Antigen-specific Degranulation Research Articles

Orvacabtagene Autoleucel (orva-cel; JCARH125): A Fully Human BCMA-Targeted Second-Generation CAR T Cell Product Characterized By a Predominant Central Memory Phenotype with High in Vitro and In Vivo Proliferative Potential and Sustained In Vivo Persistence

Background: Orva-cel is an investigational B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T cell product genetically modified with a lentiviral vector to express a CAR construct with a unique fully human single-chain variable fragment, optimized spacer, and 4-1BB costimulatory and CD3ζ activation domains. Orva-cel is currently being evaluated for efficacy and safety in the ongoing phase 1/2 EVOLVE study (NCT03430011) in heavily pretreated patients with relapsed/refractory multiple myeloma. We characterized orva-cel drug products, manufactured using the process in place for the phase 2 portion of the study and intended for commercial manufacturing, for CAR+ T cell purity, phenotype, and function. Methods: Immunophenotyping was performed by flow cytometry of both surface and intracellular markers, including CD3, CD4, CD8, CD45, CCR7, CD45RA, CD28, CD27, and active caspase 3. Cytokine production after challenge with BCMA+ target cells was assessed by intracellular cytokine staining and Luminex multiplex assay of secreted cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrB). CAR-mediated in vitro proliferative capacity was measured after anti-idiotypic antibody stimulation using the IncuCyte Live-Cell Analysis System (Sartorius, Göttingen, Germany). In vivo CAR+ T cell proliferation and persistence were assessed by quantitative polymerase chain reaction (qPCR). Results: The orva-cel manufacturing process was designed to enable consistent production of highly pure CD3+ cell products (median frequency of CD3+ T cells, 99.96%; quartiles 1-3 interquartile range, 99.9%-100.0%; n = 81). Orva-cel drug products were characterized by high frequencies of less-differentiated CAR+ T cells, leading to a dominant central memory-like population (CCR7+CD45RA- CAR+ T cells) and substantial frequencies of naïve-like cells (CCR7+CD45RA+ CAR+ T cells) (Figure). When assayed for in vitro functional activity, orva-cel drug products showed robust antigen-specific cytokine and effector molecule production (IFN-γ, TNF-α, IL-2, and GrB) upon challenge with BCMA+ tumor cells, as well as vigorous proliferation in response to CAR stimulation. Preliminary correlative analysis suggested that the early memory phenotype may be linked to increased CAR+ T cell proliferative capacity, as determined by in vitro experiments and in vivo PK parameters (ie, maximum CAR+ T cell concentration observed in the blood [Cmax], time to Cmax, area under the curve from Day 0 to 28 [AUC0-28], and CAR+ T cell persistence at Month 3 and Month 6). Consistent with the early CAR T cell memory phenotype, qPCR analysis showed robust in vivo proliferation of CAR+ T cells after infusion, with a median Cmax of 1.54 × 105 transgene copies/µg DNA and median AUC0-28 of 1.61 × 106 transgene copies/µg DNA*day, as well as long-term in vivo persistence, with CAR+ T cells detected in 69% of patients at 6 months postinfusion. Conclusions: The orva-cel manufacturing process results in drug products characterized by highly pure T cells, with high frequencies of early memory and polyfunctional CAR+ T cells. Orva-cel drug products showed robust antigen-specific degranulation, production of multiple cytokines, sustained in vitro and in vivo proliferation, and in vivo persistence. Disclosures Colonna: Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Navarro:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Devries:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Beckett:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Amsberry:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Radhakrishnan:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Piasecki:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Heipel:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Li:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Kavita:Bristol Myers Squibb Company: Current Employment, Current equity holder in publicly-traded company. Works:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Mujacic:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment.

VEGFR2 as a target for CAR T cell therapy of Ewing sarcoma

T cells engineered to express chimeric antigen receptors (CARs) are a novel modality to treat refractory cancers. The development of CAR T cells against Ewing sarcoma (EwS) is limited by a lack of targetable surface antigens. We investigated vascular endothelial growth factor receptor 2 (VEGFR2) expressed on tumor-associated blood vessels as potential CAR target in this cancer. Expression of VEGFR2 was studied by immunohistochemistry in human EwS biopsies and in murine xenografts and by flow cytometry in EwS cell lines. CARs with short, medium, and long hinge domains against either human or murine VEGFR2 were generated and expressed in human T cells by retroviral gene transfer. The capacity of the individual CARs to activate T cells in response to VEGFR2-expressing cells was compared in vitro. Tumor-associated endothelial cells in human EwS biopsies and in xenografts expressed VEGFR2. Tumor cells in the majority of EwS biopsies were also VEGFR2-positive. Following modification with anti-mouse or anti-human VEGFR2-specific CAR genes, T cells specifically lysed VEGFR2-expressing target cells of the respective species. CAR T cells with short-length or medium-length hinge domains were functionally superior over those with the long hinge region by in vitro parameters, including antigen-specific degranulation responses, lysis of tumor spheroids, tumor necrosis factor α secretion, sequential killing, and proliferation. VEGFR2 is consistently expressed on endothelial cells of the tumor stroma in EwS and thus is a candidate target for CAR T cells in this cancer. Among various VEGFR2-specific CARs, a construct with a short hinge domain was chosen to be further developed toward clinical translation.

Reprogramming Human T Cell Function and Specificity With Non–Viral Genome Targeting

TL Roth, C Puig–Saus, R Yu. Nature. 2018;559(7714):405–409 To develop and optimize nonviral CRISPR-Cas9 genome editing for primary T cells and apply this technique in part to correct a pathogenic mutation in a monogenic autoimmune disease. Using CRISPR-Cas9, primary human T cells from healthy donors and a family with monogenic autoimmune disease were engineered ex vivo along with other important proofs of concept. The CRISPR-Cas9 system was first described as a prokaryotic adaptive immune response, and it has since been widely used as a gene editing tool. CRISPR sequences identify homologous areas in the DNA, and Cas9 creates precise double-stranded breaks. Double-stranded breaks are repaired by either nonhomologous end joining or homology-directed repair (HDR). HDR is less error prone, and by using CRISPR-Cas9 with HDR templates, transgenes can be integrated into the targeted genome with precision. In this study, an electroporation method was optimized for ex vivo primary human T cells to allow for uptake of CRISPR-Cas9 ribonucleoprotein complexes and large (>1 kb) double-stranded DNA (dsDNA) templates for HDR. The technique allowed for optimized cell viability along with efficiency under …

Human melanoma-specific CD8+ T-cells from metastases are capable of antigen-specific degranulation and cytolysis directly ex vivo

The relatively low frequencies of tumor Ag-specific T-cells in PBMC and metastases from cancer patients have long precluded the analysis of their direct ex vivo cytolytic capacity. Using a new composite technique that works well with low cell numbers, we aimed at determining the functional competence of melanoma-specific CD8+ T-cells. A multiparameter flow cytometry based technique was applied to assess the cytolytic function, degranulation and IFNγ production by tumor Ag-specific CD8+ T-cells from PBMC and tumor-infiltrated lymph nodes (TILN) of melanoma patients. We found strong cytotoxicity by T-cells not only when they were isolated from PBMC but also from TILN. Cytotoxicity was observed against peptide-pulsed target cells and melanoma cells presenting the naturally processed endogenous antigen. However, unlike their PBMC-derived counterparts, T-cells from TILN produced only minimal amounts of IFNγ, while exhibiting similar levels of degranulation, revealing a critical functional dichotomy in metastatic lesions. Our finding of partial functional impairment fits well with the current knowledge that T-cells from cancer metastases are so-called exhausted, a state of T-cell hyporesponsiveness also found in chronic viral infections. The identification of responsible mechanisms in the tumor microenvironment is important for improving cancer therapies.

CD8+Foxp3+ tumor infiltrating lymphocytes accumulate in the context of an effective anti‐tumor response

The composition of tumor infiltrating lymphocytes (TIL) is heterogeneous. In addition, the ratio of various subpopulations in the tumor microenvironment is highly dependent on the nature of the host's immune response. Here, we characterize Foxp3-expressing CD8(+) T cells in the tumor that demonstrate effector function and accumulate in the context of an effective anti-tumor response. CD8(+) Foxp3(+) T cells are induced in TIL in regressing tumors of FVB/N mice treated with a GM-CSF secreting HER-2/neu targeted whole cell vaccine. Foxp3 expression in tumor antigen-specific CD8 T cells is restricted to the tumor microenvironment and influenced by cues in the tumor. Interestingly, Foxp3(+) and Foxp3(-) CD8(+) T cells have similar IFN-γ production and antigen-specific degranulation after stimulation with RNEU(420-429) , the immunodominant HER-2/neu (neu) epitope in this model. Adoptive transfer studies, using RNEU((420-429)) -specific effector T cells into neu-N mice (a model that results in immune tolerance to neu), confirm that CD8(+) Foxp3(+) T cells are present in tumors only if there is an existing pool of tumor-rejecting effector T cells. CD8(+) Foxp3(+) TILs mark the presence of tumor-rejecting antigen-specific T cells and their accumulation serves as a marker for an effective T cell response.

P02-02. Analysis of antigen specific degranulation potentials using IL-12 or IL-28B during HIV DNA Vaccination

Background A number of studies have suggested that CD8 T cells play an important role in control of HIV/SIV replication. Recently, it has been shown that CD8 T cells taken from HIV-positive long term non-progressors are more efficiently cytotoxic than those from rapid progressors. We therefore endeavored to test vaccine adjuvants for their ability to generate antigen specific CD8 T cells with a significant capacity for cytolytic degranulation. The adjuvants tested for this purpose were IL-12 and IL-28B.

Phenotypically and Functionally Distinct CD8+ Lymphocyte Populations in Long-Term Drug-Free Tolerance and Chronic Rejection in Human Kidney Graft Recipients

A substantial proportion of long-term kidney graft recipients, including those with a stable renal function in the absence of immunosuppressive therapy, present a skewed T cell receptor (TCR) Vbeta chain usage, essentially in the CD8+ subset. This study analyzed in more detail phenotypical and functional alterations of CD8+ lymphocytes in drug-free tolerant patients (DF-Tol) compared with recipients with chronic rejection (CR). Phenotyping revealed a significant increase in central memory and a decrease in effector CD8+ lymphocytes in DF-Tol versus CR. The expression of CD28+ and CD27+ on these effector cells was significantly decreased in CR. These profiles were stable over time and independent of treatment. Functionally, the CD8+CD28- lymphocytes were less sensitive to apoptosis than their CD8+CD28+ counterparts, without differences in polyclonal proliferation. The CD8+CD28- cells did not express GITR and FoxP3 but were characterized by high levels of preformed perforin and granzyme A, pointing toward a cytotoxic rather than a suppressor function. CD8+CD28- lymphocytes did not show antigen-specific degranulation when co-cultured with targets that bear donor HLA class I antigens, suggesting that the cytotoxicity is directed either to other determinants of the graft or to nongraft epitopes. Of interest, CD8+ cells from DF-Tol displayed the same profile as healthy individuals, indicating an increase in CD8+CD28- effector lymphocytes in CR rather than a decrease in DF-Tol. CD8+ lymphocytes from stable kidney recipients under conventional maintenance immunosuppression displayed a mixed profile, independent of treatment and time of sampling. Taken collectively, these data show a strong cytotoxicity-associated CD8+CD28- signature in CR and suggest a suppression of pathologic cytotoxicity in DF-Tol. Further prospective studies should assess whether serial CD8+ phenotyping may help to identify patients who are at risk for CR when immunosuppression is tapered.

Identification of a Potential Effector Function for IgE Autoantibodies in the Organ-Specific Autoimmune Disease Bullous Pemphigoid

Bullous pemphigoid (BP) is an autoimmune skin disease characterized by autoantibodies against the hemidesmosomal protein BP180. In addition to IgG autoantibodies, IgE class autoantibodies have been reported in BP patients. Because animal models utilizing only IgG antibodies do not totally replicate human BP, we examined the specificity and potential relevance of IgE autoantibodies in this disease. Thirty BP patients participated in these studies. Serum IgE was measured and the IgE specificity was determined by immunoblotting. Double labeling Immunofluorescence was performed using combinations of specific antibodies to human mast cell tryptase, IgE and BP180. BP180-stimulated histamine release was measured from basophils of untreated BP patients (n=9), BP patients undergoing immunosuppressive therapy (n=9) and controls (n=16). Elevated IgE levels were found In 70% of untreated BP patients. IgE autoantibodies directed against BP180 were detected in 86% of untreated patients and in all but one of these patients the IgE reacted with the NC16A domain of BP180. IgE-coated mast cells were detected in perilesional skin of the BP patients. Moreover, BP180 peptides were detected on these mast cells. BP180-stimulated histamine release was significantly higher in basophils obtained from untreated BP patients compared with control basophils (p=0.006) or from treated BP patients (p=0.01). These findings support the hypothesis that IgE autoantibodies are involved in the pathogenesis of BP. IgE and IgG BP autoantibodies share the same antigenic specificity. Antigen-specific degranulation of basophils and/or mast cells from BP patients suggests a mechanism by which IgE may contribute to lesion development.

Anti-schistosome monoclonal antibodies of different isotypes--correlation with cytotoxicity.

Five monoclonal antibodies specific towards Schistosoma mansoni antigens were prepared by fusion of spleen cells of infected and immunized mouse with the murine myeloma NS-1 cells. Three of the five antibodies belonged to the IgG1 class, one was an IgM and the fifth one was an IgE. The IgE monoclonal antibody designated 54.10, induced antigen-specific degranulation of rat basophilic cell line, a property which served as the basis for the screening assay. Its biological function was demonstrated by a specific macrophage activation that led to killing of schistosomula; no such killing was obtained with anti-schistosome antibodies of other classes or with IgE of different antigenic specificity. The second monoclonal antibody of biological significance was an IgG1, designated 27.21 which is reactive in the immunofluorescence staining of surface antigens on intact schistosomula. All three monoclonal antibodies that belonged to the IgG1 class were effective in mediating killing of schistosomula by complement, with the highest effect exerted by 27.21. It is thus apparent that the 27.21 monoclonal antibody is directed against a densely distributed surface antigen on the schistosomula membrane which is possibly involved in the protective immunity. Preliminary data showed that immunoprecipitation with the 27.21 antibodies results in the isolation of three major protein bands, of 60 kd, 50 kd, 19 kd, respectively.

Leukocyte-dependent platelet vasoactive amine release and immune complex deposition in African swine fever.

Fifteen pigs were inoculated with African swine fever virus in a study of the pathogenesis of the disease. All pigs surviving the first two weeks developed high circulating antibody titers against African swine fever virus and persistent viremia. Hemolytic complement levels declined to 50 to 70 hemolytic complement 50 (CH50) units/ml from mean preinoculation levels of 120 CH50 units/ml. Immune deposits consisting of African swine fever antigen, host immunoglobulin G, and native C3 were found in the glomeruli of surviving pigs. All pigs surviving two weeks after inoculation developed leukocyte-bound antibody functionally characteristic of immunoglobulin E (IgE). Antigen-specific degranulation of antibody-coated leukocytes produced secondary platelet aggregation and vasoactive amine release. The results suggest that the IgE-basophil-platelet loop acting via amplification by leukocyte-derived platelet-activating factor participates in the immune complex deposition process in African swine fever.