Chimeric Ag Research Articles

The evolutionary modifications of a GoLoco motif in the AGS protein facilitate micromere formation in the sea urchin embryo.

The evolutionary introduction of asymmetric cell division (ACD) into the developmental program facilitates the formation of a new cell type, contributing to developmental diversity and, eventually, to species diversification. The micromere of the sea urchin embryo may serve as one of those examples: An ACD at the 16-cell stage forms micromeres unique to echinoids among echinoderms. We previously reported that a polarity factor, Activator of G-protein Signaling (AGS), plays a crucial role in micromere formation. However, AGS and its associated ACD factors are present in all echinoderms and across most metazoans, leaving a question of what evolutionary modification of AGS protein or its surrounding molecular environment contributed to the evolutionary acquisition of micromeres only in echinoids. In this study, we learned that the GoLoco motifs at the AGS C-terminus play critical roles in regulating micromere formation in sea urchin embryos. Further, other echinoderms AGS or chimeric AGS that contain the C-terminus of AGS orthologs from various organisms showed varied localization and function in micromere formation. In contrast, the sea star or the pencil urchin orthologs of other ACD factors were consistently localized at the vegetal cortex in the sea urchin embryo, suggesting that AGS may be a unique variable factor that facilitates ACD diversity among echinoderms. Consistently, sea urchin AGS appears to facilitate micromere-like cell formation and accelerate the enrichment timing of the germline factor Vasa during early embryogenesis of the pencil urchin, an ancestral type of sea urchin. Based on these observations, we propose that the molecular evolution of a single polarity factor facilitates ACD diversity while preserving the core ACD machinery among echinoderms and beyond during evolution.

Chick Embryo Chorioallantoic Membrane as a Platform for Assessing the InVivo Efficacy of Chimeric Antigen Receptor T-cell Therapy in Solid Tumors.

The fertilized chicken egg chorioallantoic membrane (CAM), a highly vascularized membrane nourishing the developing embryo, also supports rapid growth of three-dimensional vascularized tumors from engrafted cells and tumor explants. Because murine xenograft models suffer limitations of time, cost, and scalability, we propose CAM tumors as a rapid, efficient screening tool for assessing anti-tumor efficacy of chimeric Ag receptor (CAR) T cells against solid tumors. We tested the efficacy of human epidermal growth factor receptor 2 (HER2)-specific CAR T cells against luminescent, HER2-expressing (FaDu, SCC-47) or HER2-negative (MDA-MB-468) CAM-engrafted tumors. Three days after tumor engraftment, HER2-specific CAR T cells were applied to tumors grown on the CAM. Four days post-CAR T cell treatment, HER2-expressing FaDu and SCC-47 tumors treated with CAR T showed reduced viable cancer cells as assessed by luciferase activity. This reduction in viable tumor cells was confirmed by histology, with lower Ki-67 staining observed in CAR T cell-treated tumors relative to T cell-treated controls. Persistence of CAR T in CAM and tumor tissue 4 days post-treatment was confirmed by CD3 staining. Altogether, our findings support further development of the chick CAM as an invivo system for rapid, scalable screening of CAR T cell efficacy against human solid tumors.

T Cell Activators Exhibit Distinct Downstream Effects on Chimeric Antigen Receptor T Cell Phenotype and Function.

T cell activation is an essential step in chimeric Ag receptor (CAR) T (CAR T) cell manufacturing and is accomplished by the addition of activator reagents that trigger the TCR and provide costimulation. We explore several T cell activation reagents and examine their effects on key attributes of CAR T cell cultures, such as activation/exhaustion markers, cell expansion, gene expression, and transduction efficiency. Four distinct activators were examined, all using anti-CD3 and anti-CD28, but incorporating different mechanisms of delivery: Dynabeads (magnetic microspheres), TransAct (polymeric nanomatrix), Cloudz (alginate hydrogel), and Microbubbles (lipid membrane containing perfluorocarbon gas). Clinical-grade lentiviral vector was used to transduce cells with a bivalent CD19/CD22 CAR, and cell counts and flow cytometry were used to monitor the cells throughout the culture. We observed differences in CD4/CD8 ratio when stimulating with the Cloudz activator, where there was a significant skewing toward CD8 T cells. The naive T cell subset expressing CD62L+CCR7+CD45RA+ was the highest in all donors when stimulating with Dynabeads, whereas effector/effector memory cells were highest when using the Cloudz. Functional assays demonstrated differences in killing of target cells and proinflammatory cytokine secretion, with the highest killing from the Cloudz-stimulated cells among all donors. This study demonstrates that the means by which these stimulatory Abs are presented to T cells contribute to the activation, resulting in differing effects on CAR T cell function. These studies highlight important differences in the final product that should be considered when manufacturing CAR T cells for patients in the clinic.

Granzyme F: Exhaustion Marker and Modulator of Chimeric Antigen Receptor T Cell-Mediated Cytotoxicity.

Granzymes are a family of proteases used by CD8 T cells to mediate cytotoxicity and other less-defined activities. The substrate and mechanism of action of many granzymes are unknown, although they diverge among the family members. In this study, we show that mouse CD8+ tumor-infiltrating lymphocytes (TILs) express a unique array of granzymes relative to CD8 T cells outside the tumor microenvironment in multiple tumor models. Granzyme F was one of the most highly upregulated genes in TILs and was exclusively detected in PD1/TIM3 double-positive CD8 TILs. To determine the function of granzyme F and to improve the cytotoxic response to leukemia, we constructed chimeric Ag receptor T cells to overexpress a single granzyme, granzyme F or the better-characterized granzyme A or B. Using these doubly recombinant T cells, we demonstrated that granzyme F expression improved T cell-mediated cytotoxicity against target leukemia cells and induced a form of cell death other than chimeric Ag receptor T cells expressing only endogenous granzymes or exogenous granzyme A or B. However, increasing expression of granzyme F also had a detrimental impact on the viability of the host T cells, decreasing their persistence in circulation invivo. These results suggest a unique role for granzyme F as a marker of terminally differentiated CD8 T cells with increased cytotoxicity, but also increased self-directed cytotoxicity, suggesting a potential mechanism for the end of the terminal exhaustion pathway.

Efficacy against Lung Cancer Is Augmented by Combining Aberrantly N-Glycosylated T Cells with a Chimeric Antigen Receptor Targeting Fragile X Mental Retardation 1 Neighbor.

The adaptive transfer of T cells redirected to cancer cells via chimeric Ag receptors (CARs) has produced clinical benefits for the treatment of hematologic diseases. To extend this approach to solid cancer, we screened CARs targeting surface Ags on human lung cancer cells using (to our knowledge) novel expression cloning based on the Ag receptor-induced transcriptional activation of IL-2. Isolated CARs were directed against fragile X mental retardation 1 neighbor (FMR1NB), a cancer-testis Ag that is expressed by malignant cells and adult testicular germ cells. Anti-FMR1NB CAR human T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in mouse xenograft models of lung cancer. Furthermore, to protect CAR T cells from immune-inhibitory molecules, which are present in the tumor microenvironment, we introduced anti-FMR1NB CARs into 2-deoxy-glucose (2DG)-treated human T cells. These cells exhibited reduced binding affinity to immune-inhibitory molecules, and the suppressive effects of these molecules were resisted through blockade of the N-glycosylation of their receptors. Anti-FMR1NB CARs in 2DG-treated human T cells augmented target-specific cytotoxicity invitro and invivo. Thus, our findings demonstrated the feasibility of eradicating lung cancer cells using 2DG-treated human T cells, which are able to direct tumor-specific FMR1NB via CARs and survive in the suppressive tumor microenvironment.

Chimeric Antigen Cytotoxic Receptors for In Vivo Engineering of Tumor-Targeting NK Cells.

Chimeric Ag receptor (CAR) NK cells are challenging to manufacture and fail to achieve consistent tumor infiltration and sustained cytolytic function in the tumor microenvironment. In vivo engineering of NK cells using mRNA-based CAR delivery may overcome these issues. In this study, we developed an invivo programming method by designing CARs that leverage the biology of NK cell receptors for cell type-specific expression and function. These CARs were engineered by fusion of a tumor recognition domain with the natural cytotoxic receptor family including NKp30, NKp44, and NKp46. Our results demonstrated that these natural cytotoxic receptor-based CARs can engage endogenous signaling adaptors to effectively activate human NK cells for tumor lysis and cytokine production. Specifically, we discovered that stable expression of an NKp44-based CAR was contingent on the presence of the immune cell-specific signaling adaptor DAP12. This innovative strategy facilitates direct in situ programming of NK cells, enhancing safety and minimizing off-target effects in nontargeted, healthy tissues.

Assessing of the use of proteins A, G, and chimeric protein AG to detect marine mammal immunoglobulins.

In recent years, there has been an increase in infectious diseases in marine mammals, including brucellosis, infections of morbillivirus, herpesvirus, and poxvirus. Several serological diagnostic methods, including enzyme-linked immunosorbent assays, immunofluorescence assays (ELISA), and western blotting, have been used to detect antibodies against pathogens in marine mammals. However, options for commercial secondary antibodies used to detect antibodies in marine mammals are limited; therefore, the use of proteins A, G, or chimeric protein AG may provide a suitable alternative. This study aimed to assess the use of proteins A, G, and chimeric protein AG to detect marine mammal immunoglobulins. Currently, there are no comparative studies on the use of proteins A, G, and chimeric protein AG for the detection of immunoglobulins in marine mammals. In this study, we used ten pinnipeds' species (Baikal seal, California sea lion, harbor seal, northern fur seal, ringed seal, South American fur seal, South American sea lion, spotted seal, Steller sea lion, and walrus) and five cetacean species (beluga whale, bottlenose dolphin, harbor porpoise, killer whale, and Pacific white-sided dolphin) and compare binding ability to proteins A, G, or chimeric protein AG by ELISA. The results revealed that the immunoglobulins from pinniped and cetacean species reacted more strongly to protein A than protein G. In addition, the immunoglobulins of pinnipeds and cetaceans showed a strong binding ability to chimeric protein AG. These results suggest that proteins A, G, and chimeric protein AG would be used to help further develop serological assays.

Engineering Human MAIT Cells with Chimeric Antigen Receptors for Cancer Immunotherapy.

Engineering immune cells with chimeric Ag receptors (CARs) is a promising technology in cancer immunotherapy. Besides classical cytotoxic CD8+ T cells, innate cell types such as NK cells have also been used to generate CAR-T or CAR-NK cells. In this study, we devised an approach to program a nonclassical cytotoxic T cell subset called mucosal-associated invariant T (MAIT) cells into effective CAR-T cells against B cell lymphoma and breast cancer cells. Accordingly, we expressed anti-CD19 and anti-Her2 CARs in activated primary human MAIT cells and CD8+ T cells, expanded them in vitro, and compared their cytotoxicity against tumor cell targets. We show upon activation through CARs that CAR-MAIT cells exhibit high levels of cytotoxicity toward target cells, comparable to CD8+ CAR-T cells, but interestingly expressed lower levels of IFN-γ than conventional CAR CD8+ T cells. Additionally, in the presence of vitamin B2 metabolite 5-ARU (5-amino-4-d-ribitylaminouracil dihydrochloride), which is a conserved compound that activates MAIT cells through MHC class I-related (MR1) protein, MAIT cells killed MR1-expressing target breast cancer and B cell lymphoma cell lines in a dose-dependent manner. Thus, MAIT cells can be genetically edited as CAR-T cells or mobilized and expanded by MR1 ligands as an off-the-shelf novel approach to cell-based cancer immunotherapy strategies while being comparable to conventional methods in effectivity.

HIV-Specific CAR T Cells with CD28 or 4-1BB Signaling Domains Are Phenotypically and Functionally Distinct and Effective at Suppressing HIV and Simian Immunodeficiency Virus.

Despite mounting a robust antiviral CD8 T cell response to HIV infection, most infected individuals are unable to control HIV viral load without antiretroviral therapy (ART). Chimeric Ag receptor (CAR) T cell treatment is under intensive investigation as an alternative therapy for ART-free remission of chronic HIV infection. However, achieving durable remission of HIV will require a successful balance between CAR T cell effector function and persistence. CAR T cells with CD28 costimulatory domains have robust effector function but limited persistence in vivo, whereas CAR T cells with 4-1BB costimulatory domains present a more undifferentiated phenotype and greater in vivo persistence. We compared the in vitro phenotype and function of rhesus macaque and human CAR T cells that contained either the CD28 or 4-1BB costimulatory domain; both constructs also included CARs that are bispecific for gp120 of HIV or SIV and the CXCR5 moiety to promote in vivo homing of CAR/CXCR5 T cells to B cell follicles. Cells were transduced using a gammaretroviral vector and evaluated using flow cytometry. 4-1BB-CAR/CXCR5 T cells were phenotypically distinct from CD28-CAR/CXCR5 T cells and showed increased expression of CAR and CD95. Importantly, both CD28- and 4-1BB-CAR/CXCR5 T cells retained equal capacity to recognize and suppress SIV in vitro. These studies provide new insights into rhesus macaque and human 4-1BB- and CD28-bearing CAR T cells.

STAP-2 Is a Novel Positive Regulator of TCR-Proximal Signals.

TCR ligation with an Ag presented on MHC molecules promotes T cell activation, leading to the selection, differentiation, and proliferation of T cells and cytokine production. These immunological events are optimally arranged to provide appropriate responses against a variety of pathogens. We here propose signal-transducing adaptor protein-2 (STAP-2) as a new positive regulator of TCR signaling. STAP-2-deficient T cells showed reduced, whereas STAP-2-overexpressing T cells showed enhanced, TCR-mediated signaling and downstream IL-2 production. For the mechanisms, STAP-2 associated with TCR-proximal CD3ζ immunoreceptor tyrosine activation motifs and phosphorylated LCK, resulting in enhancement of their binding after TCR stimulation. In parallel, STAP-2 expression is required for full activation of downstream TCR signaling. Importantly, STAP-2-deficient mice exhibited slight phenotypes of CD4+ T-cell-mediated inflammatory diseases, such as experimental autoimmune encephalomyelitis, whereas STAP-2-overexpressing transgenic mice showed severe phenotypes of these diseases. Together, STAP-2 is an adaptor protein to enhance TCR signaling; therefore, manipulating STAP-2 will have an ability to improve the treatment of patients with autoimmune diseases as well as the chimeric Ag receptor T cell therapy.

Preclinical Analysis of Candidate Anti-Human CD79 Therapeutic Antibodies Using a Humanized CD79 Mouse Model.

The BCR comprises a membrane-bound Ig that is noncovalently associated with a heterodimer of CD79A and CD79B. While the BCR Ig component functions to sense extracellular Ag, CD79 subunits contain cytoplasmic ITAMs that mediate intracellular propagation of BCR signals critical for B cell development, survival, and Ag-induced activation. CD79 is therefore an attractive target for Ab and chimeric Ag receptor T cell therapies for autoimmunity and B cell neoplasia. Although the mouse is an attractive model for preclinical testing, due to its well-defined immune system, an obstacle is the lack of cross-reactivity of candidate therapeutic anti-human mAbs with mouse CD79. To overcome this problem, we generated knockin mice in which the extracellular Ig-like domains of CD79A and CD79B were replaced with human equivalents. In this study, we describe the generation and characterization of mice expressing chimeric CD79 and report studies that demonstrate their utility in preclinical analysis of anti-human CD79 therapy. We demonstrate that human and mouse CD79 extracellular domains are functionally interchangeable, and that anti-human CD79 lacking Fc region effector function does not cause significant B cell depletion, but induces 1) decreased expression of plasma membrane-associated IgM and IgD, 2) uncoupling of BCR-induced tyrosine phosphorylation and calcium mobilization, and 3) increased expression of PTEN, consistent with the levels observed in anergic B cells. Finally, anti-human CD79 treatment prevents disease development in two mouse models of autoimmunity. We also present evidence that anti-human CD79 treatment may inhibit Ab secretion by terminally differentiated plasmablasts and plasma cells in vitro.

Long Noncoding RNA lncNDEPD1 Regulates PD-1 Expression via miR-3619-5p in CD8+ T Cells.

Therapies targeting programmed cell death protein 1 (PD-1) have gained great success in patients with multiple types of cancer. The regulatory mechanisms underlying PD-1 expression have been extensively explored. However, the impact of long noncoding RNAs on PD-1 expression remains elusive. In this study, we identified the Notch1/lncNDEPD1 axis, which plays a critical role in PD-1 expression in human CD8+ T cells. RNA sequencing and quantitative reverse transcription PCR data showed that lncNDEPD1 was upregulated in activated T cells, especially in PD-1high subsets. Fluorescence in situ hybridization demonstrated that lncNDEPD1 was localized in the cytoplasm. A mechanistic study showed that lncNDEPD1 could bind with miR-3619-5p and PDCD1 mRNA to prevent PDCD1 mRNA degradation and then upregulate PD-1 expression. A chromatin immunoprecipitation assay showed that Notch1 directly binds to the promoter of lncNDEPD1 instead of PDCD1 Furthermore, chimeric Ag receptor T cells expressing lncNDEPD1-specific short hairpin RNAs were generated. Chimeric Ag receptor T cells with decreased lncNDEPD1 expression showed enhanced tumoricidal effects when PD-L1 was present. Our work uncovered a new regulatory mechanism of PD-1 expression and thus provided a potential target to decrease PD-1 without affecting T cell function.

Sweet Immune Checkpoint Targets to Enhance T Cell Therapy

Abstract Despite tremendous success against hematological malignancies, the performance of chimeric Ag receptor T cells against solid tumors remains poor. In such settings, the lack of success of this groundbreaking immunotherapy is in part mediated by ligand engagement of immune checkpoint molecules on the surface of T cells in the tumor microenvironment. Although CTLA-4 and programmed death-1 (PD-1) are well-established checkpoints that inhibit T cell activity, the engagement of glycans and glycan-binding proteins are a growing area of interest due to their immunomodulatory effects. This review discusses exemplary strategies to neutralize checkpoint molecules through an in-depth overview of genetic engineering approaches aimed at overcoming the inhibitory programmed death ligand-1 (PD-L1)/PD-1 axis in T cell therapies and summarizes current knowledge on glycoimmune interactions that mediate T cell immunosuppression.

CD8+ T Cells Expressing an HLA-DR1 Chimeric Antigen Receptor Target Autoimmune CD4+ T Cells in an Antigen-Specific Manner and Inhibit the Development of Autoimmune Arthritis.

Ag-specific immunotherapy is a long-term goal for the treatment of autoimmune diseases; however developing a means of therapeutically targeting autoimmune T cells in an Ag-specific manner has been difficult. Through the engineering of an HLA-DR1 chimeric Ag receptor (CAR), we have produced CD8+ CAR T cells that target CD4+ T cells in an Ag-specific manner and tested their ability to inhibit the development of autoimmune arthritis in a mouse model. The DR1 CAR molecule was engineered to contain CD3ζ activation and CD28 signaling domains and a covalently linked autoantigenic peptide from type II collagen (CII; DR1-CII) to provide specificity for targeting the autoimmune T cells. Stimulation of the DR1-CII CAR T cells by an anti-DR Ab induced cytokine production, indicating that the DR1-CAR functions as a chimeric molecule. In vitro CTL assays using cloned CD4+ T cells as target cells demonstrated that the DR1-CII CAR T cells efficiently recognize and kill CD4+ T cells that are specific for the CII autoantigen. The CTL function was highly specific, as no killing was observed using DR1-restricted CD4+ T cells that recognize other Ags. When B6.DR1 mice, in which autoimmune arthritis had been induced, were treated with the DR1-CII CAR T cells, the CII-specific autoimmune CD4+ T cell response was significantly decreased, autoantibody production was suppressed, and the incidence and severity of the autoimmune arthritis was diminished. These data demonstrate that HLA-DR CAR T cells have the potential to provide a highly specific therapeutic approach for the treatment of autoimmune disease.

Stem cell-like memory T cells: The generation and application.

Stem cell-like memory T cells (Tscm), are a newly defined memory T cell subset with characteristics of long life span, consistent self-renewing, rapid differentiation into effector T cells, and apoptosis resistance. These features indicate that Tscm have great therapeutic or preventive purposes, including being applied in chimeric Ag receptor-engineered T cells, TCR gene-modified T cells, and vaccines. However, the little knowledge about Tscm development restrains their applications. Strength and duration of TCR signaling, cytokines and metabolism in the T cells during activation all influence the Tscm development via regulating transcriptional factors and cell signaling pathways. Here, we summarize the molecular and cellular pathways involving Tscm differentiation, and its clinical application for cancer immunotherapy and prevention.

T Cells Retain Pivotal Antitumoral Functions under Tumor-Treating Electric Fields.

Tumor-treating fields (TTFields) are a localized, antitumoral therapy using alternating electric fields, which impair cell proliferation. Combining TTFields with tumor immunotherapy constitutes a rational approach; however, it is currently unknown whether TTFields' locoregional effects are compatible with T cell functionality. Healthy donor PBMCs and viably dissociated human glioblastoma samples were cultured under either standard or TTFields conditions. Select pivotal T cell functions were measured by multiparametric flow cytometry. Cytotoxicity was evaluated using a chimeric Ag receptor (CAR)-T-based assay. Glioblastoma patient samples were acquired before and after standard chemoradiation or standard chemoradiation + TTFields treatment and examined by immunohistochemistry and by RNA sequencing. TTFields reduced the viability of proliferating T cells, but had little or no effect on the viability of nonproliferating T cells. The functionality of T cells cultured under TTFields was retained: they exhibited similar IFN-γ secretion, cytotoxic degranulation, and PD1 upregulation as controls with similar polyfunctional patterns. Glioblastoma Ag-specific T cells exhibited unaltered viability and functionality under TTFields. CAR-T cells cultured under TTFields exhibited similar cytotoxicity as controls toward their CAR target. Transcriptomic analysis of patients' glioblastoma samples revealed a significant shift in the TTFields-treated versus the standard-treated samples, from a protumoral to an antitumoral immune signature. Immunohistochemistry of samples before and after TTFields treatment showed no reduction in T cell infiltration. T cells were found to retain key antitumoral functions under TTFields settings. Our data provide a mechanistic insight and a rationale for ongoing and future clinical trials that combine TTFields with immunotherapy.

Spacer Length Modification Facilitates Discrimination between Normal and Neoplastic Cells and Provides Clinically Relevant CD37 CAR T Cells.

Despite the remarkable initial efficacy of CD19 chimeric Ag receptor T (CAR-T) cell therapy, a high incidence of relapse has been observed. To further increase treatment efficacy and reduce the rate of escape of Ag-negative cells, we need to develop CAR-T cells that target other Ags. Given its restricted expression pattern, CD37 was considered a preferred novel target for immunotherapy in hematopoietic malignancies. Therefore, we designed a CD37-targeting CAR-T (CD37CAR-T) using the single-chain variable fragment of a humanized anti-CD37 Ab, transmembrane and intracellular domains of CD28, and CD3ζ signaling domains. High levels of CD37 expression were confirmed in B cells from human peripheral blood and bone marrow B cell precursors at late developmental stages; by contrast, more limited expression of CD37 was observed in early precursor B cells. Furthermore, we found that human CD37CAR-T cells with longer spacer lengths exhibited high gene transduction efficacy but reduced capacity to proliferate; this may be due to overactivation and fratricide. Spacer length optimization resulted in a modest transduction efficiency together with robust capacity to proliferate. CD37CAR-T cells with optimized spacer length efficiently targeted various CD37+ human tumor cell lines but had no impact on normal leukocytes both in vitro and in vivo. CD37CAR-T cells effectively eradicated Raji cells in xenograft model. Collectively, these results suggested that spacer-optimized CD37CAR-T cells could target CD37-high neoplastic B cells both in vitro and in vivo, with only limited interactions with their normal leukocyte lineages, thereby providing an additional promising therapeutic intervention for patients with B cell malignancies.

Systems Approaches to Unravel T Cell Function and Therapeutic Potential in Autoimmune Disease

Producing Ag-specific immune responses constrained to target tissues or cells that can be engaged or disengaged at will is predicated on understanding the network of genes governing immune cell function, defining the rules underlying Ag specificity, and synthesizing the tools to engineer them. The successes and limitations of chimeric Ag receptor (CAR) T cells emphasize this goal, and advances in high-throughput sequencing, large-scale genomic screens, single-cell profiling, and genetic modification are providing the necessary data to bring it to fruition-including a broader application into the treatment of autoimmune diseases. In this review, we delve into the implementation of these developments, survey the relevant works, and propose a framework for generating the next generation of synthetic T cells informed by the principles learned from these systems approaches.

A Chimeric Antigen Receptor That Binds to a Conserved Site on MICA.

The NKG2D ligand MHC class I chain-related protein A (MICA) is expressed on many varieties of malignant cells but is absent from most normal tissues, and thus represents a potential target for chimeric Ag receptor (CAR) T cell-based therapeutics. However, there are more than 100 alleles of MICA, so the ability to target a conserved site is needed for a therapy to be used in most patients. In this study, we describe a fully human anti-MICA CAR created by fusing the single-chain fragment variable B2 to the full length DAP10 protein and the traditional CD3ζ signaling domain. Human T cells expressing the B2 CAR killed MICA-positive tumor cells, produced IFN-γ when in contact with MICA-positive tumor cells or plate-bound MICA protein, and inhibited PANC-1 growth in a mouse xenograft model. To localize B2's epitope on MICA, we used novel computational methods to model potential binding modes and to design mutational variants of MICA testing these hypotheses. Flow cytometry using a commercial anti-MICA/MICB Ab indicated that the variant proteins were expressed at high levels on transduced P815 cell lines. One variant protein (R38S/K40T/K57E) showed reduced staining with a B2-IgG1 fusion protein compared with controls and did not induce IFN-γ production by human T cells expressing the B2 CAR. These results show antitumor activity of MICA-specific CAR T cells and indicate an essential role for a conserved site in the exposed loop involving aa 38-57 of MICA. This study describes a novel MICA-specific CAR and discusses its potential use as a cancer therapeutic.

Tetramer Immunization and Selection Followed by CELLISA Screening to Generate Monoclonal Antibodies against the Mouse Cytomegalovirus m12 Immunoevasin.

The generation of reliable mAb of unique and desired specificities serves as a valuable technology to study protein expression and function. However, standard approaches to mAb generation usually involve large-scale protein purification and intensive screening. In this study, we describe an optimized high-throughput proof-of-principle method for the expanded generation, enrichment, and screening of mouse hybridomas secreting mAb specific for a protein of interest. Briefly, we demonstrate that small amounts of a biotinylated protein of interest can be used to generate tetramers for use as prime-boost immunogens, followed by selective enrichment of Ag-specific B cells by magnetic sorting using the same tetramers prior to hybridoma generation. This serves two purposes: 1) to effectively expand both low- and high-affinity B cells specific for the antigenic bait during immunization and 2) to minimize subsequent laborious hybridoma efforts by positive selection of Ag-specific, Ab-secreting cells prior to hybridoma fusion and validation screening. Finally, we employ a rapid and inexpensive screening technology, CELLISA, a high-throughput validation method that uses a chimeric Ag fused to the CD3ζ signaling domain expressed on enzyme-generating reporter cells; these reporters can detect specific mAb in hybridoma supernatants via plate-bound Ab-capture arrays, thereby easing screening. Using this strategy, we generated and characterized novel mouse mAb specific for a viral immunoevasin, the mouse CMV m12 protein, and suggest that these mAb may protect mice from CMV infection via passive immunity.