recurrence and resistance to conventional therapies,including radiotherapy and chemotherapy.Chimeric antigen receptor(CAR)-T cells are engineered T cells which express an artificial receptor specific for tumor associated antigens(TAAs)by which they accurately target and kill cancer cells.In recent years,CAR-T cell therapy has shown more efficiency in cancer treatment,particularly regarding blood cancers.The expression of specific markers such as TAAs on CSCs in varied cancer types makes them as potent tools for CAR-T cell therapy.Here we review the CSC markers that have been previously targeted with CAR-T cells,as well as the CSC markers that may be used as possible targets for CAR-T cell therapy in the future.Furthermore,we will detail the most important obstacles against CART cell therapy and suggest solutions.