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1.
Chem Biol ; 20(2): 161-7, 2013 Feb 21.
Article in English | MEDLINE | ID: mdl-23438745

ABSTRACT

Antibody drug conjugates (ADCs) are a therapeutic class offering promise for cancer therapy. The attachment of cytotoxic drugs to antibodies can result in an effective therapy with better safety potential than nontargeted cytotoxics. To understand the role of conjugation site, we developed an enzymatic method for site-specific antibody drug conjugation using microbial transglutaminase. This allowed us to attach diverse compounds at multiple positions and investigate how the site influences stability, toxicity, and efficacy. We show that the conjugation site has significant impact on ADC stability and pharmacokinetics in a species-dependent manner. These differences can be directly attributed to the position of the linkage rather than the chemical instability, as was observed with a maleimide linkage. With this method, it is possible to produce homogeneous ADCs and tune their properties to maximize the therapeutic window.


Subject(s)
Antibodies/chemistry , Antineoplastic Agents/chemistry , Immunoconjugates/chemistry , Animals , Antibodies/immunology , Half-Life , Humans , Immunoconjugates/pharmacokinetics , Immunoconjugates/therapeutic use , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/immunology , Mice , Neoplasms/drug therapy , Rats , Transglutaminases/metabolism , Tubulin Modulators/chemistry
2.
J Immunol ; 190(4): 1481-90, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23303672

ABSTRACT

Mice transgenic for human Ig loci are an invaluable resource for the production of human Abs. However, such mice often do not yield human mAbs as effectively as conventional mice yield mouse mAbs. Suboptimal efficacy in delivery of human Abs might reflect imperfect interaction between the human membrane IgH chains and the mouse cellular signaling machinery. To obviate this problem, in this study we generated a humanized rat strain (OmniRat) carrying a chimeric human/rat IgH locus (comprising 22 human V(H)s, all human D and J(H) segments in natural configuration linked to the rat C(H) locus) together with fully human IgL loci (12 Vκs linked to Jκ-Cκ and 16 Vλs linked to Jλ-Cλ). The endogenous Ig loci were silenced using designer zinc finger nucleases. Breeding to homozygosity resulted in a novel transgenic rat line exclusively producing chimeric Abs with human idiotypes. B cell recovery was indistinguishable from wild-type animals, and human V(D)J transcripts were highly diverse. Following immunization, the OmniRat strain performed as efficiently as did normal rats in yielding high-affinity serum IgG. mAbs, comprising fully human variable regions with subnanomolar Ag affinity and carrying extensive somatic mutations, are readily obtainable, similarly to conventional mAbs from normal rats.


Subject(s)
Binding Sites, Antibody , IgG Deficiency/genetics , IgG Deficiency/immunology , Immunoglobulin Constant Regions/genetics , Immunoglobulin G/biosynthesis , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin kappa-Chains/genetics , Immunoglobulin lambda-Chains/genetics , Animals , Binding Sites, Antibody/genetics , Chromosomes, Artificial, Bacterial/genetics , Chromosomes, Artificial, Yeast/genetics , Genes, Overlapping/genetics , Germ Cells/immunology , Germ Cells/metabolism , Humans , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Immunoglobulin Heavy Chains/genetics , Mice , Mice, Transgenic , Rats , Rats, Transgenic
3.
Proc Natl Acad Sci U S A ; 109(31): 12674-9, 2012 Jul 31.
Article in English | MEDLINE | ID: mdl-22733769

ABSTRACT

Genetic variation in the IL-7 receptor-α (IL-7R) gene is associated with susceptibility to human type 1 diabetes (T1D). Here we investigate the therapeutic efficacy and mechanism of IL-7Rα antibody in a mouse model of T1D. IL-7Rα antibody induces durable, complete remission in newly onset diabetic mice after only two to three injections. IL-7 increases, whereas IL-7Rα antibody therapy reduces, the IFN-γ-producing CD4(+) (T(H)1) and IFN-γ-producing CD8(+) T cells. Conversely, IL-7 decreases and IL-7Rα antibody enhances the inhibitory receptor Programmed Death 1 (PD-1) expression in the effector T cells. Programmed Death 1 blockade reversed the immune tolerance mediated by the IL-7Rα antibody therapy. Furthermore, IL-7Rα antibody therapy increases the frequency of regulatory T cells without affecting their suppressor activity. The durable efficacy and the multipronged tolerogenic mechanisms of IL-7Rα antibody therapy suggest a unique disease-modifying approach to T1D.


Subject(s)
Antibodies/pharmacology , Diabetes Mellitus, Type 1/immunology , Interleukin-7/immunology , Receptors, Interleukin-7/antagonists & inhibitors , T-Lymphocytes, Regulatory/immunology , Th1 Cells/immunology , Animals , Antibodies/immunology , Antigens, Differentiation/immunology , Diabetes Mellitus, Type 1/pathology , Diabetes Mellitus, Type 1/therapy , Disease Models, Animal , Female , Humans , Interferon-gamma/immunology , Mice , Mice, Inbred NOD , Programmed Cell Death 1 Receptor , Receptors, Interleukin-7/immunology , T-Lymphocytes, Regulatory/pathology , Th1 Cells/pathology
4.
J Pharmacol Exp Ther ; 340(2): 228-36, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22019884

ABSTRACT

Proprotein convertase substilisin/kexin type 9 (PCSK9) promotes the degradation of low-density lipoprotein (LDL) receptor (LDLR) and thereby increases serum LDL-cholesterol (LDL-C). We have developed a humanized monoclonal antibody that recognizes the LDLR binding domain of PCSK9. This antibody, J16, and its precursor mouse antibody, J10, potently inhibit PCSK9 binding to the LDLR extracellular domain and PCSK9-mediated down-regulation of LDLR in vitro. In vivo, J10 effectively reduces serum cholesterol in C57BL/6 mice fed normal chow. J16 reduces LDL-C in healthy and diet-induced hypercholesterolemic cynomologous monkeys, but does not significantly affect high-density lipoprotein-cholesterol. Furthermore, J16 greatly lowered LDL-C in hypercholesterolemic monkeys treated with the HMG-CoA reductase inhibitor simvastatin. Our data demonstrate that anti-PCSK9 antibody is a promising LDL-C-lowering agent that is both efficacious and potentially additive to current therapies.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Cholesterol, LDL/drug effects , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Primates , Proprotein Convertases/antagonists & inhibitors , Animals , Antibodies, Monoclonal, Humanized/adverse effects , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Monoclonal, Humanized/therapeutic use , Catalytic Domain/immunology , Cell Line, Tumor , Cholesterol/blood , Cholesterol, Dietary/administration & dosage , Cholesterol, Dietary/pharmacology , Cholesterol, HDL/blood , Cholesterol, HDL/drug effects , Cholesterol, LDL/blood , Dietary Fats/administration & dosage , Dietary Fats/pharmacology , Dose-Response Relationship, Drug , Drug Therapy, Combination/methods , Epitopes/immunology , Female , Fluorobenzenes/pharmacology , Fluorobenzenes/therapeutic use , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Hypercholesterolemia/blood , Hypercholesterolemia/chemically induced , Hypercholesterolemia/drug therapy , Liver/drug effects , Liver/metabolism , Macaca fascicularis , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Proprotein Convertase 9 , Proprotein Convertases/immunology , Proprotein Convertases/pharmacology , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Receptors, LDL/metabolism , Rosuvastatin Calcium , Serine Endopeptidases/blood , Serine Endopeptidases/immunology , Serine Endopeptidases/pharmacology , Simvastatin/pharmacology , Simvastatin/therapeutic use , Sulfonamides/pharmacology , Sulfonamides/therapeutic use
5.
Sci Transl Med ; 3(93): 93ra68, 2011 Jul 27.
Article in English | MEDLINE | ID: mdl-21795588

ABSTRACT

The interleukin-7 receptor α chain (IL-7Rα) gene was identified as a top non-major histocompatibility complex-linked risk locus for multiple sclerosis (MS). Recently, we showed that a T helper 1 (T(H)1)-driven, but not a T(H)17-driven, form of MS exhibited a good clinical response to interferon-ß (IFN-ß) therapy. We now demonstrate that high serum levels of IL-7, particularly when paired with low levels of IL-17F, predict responsiveness to IFN-ß and hence a T(H)1-driven subtype of MS. We also show that although IL-7 signaling is neither necessary nor sufficient for the induction or expansion of T(H)17 cells, IL-7 can greatly enhance both human and mouse T(H)1 cell differentiation. IL-7 alone is sufficient to induce human T(H)1 differentiation in the absence of IL-12 or other cytokines. Furthermore, targeting IL-7/IL-7Rα is beneficial in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Mice treated with IL-7Rα-blocking antibodies before or after onset of paralysis exhibited reduced clinical signs of EAE, with reduction in peripheral naïve and activated T cells, whereas central memory T, regulatory T, B, and natural killer cell populations were largely spared. IL-7Rα antibody treatment markedly reduced lymphocyte infiltration into the central nervous system in mice with EAE. Thus, a serum profile of high IL-7 may signify a T(H)1-driven form of MS and may predict outcome in MS patients undergoing IFN-ß therapy. Blockade of IL-7 and the IL-7Rα pathway may have therapeutic potential in MS and other autoimmune diseases.


Subject(s)
Interferon-beta/therapeutic use , Interleukin-7/blood , Interleukin-7/immunology , Multiple Sclerosis/blood , Multiple Sclerosis/drug therapy , Th1 Cells/cytology , Th1 Cells/immunology , Animals , Antibodies/pharmacology , Antibody Specificity/drug effects , Cell Differentiation/drug effects , Encephalomyelitis, Autoimmune, Experimental/blood , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Humans , Immunologic Memory/drug effects , Interferon-beta/immunology , Mice , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Receptors, Interleukin-7/antagonists & inhibitors , Th1 Cells/drug effects , Treatment Outcome
6.
Proc Natl Acad Sci U S A ; 108(28): E279-87, 2011 Jul 12.
Article in English | MEDLINE | ID: mdl-21690377

ABSTRACT

Age-related macular degeneration (AMD) is a leading cause of visual dysfunction worldwide. Amyloid ß (Aß) peptides, Aß1-40 (Aß40) and Aß1-42 (Aß42), have been implicated previously in the AMD disease process. Consistent with a pathogenic role for Aß, we show here that a mouse model of AMD that invokes multiple factors that are known to modify AMD risk (aged human apolipoprotein E 4 targeted replacement mice on a high-fat, cholesterol-enriched diet) presents with Aß-containing deposits basal to the retinal pigmented epithelium (RPE), histopathologic changes in the RPE, and a deficit in scotopic electroretinographic response, which is reflective of impaired visual function. Strikingly, these electroretinographic deficits are abrogated in a dose-dependent manner by systemic administration of an antibody targeting the C termini of Aß40 and Aß42. Concomitant reduction in the levels of Aß and activated complement components in sub-RPE deposits and structural preservation of the RPE are associated with anti-Aß40/42 antibody immunotherapy and visual protection. These observations are consistent with the reduction in amyloid plaques and improvement of cognitive function in mouse models of Alzheimer's disease treated with anti-Aß antibodies. They also implicate Aß in the pathogenesis of AMD and identify Aß as a viable therapeutic target for its treatment.


Subject(s)
Amyloid beta-Peptides/antagonists & inhibitors , Macular Degeneration/therapy , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathology , Amyloid beta-Peptides/immunology , Amyloid beta-Peptides/metabolism , Animals , Antibodies, Bispecific/administration & dosage , Antibodies, Bispecific/therapeutic use , Apolipoprotein E4/genetics , Complement System Proteins/metabolism , Dietary Fats/administration & dosage , Disease Models, Animal , Dose-Response Relationship, Immunologic , Female , Humans , Immunotherapy , Macular Degeneration/etiology , Macular Degeneration/pathology , Macular Degeneration/physiopathology , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Transgenic , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/immunology , Vision, Low/physiopathology , Vision, Low/prevention & control
7.
PLoS One ; 3(4): e1900, 2008 Apr 02.
Article in English | MEDLINE | ID: mdl-18382675

ABSTRACT

Loss of function mutations in the receptor tyrosine kinase TrkB pathway resulted in hyperphagia and morbid obesity in human and rodents. Conversely, peripheral or central stimulation of TrkB by its natural ligands BDNF or NT4 reduced body weight and food intake in mice, supporting the idea that TrkB is a key anorexigenic signal downstream of the melanocortin-4 receptor (Mc4r) system. Here we show that in non-human primates TrkB agonists were anorexigenic when applied centrally, but surprisingly orexigenic, leading to gain in appetite, body weight, fat deposits and serum leptin levels, when given peripherally. The orexigenic and pro-obesity effects of peripherally administered TrkB agonists appear to be dose dependent, not associated with fluid retention nor with evidence of receptor down regulation. Our findings revealed that TrkB signaling exerts dual control on energy homeostasis in the primates that could be targeted for the treatment of either wasting disorders or obesity.


Subject(s)
Appetite , Mutation , Receptor, trkB/agonists , Weight Gain , Animals , Body Weight , Brain-Derived Neurotrophic Factor/metabolism , Gene Deletion , Leptin/blood , Ligands , Macaca mulatta , Obesity/metabolism , Receptor, Melanocortin, Type 4/metabolism , Recombinant Proteins/metabolism , Signal Transduction
8.
Cancer Gene Ther ; 12(8): 715-22, 2005 Aug.
Article in English | MEDLINE | ID: mdl-15832172

ABSTRACT

Conditionally replicating adenoviruses that selectively replicate in tumor cells, but not in normal cells, are being explored as virotherapeutic agents for cancer. A prostate-specific oncolytic adenovirus, CG7870 is currently being evaluated in phase 1/2 clinical trials for the treatment of prostate cancer. To decrease the effective dose and further increase the therapeutic efficacy of CG7870, the combination of virotherapy with radiation therapy was explored in this study. CG7870 is an oncolytic adenovirus in which tumor-specific promoters are driving the expression of E1A and E1B proteins. The effects of combined treatment with CG7870 and radiation on cultured cells were determined in cytotoxicity and virus yield assays. The antitumor efficacy of CG7870 (1 x 10(7) particles/mm3 of tumor), 10 Gy of local radiation or both was evaluated in established subcutaneous LNCaP xenografts in nude mice. In vitro, the dual agent treatment resulted in synergistically enhanced potency at suboptimal doses of radiation and virus. Virus yield in irradiated cells increased relative to yield in nonirradiated cells without compromising the specificity of the vector for its target cell types. In vivo, CG7870 treatment alone suppressed tumor growth and extended tumor nonprogression time. The average tumor-volume of the groups treated with CG7870 only and radiation only was 121 and 126% of baseline, respectively, 39 days after treatment. The average tumor-volume of the combination group was 34% of baseline 39 days after a single dose of treatment. No significant body weight loss was observed in any treatment group. There was a significant drop in serum level of prostate-specific antigen (PSA) in the combination group compared to the group treated with either agent alone. In mice treated with CG7870 only or radiation only, serum PSA levels changed to 26 and 383% of baseline, respectively, by study day 46. In contrast, PSA levels in mice treated with CG7870 plus radiation decreased to less than 11% of baseline by study day 46. Histological analysis of tumor sections collected from the combination group revealed enhanced necrosis and more apoptotic cells. Combination of CG7870 with radiotherapy significantly increased antitumor efficacy compared to either agent alone. These results suggest that CG7870 in combination with radiation has improved antitumor efficacy at lower doses and with no additional side effects.


Subject(s)
Adenoviridae/genetics , Prostatic Neoplasms/therapy , Animals , Apoptosis , Combined Modality Therapy , Genetic Therapy , Humans , Male , Mice , Prostate-Specific Antigen/blood , Prostatic Neoplasms/radiotherapy , Virus Replication , Xenograft Model Antitumor Assays
9.
Mol Cancer Ther ; 2(10): 1003-9, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14578465

ABSTRACT

Human carcinoembryonic antigen (CEA) is overexpressed in most colorectal cancers and has been widely used as a clinical marker for the management of colon cancer patients. The transcriptional regulatory elements (TREs) of CEA include two enhancer elements and a promoter in the 5'-flanking region of the CEA gene. By using these elements in different combinations to control reporter gene expression and the replication of adenovirus variants in various tumor cells, we have identified an optimal CEA regulatory cassette that tightly controls gene expression and viral replication in CEA-producing colon cancer cells. One of these variants, OV798, in which this regulatory cassette controls E1A expression, was further characterized. OV798 preferentially replicates in and kills CEA-producing colorectal cancer cell lines such as LoVo and SW1463, but its replication is attenuated by 1000-fold in the CEA-negative cell lines Colo-320DM (colon cancer), PA-1 (ovarian cancer), G361 (melanoma), U118 MG (glioma), and HBL-100 (human breast epithelial cell). The antitumor activity of OV798 was further examined in BALB/c nu/nu mice carrying s.c. human colon tumor xenografts. A single intratumoral administration of OV798 resulted in growth inhibition of human LoVo colon cancer xenografts. Six weeks after treatment, relative tumor volume decreased to 90% of baseline for the OV798 treatment group, compared to an increase to 1200% of baseline at 4 weeks for the vehicle-treated group. In vitro and in vivo characterization indicate that OV798 could be used as a therapy for human colon cancer.


Subject(s)
Adenoviridae/genetics , Carcinoembryonic Antigen/metabolism , Colorectal Neoplasms/genetics , Colorectal Neoplasms/therapy , Animals , Cell Line , Cell Line, Tumor , DNA/chemistry , DNA, Viral/genetics , Humans , Luciferases/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Promoter Regions, Genetic , Time Factors , Transcription, Genetic , Transfection
10.
Cancer Res ; 62(13): 3743-50, 2002 Jul 01.
Article in English | MEDLINE | ID: mdl-12097284

ABSTRACT

Uroplakins (UPs) are a group of integral membrane proteins that are synthesized as the major differentiation products of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but very little is known about its transcription response elements. To identify the promoter elements, a DNA fragment of 2239 bp upstream of the UPII gene was amplified by PCR and linked to a promoterless firefly luciferase reporter gene. Transient transfection experiments showed that the DNA segment located between -1809 and +1 bp resulted in preferential expression in bladder carcinoma cells with negligible expression in nonurothelial cells. This promoter was engineered into adenovirus (Ad) type 5 to drive the expression of the E1A and E1B genes and to create an attenuated replication-competent Ad variant, termed CG8840. Viral replication and the cytopathic effect of CG8840 were evaluated by virus yield and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in bladder transitional cell carcinoma (TCC) cell lines RT4 and SW780; nonbladder cancer cell lines G361 (melanoma), LNCaP (prostate cancer), PA-1 (ovarian cancer), and U118 (brain cancer); and human primary cells including lung fibroblasts, bladder smooth muscle cells, and mammary epithelial cells. CG8840 replicated in and eliminated bladder TCC efficiently with high specificity (10,000:1) in comparison with nonbladder cells. The antitumor activity of CG8840 was examined in BALB/c nu/nu mice carrying s.c. human TCC xenografts. Intratumoral and i.v. administration of CG8840 in RT4 human bladder cancer xenografts caused significant (P < 0.01) inhibition of tumor growth. Synergistic antitumor efficacy was observed when CG8840 was combined with docetaxel, resulting in significant regression of RT4 bladder cancer xenograft tumors within 6 weeks after i.v. administration of CG8840 (3.33 x 10(9) plaque-forming units/animal on day 1) and docetaxel (20 mg/kg on days 2, 6, and 9). These results demonstrate the utility of the UPII promoter in the generation of urothelium-specific adenoviral vectors and provide a potential foundation for the development of bladder tumor-specific oncolytic viral therapies.


Subject(s)
Adenoviruses, Human/physiology , Antineoplastic Agents, Phytogenic/pharmacology , Carcinoma, Transitional Cell/therapy , Membrane Proteins/genetics , Paclitaxel/analogs & derivatives , Paclitaxel/pharmacology , Taxoids , Urinary Bladder Neoplasms/therapy , Adenovirus E1B Proteins/genetics , Adenoviruses, Human/genetics , Animals , Carcinoma, Transitional Cell/drug therapy , Carcinoma, Transitional Cell/genetics , Carcinoma, Transitional Cell/virology , Combined Modality Therapy , Cytopathogenic Effect, Viral , Docetaxel , Drug Synergism , Gene Deletion , Genes, Regulator , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Promoter Regions, Genetic/genetics , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/virology , Uroplakin II , Virus Replication/genetics , Virus Replication/physiology , Xenograft Model Antitumor Assays
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