Co-Introduced Functional CCR2 Potentiates In Vivo Anti-Lung Cancer Functionality Mediated by T Cells Double Gene-Modified to Express WT1-Specific T-Cell Receptor

Background and Purpose

Although gene-modification of T cells to express tumor-related antigen-specific T-cell receptor (TCR) or chimeric antigen receptor (CAR) has clinically proved promise, there still remains room to improve the clinical efficacy of re-directed T-cell based antitumor adoptive therapy. In order to achieve more objective clinical responses using ex vivo-expanded tumor-responsive T cells, the infused T cells need to show adequate localized infiltration into the tumor.

Methodology/Principal Findings

Human lung cancer cells variously express a tumor antigen, Wilms'' Tumor gene product 1 (WT1), and an inflammatory chemokine, CCL2. However, CCR2, the relevant receptor for CCL2, is rarely expressed on activated T-lymphocytes. A HLA-A2402⁺ human lung cancer cell line, LK79, which expresses high amounts of both CCL2 and WT1 mRNA, was employed as a target. Normal CD8⁺ T cells were retrovirally gene-modified to express both CCR2 and HLA-A*2402-restricted and WT1_235–243 nonapeptide-specific TCR as an effector. Anti-tumor functionality mediated by these effector cells against LK79 cells was assessed both in vitro and in vivo. Finally the impact of CCL2 on WT1 epitope-responsive TCR signaling mediated by the effector cells was studied. Introduced CCR2 was functionally validated using gene-modified Jurkat cells and human CD3⁺ T cells both in vitro and in vivo. Double gene-modified CD3⁺ T cells successfully demonstrated both CCL2-tropic tumor trafficking and cytocidal reactivity against LK79 cells in vitro and in vivo. CCL2 augmented the WT1 epitope-responsive TCR signaling shown by relevant luciferase production in double gene-modified Jurkat/MA cells to express luciferase and WT1-specific TCR, and CCL2 also dose-dependently augmented WT1 epitope-responsive IFN-γ production and CD107a expression mediated by these double gene-modifiedCD3⁺ T cells.

Conclusion/Significance

Introduction of the CCL2/CCR2 axis successfully potentiated in vivo anti-lung cancer reactivity mediated by CD8⁺ T cells double gene-modified to express WT1-specific TCR and CCR2 not only via CCL2-tropic tumor trafficking, but also CCL2-enhanced WT1-responsiveness.     

CD19 target-engineered T-cells accumulate at tumor lesions in human B-cell lymphoma xenograft mouse models

Adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) is promising for treatment of advanced B-cell malignancies. Tumor targeting of CAR-modified T-cells is likely to contribute therapeutic potency; therefore we examined the relationship between the ability of CD19-specific CAR (CD19-CAR)-transduced T-cells to accumulate at CD19⁺ tumor lesions, and their ability to provide anti-tumor effects in xenograft mouse models. Normal human peripheral blood lymphocytes, activated with immobilized RetroNectin and anti-CD3 antibodies, were transduced with retroviral vectors that encode CD19-CAR. Expanded CD19-CAR T-cells with a high transgene expression level of about 75% produced IL-2 and IFN-γ in response to CD19, and lysed both Raji and Daudi CD19⁺ human B-cell lymphoma cell lines. Furthermore, these cells efficiently accumulated at Raji tumor lesions where they suppressed tumor progression and prolonged survival in tumor-bearing Rag2^−/−γc^−/− immunodeficient mice compared to control cohorts. These results show that the ability of CD19-CAR T-cells to home in on tumor lesions is pivotal for their anti-tumor effects in our xenograft models, and therefore may enhance the efficacy of adoptive T-cell therapy for refractory B-cell lymphoma.     

Dose-dependent thresholds of dexamethasone destabilize CAR T-cell treatment efficacy

Chimeric antigen receptor (CAR) T-cell therapy is potentially an effective targeted immunotherapy for glioblastoma, yet there is presently little known about the efficacy of CAR T-cell treatment when combined with the widely used anti-inflammatory and immunosuppressant glucocorticoid, dexamethasone. Here we present a mathematical model-based analysis of three patient-derived glioblastoma cell lines treated in vitro with CAR T-cells and dexamethasone. Advanced in vitro experimental cell killing assay technologies allow for highly resolved temporal dynamics of tumor cells treated with CAR T-cells and dexamethasone, making this a valuable model system for studying the rich dynamics of nonlinear biological processes with translational applications. We model the system as a nonautonomous, two-species predator-prey interaction of tumor cells and CAR T-cells, with explicit time-dependence in the clearance rate of dexamethasone. Using time as a bifurcation parameter, we show that (1) dexamethasone destabilizes coexistence equilibria between CAR T-cells and tumor cells in a dose-dependent manner and (2) as dexamethasone is cleared from the system, a stable coexistence equilibrium returns in the form of a Hopf bifurcation. With the model fit to experimental data, we demonstrate that high concentrations of dexamethasone antagonizes CAR T-cell efficacy by exhausting, or reducing the activity of CAR T-cells, and by promoting tumor cell growth. Finally, we identify a critical threshold in the ratio of CAR T-cell death to CAR T-cell proliferation rates that predicts eventual treatment success or failure that may be used to guide the dose and timing of CAR T-cell therapy in the presence of dexamethasone in patients.     

Toll-like receptor (TLR) expression on CD4 and CD8 T-cells in patients chronically infected with hepatitis C virus

Toll-like receptor (TLR) expression on T-cells and the signalling pathways that lead to the production of cytokines may limit antigen-specific T-cell responses. Here, expression of TLR and retinoic acid inducible gene I (RIG-I) on T-cells were evaluated in patients chronically infected with hepatitis C virus (HCV), before and during pegylated interferon-α and ribavirin therapy. Expression of TLR2,3,4,7,9 and retinoic acid inducible gene (RIG)-I on different CD4⁺ and CD8⁺ T-cell sub-populations (naïve: CD45RA⁺CD57⁻; central memory: T_CM CD45RA⁻CD57⁻; effector memory: T_EM CD45RA⁻CD57⁺ and terminally differentiated effector memory: T_EMRA CD45RA⁺CD57⁺) were measured by flow cytometry. TLR7, TLR9 and RIG-I expression on CD4⁺ T-cells and RIG-I expression on CD8⁺ T-cells was higher in patients than healthy controls. Therapy increased expression of TLR2, TLR4 and TLR9 and this was observed for all T-cell sub-populations. Evaluation of TLR expression at baseline did not identify patients able to achieve sustained virological response following therapy.     

Distinct APC Subtypes Drive Spatially Segregated CD4+ and CD8+ T-Cell Effector Activity during Skin Infection with HSV-1

Efficient infection control requires potent T-cell responses at sites of pathogen replication. However, the regulation of T-cell effector function in situ remains poorly understood. Here, we show key differences in the regulation of effector activity between CD4⁺ and CD8⁺ T-cells during skin infection with HSV-1. IFN-γ-producing CD4⁺ T cells disseminated widely throughout the skin and draining lymph nodes (LN), clearly exceeding the epithelial distribution of infectious virus. By contrast, IFN-γ-producing CD8⁺ T cells were only found within the infected epidermal layer of the skin and associated hair follicles. Mechanistically, while various subsets of lymphoid- and skin-derived dendritic cells (DC) elicited IFN-γ production by CD4⁺ T cells, CD8⁺ T cells responded exclusively to infected epidermal cells directly presenting viral antigen. Notably, uninfected cross-presenting DCs from both skin and LNs failed to trigger IFN-γ production by CD8⁺ T-cells. Thus, we describe a previously unappreciated complexity in the regulation of CD4⁺ and CD8⁺ T-cell effector activity that is subset-specific, microanatomically distinct and involves largely non-overlapping types of antigen-presenting cells (APC).     

Prognostic impact of peripheral eosinophil counts in patients with diffuse large B-cell lymphoma receiving chimeric antigen receptor T-cell therapy

Background aimsChimeric antigen receptor (CAR) T-cell therapy is a breakthrough treatment for patients with relapsed or refractory diffuse large B-cell lymphoma. However, many patients do not achieve remission or relapse after remission. Previous studies have demonstrated that eosinophils have synergistic anti-tumor effects with CD8⁺T cells and pre-CAR T-eosinophil counts are associated with the efficacy of CAR T cells.MethodsWe retrospectively analyzed the eosinophil counts of patients with diffuse large B-cell lymphoma and found it changed remarkably pre- and post-CAR T-cell therapy.ResultsPatients who achieved complete remission after CAR T-cell infusion had greater post-CAR T-eosinophil counts than those who did not. Kaplan–Meier curves showed that patients with greater eosinophil counts during the second month after CAR T-cell infusion had superior progression-free survival and overall survival compared with those with lower eosinophil counts.ConclusionsFor patients who responded to CAR T-cell therapy, eosinophil counts also can be used to predict 6-month duration of response. In conclusion, the post-CAR T-eosinophil count is associated with the prognosis of patients treated with CAR T-cell therapy and can be used to clinically identify patients who can achieve longer remission after CAR T-cell infusion.     

Manufacturing development and clinical production of NKG2D chimeric antigen receptor–expressing T cells for autologous adoptive cell therapy

Background aims

Adoptive cell therapy employing natural killer group 2D (NKG2D) chimeric antigen receptor (CAR)-modified T cells has demonstrated preclinical efficacy in several model systems, including hematological and solid tumors. We present comprehensive data on manufacturing development and clinical production of autologous NKG2D CAR T cells for treatment of acute myeloid leukemia and multiple myeloma (ClinicalTrials.gov Identifier: NCT02203825). An NKG2D CAR was generated by fusing native full-length human NKG2D to the human CD3ζ cytoplasmic signaling domain. NKG2D naturally associates with native costimulatory molecule DAP10, effectively generating a second-generation CAR against multiple ligands upregulated during malignant transformation including MIC-A, MIC-B and the UL-16 binding proteins.

Complementary Role of HCV and HIV in T-Cell Activation and Exhaustion in HIV/HCV Coinfection

Objectives

To investigate whether T-cell activation and exhaustion is linked to HCV- and HIV disease parameters in HIV/HCV infected individuals, we studied T-cell characteristics in HIV/HCV coinfected patients and controls.

Methods

14 HIV/HCV coinfected, 19 HCV monoinfected, 10 HIV monoinfected patients and 15 healthy controls were included in this cross-sectional study. Differences in expression of activation and exhaustion markers (HLA-DR, CD38, PD-1, Tim-3 and Fas) and phenotypic markers on CD4⁺ and CD8⁺ T-cells were analysed by flow cytometry and were related to HCV disease parameters (HCV-viremia, ALT and liver fibrosis).

Results

Frequencies of activated CD4⁺ and CD8⁺ T-cells were higher in HIV/HCV-coinfected compared to healthy controls and HCV or HIV mono-infected individuals. Coinfected patients also showed high expression of the exhaustion marker PD-1 and death receptor Fas. In contrast, the exhaustion marker Tim-3 was only elevated in HIV-monoinfected patients. T-cell activation and exhaustion were correlated with HCV-RNA, suggesting that viral antigen influences T-cell activation and exhaustion. Interestingly, increased percentages of effector CD8⁺ T-cells were found in patients with severe (F3–F4) liver fibrosis compared to those with no to minimal fibrosis (F0–F2).

Conclusions

HIV/HCV coinfected patients display a high level of T-cell activation and exhaustion in the peripheral blood. Our data suggest that T-cell activation and exhaustion are influenced by the level of HCV viremia. Furthermore, high percentages of cytotoxic/effector CD8⁺ T-cells are associated with liver fibrosis in both HCV monoinfected and HIV/HCV coinfected patients.     

Identification of MAGE-C1 (CT-7) epitopes for T-cell therapy of multiple myeloma

Multiple myeloma is incurable with standard therapies but is susceptible to a T-cell-mediated graft versus myeloma effect after allogeneic stem cell transplantation. We sought to identify myeloma-specific antigens that might be used for T-cell immunotherapy of myeloma. MAGE-C1 (CT-7) is a cancer-testis antigen that is expressed by tumor cells in >70% of myeloma patients and elicits a humoral response in up to 93% of patients with CT-7⁺ myeloma. No CD8⁺ T-cell epitopes have been described for CT-7, so we used a combination of reverse immunology and immunization of HLA-A2 transgenic mice with a novel cell-based vaccine to identify three immunogenic epitopes of CT-7 that are recognized by human CD8⁺ T-cells. CT-7-specific T-cells recognizing two of these peptides are able to recognize myeloma cells as well as CT-7 gene-transduced tumor cells, demonstrating that these epitopes are naturally processed and presented by tumor cells. This is the first report of the identification of immunogenic CD8⁺ T-cell epitopes of MAGE-C1 (CT-7), which is the most commonly expressed cancer-testis antigen found in myeloma, and these epitopes may be promising candidate targets for vaccination or T-cell therapy of myeloma or other CT-7⁺ malignancies.     

Adaptive immune responses during Shigella dysenteriae type 1 infection: an in vitro stimulation with 57 kDa major antigenic OMP in the presence of anti-CD3 antibody

An effort was made to understand the role of the 57 kDa major antigenic fraction of Shigella outer membrane protein (OMP) in the presence of T-cell antigen receptor in activation of adaptive immune responses of the cell mediated immune (CMI) restored patients. The expression of HLA-DR/CD4 out of CD3⁺ T-cells was significantly dominant over the HLA-DR/CD8 and comparable to unstimulated cells of infected or healthy controls. CD4⁺ T-cell activation together with HLA-DR is associated with the expression of CD25⁺ (IL2Rα) for IL-2 growth factors with decreased IL-4 levels, required for maintaining the homeostasis of CD4⁺ T cell. Furthermore, the positive expression of the CD45 antigen is possibly required for acquiring the memory for CD4⁺ cells signals and facilitates the interaction with CD54 antigen. As a result, antigen-specific secondary signal is generated for B-cell activation to produce IgG2a and IgG2b. This suggests that antibody mediated-adaptive immune responses are generated due to anti-CD3 induced helper T-cell activity. The above mentioned findings reflect that the antigen alone might not exacerbate the selective T-cell responses. But these antigens in the presence of anti-CD3 antibody might help to elicit adaptive immune response via T-cell receptor (TCR) activation.     

Generation of Zika virus–specific T cells from seropositive and virus-naïve donors for potential use as an autologous or “off-the-shelf” immunotherapeutic

BackgroundZika virus (ZIKV) infection can cause severe birth defects in newborns with no effective currently available treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the prevention or treatment of many viral infections, and could represent a novel treatment approach for patients with ZIKV infection. However, extending this strategy to the ZIKV setting has been hampered by limited data on immunogenic T-cell antigens within ZIKV. Hence, we have generated ZIKV-specific T cells and characterized the cellular immune responses against ZIKV antigens.MethodsT-cell products were generated from peripheral blood of ZIKV-exposed donors, ZIKV-naive adult donors and umbilical cord blood by stimulation with pentadecamer (15mer) overlapping peptide libraries spanning four ZIKV polyproteins (C, M, E and NS1) using a Good Manufacturing Practice–compliant protocol.ResultsWe successfully generated T cells targeting ZIKV antigens with clinically relevant numbers. The ex vivo–expanded T cells comprised both CD4⁺ and CD8⁺ T cells that were able to produce Th1-polarized effector cytokines and kill ZIKV-infected HLA-matched monocytes, confirming functionality of this unique T-cell product as a potential “off-the-shelf” therapeutic. Epitope mapping using peptide arrays identified several novel HLA class I and class II–restricted epitopes within NS1 antigen, which is essential for viral replication and immune evasion.DiscussionOur findings demonstrate that it is feasible to generate potent ZIKV-specific T cells from a variety of cell sources including virus naïve donors for future clinical use in an “off-the-shelf” setting.     

A hub-and-spoke model to deliver effective access to chimeric antigen receptor T-cell therapy in a public health network: the Catalan Blood and Tissue Bank experience

Background aimsTo describe and analyze whether a hub-and-spoke organizational model could efficiently provide access to chimeric antigen receptor (CAR) T-cell therapy within a network of academic hospitals and address the growing demands of this complex and specialized activity.MethodsThe authors performed a retrospective evaluation of activity within the Catalan Blood and Tissue Bank network, which was established for hematopoietic stem cell transplantation to serve six CAR T-cell programs in academic hospitals of the Catalan Health Service. Procedures at six hospitals were followed from 2016 to 2021. Collection shipments of starting materials, CAR T-cell returns for storage and infusions for either clinical trials or commercial use were evaluated.ResultsA total of 348 leukocytapheresis procedures were performed, 39% of which were delivered fresh and 61% of which were cryopreserved. The network was linked to seven advanced therapy medicinal product manufacturers. After production, 313 CAR T-cell products were shipped back to the central cryogenic medicine warehouse located in the hub. Of the units received, 90% were eventually administered to patients. A total of 281 patients were treated during this period, 45% in clinical trials and the rest with commercially available CAR T-cell therapies.ConclusionsA hub-and-spoke organizational model based on an existing hematopoietic stem cell transplantation program is efficient in incorporating CAR T-cell therapy into a public health hospital network. Rapid access and support of growing activity enabled 281 patients to receive CAR T cells during the study period.     

Quality risk management of the chimeric antigen receptor T cell pharmaceutical circuit in one of the first qualified European centers

Background aimsAccording to European Directive 2001/83/EC, chimeric antigen receptor T (CAR T) cells belong to a new class of medicines referred to as advanced therapy medicinal products (ATMPs). The specific features and complexity of these products require a total reorganization of the hospital circuit, from cell collection from the patient to administration of the final medicinal product. In France, at the cell stage, products are under the responsibility of a cell therapy unit (CTU) that controls, manipulates (if necessary) and ships cells to the manufacturing site. However, the final product is a medicinal product, and as with any other medicine, ATMPs have to be received, stored and further reconstituted for final distribution under the responsibility of the hospital pharmacy. The aim of our work was to perform a risk analysis of this circuit according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Q9 guidelines on quality risk management.MethodsWe evaluated the activities carried out by the Saint-Louis Hospital CTU and pharmacy. Process mapping was established to trace all the steps of the circuit and to identify potential risks or failures. The risk analysis was performed according to failure mode, effects and criticality analysis. The criticality of each risk (minor [Mi], moderate [Mo], significant [S] or major [Ma]) was scored, and corrective actions or preventive actions (CAPAs) for Mo, S and Ma risks were proposed.ResultsWe identified five Mo, six S and no Ma risks for the CTU part of the process. The most frequent risk was traceability failure. To reduce its frequency, we developed and validated software dedicated to ATMP activities. Another S risk was non-compliance of CAR T cell-specific steps due to the significant variability between companies. Our CAPA process was to implement procedures and design information sheets specific to each CAR T-cell program. In addition, critical steps were added to the ATMP software. Our CAPA process allowed us to reduce the criticality of identified risks to one Mi, seven Mo and three S. For the pharmacy part of the process, five Mo, two S and one Ma risk were identified. The most critical risk was compromised integrity of the CAR T-cell bag at the time of thawing. In case of unavailability of a backup bag, we designed and validated a degraded mode of operation allowing product recovery. In this exceptional circumstance, an agreement has to be signed between the physician, pharmacy, CTU and sponsor or marketing authorization holder. The implemented CAPA process allowed us to reduce the criticality of risks to three Mi and five Mo.ConclusionsOur risk analysis identified several Mo and S risks but only one Ma risk. The implementation of the CAPA process allowed for controlling some risks by decreasing their frequency and/or criticality or by increasing their detectability. The close collaboration between the CTU and pharmacy allows complete traceability of the CAR T-cell circuit, which is essential to guarantee safe use.     

Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR⁺ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19⁺ tumor targets. This clone can be used to detect CD19-specific CAR⁺ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR⁺ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.     

Targeting of low ALK antigen density neuroblastoma using AND logic-gate engineered CAR-T cells

Background aimsThe targeting of solid cancers with chimeric antigen receptor (CAR) T cells faces many technological hurdles, including selection of optimal target antigens. Promising pre-clinical and clinical data of CAR T-cell activity have emerged from targeting surface antigens such as GD2 and B7H3 in childhood cancer neuroblastoma. Anaplastic lymphoma kinase (ALK) is expressed in a majority of neuroblastomas at low antigen density but is largely absent from healthy tissues.MethodsTo explore an alternate target antigen for neuroblastoma CAR T-cell therapy, the authors generated and screened a single-chain variable fragment library targeting ALK extracellular domain to make a panel of new anti-ALK CAR T-cell constructs.ResultsA lead novel CAR T-cell construct was capable of specific cytotoxicity against neuroblastoma cells expressing low levels of ALK, but with only weak cytokine and proliferative T-cell responses. To explore strategies for amplifying ALK CAR T cells, the authors generated a co-CAR approach in which T cells received signal 1 from a first-generation ALK construct and signal 2 from anti-B7H3 or GD2 chimeric co-stimulatory receptors. The co-CAR approach successfully demonstrated the ability to avoid targeting single-antigen-positive targets as a strategy for mitigating on-target off-tumor toxicity.ConclusionsThese data provide further proof of concept for ALK as a neuroblastoma CAR T-cell target.     

Manufacturing chimeric antigen receptor T cells from cryopreserved peripheral blood cells: time for a collect-and-freeze model?

Background aimsChimeric antigen receptor (CAR)-modified T-cell therapy has revolutionized outcomes for patients with relapsed/refractory B-cell malignancies. Despite the exciting results, several clinical and logistical challenges limit its wide applicability. First, the apheresis requirement restricts accessibility to institutions with the resources to collect and process peripheral blood mononuclear cells (PBMCs). Second, even when utilizing an apheresis product, failure to manufacture CAR T cells is a well-established problem in a significant subset. In heavily pre-treated patients, prior chemotherapy may impact T-cell quality and function, limiting the ability to manufacture a potent CAR T-cell product. Isolation and storage of T cells shortly after initial cancer diagnosis or earlier in life while an individual is still healthy are an alternative to using T cells from heavily pre-treated patients. The goal of this study was to determine if a CAR T-cell product could be manufactured from a small volume (50 mL) of healthy donor blood.MethodsCollaborators at Cell Vault collected 50 mL of whole peripheral venous blood from three healthy donors. PBMCs were isolated, cryopreserved and shipped to the Medical College of Wisconsin. PBMCs for each individual donor were thawed, and CAR T cells were manufactured using an 8-day process on the CliniMACS Prodigy device with a CD19 lentiviral vector.ResultsStarting doses of enriched T-cell numbers ranged from 4.0 × 10⁷ cells to 4.8 × 10⁷ cells, with a CD4/CD8 purity of 74–79% and an average CD4:CD8 ratio of 1.4. On the day of harvest, total CD3 cells in the culture expanded to 3.6–4.6 × 10⁹ cells, resulting in a 74- to 115-fold expansion, an average CD4:CD8 ratio of 2.9 and a CD3 frequency of greater than 99%. Resulting CD19 CAR expression varied from 19.2% to 48.1%, with corresponding final CD19+ CAR T-cell counts ranging from 7.82 × 10⁸ cells to 2.21 × 10⁹ cells. The final CAR T-cell products were phenotypically activated and non-exhausted and contained a differentiated population consisting of stem cell-like memory T cells.ConclusionsOverall, these data demonstrate the ability to successfully generate CAR T-cell products in just 8 days using cryopreserved healthy donor PBMCs isolated from only 50 mL of blood. Notably, numbers of CAR T cells were more than adequate for infusion of an 80-kg patient at dose levels used for products currently approved by the Food and Drug Administration. The authors offer proof of principle that cryopreservation of limited volumes of venous blood with an adequate starting T-cell count allows later successful manufacture of CAR T-cell therapy.     

CAR T-cells for cancer therapy

Chimeric antigen receptor (CAR) T-cells are redirected T-cells that can recognize cancer antigens in a major histocompatibility complex (MHC)-independent fashion. A typical CAR is comprised of two main functional domains: an extracellular antigen recognition domain, called a single-chain variable fragment (scFv), and an intracellular signaling domain. Based on the number of intracellular signaling molecules, CARs are categorized into four generations. CAR T-cell therapy has become a promising treatment for hematologic malignancies. However, results of its clinical trials on solid tumors have not been encouraging. Here, we described the structure of CARs and summarized the clinical trials of CD19-targeted CAR T-cells. The side effects, safety management, challenges, and future prospects of CAR T-cells for the treatment of cancer, particularly for solid tumors, were also discussed.     

Media evaluation for production and expansion of anti-CD19 chimeric antigen receptor T cells

Background

The use of CD19 chimeric antigen receptor (CAR) T cells to treat B-cell malignancies has proven beneficial. Several groups use serum to produce CD19 CAR T cells. Today, ready-to-use serum-free media that require no addition of serum are commercially available. Therefore, it becomes important to evaluate the production of CD19 CAR T cells with and without the addition of serum.

Vaccination with Single Chain Antigen Receptors for Islet-Derived Peptides Presented on I-Ag7 Delays Diabetes in NOD Mice by Inducing Anergy in Self-Reactive T-Cells

To develop a vaccination approach for prevention of type 1 diabetes (T1D) that selectively attenuates self-reactive T-cells targeting specific autoantigens, we selected phage-displayed single chain antigen receptor libraries for clones binding to a complex of the NOD classII MHC I-A^g7 and epitopes derived from the islet autoantigen RegII. Libraries were generated from B-cell receptor repertoires of classII-mismatched mice immunized with RegII-pulsed NOD antigen presenting cells or from T-cell receptor repertoires in pancreatic lymph nodes of NOD mice. Both approaches yielded clones recognizing a RegII-derived epitope in the context of I-A^g7, which activated autoreactive CD4⁺ T-cells. A receptor with different specificity was obtained by converting the BDC2.5 TCR into single chain form. B- but not T-cells from donors vaccinated with the clones transferred protection from diabetes to NOD-SCID recipients if the specificity of the diabetes inducer cell and the single chain receptor were matched. B-cells and antibodies from donors vaccinated with the BDC2.5 single chain receptor induced a state of profound anergy in T-cells of BDC2.5 TCR transgenic NOD recipients while B-cells from donors vaccinated with a single chain receptor specific for I-A^g7 RegII peptide complexes induced only partial non-responsiveness. Vaccination of normal NOD mice with receptors recognizing I-A^g7 RegII peptide complexes or with the BDC2.5 single chain receptor delayed onset of T1D. Thus anti-idiotypic vaccination can be successfully applied to T1D with vaccines either generated from self-reactive T-cell clones or derived from antigen receptor libraries.