ABSTRACT
In chronic stages of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalitis (EAE), connexin (Cx)43 gap junction channel proteins are overexpressed because of astrogliosis. To elucidate the role of increased Cx43, the central nervous system (CNS)-permeable Cx blocker INI-0602 was therapeutically administered. C57BL6 mice with chronic EAE initiated by MOG35-55 received INI-0602 (40 mg/kg) or saline intraperitoneally every other day from days post-immunization (dpi) 17-50. Primary astroglia were employed to observe calcein efflux responses. In INI-0602-treated mice, EAE clinical signs improved significantly in the chronic phase, with reduced demyelination and decreased CD3+ T cells, Iba-1+ and F4/80+ microglia/macrophages, and C3+GFAP+ reactive astroglia infiltration in spinal cord lesions. Flow cytometry analysis of CD4+ T cells from CNS tissues revealed significantly reduced Th17 and Th17/Th1 cells (dpi 24) and Th1 cells (dpi 50). Multiplex array of cerebrospinal fluid showed significantly suppressed IL-6 and significantly increased IL-10 on dpi 24 in INI-0602-treated mice, and significantly suppressed IFN-γ and MCP-1 on dpi 50 in the same group. In vitro INI-0602 treatment inhibited ATP-induced calcium propagations of Cx43+/+ astroglial cells to similar levels of those of Cx43-/- cells. Astroglial Cx43 hemichannels represent a novel therapeutic target for chronic EAE and MS.
Subject(s)
Astrocytes , Connexin 43 , Encephalomyelitis, Autoimmune, Experimental , Multiple Sclerosis , Animals , Mice , Astrocytes/metabolism , Astrocytes/drug effects , Astrocytes/pathology , Connexin 43/metabolism , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/pathology , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Mice, Inbred C57BL , Multiple Sclerosis/drug therapy , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , Heterocyclic Compounds, 4 or More Rings/pharmacologyABSTRACT
CD40 is a member of the tumor necrosis factor receptor superfamily, and it is widely expressed on immune and non-immune cell types. The interaction between CD40 and the CD40 ligand (CD40L) plays an essential function in signaling, and the CD40/CD40L complex works as an immune checkpoint molecule. CD40 has become a therapeutic target, and a variety of agonistic/antagonistic anti-CD40 monoclonal antibodies (mAbs) have been developed. To better understand the mode of action of anti-CD40 mAbs, we determined the X-ray crystal structures of dacetuzumab (agonist) and bleselumab (antagonist) in complex with the extracellular domain of human CD40, respectively. The structure reveals that dacetuzumab binds to CD40 on the top of cysteine-rich domain 1 (CRD1), which is the domain most distant from the cell surface, and it does not compete with CD40L binding. The binding interface of bleselumab spread between CRD2 and CRD1, overlapping with the binding surface of the ligand. Our results offer important insights for future structural and functional studies of CD40 and provide clues to understanding the mechanism of biological response. These data can be applied to developing new strategies for designing antibodies with more therapeutic efficacy.
Subject(s)
Antibodies, Monoclonal, Humanized , CD40 Antigens , Humans , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Monoclonal, Humanized/immunology , Binding Sites , CD40 Antigens/chemistry , CD40 Antigens/immunology , CD40 Antigens/metabolism , CD40 Ligand/chemistry , CD40 Ligand/metabolism , CD40 Ligand/immunology , Crystallography, X-Ray , Models, Molecular , Protein Binding , Protein ConformationABSTRACT
Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we develop BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of antibody variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulate the size of BpAb-TNFR2 immunocomplexes to result in controlled agonistic activities. Our series of results indicate that the relative positions of the two epitopes recognized by the BpAb are critical for controlling its signaling activity. One particular antagonist, Bp109-92, binds TNFR2 in a 1:1 manner without unwanted signal transduction, and its structural basis is determined using cryo-electron microscopy. This antagonist suppresses the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.
Subject(s)
Antibodies , Receptors, Tumor Necrosis Factor, Type II , Epitopes , Cryoelectron Microscopy , Signal TransductionABSTRACT
We previously reported a novel secondary progressive multiple sclerosis (SPMS) model, progressive experimental autoimmune encephalomyelitis (pEAE), in oligodendroglia-specific Cx47-inducible conditional knockout (Cx47 icKO) mice. Based on our prior study showing the efficacy of iguratimod (IGU), an antirheumatic drug, for acute EAE treatment, we aimed to elucidate the effect of IGU on the SPMS animal model. We induced pEAE by immunizing Cx47 icKO mice with myelin oligodendrocyte glycoprotein peptide 35-55. IGU was orally administered from 17 to 50 days post-immunization. We also prepared a primary mixed glial cell culture and measured cytokine levels in the culture supernatant after stimulation with designated cytokines (IL-1α, C1q, TNF-α) and lipopolysaccharide. A migration assay was performed to evaluate the effect of IGU on the migration ability of T cells toward mixed glial cell cultures. IGU treatment ameliorated the clinical signs of pEAE, decreased the demyelinated area, and attenuated glial inflammation on immunohistochemical analysis. Additionally, IGU decreased the intrathecal IL-6 level and infiltrating Th17 cells. The migration assay revealed reduced Th17 cell migration and IL-6 levels in the culture supernatant after IGU treatment. Collectively, IGU successfully mitigated the clinical signs of pEAE by suppressing Th17 migration through inhibition of IL-6 production by proinflammatory-activated glial cells.
ABSTRACT
Proximity-induced chemical reactions are site-specific and rapid by taking advantage of their high affinity and highly selective interactions with the template. However, reactions induced solely by antibody-antigen interactions have not been developed. Herein, we propose a biepitopic antigen-templated chemical reaction (BATER) as a novel template reaction. In BATER, reactive functional groups are conjugated to two antibodies that interact with two epitopes of the same antigen to accelerate the reaction. We developed a method for visualizing the progress of BATER using fluorogenic click chemistry for optimal antibody selection and linker design. The reaction is accelerated in the presence of a specific antigen in a linker length-dependent manner. The choice of the antibody epitope is important for a rapid reaction. This design will lead to various applications of BATER in living systems.
Subject(s)
Epitopes , Epitopes/chemistry , Epitopes/immunology , Antibodies/chemistry , Antibodies/immunology , Antigens/chemistry , Antigens/immunology , Fluorescent Dyes/chemistryABSTRACT
The PD-1 receptor triggers a negative immunoregulatory mechanism that prevents overactivation of immune cells and subsequent inflammatory diseases. Because of its biological significance, PD-1 has been a drug target for modulating immune responses. Immunoenhancing anti-PD-1 blocking antibodies have become a widely used cancer treatment; however, little is known about the required characteristics for anti-PD-1 antibodies to be capable of stimulating immunosuppressive activity. Here, we show that PD-1 agonists exist in the group of anti-PD-1 antibodies recognizing the membrane-proximal extracellular region in sharp contrast to the binding of the membrane-distal region by blocking antibodies. This trend was consistent in an analysis of 81 anti-human PD-1 monoclonal antibodies. Because PD-1 agonist antibodies trigger immunosuppressive signaling by cross-linking PD-1 molecules, Fc engineering to enhance FcγRIIB binding of PD-1 agonist antibodies notably improved human T cell inhibition. A PD-1 agonist antibody suppressed inflammation in murine disease models, indicating its clinical potential for treatment of various inflammatory disorders, including autoimmune diseases.
Subject(s)
Antibodies, Monoclonal , T-Lymphocytes , Animals , Mice , Antibodies, Blocking/metabolism , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal/metabolism , HumansABSTRACT
Angiotensin-converting enzyme 2 (ACE2) is a receptor for cell entry of SARS-CoV-2, and recombinant soluble ACE2 protein inhibits SARS-CoV-2 infection as a decoy. ACE2 is a carboxypeptidase that degrades angiotensin II, thereby improving the pathologies of cardiovascular disease or acute lung injury. Here we show that B38-CAP, an ACE2-like enzyme, is protective against SARS-CoV-2-induced lung injury. Endogenous ACE2 expression is downregulated in the lungs of SARS-CoV-2-infected hamsters, leading to elevation of angiotensin II levels. Recombinant Spike also downregulates ACE2 expression and worsens the symptoms of acid-induced lung injury. B38-CAP does not neutralize cell entry of SARS-CoV-2. However, B38-CAP treatment improves the pathologies of Spike-augmented acid-induced lung injury. In SARS-CoV-2-infected hamsters or human ACE2 transgenic mice, B38-CAP significantly improves lung edema and pathologies of lung injury. These results provide the first in vivo evidence that increasing ACE2-like enzymatic activity is a potential therapeutic strategy to alleviate lung pathologies in COVID-19 patients.
Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19 Drug Treatment , COVID-19/prevention & control , Lung Injury/prevention & control , SARS-CoV-2/drug effects , Virus Internalization/drug effects , Acute Lung Injury , Angiotensin II , Animals , COVID-19/pathology , Carboxypeptidases , Chlorocebus aethiops , Cricetinae , Disease Models, Animal , Female , Humans , Lung/pathology , Male , Mice , Mice, Transgenic , Pulmonary Edema/pathology , Pulmonary Edema/prevention & control , Spike Glycoprotein, Coronavirus/drug effects , Vero CellsABSTRACT
A major class of bispecific antibodies (BsAbs) utilizes heterodimeric Fc to produce the native immunoglobulin G (IgG) structure. Because appropriate pairing of heavy and light chains is required, the design of BsAbs produced through recombination or reassembly of two separately-expressed antigen-binding fragments is advantageous. One such method uses intein-mediated protein trans-splicing (IMPTS) to produce an IgG1-based structure. An extra Cys residue is incorporated as a consensus sequence for IMPTS in successful examples, but this may lead to potential destabilization or disturbance of the assay system. In this study, we designed a BsAb linked by IMPTS, without the extra Cys residue. A BsAb binding to both TNFR2 and CD30 was successfully produced. Cleaved side product formation was inevitable, but it was minimized under the optimized conditions. The fine-tuned design is suitable for the production of IgG-like BsAb with high symmetry between the two antigen-binding fragments that is advantageous for screening BsAbs.
Subject(s)
Antibodies, Bispecific/isolation & purification , Immunoglobulin Fab Fragments/immunology , Immunoglobulin G/immunology , Protein Engineering/methods , Recombinant Fusion Proteins/isolation & purification , HumansABSTRACT
The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and life. A useful pathological animal model accurately reflecting human pathology is needed to overcome the COVID-19 crisis. In the present study, COVID-19 cynomolgus monkey models including monkeys with underlying diseases causing severe pathogenicity such as metabolic disease and elderly monkeys were examined. Cynomolgus macaques with various clinical conditions were intranasally and/or intratracheally inoculated with SARS-CoV-2. Infection with SARS-CoV-2 was found in mucosal swab samples, and a higher level and longer period of viral RNA was detected in elderly monkeys than in young monkeys. Pneumonia was confirmed in all of the monkeys by computed tomography images. When monkeys were readministrated SARS-CoV-2 at 56 d or later after initial infection all of the animals showed inflammatory responses without virus detection in swab samples. Surprisingly, in elderly monkeys reinfection showed transient severe pneumonia with increased levels of various serum cytokines and chemokines compared with those in primary infection. The results of this study indicated that the COVID-19 cynomolgus monkey model reflects the pathophysiology of humans and would be useful for elucidating the pathophysiology and developing therapeutic agents and vaccines.
Subject(s)
COVID-19/immunology , Disease Models, Animal , Macaca fascicularis/immunology , Primate Diseases/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/virology , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Lung/diagnostic imaging , Lung/immunology , Lung/virology , Macaca fascicularis/virology , Male , Primate Diseases/virology , SARS-CoV-2/physiology , Tomography, X-Ray Computed/methods , Virus Shedding/immunology , Virus Shedding/physiologyABSTRACT
COVID-19, caused by SARS-CoV-2, has spread worldwide with dire consequences. To urgently investigate the pathogenicity of COVID-19 and develop vaccines and therapeutics, animal models that are highly susceptible to SARS-CoV-2 infection are needed. In the present study, we established an animal model highly susceptible to SARS-CoV-2 via the intratracheal tract infection in CAG promoter-driven human angiotensin-converting enzyme 2-transgenic (CAG-hACE2) mice. The CAG-hACE2 mice showed several severe symptoms of SARS-CoV-2 infection, with definitive weight loss and subsequent death. Acute lung injury with elevated cytokine and chemokine levels was observed at an early stage of infection in CAG-hACE2 mice infected with SARS-CoV-2. Analysis of the hACE2 gene in CAG-hACE2 mice revealed that more than 15 copies of hACE2 genes were integrated in tandem into the mouse genome, supporting the high susceptibility to SARS-CoV-2. In the developed model, immunization with viral antigen or injection of plasma from immunized mice prevented body weight loss and lethality due to infection with SARS-CoV-2. These results indicate that a highly susceptible model of SARS-CoV-2 infection in CAG-hACE2 mice via the intratracheal tract is suitable for evaluating vaccines and therapeutic medicines.
Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Animals , COVID-19/pathology , Disease Models, Animal , Disease Susceptibility , Female , Male , Mice, Inbred C57BL , Mice, Transgenic , Promoter Regions, Genetic , SARS-CoV-2/isolation & purificationABSTRACT
BACKGROUND: Brain astroglia are activated preceding the onset of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We characterized the effects of brain astroglia on spinal cord inflammation, focusing on astroglial connexin (Cx)43, because we recently reported that Cx43 has a critical role in regulating neuroinflammation. METHODS: Because glutamate aspartate transporter (GLAST)+ astroglia are enriched in the brain gray matter, we generated Cx43fl/fl;GLAST-CreERT2/+ mice that were brain gray matter astroglia-specific Cx43 conditional knockouts (Cx43 icKO). EAE was induced by immunization with myelin oligodendroglia glycoprotein (MOG) 35-55 peptide 10 days after tamoxifen injection. Cx43fl/fl mice were used as controls. RESULTS: Acute and chronic EAE signs were significantly milder in Cx43 icKO mice than in controls whereas splenocyte MOG-specific responses were unaltered. Histologically, Cx43 icKO mice showed significantly less demyelination and fewer CD45+ infiltrating immunocytes, including F4/80+ macrophages, and Iba1+ microglia in the spinal cord than controls. Microarray analysis of the whole cerebellum revealed marked upregulation of anti-inflammatory A2-specific astroglia gene sets in the pre-immunized phase and decreased proinflammatory A1-specific and pan-reactive astroglial gene expression in the onset phase in Cx43 icKO mice compared with controls. Astroglia expressing C3, a representative A1 marker, were significantly decreased in the cerebrum, cerebellum, and spinal cord of Cx43 icKO mice compared with controls in the peak phase. Isolated Cx43 icKO spinal microglia showed more anti-inflammatory and less proinflammatory gene expression than control microglia in the pre-immunized phase. In particular, microglial expression of Ccl2, Ccl5, Ccl7, and Ccl8 in the pre-immunized phase and of Cxcl9 at the peak phase was lower in Cx43 icKO than in controls. Spinal microglia circularity was significantly lower in Cx43 icKO than in controls in the peak phase. Significantly lower interleukin (IL)-6, interferon-γ, and IL-10 levels were present in cerebrospinal fluid from Cx43 icKO mice in the onset phase compared with controls. CONCLUSIONS: The ablation of Cx43 in brain gray matter astroglia attenuates EAE by promoting astroglia toward an anti-inflammatory phenotype and suppressing proinflammatory activation of spinal microglia partly through depressed cerebrospinal fluid proinflammatory cytokine/chemokine levels. Brain astroglial Cx43 might be a novel therapeutic target for MS.
Subject(s)
Astrocytes/metabolism , Connexin 43/deficiency , Connexin 43/genetics , Demyelinating Diseases , Gray Matter/metabolism , Neuroinflammatory Diseases , Spinal Cord/pathology , Animals , Astrocytes/pathology , Chemokines/cerebrospinal fluid , Connexin 43/metabolism , Cytokines/cerebrospinal fluid , Demyelinating Diseases/etiology , Demyelinating Diseases/physiopathology , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/genetics , Gene Expression , Gray Matter/pathology , Mice , Mice, Knockout , Multiple Sclerosis/physiopathology , Neuroinflammatory Diseases/etiology , Neuroinflammatory Diseases/genetics , Neuroinflammatory Diseases/physiopathology , Spinal Cord/immunologyABSTRACT
In multiple sclerosis plaques, oligodendroglial connexin (Cx) 47 constituting main gap junction channels with astroglial Cx43 is persistently lost. As mice with Cx47 single knockout exhibit no demyelination, the roles of Cx47 remain undefined. We aimed to clarify the effects of oligodendroglia-specific Cx47 inducible conditional knockout (icKO) on experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein peptide (MOG35-55) in PLP/CreERT;Cx47fl/fl mice at 14 d after tamoxifen injection. Cx47 icKO mice demonstrated exacerbation of acute and chronic relapsing EAE with more pronounced demyelination than Cx47 flox (fl)/fl littermates. CD3+ T cells more abundantly infiltrated the spinal cord in Cx47 icKO than in Cx47 fl/fl mice throughout the acute to chronic phases. CXCR3-CCR6+CD4+ and IL17+IFNγ-CD4+ helper T (Th) 17 cells isolated from spinal cord and brain tissues were significantly increased in Cx47 icKO mice compared with Cx47 fl/fl mice, while MOG35-55-specific proliferation and proinflammatory cytokine production of splenocytes were unaltered. Microarray analysis of isolated microglia revealed stronger microglial activation toward proinflammatory and injury-response phenotypes with increased expressions of chemokines that can attract Th17 cells, including Ccl2, Ccl3, Ccl4, Ccl7, and Ccl8, in Cx47 icKO mice compared with Cx47 fl/fl mice. In Cx47 icKO mice, NOS2+ and MHC class II+ microglia were more enriched immunohistochemically, and A1-specific astroglial gene expressions and astroglia immunostained for C3, a representative A1 astrocyte marker, were significantly increased at the acute phase, compared with Cx47 fl/fl mice. These findings suggest that oligodendroglia-specific Cx47 ablation induces severe inflammation upon autoimmune demyelination, underscoring a critical role for Cx47 in regulating neuroinflammation.
Subject(s)
Connexins/immunology , Multiple Sclerosis/immunology , Oligodendroglia/immunology , Animals , Chemokines/genetics , Chemokines/immunology , Connexins/genetics , Demyelinating Diseases , Disease Models, Animal , Female , Humans , Mice , Mice, Knockout , Multiple Sclerosis/genetics , Myelin Sheath/genetics , Myelin Sheath/immunology , Spinal Cord/immunology , Th17 Cells/immunologyABSTRACT
BACKGROUND: Nuclear antigen released from central nervous system (CNS) cells undergoing destruction may induce production of antinuclear antibodies (ANA). We characterized the CNS-specific production of ANA in multiple sclerosis (MS). METHODS: We assessed CNS-ANA binding to mouse cerebellar cell nuclei by immunofluorescence assay (IFA) with sera from 104 MS patients (91 relapsing-remitting; 13 secondary progressive), 30 patients with neuromyelitis optica spectrum disorders (NMOSD), and 30 healthy controls (HCs). Conventional ANA (cANA) was detected by IFA using human epithelial type-2 cells. CNS-ANA-positive cANA-negative patients were termed CNS-specific ANA-positive. Western blotting (WB) was performed using mouse cerebellar nuclear fractions. RESULTS: CNS-specific ANA were more frequent in MS than in NMOSD patients or HCs (13.5% vs 0% for both comparisons, both p < .05) and were associated with HLA-DRB1*15:01 (p = .0174). WB revealed a common 55 kDa band in seven MS patients. Compared with CNS-specific ANA-negative MS patients, those with 55 kDa band-immunoreactive CNS-specific ANA showed a higher frequency of secondary progressive MS (42.9% vs 10.0%, p = .0387) and greater Expanded Disability Status Scale scores (4.50 ± 2.02 vs 2.92 ± 2.27, p = .0506). CONCLUSIONS: The CNS-specific ANA was more frequently detected in MS patients than NMOSD patients or HCs. 55 kDa band-reactive CNS-specific ANA may reflect clinical disease progression in MS.
Subject(s)
Antibodies, Antinuclear/blood , Multiple Sclerosis, Chronic Progressive/blood , Multiple Sclerosis, Relapsing-Remitting/blood , Neuromyelitis Optica/blood , Adult , Aged , Animals , Female , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Multiple Sclerosis/blood , Multiple Sclerosis/diagnostic imaging , Multiple Sclerosis, Chronic Progressive/diagnostic imaging , Multiple Sclerosis, Relapsing-Remitting/diagnostic imaging , Neuromyelitis Optica/diagnostic imaging , Retrospective StudiesABSTRACT
Malaria is a deadly infectious disease associated with fundamental changes in the composition of the memory B cell (MBC) compartment, most notably a large expansion of T-bet+ MBCs, termed atypical MBCs. However, we know little about the precursors of atypical MBCs and the conditions that drive their differentiation. We compared the responses of human tonsil naïve B cells, MBCs, and germinal center B cells to a variety of stimulatory conditions. We determined that prolonged antigen presentation in the presence of CpG and IFN-γ induced maximal expression of T-bet and other phenotypic markers of malaria-associated atypical MBCs primarily in naïve B cells in vitro. Importantly T-bet+ naïve-derived B cells resembled atypical MBCs in their hypo-responsiveness to signaling through their B cell receptors. Thus, naïve B cells can be induced to differentiate into phenotypically and functionally atypical-like MBCs in vitro under conditions that may prevail in chronic infectious diseases in vivo.
Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Immunologic Memory , Malaria/immunology , Malaria/metabolism , Palatine Tonsil/cytology , Palatine Tonsil/immunology , T-Box Domain Proteins/metabolism , Antigens/immunology , Biomarkers , Gene Expression , Host-Parasite Interactions/immunology , Humans , Interferon-gamma/metabolism , Interferon-gamma/pharmacology , Lymphocyte Activation , Malaria/parasitology , Oligodeoxyribonucleotides/immunology , Plasmodium falciparum/immunology , Receptors, Antigen, B-Cell/metabolism , Signal Transduction , T-Box Domain Proteins/geneticsABSTRACT
Multiple myeloma (MM) is a B cell malignancy for which new treatments are urgently needed. The B cell maturation antigen (BCMA) is a lineage-restricted differentiation protein highly expressed on myeloma. Recombinant immunotoxins (RITs) are proteins composed of the Fv or Fab portion of an antibody fused to a bacterial toxin. We previously treated H929 myeloma s.c. tumors with anti-BCMA immunotoxins, very active on killing cultured cells, and observed tumor growth inhibition but not complete tumor responses. To determine if immunotoxins were more active against cells growing in the bone marrow (BM), the normal location of myeloma cells, we developed a BM mouse model that is more relevant to human disease. H929 cells were transfected with luciferase and GFP, enriched by flow, recycled through the BM of a mouse, and injected IV into nonobese diabetic scid γ mice (NSG) mice. A second myeloma mouse model used the MM.1S-GFP-luc cell line. Mice were treated IV with immunotoxins, and the tumor burden was assessed using bioluminescence imaging. We achieved complete durable remissions when treating mice with H929-GFP-luc cells with anti-BCMA RITs both leptomycin B-75 (LMB-75) [anti-BCMA-disulfide-stabilized (ds)-Fv-PE24] (where PE represents Pseudomonas exotoxin A) or LMB-70 (anti-BCMA-Fab-PE24) given every other day for 5-d (QOD×5) doses beginning on day 4 or day 8. Mice were disease free at 3 months; untreated mice became moribund around day 40. We also achieved long-term responses using the MM.1S-GFP-luc myeloma cell line. Treatment with an 1.5 mg/kg LMB-75 QOD×5 anti-BCMA RIT beginning on day 4 caused the complete disappearance of tumors for 80 days. To summarize, LMB-75 and LMB-70, our anti-BCMA RITs, induced complete durable responses in two myeloma models.
Subject(s)
Antibodies, Monoclonal/administration & dosage , B-Cell Maturation Antigen/immunology , Immunotherapy , Immunotoxins/administration & dosage , Multiple Myeloma/therapy , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Multiple Myeloma/genetics , Multiple Myeloma/immunology , Multiple Myeloma/physiopathology , Remission InductionABSTRACT
BACKGROUND: Disulfide-linked knobs-into-holes (dKiH) mutation is a well-validated antibody engineering technique to force heterodimer formation of different Fcs for efficient production of bispecific antibodies. An artificial disulfide bond is created between mutated cysteine residues in CH3 domain of human IgG1 Fc whose positions are 354 of the "knob" and 349 of the "hole" heavy chains. The disulfide bond is located adjacent to the exposed loop with allotypic variations at positions 356 and 358. Effects of the variation on the biophysical property of the Fc protein with dKiH mutations have not been reported. METHODS: We produced dKiH Fc proteins of high purity by affinity-tag fusion to the hole chain and IdeS treatment, which enabled removal of mispaired side products. Thermal stability was analyzed in a differential scanning calorimetry instrument. RESULTS: We firstly analyzed the effect of the difference in allotypes of the Fcs on the thermal stability of the heterodimeric Fc. We observed different melting profiles of the two allotypes (G1m1 and nG1m1) showing slightly higher melting temperature of G1m1 than nG1m1. Additionally, we showed different characteristics among heterodimers with different combinations of the allotypes in knob and hole chains. CONCLUSION: Allotypic variations affected melting profiles of dKiH Fc proteins possibly with larger contribution of variations adjacent to the disulfide linkage.
ABSTRACT
BACKGROUND: Multiple myeloma (MM) is a B-cell malignancy that is incurable for the majority of patients. New treatments are urgently needed. Recombinant immunotoxins (RITs) are chimeric proteins that are composed of the Fv or Fab portion of an antibody fused to a bacterial toxin. B-cell maturation antigen (BCMA) is a lineage-restricted differentiation protein and an ideal target for antibody-based treatments for MM. METHODS: RITs were produced by expressing plasmids encoding the components of the anti-BCMA RITs in Escherichia coli followed by inclusion body preparation, solubilization, renaturation, and purification by column chromatography. The cytotoxic activity of RITs was tested in vitro by WST-8 assays. We also measured their binding to human and mouse serum albumins and to BCMA and measured their serum half-life in mice. RESULTS: Using Fvs from different anti-BCMA antibodies, we produced RITs that specifically kill BCMA-expressing MM cells in vitro. To increase the serum half-life in vivo, we generated RITs that are fused with albumin-binding domains (ABDs). All RITs with ABDs have some decreased activity compared to the parent RIT, which is not due to decreased binding to BCMA. CONCLUSIONS: Various new anti-BCMA immunotoxins were produced and evaluated. None of these were better than LMB-75 (anti-BCMA BM306-disulfide-stabilized Fv-LRggs) supporting the further preclinical development of LMB-75.
ABSTRACT
A 20-year-old female was hospitalized due to generalized seizure two weeks after an infection. She reported disorientation, neck stiffness and weakness in her legs. MRI FLAIR images and T2WI on her first visit to our hospital showed hyperintense lesions in the bilateral cingulate gyrus and the medial region of the superior frontal gyrus. Gadolinium (Gd)-enhanced T1WI showed enhancement in the upper part of the corpus callosum. Examination of her cerebrospinal fluid (CSF) revealed mildly elevated leucocytes. After the administration of high-dose intravenous methylprednisolone, her symptoms partially improved. However, MRI T2WI at 16 days after admission showed a lesion with a peripheral hypointense rim in the left side of the cingulate gyrus, which had ring enhancement on contrast CT. FLAIR images at 28 days after admission showed the hyperintense lesion spreading in the subcallosal area and the brainstem, and coronal short inversion time inversion recovery (STIR) images demonstrated bilateral optic neuritis. She was treated with steroid pulse therapy and plasma exchange. Thereafter her symptoms improved. The patient's CSF at 27 days after admission tested positive for anti-myelin oligodendrocyte glycoprotein (anti-MOG) antibodies and anti-N-methyl-D-aspartate (anti-NMDA) receptor antibodies. Encephalitis with optic neuritis in a patient with both anti-MOG and anti-NMDA receptor antibodies is very rare. Coexistence of multiple antibodies in the same patient may contribute to the diversity of autoimmune diseases associated with anti-MOG antibodies or anti-NMDA receptor antibodies.
Subject(s)
Autoantibodies/blood , Encephalitis/diagnosis , Encephalitis/immunology , Myelin-Oligodendrocyte Glycoprotein/immunology , Optic Neuritis/diagnosis , Optic Neuritis/immunology , Receptors, N-Methyl-D-Aspartate/immunology , Adult , Biomarkers/blood , Biomarkers/cerebrospinal fluid , Brain/diagnostic imaging , Encephalitis/etiology , Encephalitis/therapy , Female , Hemofiltration , Humans , Immunoglobulins, Intravenous/administration & dosage , Levetiracetam , Magnetic Resonance Imaging , Methylprednisolone/administration & dosage , Neuroimaging , Optic Neuritis/etiology , Piracetam/administration & dosage , Piracetam/analogs & derivatives , Pulse Therapy, Drug , Treatment Outcome , Young AdultABSTRACT
BACKGROUND: Metastatic breast cancer (BCa) is most often diagnosed months after completion of treatment of the primary tumor when a patient reports physical symptoms. Besides a physical examination, no other alternative recurrence screening method is recommended for routine follow-up care. Detection of autoantibodies against tumor-associated antigens (TAAs) has demonstrated promise for distinguishing healthy women from patients diagnosed with primary BCa. However, it is unknown what changes occur to patient autoantibody levels during and after treatment. METHODS: Three serial blood draws were collected from 200 BCa patients: before treatment, 6 and 12 months after surgery. Patients were categorized according to treatment regimen, including surgery, chemotherapy, radiation, trastuzumab and hormonal therapies. The longitudinal samples were assayed for autoantibody responses against 32 conformation-carrying TAAs using a Luminex multiplex bead assay. RESULTS: The treatment modality groups that had the greatest decrease in autoantibody response levels were radiation + hormonal therapy; radiation + chemotherapy; and radiation + hormonal therapy + chemotherapy. For these three treatment groups, autoantibody responses against 9 TAAs (A1AT, ANGPTL4, CAPC, CST2, DKK1, GFRA1, GRN, LGALS3 and LRP10) were significantly reduced at 12 months after surgery compared to before treatment. One TAA, GRP78, had a significantly increased autoantibody response after 12 months. CONCLUSIONS: Single treatment regimens alone did not significantly alter autoantibodies levels against the studied TAAs. Radiation treatment was the common denominator of the three most affected groups for significant changes in autoantibody response levels.