ABSTRACT
Harnessing the immunomodulatory activity of cytokines is a focus of therapies targeting inflammatory disease. The interleukin (IL)-1 superfamily contains pro-inflammatory and anti-inflammatory members that help orchestrate the immune response in adaptive and innate immunity. Of these molecules, IL-37 has robust anti-inflammatory activity across a range of disease models through inhibition of pro-inflammatory signaling cascades downstream of tumor necrosis factor, IL-1, and toll-like receptor pathways. We find that IL-37 is unstable with a poor pharmacokinetic and manufacturing profile. Here, we present the engineering of IL-37 from an unstable cytokine into an anti-inflammatory molecule with an excellent therapeutic likeness. We overcame these shortcomings through site-directed mutagenesis, the addition of a non-native disulfide bond, and the engineering of IL-37 as an Fc-fusion protein. Our results provide a platform for preclinical testing of IL-37 Fc-fusion proteins. The engineering approaches undertaken herein will apply to the conversion of similar potent yet short-acting cytokines into therapeutics.
Subject(s)
Anti-Inflammatory Agents , Cytokines , Cytokines/metabolism , Immunity, Innate , Immunomodulation , Protein EngineeringABSTRACT
Necrotizing enterocolitis (NEC) is a severe, currently untreatable intestinal disease that predominantly affects preterm infants and is driven by poorly characterized inflammatory pathways. Here, human and murine NEC intestines exhibit an unexpected predominance of type 3/TH17 polarization. In murine NEC, pro-inflammatory type 3 NKp46-RORγt+Tbet+ innate lymphoid cells (ILC3) are 5-fold increased, whereas ILC1 and protective NKp46+RORγt+ ILC3 are obliterated. Both species exhibit dysregulation of intestinal TLR repertoires, with TLR4 and TLR8 increased, but TLR5-7 and TLR9-12 reduced. Transgenic IL-37 effectively protects mice from intestinal injury and mortality, whilst exogenous IL-37 is only modestly efficacious. Mechanistically, IL-37 favorably modulates immune homeostasis, TLR repertoires and microbial diversity. Moreover, IL-37 and its receptor IL-1R8 are reduced in human NEC epithelia, and IL-37 is lower in blood monocytes from infants with NEC and/or lower birthweight. Our results on NEC pathomechanisms thus implicate type 3 cytokines, TLRs and IL-37 as potential targets for novel NEC therapies.
Subject(s)
Enterocolitis, Necrotizing/drug therapy , Enterocolitis, Necrotizing/immunology , Adaptive Immunity , Animals , Animals, Newborn , Biomarkers/metabolism , Enterocolitis, Necrotizing/blood , Enterocolitis, Necrotizing/pathology , Homeostasis , Humans , Immunity, Innate , Infant, Newborn , Inflammation Mediators/metabolism , Interleukin-1 , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Lymphocytes/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Toll-Like Receptors/metabolismABSTRACT
In this review we summarize the principles of anti-metastatic therapy with selected serpin family proteins, such as pigment epithelial-derived factor (PEDF) and maspin, as well as inter α-trypsin inhibitor (IαIs) light chains (bikunin) and heavy chains (ITIHs). Case-by-case, antimetastatic activity may be dependent or independent of the protease-inhibitory activity of the corresponding proteins. We discuss the incidence of target deregulation in different tumor entities, mechanisms of deregulation, context-dependent functional issues as well as in vitro and in vivo target validation studies with transfected tumor cells or recombinant protein as anti-metastatic agents. Finally, we comment on possible clinical evaluation of these proteins in adjuvant therapy.
Subject(s)
Neoplasm Metastasis/drug therapy , Neoplasms/drug therapy , Serpins/therapeutic use , Trypsin Inhibitors/therapeutic use , Alpha-Globulins/therapeutic use , Humans , Neoplasms/pathology , PrognosisABSTRACT
Receptors show promise for the transport of monoclonal antibodies (mAbs) across the blood-brain barrier. However, safety liabilities associated with peripheral receptor binding and Fc effector function have been reported. We present the Brain Shuttle-mAb (BS-mAb) technology, and we investigate the role of Fc effector function in vitro and in an Fcγ receptor (FcγR)-humanized mouse model. Strong first infusion reactions (FIRs) were observed for a conventional mAb against transferrin receptor (TfR) with a wild-type immunoglobulin G1 (IgG1) Fc. Fc effector-dead constructs completely eliminated all FIRs. Remarkably, no FIR was observed for the BS-mAb construct with a native IgG1 Fc function. Using various BS-mAb constructs, we show that TfR binding through the C-terminal BS module attenuates Fc-FcγR interactions, primarily because of steric hindrance. Nevertheless, BS-mAbs maintain effector function activity when binding their brain target. Thus, mAbs with full effector function can be transported in a stealth mode in the periphery while fully active when engaged with their brain target.
Subject(s)
Alzheimer Disease/metabolism , Antibodies, Monoclonal , Blood-Brain Barrier/metabolism , Drug Delivery Systems , Immunoglobulin G/pharmacology , Receptors, IgG/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Animals , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal/pharmacology , Blood-Brain Barrier/pathology , CHO Cells , Cricetulus , Humans , Male , Mice , Mice, Transgenic , Receptors, IgG/geneticsABSTRACT
Formation of metastases from various tumor entities in the brain is a major problem for the treatment of advanced cancer. We describe target molecules and tools for the delivery of small molecules or proteins across the blood-brain barrier (BBB), and the treatment of brain tumors and metastases with antibody-related moieties. In addition, drugs preventing formation of metastases or interfering with the growth of established metastases are described, as well as pre-clinical metastasis models and corresponding clinical data. Furthermore, we discuss the delivery of effector proteins and antibody-based moieties fused with an antibody-based scaffold across the BBB in several model systems which might be applicable for the treatment of brain metastases.
Subject(s)
Blood-Brain Barrier/pathology , Brain Neoplasms/drug therapy , Brain Neoplasms/pathology , Animals , Blood-Brain Barrier/drug effects , Disease Models, Animal , Drug Delivery Systems , Humans , Neoplasm Metastasis , Small Molecule Libraries/therapeutic useABSTRACT
Tumor-related antigens can be presented as peptides forming complexes with major histocompatibility complex (MHC) molecules that interact with T-cell receptors, thus generating an immunologic anti-tumor response. Unfortunately, however, this response can be decreased by many effectors and pathways. On the other hand, such peptide-MHC complexes are unique starting points for therapeutic intervention. We present strategies for eliciting an anti-tumoral response by T-cell receptor-based fusion proteins with interleukin (IL)2 and antibody constant region domains, superantigens, and T-cell recruiting antibodies, as well as using genetically modified autologous T-cells as effectors. Another strategy is to direct peptide-MHC complexes to tumors as fusion proteins with an antibody-derived targeting moiety. Finally, we describe T-cell receptor-mimicking antibodies and antibody conjugates as anti tumoral agents.
Subject(s)
Antineoplastic Agents/pharmacology , Histocompatibility Antigens/metabolism , Peptides/metabolism , Receptors, Antigen, T-Cell/metabolism , Animals , Humans , Protein Binding/drug effects , Recombinant Fusion Proteins/metabolismABSTRACT
Proteases are often overexpressed in tumor cells and/or the stromal compartment and can thus be exploited in tumor therapy to activate cytotoxic prodrugs as, for example, in cytolytic fusion proteins, and for tumor imaging. Specifically, we discuss cathepsin B-activated prodrug conjugates, antibody-directed prodrug therapy, protease-activated peptide-thapsigargin conjugates, protease-activated cytotoxic receptor ligands and other cytotoxic proteins, protease-mediated activation of anthrax toxin, granzyme B as a therapeutic principle in cytolytic fusion proteins, and tumor-imaging based on deregulated proteases.
Subject(s)
Antineoplastic Agents/metabolism , Neoplasms/drug therapy , Prodrugs/metabolism , Animals , Antineoplastic Agents/therapeutic use , Cathepsin B/physiology , Granzymes/physiology , Humans , Matrix Metalloproteinases/physiology , Prodrugs/therapeutic use , Proteolysis , Serine Endopeptidases/physiology , Urokinase-Type Plasminogen Activator/physiologyABSTRACT
Bacterial- and plant-derived immunotoxins have documented potential for treatment of cancer. We discuss Anthrax toxin, ribosome inactivating-toxins, such as saporin and ricin, and ADP-ribosylating toxins such as Diphtheria toxin and Pseudomonas exotoxin, with focus on the latter, which has been most thoroughly investigated. Regarding their potential as anticancer agents, critical issues such as immunogenicity and toxicity are outlined. We describe different generations of immunotoxins, the pathways for the delivery of the cytotoxic 'warheads', molecular parameters modulating efficacy, and combination therapy with other anticancer agents. Finally, we discuss deimmunization strategies based on the removal of B- and T-cell epitopes from the cytotoxic component, and highlight promising clinical proof-of-concept studies.
Subject(s)
Antineoplastic Agents/therapeutic use , Bacterial Proteins/therapeutic use , Immunotoxins/therapeutic use , Neoplasms/drug therapy , Plant Proteins/therapeutic use , Animals , Antineoplastic Agents/pharmacology , Bacterial Proteins/pharmacology , Humans , Immunotoxins/pharmacology , Plant Proteins/pharmacologyABSTRACT
We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/blood , B-Lymphocytes/metabolism , High-Throughput Screening Assays/methods , Recombinant Proteins/biosynthesis , Recombinant Proteins/blood , Amino Acid Sequence , Animals , Antibody Specificity/immunology , Cell Separation , Cells, Cultured , Clone Cells , Epitopes/immunology , HEK293 Cells , Humans , Immunization , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/immunology , Immunoglobulin Variable Region/immunology , Interleukin-1 Receptor-Like 1 Protein , Interleukin-33 , Interleukins/metabolism , Protein Binding , Rabbits , Receptors, Cell Surface/metabolismABSTRACT
Investigation of protein-protein interactions (PPIs) and protein phosphorylation in clinical tissue samples can offer valuable information about the activation status and function of proteins involved in disease progression. However, existing antibody-based methods for phosphorylation detection have been found to lack specificity, and methods developed for examining PPIs in vitro cannot be easily adapted for tissues samples. In this study, we eliminated some of these limitations by developing a specific immunohistochemical staining method that uses "dual binders" (DBs), which are bispecific detection agents consisting of two Fab fragment molecules joined by a flexible linker, to detect PPIs and protein phosphorylation. We engineered DBs by selecting Fab fragments with fast off-rate kinetics, which allowed us to demonstrate that stable target binding was achieved only upon simultaneous, cooperative binding to both epitopes. We show that DBs specifically detect the activated HER2/HER3 complex in formalin-fixed, paraffin-embedded cancer cells and exhibit superior detection specificity for phospho-HER3 compared to the corresponding monoclonal antibody. Overall, the performance of DBs makes them attractive tools for future development for clinical applications.
Subject(s)
Immunohistochemistry , Proteins/metabolism , Receptor, ErbB-2/analysis , Receptor, ErbB-3/analysis , Animals , Antibodies/chemistry , Antibodies/immunology , Cell Line, Tumor , Dimerization , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/metabolism , MCF-7 Cells , Mice , Oligonucleotides/chemical synthesis , Oligonucleotides/chemistry , Phosphorylation , Protein Interaction Domains and Motifs , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/genetics , Receptor, ErbB-3/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
Antibody-based molecules can be delivered into cells either by intracellular expression or through cellular uptake. We describe technologies for identification and expression of intracellular antibodies for target validation, intracellular immunization and tumor therapy, such as intracellular antibody capture technology, suicide or silencing technology, antigen-antibody interaction dependent apoptosis and their application for inhibition of oncogenic intracellular proteins and induction of apoptosis. These strategies have to be viewed in the context that inhibition of protein-protein interactions by small molecules is often limited due to their large interaction surface. We summarize antibodies with the ability to penetrate cells and strategies to induce uptake of antibodies after modification with protein transduction domains. Interference in oncogenic pathways is described for moieties based on antibody 3E10, which translocates into the nucleus after extracellular administration. Finally, we discuss examples of tumor immunotherapy and vaccination against intracellular antigens, and possible interactions mediating their mode of action.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Neoplasm/immunology , Immunotherapy/methods , Neoplasms/immunology , Neoplasms/therapy , Animals , Antibodies, Neoplasm/biosynthesis , Humans , Molecular Targeted TherapyABSTRACT
In order to overcome limitations of monoclonal antibodies, new protein-based scaffolds have been designed and evaluated pre-clinically, and some of them are in clinical studies for the treatment of cancer. These entities can be placed into two categories: scaffolds which bind ligands via amino acids in exposed loops and those in which ligand binding is mediated by amino acids in secondary structures, such as ß-sheet modules. Accordingly, we discuss adnectins, lipocalins, Kunitz domain-based binders, avimers, knottins, fynomers, atrimers and cytotoxic T-lymphocyte associated protein-4 (CTLA4)-based binders which fall into the first category, while darpins, affibodies, affilins and armadillo repeat protein-based scaffolds are members of the second category. In addition, we also discuss the new molecular entities as imaging tools and outline their unique characteristics in the context of multimeric and multivalent binding.
Subject(s)
Amino Acids/chemistry , Antibodies, Monoclonal/therapeutic use , Neoplasms/drug therapy , Proteins/administration & dosage , Amino Acids/therapeutic use , Antibodies, Monoclonal/chemistry , CTLA-4 Antigen/administration & dosage , CTLA-4 Antigen/chemistry , Humans , Ligands , Neoplasms/genetics , Neoplasms/immunology , Protein Binding , Protein Structure, Secondary , Proteins/chemistryABSTRACT
The advent of various technologies for the generation of bi- and multispecific recombinant antibody-based molecules brought with it a multitude of formats for selecting target combinations. Some of the format options are outlined from a technical point of view. We focus on the achievements and prospects of the underlying technologies for generating bi- and multispecific antibodies to i) target immune effector cells and/or cytokines to tumors, ii) engage death receptors on tumor cells simultaneously, iii) improve antiangiogenic intervention by blocking complementary pathways of angiogenesis and iv) achieve more efficient targeting of human epidermal growth factor-related and other receptor tyrosine kinase-related pathways. Many of the outlined approaches, in addition to potential improvement of therapeutic efficacy in comparison to single agent intervention, also offer the potential to counteract therapy resistance.
Subject(s)
Antibodies, Bispecific/therapeutic use , Antineoplastic Agents/therapeutic use , Neoplasms/drug therapy , Animals , Antibodies, Bispecific/pharmacology , Antibody-Dependent Cell Cytotoxicity , Antigens, Neoplasm/immunology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , CD28 Antigens/immunology , Humans , Models, Molecular , Neoplasms/blood supply , Neoplasms/immunology , Neovascularization, Pathologic/drug therapy , Protein Structure, Tertiary , T-Lymphocytes/chemistry , T-Lymphocytes/immunologyABSTRACT
UNLABELLED: During antiviral therapy, specific delivery of interferon-α (IFNα) to infected cells may increase its antiviral efficacy, trigger a localized immune reaction, and reduce the side effects caused by systemic administration. Two T-cell receptor-like antibodies (TCR-L) able to selectively bind hepatitis B virus (HBV)-infected hepatocytes of chronic hepatitis B patients and recognize core (HBc18-27) and surface (HBs183-91) HBV epitopes associated with different human leukocyte antigen (HLA)-A*02 alleles (A*02:01, A*02:02, A*02:07, A*02:11) were generated. Each antibody was genetically linked to two IFNα molecules to produce TCR-L/IFNα fusion proteins. We demonstrate that the fusion proteins triggered an IFNα response preferentially on the hepatocytes presenting the correct HBV-peptide HLA-complex and that the mechanism of the targeted IFNα response was dependent on the specific binding of the fusion proteins to the HLA/HBV peptide complexes through the TCR-like variable regions of the antibodies. CONCLUSION: TCR-L antibodies can be used to target cytokines to HBV-infected hepatocytes in vitro. Fusion of IFNα to TCR-L decreased the intrinsic biological activity of IFNα but preserved the overall specificity of the protein for the cognate HBV peptide/HLA complexes. This induction of an effective IFNα response selectively in HBV-infected cells might have a therapeutic advantage in comparison to the currently used native or pegylated IFNα.
Subject(s)
Antibodies, Viral/pharmacology , Antiviral Agents/pharmacology , HLA-A Antigens/immunology , Hepatitis B virus/immunology , Hepatitis B/immunology , Interferon-alpha/pharmacology , Recombinant Fusion Proteins/pharmacology , Animals , Antibodies, Viral/immunology , Artificial Gene Fusion , CD8-Positive T-Lymphocytes/drug effects , Chemokines/metabolism , Drug Carriers/pharmacology , Hep G2 Cells , Hepatitis B/drug therapy , Hepatitis B/virology , Hepatitis B virus/genetics , Humans , Lymphocyte Activation/drug effects , MiceABSTRACT
PURPOSE: A major impediment in the optimal selection of cancer patients for the most effective therapy is the lack of suitable biomarkers that foretell the response of a patient to a given drug. In the present study, we have used large-scale RNA interference-based genetic screens to find candidate biomarkers of resistance to a new acyl sulfonamide derivative, R3200. This compound inhibits the proliferation of tumor cells in vitro and in vivo, but its mechanism of action is unknown. EXPERIMENTAL DESIGN: We used a large-scale RNA interference genetic screen to identify modulators of the efficacy of R3200. We searched for genes whose suppression in an in vitro cell system could cause resistance to the anticancer effects of R3200. RESULTS: We report here that knockdown of either RBX1 or DDB1 causes resistance to the anticancer effects of R3200, raising the possibility that these two genes may have utility as biomarkers of response to this drug in a clinical setting. Interestingly, both RBX1 and DDB1 are part of an E3 ubiquitin ligase complex. CONCLUSIONS: We propose that suppression of the activity of a RBX1 and DDB1-containing E3 ligase complex leads to the stabilization of certain proteins, the increased abundance of which is in turn responsible for resistance to R3200. Moreover, our data suggest that RBX1 and DDB1 could potentially be developed into biomarkers of resistance to acyl sulfonamide-based cancer drugs. This will require clinical validation in a series of patients treated with R3200.