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1.
Cancer Chemother Pharmacol ; 89(4): 515-527, 2022 04.
Article in English | MEDLINE | ID: mdl-35298699

ABSTRACT

PURPOSE: Preclinical characterization of cetrelimab (JNJ-63723283), a fully humanized immunoglobulin G4 kappa monoclonal antibody targeting programmed cell death protein-1 (PD-1), in human cancer models. METHODS: Cetrelimab was generated by phage panning against human and cynomolgus monkey (cyno) PD-1 extracellular domains (ECDs) and affinity maturation. Binding to primate and rodent PD-1 ECDs, transfected and endogenous cell-surface PD-1, and inhibition of ligand binding were measured. In vitro activity was evaluated using cytomegalovirus recall, mixed lymphocyte reaction, staphylococcal enterotoxin B stimulation, and Jurkat-PD-1 nuclear factor of activated T cell reporter assays. In vivo activity was assessed using human PD-1 knock-in mice implanted with MC38 tumors and a lung patient-derived xenograft (PDX) model (LG1306) using CD34 cord-blood-humanized NSG mice. Pharmacodynamics, toxicokinetics, and safety were assessed in cynos following single and/or repeat intravenous dosing. RESULTS: Cetrelimab showed high affinity binding to human (1.72 nM) and cyno (0.90 nM) PD-1 and blocked binding of programmed death-ligand 1 (PD-L1; inhibitory concentration [IC] 111.7 ng/mL) and PD-L2 (IC 138.6 ng/mL). Cetrelimab dose-dependently increased T cell-mediated cytokine production and stimulated cytokine expression. Cetrelimab 10 mg/kg reduced mean MC38 tumor volume in PD-1 knock-in mice at Day 21 (P < 0.0001) versus control. In a PDX lung model, 10 mg/kg cetrelimab (every 5 days for six cycles) increased frequency of peripheral T cells and reduced (P < 0.05) mean tumor volume versus control. Activity was consistent with that of established PD-1 inhibitors. Cetrelimab dosing was well tolerated in cynos and mean drug exposure increase was dose-dependent. CONCLUSION: Cetrelimab potently inhibits PD-1 in vitro and in vivo, supporting its clinical evaluation.


Subject(s)
Antibodies, Monoclonal, Humanized , Antibodies, Monoclonal , Immune Checkpoint Inhibitors , Neoplasms , Programmed Cell Death 1 Receptor , Animals , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized/therapeutic use , Cytokines/metabolism , Disease Models, Animal , Humans , Immune Checkpoint Inhibitors/therapeutic use , Macaca fascicularis , Mice , Neoplasms/drug therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors
2.
Blood Adv ; 4(18): 4538-4549, 2020 09 22.
Article in English | MEDLINE | ID: mdl-32956453

ABSTRACT

B-cell maturation antigen (BCMA), a member of the tumor necrosis factor family of receptors, is predominantly expressed on the surface of terminally differentiated B cells. BCMA is highly expressed on plasmablasts and plasma cells from multiple myeloma (MM) patient samples. We developed a BCMAxCD3 bispecific antibody (teclistamab [JNJ-64007957]) to recruit and activate T cells to kill BCMA-expressing MM cells. Teclistamab induced cytotoxicity of BCMA+ MM cell lines in vitro (H929 cells, 50% effective concentration [EC50] = 0.15 nM; MM.1R cells, EC50 = 0.06 nM; RPMI 8226 cells, EC50 = 0.45 nM) with concomitant T-cell activation (H929 cells, EC50 = 0.21 nM; MM.1R cells, EC50 = 0.1 nM; RPMI 8226 cells, EC50 = 0.28 nM) and cytokine release. This activity was further increased in the presence of a γ-secretase inhibitor (LY-411575). Teclistamab also depleted BCMA+ cells in bone marrow samples from MM patients in an ex vivo assay with an average EC50 value of 1.7 nM. Under more physiological conditions using healthy human whole blood, teclistamab mediated dose-dependent lysis of H929 cells and activation of T cells. Antitumor activity of teclistamab was also observed in 2 BCMA+ MM murine xenograft models inoculated with human T cells (tumor inhibition with H929 model and tumor regression with the RPMI 8226 model) compared with vehicle and antibody controls. The specific and potent activity of teclistamab against BCMA-expressing cells from MM cell lines, patient samples, and MM xenograft models warrant further evaluation of this bispecific antibody for the treatment of MM. Phase 1 clinical trials (monotherapy, #NCT03145181; combination therapy, #NCT04108195) are ongoing for patients with relapsed/refractory MM.


Subject(s)
Antibodies, Bispecific , Multiple Myeloma , Animals , Antibodies, Bispecific/pharmacology , B-Cell Maturation Antigen , Humans , Lymphocyte Activation , Mice , Multiple Myeloma/drug therapy , T-Lymphocytes
3.
Sci Rep ; 10(1): 7557, 2020 05 05.
Article in English | MEDLINE | ID: mdl-32372058

ABSTRACT

Generation of bispecific antibodies (BsAbs) having two unique Fab domains requires heterodimerization of the two heavy chains and pairing of each heavy chain with its cognate light chain. An alternative bispecific scaffold (Bipod) comprising an scFv and a Fab on a heterodimeric Fc eliminates the possibility of light chain mispairing. However, unpredictable levels of chain expression and scFv-induced aggregation can complicate purification and reduce the yield of desired Bipod. Here, we describe a high-throughput method for generation of Bipods based on protein A and CH1 domain affinity capture. This method exploits over-expression of the scFv chain to maximize heterodimer yield. Bipods purified by this method have purity suitable for cell-based functional assays and in vivo studies.


Subject(s)
Antibodies, Bispecific/chemistry , Immunoglobulin Fab Fragments/chemistry , Protein Engineering/methods , Single-Chain Antibodies/chemistry , Animals , Biological Products/therapeutic use , CHO Cells , Cricetulus , DNA/chemistry , Dimerization , Drug Evaluation, Preclinical , Drug Screening Assays, Antitumor , Epitopes/chemistry , Humans , Immunoglobulin G/genetics , Immunosuppressive Agents/therapeutic use , Mutation , Neoplasms/therapy , Plasmids , Protein Domains
4.
Front Pharmacol ; 5: 225, 2014.
Article in English | MEDLINE | ID: mdl-25339905

ABSTRACT

The neonatal Fc receptor (FcRn) in intestinal epithelium is the primary mechanism for transfer of maternal immunoglobulin G (IgG) from suckled milk to serum; but the factors contributing to the rapid uptake of IgG are poorly understood. These studies help to determine the contribution of cell surface FcRn in IgG uptake in 2-week-old rat pups by varying local pH and binding conditions. Variants of a human wild-type (WT) IgG monoclonal antibody (mAb WT) were assessed for binding affinity (KD) to rat (r)FcRn at pH 6.0 and subsequent off-rate at pH 7.4 (1/s) by surface plasmon resonance. Selected mAbs were administered intra-intestinally in isoflurane-anesthetized 2-week rat pups. Full length mAb in serum was quantified by immunoassay, (r)FcRn mRNA expression by reverse transcription polymerase chain reaction, and mAb epithelial localization was visualized by immunohistochemistry. After duodenal administration, serum levels of mAb variants correlated with their rFcRn off-rate at pH 7.4, but not their affinity at pH 6.0. The greatest serum levels of IgG were measured when mAb was administered in the duodenum where rFcRn mRNA expression is greatest, and was increased further by duodenal administration in pH 6.0 buffer. More intense human IgG immunostaining was detected in epithelium than the same variant administered at higher pH. These data suggest an increased contribution for cell surface receptor. We conclude that, in the neonate duodenum, receptor off-rates are as important as affinities for FcRn mediated uptake, and cell surface binding of IgG to rFcRn plays contributes to IgG uptake alongside pinocytosis; both of which responsible for increased IgG uptake.

5.
Eur J Immunol ; 44(8): 2331-9, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24810893

ABSTRACT

NK cells that mediate ADCC play an important role in tumor-specific immunity. We have examined factors limiting specific lysis of tumor cells by CD16.NK-92 cells induced by CNTO 95LF antibodies recognizing αV integrins that are overexpressed on many tumor cells. Although all tested tumor cells were killed by CD16.NK-92 effectors in the presence of the antibodies, the killing of target cells with a low level of ICAM-1 expression revealed a dramatic decrease in their specific lysis at high antibody concentration, revealing a dose limiting effect. A similar effect was also observed with primary human NK cells. The effect was erased after IFN-γ treatment of tumor cells resulting in upregulation of ICAM-1. Furthermore, killing of the same tumor cells induced by Herceptin antibody was significantly impaired in the presence of CNTO 95Ala-Ala antibody variant that blocks αV integrins but is incapable of binding to CD16. These data suggest that αV integrins on tumor cells could compensate for the loss of ICAM-1 molecules, thereby facilitating ADCC by NK cells. Thus, NK cells could exercise cytolytic activity against ICAM-1 deficient tumor cells in the absence of proinflammatory cytokines, emphasizing the importance of NK cells in tumor-specific immunity at early stages of cancer.


Subject(s)
Antibodies/immunology , Integrin alphaV/immunology , Killer Cells, Natural/immunology , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized , Cytokines/immunology , Cytotoxicity, Immunologic , GPI-Linked Proteins/immunology , Humans , Inflammation/immunology , Intercellular Adhesion Molecule-1/immunology , Interferon-gamma/immunology , Receptors, IgG/immunology , Tumor Cells, Cultured , Up-Regulation/immunology
6.
Pharm Res ; 31(4): 908-22, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24072267

ABSTRACT

PURPOSE: To evaluate transcytosis of immunoglobulin G (IgG) by the neonatal Fc receptor (FcRn) in adult primate intestine to determine whether this is a means for oral delivery of monoclonal antibodies (mAbs). METHODS: Relative regional expression of FcRn and localization in human intestinal mucosa by RT-PCR, ELISA & immunohistochemistry. Transcytosis of full-length mAbs (sandwich ELISA-based detection) across human intestinal segments mounted in Ussing-type chambers, human intestinal (caco-2) cell monolayers grown in transwells, and serum levels after regional intestinal delivery in isoflurane-anesthetized cynomolgus monkeys. RESULTS: In human intestine, there was an increasing proximal-distal gradient of mucosal FcRn mRNA and protein expression. In cynomolgus, serum mAb levels were greater after ileum-proximal colon infusion than after administration to stomach or proximal small intestine (1-5 mg/kg). Serum levels of wild-type mAb dosed into ileum/proximal colon (2 mg/kg) were 124 ± 104 ng/ml (n = 3) compared to 48 ± 48 ng/ml (n = 2) after a non-FcRn binding variant. In vitro, mAb transcytosis in polarized caco-2 cell monolayers and was not enhanced by increased apical cell surface IgG binding to FcRn. An unexpected finding in primate small intestine, was intense FcRn expression in enteroendocrine cells (chromagranin A, GLP-1 and GLP-2 containing). CONCLUSIONS: In adult primates, FcRn is expressed more highly in distal intestinal epithelial cells. However, mAb delivery to that region results in low serum levels, in part because apical surface FcRn binding does not influence mAb transcytosis. High FcRn expression in enteroendocrine cells could provide a novel means to target mAbs for metabolic diseases after systemic administration.


Subject(s)
Gene Expression Regulation , Histocompatibility Antigens Class I/biosynthesis , Immunoglobulin G/metabolism , Intestinal Mucosa/metabolism , Receptors, Fc/biosynthesis , Transcytosis/physiology , Adult , Animals , Caco-2 Cells , Female , Humans , Macaca fascicularis , Male , Middle Aged , Organ Culture Techniques , RNA, Messenger/biosynthesis , Rats , Young Adult
7.
Proc Natl Acad Sci U S A ; 108(48): E1236-43, 2011 Nov 29.
Article in English | MEDLINE | ID: mdl-22074846

ABSTRACT

Many different systems of bacterial interactions have been described. However, relatively few studies have explored how interactions between different microorganisms might influence bacterial development. To explore such interspecies interactions, we focused on Bacillus subtilis, which characteristically develops into matrix-producing cannibals before entering sporulation. We investigated whether organisms from the natural environment of B. subtilis--the soil--were able to alter the development of B. subtilis. To test this possibility, we developed a coculture microcolony screen in which we used fluorescent reporters to identify soil bacteria able to induce matrix production in B. subtilis. Most of the bacteria that influence matrix production in B. subtilis are members of the genus Bacillus, suggesting that such interactions may be predominantly with close relatives. The interactions we observed were mediated via two different mechanisms. One resulted in increased expression of matrix genes via the activation of a sensor histidine kinase, KinD. The second was kinase independent and conceivably functions by altering the relative subpopulations of B. subtilis cell types by preferentially killing noncannibals. These two mechanisms were grouped according to the inducing strain's relatedness to B. subtilis. Our results suggest that bacteria preferentially alter their development in response to secreted molecules from closely related bacteria and do so using mechanisms that depend on the phylogenetic relatedness of the interacting bacteria.


Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/metabolism , Biofilms/growth & development , Extracellular Matrix/metabolism , Gene Expression Regulation, Bacterial/physiology , Quorum Sensing/physiology , Soil Microbiology , Base Sequence , Fluorescence , Likelihood Functions , Microbial Viability , Models, Genetic , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Species Specificity
8.
Protein Expr Purif ; 79(1): 7-15, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21640830

ABSTRACT

Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a 'tag after stop codon' system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS-PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels. In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity.


Subject(s)
Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays/methods , Membrane Proteins/genetics , Peptide Library , Proteins/genetics , Animals , Gene Expression , HEK293 Cells , Humans , Membrane Proteins/metabolism , Proteins/metabolism , Proteomics/methods , Transfection
9.
Hybridoma (Larchmt) ; 30(2): 153-62, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21529288

ABSTRACT

ST2L is a transmembrane receptor that belongs to the IL-1 receptor family. The receptor is expressed on various cell types including Th2 cells, mast cells, basophils, growth-activated fibroblasts, and vascular endothelial cells. ST2L activation by its ligand IL-33 has been implicated in Th2-mediated immunity, inflammation, and allergic responses in vivo. Inhibition of ST2L activity can attenuate Th2-dominated immune responses such as lung eosinophilia, airway hyper-responsiveness, and arthritis in animal models. Here we report the generation and in vitro characterization of a panel of rat anti-mouse ST2L monoclonal antibodies. We demonstrate that the antibodies specifically bind to recombinant receptor protein and that a subset of the binders inhibits mouse ST2L activity in multiple in vitro assays. Four of the identified anti-mouse ST2L antibodies were shown to prevent IL-33 from binding to ST2L, down-regulate IL-33-induced NF-κB signaling, and neutralize the ability of IL-33 to stimulate mouse Th2 cell proliferation. The characterized monoclonal antibodies are important tools that will be used to study mouse ST2L receptor functionality in vivo.


Subject(s)
Antibodies, Monoclonal , Interleukins/immunology , Receptors, Interleukin-1/immunology , Recombinant Proteins/immunology , Signal Transduction/drug effects , Th2 Cells/drug effects , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/pharmacology , Antibody Specificity , Biotin/chemistry , Biotin/metabolism , Cell Proliferation/drug effects , Down-Regulation , HEK293 Cells , Humans , Hybridomas/immunology , Hybridomas/metabolism , Hypersensitivity/drug therapy , Hypersensitivity/immunology , Immunoconjugates/chemistry , Immunoconjugates/metabolism , Inflammation/drug therapy , Inflammation/immunology , Interleukin-1/immunology , Interleukin-33 , Interleukins/metabolism , Mice , Mice, Inbred BALB C , NF-kappa B/metabolism , Plasmids/genetics , Plasmids/metabolism , Rats , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction/immunology , Th2 Cells/immunology , Transfection
10.
Assay Drug Dev Technol ; 9(4): 420-9, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21294636

ABSTRACT

Interleukin-6 (IL-6) family of cytokines are multifunctional proteins that play an important role in host defenses, acute phase reactions, immune responses, hematopoiesis, and tumorigenesis. The cytokines are produced by various lymphoid and nonlymphoid cells and mediate their biological activity through initial low-affinity binding to cell surface receptors, which are specific for their respective ligands. Ligand-specific receptor binding results in the receptor heterodimerization with ubiquitously expressed signal-transducing transmembrane component gp130 followed by activation of the gp130-associated Janus kinase, which, in turn, phosphorylates signal transducer and activator of transcription 3 (STAT3). Phosphorylated STAT3 (pSTAT3) dimerizes and translocates to the nucleus, where it activates gene transcription. Activation of STAT3 is essential to IL-6 family-associated physiological effects. Therefore, the ability to assess STAT3 phosphorylation is important for drug discovery efforts targeting IL-6 family cytokines. Various reagents and technologies are available to detect the effect of IL-6 type cytokines in treated cells. The present study describes the development of two pSTAT3 detection assays: the high-throughput screening assay based on Meso-Scale Discovery technology, which utilizes electrochemoluminescent signal measurements for the detection of pSTAT3 in treated cell extracts, and the secondary characterization assay based on fluorescent imaging analysis, which monitors pSTAT3 nuclear translocation in cells after activation. We have successfully utilized these assays to screen a small library of secreted proteins and identified inducers of STAT3 phosphorylation. The results obtained in this study demonstrate that both assays are robust, reliable, and amenable to high-throughput screening applications.


Subject(s)
High-Throughput Screening Assays/methods , STAT3 Transcription Factor/analysis , Small Molecule Libraries/analysis , Cell Line, Tumor , Cytokine Receptor gp130/physiology , Drug Discovery , Fluorescence , Humans , Interleukin-6/physiology , Janus Kinases/physiology , Phosphorylation , Proteins/analysis , Proteins/metabolism , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology
11.
Biologicals ; 39(1): 9-22, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20888784

ABSTRACT

To assess the impact of manufacturing changes on antibody structure and function during the course of product development, three comparability studies were performed for each of two different IgG1 monoclonal antibody product candidates. Comparability study #1 evaluated the effect of changing the cell line and bulk drug substance manufacturing process for cell culture and purification. Results indicated that these process changes led to differences in sialylation of N-glycans and/or C-terminal lysine levels. Comparability study #2 results confirmed that scale-up of the bulk process and transfer to the commercial site, combined with changing from a lyophilized to a liquid dosage form, did not impact the structural or functional integrity of the antibodies. Comparability study #3 examined possible differences arising when the liquid formulation filled into pre-filled syringes and vials. Results indicated nearly identical molecular structure, biological activity, and degradation profiles except for a small yet statistically significant increase in the levels of subvisible particles in pre-filled syringes. These results from comparability studies with two different monoclonal antibodies are discussed with respect to the timing of the manufacturing changes and overall comparability strategies to assure safety and efficacy during development.


Subject(s)
Antibodies, Monoclonal/analysis , Drug Industry/standards , Immunoglobulin G/immunology , Technology, Pharmaceutical/standards , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Cell Line , Chromatography, High Pressure Liquid , Circular Dichroism , Drug Industry/methods , Electrophoresis, Polyacrylamide Gel , Humans , K562 Cells , Protein Binding , Receptors, IgG/metabolism , Technology, Pharmaceutical/methods
12.
Birth Defects Res B Dev Reprod Toxicol ; 89(2): 116-23, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20151458

ABSTRACT

BACKGROUND: Intetumumab is a human IgG1 anti-alphav-integrin monoclonal antibody that inhibits angiogenesis. Integrin binding and angiogenesis are important in reproduction including fertilization, implantation, and embryofetal development. These studies were designed to determine the pharmacological relevance of the rabbit for the evaluation of potential effects on embryofetal development and to evaluate the placental transfer of intetumumab in rabbits. METHODS: In vitro pharmacology studies evaluated the binding of intetumumab to rabbit cells and the inhibition of vessel sprouting from rabbit aorta. For the evaluation of placental transfer, pregnant rabbits (8/group) were injected intravenously with intetumumab 50 or 100 mg/kg every 2 days from Gestation Day (GD)7 to GD19. Maternal sera, fetal homogenates/sera, and amniotic fluid were collected at necropsy on GD19 or GD28 for evaluation of intetumumab concentrations. Clinical condition of the dams was monitored and fetuses were screened for abnormalities. RESULTS: Intetumumab (5-40 microg/mL) inhibited aortic cell adhesion to vitronectin and vessel sprouting from rabbit aortic rings. Immunohistochemical staining of rabbit tissues demonstrated binding of intetumumab to placenta. Administration of intetumumab to pregnant rabbits was well tolerated by the dams and the fetuses did not show major abnormalities. Fetal exposure to intetumumab relative to maternal exposure was <0.1% on GD19 and 100-130% on GD29. CONCLUSIONS: The rabbit is a pharmacologically relevant species for evaluation of potential developmental effects of intetumumab. Intetumumab crosses the rabbit placenta during the fetal period (GD 19-28).


Subject(s)
Angiogenesis Inhibitors/pharmacokinetics , Antibodies, Monoclonal/pharmacokinetics , Integrin alpha5/immunology , Maternal-Fetal Exchange/drug effects , Placenta/drug effects , Amniotic Fluid/drug effects , Amniotic Fluid/metabolism , Angiogenesis Inhibitors/administration & dosage , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Humanized , Aorta, Thoracic/cytology , Aorta, Thoracic/drug effects , Blood Vessels/drug effects , Blood Vessels/growth & development , Cell Adhesion/drug effects , Cells, Cultured , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , Embryonic Development/drug effects , Embryonic Development/physiology , Endothelial Cells/drug effects , Female , Fetal Development/drug effects , Fetal Development/physiology , Fetus/drug effects , Fetus/metabolism , Injections, Intravenous , Maternal Exposure , Maternal-Fetal Exchange/physiology , Neovascularization, Physiologic/drug effects , Placenta/metabolism , Pregnancy , Rabbits
13.
Proc Natl Acad Sci U S A ; 106(42): 17864-9, 2009 Oct 20.
Article in English | MEDLINE | ID: mdl-19815504

ABSTRACT

The successful elimination of pathogenic cells and microorganisms by the humoral immune system relies on effective interactions between host immunoglobulins and Fc gamma receptors on effector cells, in addition to the complement system. Essential Ig motifs that direct those interactions reside within the conserved IgG lower hinge/CH2 interface. We noted that a group of tumor-related and microbial proteases cleaved human IgG1s in that region, and the "nick" of just one of the heavy chains profoundly inhibited IgG1 effector functions. We focused on IgG1 monoclonal antibodies (mAbs) since IgG1 is the most abundant human subclass and demonstrates robust Fc-mediated effector functions. The loss of Fc-mediated cell killing activities was correlated with diminished binding to the Fc gamma family of receptors, but a similar decrease in affinity was not observed toward the FcRn receptor that maintains IgG in circulation. Endogenous human IgG cleavage products of comparable size to mAbs with the single cleavage were detected by Western blot analysis in synovial fluid from patients with rheumatoid arthritis and in breast carcinoma extracts. Their detection is problematic under physiological conditions, since there is no loss of structure, and antigen-binding capability is unaffected. These findings suggest that within the hostile proteolytic microenvironments associated with many diseases, key effector functions of host IgGs, or therapeutic Abs, may be compromised.


Subject(s)
Immunoglobulin G/chemistry , Immunoglobulin G/metabolism , Peptide Hydrolases/metabolism , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Antibody Affinity , Antibody-Dependent Cell Cytotoxicity , Bacterial Proteins/metabolism , Binding Sites , Breast Neoplasms/enzymology , Cell Membrane/immunology , Female , Humans , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/metabolism , In Vitro Techniques , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Rats , Receptors, IgG/metabolism , Serine Endopeptidases/metabolism , Staphylococcus aureus/enzymology , Streptococcus pyogenes/enzymology
14.
Hum Antibodies ; 15(4): 155-62, 2006.
Article in English | MEDLINE | ID: mdl-17522437

ABSTRACT

The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the therapeutic mAb candidate in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Previously, we described a novel immunization method using type 1 interferons (IFNs) coupled with an agonistic anti-CD40 mAb to drive immune responses (Staquet et al., Human Antibodies 15 (2006), 61-69). This protocol allows for rapid and robust generation of large panels of anti-variable region mAbs. In order to quickly characterize and efficiently identify optimal anti-variable region antibody pairs early in the hybridoma process using crude supernatants, an inexpensive, high-throughput ELISA method was developed. The ability to rapidly identify appropriate mAb pairs will save resources by eliminating the time-consuming and laborious process of subcloning irrelevant hybridomas.


Subject(s)
Antibodies, Monoclonal/biosynthesis , CD40 Antigens/immunology , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin Variable Region/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal/therapeutic use , B-Lymphocytes , Biotechnology/methods , Female , Humans , Immunization , Immunoglobulin G/administration & dosage , Immunoglobulin G/immunology , Interferon Type I , Mice , Mice, Inbred BALB C , Time Factors
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