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1.
J Immunol ; 208(7): 1566-1584, 2022 04 01.
Article in English | MEDLINE | ID: mdl-35321883

ABSTRACT

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.


Subject(s)
B-Lymphocytes , Lymphocyte Activation , Animals , Antibodies, Monoclonal/therapeutic use , Clonal Anergy , Disease Models, Animal , Mice
2.
Immunotherapy ; 12(1): 89-103, 2020 01.
Article in English | MEDLINE | ID: mdl-31902264

ABSTRACT

Recently, two chimeric antigen receptor (CAR) T cell therapies were approved based on their remarkable efficacy in patients with hematological malignancies. By contrast, CAR-T cell therapies results in solid tumors have been less promising. To develop the next generation of T cell therapies a better understanding of T cell receptor (TCR) biology and its implication for the design of synthetic receptors is critical. Here, we review current and newly developed forms of T cell therapies and how their utilization of different components of the TCR signaling machinery and their requirement for engagement (or not) of human leukocyte antigen impacts their design, efficacy and applicability as cancer drugs. Notably, we highlight the development of human leukocyte antigen-independent T cell platforms that utilize the full TCR complex as having promise to overcome some of the limitations of existing T cell therapies.


Subject(s)
Cancer Vaccines/immunology , Immunotherapy, Adoptive/methods , Neoplasms/therapy , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolism , Animals , Humans , Molecular Targeted Therapy , Neoplasms/immunology , Protein Engineering , Receptors, Antigen, T-Cell/genetics , Signal Transduction , T-Lymphocytes/immunology
3.
Proc Natl Acad Sci U S A ; 115(46): E10898-E10906, 2018 11 13.
Article in English | MEDLINE | ID: mdl-30373813

ABSTRACT

Chimeric antigen receptor (CAR) T cells with a long-lived memory phenotype are correlated with durable, complete remissions in patients with leukemia. However, not all CAR T cell products form robust memory populations, and those that do can induce chronic B cell aplasia in patients. To address these challenges, we previously developed a switchable CAR (sCAR) T cell system that allows fully tunable, on/off control over engineered cellular activity. To further evaluate the platform, we generated and assessed different murine sCAR constructs to determine the factors that afford efficacy, persistence, and expansion of sCAR T cells in a competent immune system. We find that sCAR T cells undergo significant in vivo expansion, which is correlated with potent antitumor efficacy. Most importantly, we show that the switch dosing regimen not only allows control over B cell populations through iterative depletion and repopulation, but that the "rest" period between dosing cycles is the key for induction of memory and expansion of sCAR T cells. These findings introduce rest as a paradigm in enhancing memory and improving the efficacy and persistence of engineered T cell products.


Subject(s)
Bioengineering/methods , Immunotherapy, Adoptive/methods , Animals , Antigens, CD19/immunology , B-Lymphocytes/immunology , Cytokines/metabolism , Cytotoxicity, Immunologic/immunology , Female , Immunoglobulin Switch Region/genetics , Immunoglobulin Switch Region/immunology , Lymphocyte Activation/physiology , Mice , Mice, Inbred C57BL , Models, Animal , Models, Biological , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology
4.
Proc Natl Acad Sci U S A ; 113(4): E459-68, 2016 Jan 26.
Article in English | MEDLINE | ID: mdl-26759369

ABSTRACT

Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive results in clinical trials for B-cell malignancies. However, safety concerns related to the inability to control CAR-T cells once infused into the patient remain a significant challenge. Here we report the engineering of recombinant antibody-based bifunctional switches that consist of a tumor antigen-specific Fab molecule engrafted with a peptide neo-epitope, which is bound exclusively by a peptide-specific switchable CAR-T cell (sCAR-T). The switch redirects the activity of the bio-orthogonal sCAR-T cells through the selective formation of immunological synapses, in which the sCAR-T cell, switch, and target cell interact in a structurally defined and temporally controlled manner. Optimized switches specific for CD19 controlled the activity, tissue-homing, cytokine release, and phenotype of sCAR-T cells in a dose-titratable manner in a Nalm-6 xenograft rodent model of B-cell leukemia. The sCAR-T-cell dosing regimen could be tuned to provide efficacy comparable to the corresponding conventional CART-19, but with lower cytokine levels, thereby offering a method of mitigating cytokine release syndrome in clinical translation. Furthermore, we demonstrate that this methodology is readily adaptable to targeting CD20 on cancer cells using the same sCAR-T cell, suggesting that this approach may be broadly applicable to heterogeneous and resistant tumor populations, as well as other liquid and solid tumor antigens.


Subject(s)
Antigens, CD19/immunology , Antigens, Neoplasm/immunology , Immunotherapy, Adoptive/methods , Leukemia, B-Cell/therapy , Receptors, Antigen, T-Cell/immunology , Sialic Acid Binding Ig-like Lectin 2/immunology , T-Cell Antigen Receptor Specificity , T-Lymphocyte Subsets/immunology , Animals , Azides , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Basic-Leucine Zipper Transcription Factors/immunology , Cell Line, Tumor , Cytokines/metabolism , Cytotoxicity, Immunologic , Dose-Response Relationship, Immunologic , Female , Genes, Reporter , Genetic Vectors , Humans , Immunotherapy, Adoptive/adverse effects , Lymphocyte Activation , Lymphopenia/etiology , Lymphopenia/prevention & control , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Phenylalanine/analogs & derivatives , Protein Engineering/methods , Receptors, Antigen, T-Cell/genetics , Recombinant Fusion Proteins/immunology , Saccharomyces cerevisiae Proteins/immunology , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , Structure-Activity Relationship , T-Lymphocyte Subsets/transplantation , Xenograft Model Antitumor Assays
5.
J Immunol ; 192(4): 1641-50, 2014 Feb 15.
Article in English | MEDLINE | ID: mdl-24442438

ABSTRACT

B cells play a major role in the pathogenesis of many autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type I diabetes mellitus, as indicated by the efficacy of B cell-targeted therapies in these diseases. Therapeutic effects of the most commonly used B cell-targeted therapy, anti-CD20 mAb, are contingent upon long-term depletion of peripheral B cells. In this article, we describe an alternative approach involving the targeting of CD79, the transducer subunit of the B cell AgR. Unlike anti-CD20 mAbs, the protective effects of CD79-targeted mAbs do not require cell depletion; rather, they act by inducing an anergic-like state. Thus, we describe a novel B cell-targeted approach predicated on the induction of B cell anergy.


Subject(s)
Autoimmune Diseases/prevention & control , B-Lymphocytes/immunology , CD79 Antigens/immunology , Clonal Anergy/immunology , Animals , Antibodies, Monoclonal/immunology , Autoimmunity/immunology , Female , Lymphocyte Activation/immunology , Lymphocyte Count , Lymphocyte Depletion , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout
6.
Invest Ophthalmol Vis Sci ; 53(1): 301-8, 2012 Jan 25.
Article in English | MEDLINE | ID: mdl-22199243

ABSTRACT

PURPOSE: To investigate additional factors in the spontaneous development of keratitis previously reported in B10.TCRδ⁻/⁻ female mice. METHODS: The study tested whether susceptible B10.TCRδ⁻/⁻ mice have dry eyes compared with resistant B6.TCRδ⁻/⁻ females and also rederived the B10.TCRδ⁻/⁻ strain to test for the role of an infectious agent. Also assessed was whether adoptive transfer of αß T cells from autoimmune mice induced keratitis in resistant mice. In addition, a potential role was examined for B cells or autoantibodies by B-cell inactivation, and the role of female hormones was tested by ovariectomy. Finally, the study investigated whether adoptive transfer of Vγ1⁺ γδ T cells confers protection. RESULTS: Tear production in B10.TCRδ⁻/⁻ females was actually higher than in B6.TCRδ⁻/⁻ controls. Rederived B10.TCRδ⁻/⁻ mice still developed keratitis. Keratitis was induced in resistant mice after adoptive transfer of αß T cells from keratitic donors. Inactivation of B cells from susceptible mice had no effect on the development of keratitis. Ovariectomy did not significantly reduce disease in B10.TCRδ⁻/⁻ females. Adoptive transfer of Vγ1⁺ cells from wild-type donors reduced keratitis in B10.TCRδ⁻/⁻ females. CONCLUSIONS: Neither low tear levels nor ovarian hormones contribute to spontaneous keratitis in B10.TCRδ⁻/⁻ female mice, nor does it appear to depend on an infectious agent carried vertically in this strain. However, αß T cells from keratitic hosts are sufficient to induce disease in the resistant B10.TCRß⁻/⁻δ⁻/⁻ strain. Autoaggressive αß T cells in the absence of Vγ1⁺ T cells in B10.TCRδ⁻/⁻ mice may be insufficiently checked to prevent disease.


Subject(s)
Autoimmune Diseases/immunology , B-Lymphocytes/immunology , Keratitis/immunology , Receptors, Antigen, T-Cell, alpha-beta/physiology , Receptors, Antigen, T-Cell, gamma-delta/deficiency , T-Lymphocytes/immunology , Adoptive Transfer , Animals , Autoantibodies/immunology , Dry Eye Syndromes/immunology , Female , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Ovariectomy , Tears/metabolism
7.
Blood ; 113(11): 2508-16, 2009 Mar 12.
Article in English | MEDLINE | ID: mdl-18981293

ABSTRACT

We have generated mouse models of non-Hodgkin lymphoma (NHL) that rely on the cooperation between MYC overexpression and B-cell antigen receptor (BCR) signaling for the initiation and maintenance of B-cell lymphomas. Using these mouse models of NHL, we have focused on the identification of BCR-derived signal effectors that are important for the maintenance of NHL tumors. In the present study, we concentrate on Spleen tyrosine kinase (Syk), a nonreceptor tyrosine kinase required to transduce BCR-dependent signals. Using a genetic approach, we showed that Syk expression is required for the survival of murine NHL-like tumors in vitro and that tumor cells deficient in Syk fail to expand in vivo. In addition, a pharmacologic inhibitor of Syk was able to induce apoptosis of transformed B cells in vitro and led to tumor regression in vivo. Finally, we show that genetic or pharmacologic inhibition of Syk activity in human NHL cell lines are generally consistent with results found in the mouse models, suggesting that targeting Syk may be a viable therapeutic strategy.


Subject(s)
Gene Targeting , Intracellular Signaling Peptides and Proteins/physiology , Lymphoma, Non-Hodgkin/genetics , Lymphoma, Non-Hodgkin/therapy , Protein-Tyrosine Kinases/physiology , Animals , Apoptosis/genetics , Disease Models, Animal , Disease Progression , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , Lymphoma, Non-Hodgkin/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , RNA Interference/physiology , RNA, Small Interfering/pharmacology , Syk Kinase , Tumor Cells, Cultured
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