CD19 chimeric antigen receptor–redirected T cells combined with epidermal growth factor receptor pathway substrate 8 peptide–derived dendritic cell vaccine in leukemia

BackgroundChimeric antigen receptor (CAR)–T cell therapy opens a new era for cancer treatment. However, in prolonged follow-up, relapse has emerged as one of the major obstacles. Dendritic cell (DC) vaccination is a promising treatment to eradicate tumor cells and prevent relapse. The epidermal growth factor receptor (EGFR) pathway substrate 8 (Eps8) gene is involved in regulating cancer progression and is considered an attractive target for specific cancer immunotherapy. The purpose of this study was to explore a combinatorial therapy using CAR-T cells and a DC vaccine such as Eps8-DCs to increase leukemia treatment efficacy.MethodsWe pulsed DCs with Eps8-derived peptides to generate Eps8-DCs, engineered T cells to express a second-generation CAR specific for CD19, and analyzed the effects of the Eps8-DCs on the in vitro expansion, phenotype and effector functions of the CD19 CAR-T cells.ResultsThe Eps8-DCs significantly reduced the activation-induced cell death and enhanced the proliferative potential of CAR-T cells during in vitro expansion. In addition, the expanded T cells co-cultured with the Eps8-DCs exhibited an increased percentage of central memory T cells (Tcms) and a decreased percentage of effector memory T cells (Tems). The Eps8-DCs enhanced CD19 CAR-T cell immune functions, including cytokine production, CD107a degranulation activity and cytotoxicity.DiscussionThis study demonstrates that Eps8-DCs exert synergistic effect on CD19 targeting CAR-T cells and paves the way for clinical trials using the combination of DC vaccination and engineered T cells in relapsed leukemia.     

Cytotoxic activity of anti-mucin 1 chimeric antigen receptor T cells expressing PD-1-CD28 switch receptor against cholangiocarcinoma cells

Background aimsCholangiocarcinoma (CCA) is a lethal bile-duct cancer that is difficult to treat by current standard procedures. This drawback has prompted us to develop adoptive T-cell therapy for CCA, which requires an appropriate target antigen for binding of chimeric antigen receptor (CAR) T cells. Mucin 1 (MUC1), an overexpressed protein in CCA cells, is a potential target antigen for the CAR T-cell development. However, MUC1 overexpression also is associated with the upregulation of programmed death-ligand 1 (PD-L1), an immune checkpoint protein that prohibits anti-tumor functions of T cells, probably causing poor overall survival of patients with CCA.MethodsTo overcome this problem, we developed anti-MUC1-CAR T cells containing PD-1-CD28 switch receptor (SR), namely αM.CAR/SR T cells, to target MUC1 and switch on the inhibitory signal of PD-1/PD-L1 interaction to activate CD28 signaling. Our lentiviral construct contains the sequences that encode anti-MUC1-single chain variable fragment, CD137 and CD3ζ, linked with P2A, PD-1 and CD28.ResultsInitially, the upregulations of MUC1 and PD-L1 proteins were confirmed in CCA cell lines. αM.CAR and SR were co-expressed in 53.53 ± 13.89% of transduced T cells, mainly CD8⁺ T cells (85.7 ± 0.75%, P<0.0001) with the effector memory phenotype (59.22 ± 16.31%, P < 0.01). αM.CAR/SR T cells produced high levels of intracellular tumor necrosis factor-α and interferon-γ in response to the activation by CCA cells expressing MUC1, including KKU-055 (27.18 ± 4.38% and 27.33 ± 5.55%, respectively, P < 0.05) and KKU-213A (47.37 ± 12.67% and 54.55 ± 8.66%, respectively, P < 0.01). Remarkably, the cytotoxic function of αM.CAR/SR T cells against KKU-213A cells expressing PD-L1 was significantly enhanced compared with the αM.CAR T cells (70.69 ± 14.38% versus 47.15 ± 8.413%, respectively; P = 0.0301), correlated with increased granzyme B production (60.6 ± 9.89% versus 43.2 ± 8.95%, respectively; P = 0.0402). Moreover, the significantly enhanced disruption of KKU-213A spheroids by αM.CAR/SR T cells (P = 0.0027), compared with αM.CAR T cells, was also observed.ConclusionTaken together, the cytotoxic function of αM.CAR/SR T cells was enhanced over the αM.CAR T cells, which are potential to be further tested for CCA treatment.     

TIGIT Marks Exhausted T Cells,Correlates with Disease Progression,and Serves as a Target for Immune Restoration in HIV and SIV Infection

HIV infection induces phenotypic and functional changes to CD8⁺ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8⁺ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT⁺ and TIGIT⁺ PD-1⁺ CD8⁺ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8⁺ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8⁺ T cell effector responses. The frequency of TIGIT⁺ CD4⁺ T cells correlated with the CD4⁺ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.     

Programmed Cell Death-1 Deficiency Exacerbates T Cell Activation and Atherogenesis despite Expansion of Regulatory T Cells in Atherosclerosis-Prone Mice

T cell activation represents a double-edged sword in atherogenesis, as it promotes both pro-inflammatory T cell activation and atheroprotective Foxp3⁺ regulatory T cell (Treg) responses. Here, we investigated the role of the co-inhibitory receptor programmed cell death-1 (PD-1) in T cell activation and CD4⁺ T cell polarization towards pro-atherogenic or atheroprotective responses in mice. Mice deficient for both low density lipoprotein receptor and PD-1 (Ldlr^−/−Pd1^−/−) displayed striking increases in systemic CD4⁺ and CD8⁺ T cell activation after 9 weeks of high fat diet feeding, associated with an expansion of both pro-atherogenic IFNγ-secreting T helper 1 cells and atheroprotective Foxp3⁺ Tregs. Importantly, PD-1 deficiency did not affect Treg suppressive function in vitro. Notably, PD-1 deficiency exacerbated atherosclerotic lesion growth and entailed a massive infiltration of T cells in atherosclerotic lesions. In addition, aggravated hypercholesterolemia was observed in Ldlr^−/−Pd1^−/− mice. In conclusion, we here demonstrate that although disruption of PD-1 signaling enhances both pro- and anti-atherogenic T cell responses in Ldlr^−/− mice, pro-inflammatory T cell activation prevails and enhances dyslipidemia, vascular inflammation and atherosclerosis.     

Improvement of in vitro potency assays by a resting step for clinical-grade chimeric antigen receptor engineered T cells

BackgroundChimeric antigen receptor engineered T (CAR-T) cell therapy is a promising approach currently revolutionizing the field of cancer immunotherapy. However, data concerning clinical-grade CAR-T cell stability and functionality after months of cryopreservation have not been released by companies so far. To investigate the effect of cryopreservation on CAR-T cells and to further optimize the potency assays, we performed this study.MethodsA third generation of CD19 CAR-T cells was manufactured according to Good Manufacturing Practice (GMP) requirements, which is applied to patients in an ongoing clinical phase 1 study. Quality control tests for sterility, endotoxin and mycoplasma were performed for each batch. Stability in terms of viability, recovery, transduction efficiency and functional capacity was determined using microscopy, multiparametric flow cytometry as well as chromium-51 release tests.ResultsUp to 90days of cryopreservation had no influence on viability, recovery and transduction efficiency of CAR-T cells. However, higher cell concentration for cryopreservation could alter the cell viability and recovery but not the transduction efficiency. Moreover, directly after thawing, both the quantity and quality of the functionality of CAR-T cells were transiently hampered by the negative effects of cryopreservation. Notably, the impaired functionality could be fully restored and even strengthened after an overnight resting process.DiscussionCryopreservation is a challenge for the functional activity of CAR-T cells. However, CAR-T cells regain their potency by overnight incubation at 37°C, which mimics the clinical application setting. Therefore, an overnight resting step should be included in in vitro potency assays.     

Bicistronic CAR-T cells targeting CD123 and CLL1 for AML to reduce the risk of antigen escape

PurposeAcute myeloid leukemia (AML) is a highly heterogeneous neoplastic disease with a poor prognosis that relapses even after its treatment with chimeric antigen receptor (CAR)-T cells targeting a single antigen. CD123 and CLL1 are expressed in most AML blasts and leukemia stem cells, and their low expression in normal hematopoietic stem cells makes them ideal targets for CAR-T. In this study, we tested the hypothesis that a new bicistronic CAR targeting CD123 and CLL1 can enhance antigenic coverage and prevent antigen escape and subsequent recurrence of AML.MethodsCD123 and CLL1 expressions were evaluated on AML cell lines and blasts. Then, in addition to concentrating on CD123 and CLL1, we introduced the marker/suicide gene RQR8 with a bicistronic CAR. Xenograft models of disseminated AML and in vitro coculture models were used to assess the anti-leukemia efficacy of CAR-T cells. The hematopoietic toxicity of CAR-T cells was evaluated in vitro by colony cell formation assays. It was demonstrated in vitro that the combination of rituximab and NK cells caused RQR8-mediated clearance of 123CL CAR-T cells.ResultsWe have successfully established bicistronic 123CL CAR-T cells that can target CD123 and CLL1. 123CL CAR-T cells effectively cleared AML cell lines and blasts. They also demonstrated appreciable anti-AML activity in animal transplant models. Moreover, 123CL CAR-T cells can be eliminated in an emergency by a natural safety switch and don't target hematopoietic stem cells.ConclusionsThe bicistronic CAR-T cells targeting CD123 and CLL1 may be a useful and secure method for treating AML.     

Optimization of manufacturing conditions for chimeric antigen receptor T cells to favor cells with a central memory phenotype

BackgroundChimeric antigen receptor (CAR)-T cells are genetically engineered to recognize tumor-associated antigens and have potent cytolytic activity against tumors. Adoptive therapy with CAR-T cells has been highly successful in B-cell leukemia and lymphoma. However, in solid tumor settings, CAR-T cells face a particularly hostile tumor microenvironment where multiple immune suppressive factors serve to thwart the anti-cancer immune response. Clinical trials of solid tumor antigen-targeted CAR-T cells have shown limited efficacy, and issues for current CAR-T cell therapies include failures of expansion and persistence, tumor entry, deletion and functional exhaustion.MethodsWe compared our standard protocol for CAR-T cell manufacturing, currently used to generate CAR-T cells for a phase 1 clinical trial, with two alternative approaches for T-cell activation and expansion. The resulting cultures were analyzed using multicolor flow cytometry, cytokine bead array and xCELLigence cytotoxicity assays.ResultsWe have found that by changing the method of activation we can promote generation of CAR-T cells with enhanced CD62L and CCR7 expression, increased interleukin (IL)-2 production and retention of cytolytic activity, albeit with slower kinetics.DiscussionWe propose that these phenotypic characteristics are consistent with a central memory phenotype that will better enable CAR-T cell survival and persistence after activation in vivo, and we aim to test this in a continuation of our current phase 1 clinical trial of CAR-T cells in patients with advanced melanoma.     

Use of CRISPR/Cas9 gene editing to improve chimeric antigen-receptor T cell therapy: A systematic review and meta-analysis of preclinical studies

BackgroundChimeric antigen-receptor T (CAR-T) cells represent great promise in cancer treatment. CRISPR/Cas9 gene editing in preclinical studies has enabled the development of enhanced CAR-T products with improved function and reduced toxicity.MethodsA systematic review of preclinical animal studies was conducted to determine the efficacy and safety of this approach.Results3753 records were identified (to September 9, 2020), with 11 studies using CRISPR/Cas9 gene editing in combination with CAR-T therapy against human cells in animal models of acute leukemia (four studies), glioma (two studies), melanoma (two studies), and other cancers (three studies). Compared with unedited controls, gene-edited CAR-T cells reduced tumor volume in treated animals and improved survival. No adverse side effects were reported. Use of allogeneic “third-party” CAR-T cells appears feasible. Improved efficacy was achieved through both knock-in and knockout gene editing of various targets implicated in immune function. Targeting multiple genes also appears feasible. Significant heterogeneity in study design and outcome reporting was observed, and potential bias was identified in all studies.Conclusion: CRISPR/Cas9 gene editing enables manufacturing of CAR-T cells with improved anti-cancer effects. Future studies should reduce unintentional bias and heterogeneity of study designs and strive to augment long-term persistence of edited cells.Protocol registration: PROSPERO; registration number CRD42020220313 registered November 30, 2020     

Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy

BackgroundChimeric antigen receptor (CAR) T cell therapy has yielded impressive clinical results in hematological malignancies and is a promising approach for solid tumor treatment. However, toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, is a concern hampering its broader use.MethodsIn selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CARs bearing a low- and high-affinity single-chain variable fragment (scFv) binding to a similar epitope and cross-reactive with murine GPC3.ResultsWhere the high-affinity CAR-T cells were toxic in vivo, the low-affinity CAR maintained cytotoxic function against antigen-positive tumor cells but did not show toxicity against normal tissues. High-affinity CAR-induced toxicity was caused by on-target, off-tumor binding, based on the observation that higher doses of the high-affinity CAR-T caused toxicity in non–tumor-bearing mice and accumulated in organs with low expression of GPC3. To explore another layer of controlling CAR-T toxicity, we developed a means to target and eliminate CAR-T cells using anti-TNF-α antibody therapy after CAR-T infusion. The antibody was shown to function by eliminating early antigen-activated, but not all, CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from antitumor efficacy with only a minor loss in tumor control. By exploring additional traits of the CAR-T cells after activation, we identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that eliminated early activated CAR-T following antigen engagement in vivo.ConclusionsBy combining the reduced-affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.     

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

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

Programmed cell death protein 1 activation preferentially inhibits CD28.CAR–T cells

Targeted adoptive immunotherapy with engineered T cells is a promising treatment for refractory hematologic malignancies. However, many patients achieving early complete remissions ultimately relapse. Immunosuppressive ligands are expressed on tumor and supportive cells in the tumor microenvironment (TME). When activated, T cells express associated “checkpoint” receptors. Binding of co-inhibitory ligands and receptors may directly contribute to T-cell functional exhaustion. It is not known whether all T cells engineered to express chimeric antigen receptors (CARs) are subject to checkpoint-mediated regulation. It is also unknown whether distinct CAR signaling moieties modulate T-cell responsiveness to these inhibitory pathways. We have, therefore, directly compared functional co-inhibition in engineered T cells identically targeted to the tumor-associated antigen CD123, but distinct in their mode of T-cell activation: via the endogenous T-cell receptor (ENG), or downstream of CD28 or 41BB-containing CARs. In all cases, we have observed antigen-independent T-cell activation associated with upregulation of the co-inhibitory receptors programmed cell death protein 1 (PD-1, CD279), Tim-3 and Lag-3. Notably, CD28.CAR T cells were uniquely susceptible to PD-1/PD-L1 mediated checkpoint inhibition. Together, our data indicate that PD-1/PD-L1 checkpoint blocking agents may be considered clinically when CD28.CAR T cells do not perform optimally in human trials.     

Coupling programmed cell death 1-positive tumor-infiltrating T cells with anti-programmed cell death 1 antibody improves the efficacy of adoptive T-cell therapy

Background aimsAdoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) has shown great success in clinical trials. Programmed cell death 1 (PD-1)-expressing TILs show high specificity to autologous tumor cells. However, limited therapeutic efficiency is observed as a result of the tumor immune microenvironment (TIME).MethodsCoupling PD-1⁺ ex vivo-derived TILs with a monoclonal antibody against anti-PD-1 (aPD-1) reinvigorated the anti-tumor response of TILs against solid tumor without altering their high tumor targeting ability.ResultsUsing a melanoma-bearing mouse model, PD-1⁺ TILs blocked with aPD-1 (PD-1⁺ TILs-aPD-1) exhibited a high capability for tumor targeting as well as improved anti-tumor response in TIME. Tumor growth was substantially delayed in the mice treated with PD-1⁺ TILs-aPD-1.ConclusionsThe strategy utilizing TIL therapy coupled with immune checkpoint antibodies may extend to other therapeutic targets of ACT.     

Recent advances in cellular therapy for malignant lymphoma

Cellular therapies for malignant lymphoma include autologous or allogeneic hematopoietic stem cell transplantation (HSCT) and adaptive cellular therapy using EBV-specific T cells, cytokine-induced killer (CIK) cells, NKT cells, NK cells, chimeric antigen receptor T (CAR-T) cells and chimeric antigen receptor NK (CAR-NK) cells. In this review we discusses recent advances of these cellular therapies and consider ways to optimize these therapies. Not only a single strategy using one of these cellular therapies, but also multi-disciplinary treatment combines with antibodies, such as an anti-tumor antibody and an immune checkpoint antibody, may be more effective for relapsed and refractory lymphoma.     

Tandem CAR-T cells targeting FOLR1 and MSLN enhance the antitumor effects in ovarian cancer

Given the heterogeneity of solid tumors, single-target CAR-T cell therapy often leads to recurrence, especially in ovarian cancer (OV). Here, we constructed a Tandem-CAR targeting two antigens with secretory activity (IL-12) to improve the effects of CAR-T cell therapy. Twenty coexpressed upregulated genes were identified from the GEO database, and we found FOLR1 (folate receptor 1) and MSLN (mesothelin) were specifically and highly expressed in cancer tissues and only 11.25% of samples were negative for both antigens. We observed an increased proliferation rate for these three CAR-T cells, and Tandem CAR-T cells could efficiently lyse antigen-positive OV cells in vitro and secrete higher levels of cytokines than single-target CAR-T cells. More importantly, in vivo experiments indicated that Tandem CAR-T cells markedly decreased tumor volume, exhibited enhanced antitumor activity, and prolonged mouse survival. Furthermore, the infiltration and persistence of T cells in the Tandem-CAR group were higher than those in the MSLN-CAR and Control-T groups but comparable to those in the FOLR1-CAR group. Collectively, this study demonstrated that Tandem CAR-T cells secreting IL-12 could enhance immunotherapeutic effects by reducing tumor antigen escape and increasing T cell functionality, which could be a promising therapeutic strategy for OV and other solid tumors.     

Efficacy of B7-H3-Redirected BiTE and CAR-T Immunotherapies Against Extranodal Nasal Natural Killer/T Cell Lymphoma

Extranodal nasal natural killer (NK)/T cell lymphoma (ENKTCL) is a rare but highly aggressive subtype of non-Hodgkin lymphoma (NHL). Nevertheless, despite extensive research, the estimated 5-year overall survival of affected patients remains low. Therefore, new treatment strategies are needed urgently. Recent advances in immunotherapy have the potential to broaden the applications of chimeric antigen receptor-modified T (CAR-T) cells and the bispecific T-cell engaging (BiTE) antibody. Here, we screened a panel of biomarkers including the B7-H3, CD70, TIM-3, VISTA, ICAM-1, and PD-1 in NKTCL cell lines. As a result, we found for the first time that B7-H3 was highly and homogeneously expressed in these cells. Consequently, we constructed a novel anti-B7-H3/CD3 BiTE antibody and B7-H3-redirected CAR-T cells, and evaluated their efficacy against NKTCL cel lines both in vitro and in vivo. Notably, we found that both anti-B7-H3/CD3 BiTE and B7-H3-redirected CAR-T cells effectively targeted and killed NKTCL cells in vitro, and suppressed the growth of NKTCL tumors in NSG mouse models. Thus, B7-H3 might be a promising therapeutic target for treating patients with NKTCL tumors.     

Synergistic Reversal of Intrahepatic HCV-Specific CD8 T Cell Exhaustion by Combined PD-1/CTLA-4 Blockade

Viral persistence is associated with hierarchical antiviral CD8 T cell exhaustion with increased programmed death-1 (PD-1) expression. In HCV persistence, HCV-specific CD8 T cells from the liver (the site of viral replication) display increased PD-1 expression and a profound functional impairment that is not reversed by PD-1 blockade alone. Here, we report that the inhibitory receptor cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is preferentially upregulated in PD-1⁺ T cells from the liver but not blood of chronically HCV-infected patients. PD-1/CTLA-4 co-expression in intrahepatic T cells was associated with a profound HCV-specific effector dysfunction that was synergistically reversed by combined PD-1/CTLA-4 blockade in vitro, but not by blocking PD-1 or CTLA-4 alone. A similar effect was observed in circulating HCV-specific CD8 T cells with increased PD-1/CTLA-4 co-expression during acute hepatitis C. The functional response to combined blockade was directly associated with CTLA-4 expression, lost with CD28-depletion and CD4-independent (including CD4⁺FoxP3⁺ Tregs). We conclude that PD-1 and CTLA-4 pathways both contribute to virus-specific T cell exhaustion at the site of viral replication by a redundant mechanism that requires combined PD-1/CTLA-4 blockade to reverse. These findings provide new insights into the mechanisms of virus-specific T cell dysfunction, and suggest that the synergistic effect by combined inhibitory receptor blockade might have a therapeutic application against chronic viral infection in vivo, provided that it does not induce autoimmunity.     

Expression of programmed cell death protein 4 (PDCD4) and miR-21 in urothelial carcinoma

BackgroundWe investigated the role of the programmed cell death 4 (PDCD4) tumor suppressor gene in specimens of transitional cell carcinoma and of healthy individuals.MethodsPDCD4 immunohistochemical expression was investigated in 294 cases in histologically proven transitional cell carcinoma in different tumorous stages (28 controls, 122 non-muscle invasive urothelial carcinoma, stages Tis-T1, 119 invasive transitional cell carcinoma stages T2–T4 and 25 metastases). MiR-21 expression, an important PDCD4 regulator, was assessed with real-time PCR analysis and showed inverse correlation to tissue PDCD4 expression.ResultsNuclear and cytoplasmatic PDCD4 immunostaining decreased significantly with histopathological progression of the tumor (p < 0001). Controls showed strong nuclear and cytoplasmatic immunohistochemical staining. MiR-21 up regulation in tissue corresponded to PDCD4 suppression.ConclusionsThese data support a decisive role for PDCD4 down regulation in transitional cell carcinoma and confirm miR-21 as a negative regulator for PDCD4. Additionally, PDCD4 immunohistochemical staining turns out to be a possible diagnostic marker for transitional cell carcinoma.     

Durable blockade of PD-1 signaling links preclinical efficacy of sintilimab to its clinical benefit

ABSTRACT

Blockade of immune checkpoint pathways by programmed cell death protein 1 (PD-1) antibodies has demonstrated broad clinical efficacy against a variety of malignancies. Sintilimab, a highly selective, fully human monoclonal antibody (mAb), blocks the interaction of PD-1 and its ligands and has demonstrated clinical benefit in various clinical studies. Here, we evaluated the affinity of sintilimab to human PD-1 by surface plasmon resonance and mesoscale discovery and evaluated PD-1 receptor occupancy and anti-tumor efficacy of sintilimab in a humanized NOD/Shi-scid-IL2rgamma (null) (NOG) mouse model. We also assessed the receptor occupancy and immunogenicity of sintilimab from clinical studies in humans (9 patients with advanced solid tumor and 381 patients from 4 clinical studies, respectively). Sintilimab bound to human PD-1 with greater affinity than nivolumab (Opdivo®, MDX-1106) and pembrolizumab (Keytruda®, MK-3475). The high affinity of sintilimab is explained by its distinct structural binding mode to PD-1. The pharmacokinetic behavior of sintilimab did not show any significant differences compared to the other two anti-PD-1 mAbs. In the humanized NOG mouse model, sintilimab showed superior PD-1 occupancy on circulating T cells and a stronger anti-tumor effect against NCI-H292 tumors. The strong anti-tumor response correlated with increased interferon-γ-secreting, tumor-specific CD8+ T cells, but not with CD4+ Tregs in tumor tissue. Pharmacodynamics testing indicated a sustained mean occupancy of ≥95% of PD-1 molecules on circulating T cells in patients following sintilimab infusion, regardless of infusion dose. Sintilimab infusion was associated with 0.52% (2/381 patients) of anti-drug antibodies and 0.26% (1/381 patients) neutralizing antibodies. These data validate sintilimab as a novel, safe, and efficacious anti-PD-1 mAb for cancer immunotherapy.     

Targeting CD276 by CAR-T cells induces regression of esophagus squamous cell carcinoma in xenograft mouse models

Esophageal cancer, including esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), has a poor prognosis and limited therapeutic options. Chimeric antigen receptor (CAR)-T cells represent a potential ESCC treatment. In this study, we examined CD276 expression in healthy and esophageal tumor tissues and explored the tumoricidal potential of CD276-targeting CAR-T cells in ESCC. CD276 was strongly and homogenously expressed in ESCC and EAC tumor lesions but mildly in healthy tissues, representing a good target for CAR-T cell therapy. We generated CD276-directed CAR-T cells with a humanized antigen-recognizing domain and CD28 or 4–1BB co-stimulation. CD276-specific CAR-T cells efficiently killed ESCC tumor cells in an antigen-dependent manner both in vitro and in vivo. In patient-derived xenograft models, CAR-T cells induced tumor regression and extended mouse survival. In addition, CAR-T cells generated from patient T cells demonstrated potent cytotoxicity against autologous tumor cells. Our study indicates that CD276 is an attractive target for ESCC therapy, and CD276-targeting CAR-T cells are worth testing in ESCC clinical trials.