Antibody drug conjugates (ADCs) are an emerging class of targeted therapeutics with the potential to improve therapeutic index over the traditional chemotherapy. However, it is difficult to control the site and stoichiometry of conjugation in mAb, typically resulting in heterogeneous mixtures of ADCs that are difficult to optimize. New methods for site-specific drug attachment allow development of more homogeneous conjugates and control of the site of drug attachment. In this article, the new literature on development of ADCs and site-specific ADCs is reviewed. In addition, we summarized the various strategies in production of site-specific ADCs.
Antibodies, Monoclonal ; chemistry ; Antibody Specificity ; Binding Sites, Antibody ; Immunoconjugates ; chemistry

Base Sequence ; Binding Sites, Antibody ; Biochemistry ; methods ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Genetic Vectors ; Immunoglobulin Fc Fragments ; genetics ; metabolism ; Immunoglobulin G ; genetics ; metabolism ; Ligands ; Molecular Sequence Data ; Plasmids ; Protein Binding ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; Staphylococcal Protein A ; genetics ; metabolism

This study was purposed to detect the alloantibodies against Factor VIII (FVIII) by ELISA for estimating the incidence of the alloantibodies against Factor VIII (FVIII) in patients with hemophilia A, and to investigate the relationship between factor VIIIC domain and alloantibodies. Total of 140 patients with hemophilia A and 80 normal controls were enrolled in this study, among them plasma FVIII level of 84 patients was less than 1%, plasma FVIII level of 34 patients was between 1% and 5%, and plasma FVIII level of 22 patients was more than 5%. All patients were treated with plasma-derived FVIII concentrate or plasma before. The ELISA plate was coated with McAb (SZ-132) against FVIII prepared in our laboratory, then human recombinant FVIII concentrates were applied. After incubation in room temperature for 2 hours, diluted plasma samples and HRP-conjugated goat anti-human IgG were added successively, finally A490 was recorded. The threshold of alloantibody of patient plasma was set as mean value>3 SD more than control. The plate was coated with antibody against His, then human recombinant FVIII-C1C2 prepared in our laboratory was added. After incubation in room temperature for 2 hours, diluted plasma samples and HRP-conjugated goat anti-human IgG were added successively, finally A490 were recorded. The threshold was set as the mean value>3 SD more than control. The results showed that the alloantibodies against FVIII were found in 56 patients (40%) by ELISA, and 82.1% (46/56) of this kind of alloantibody could interact with the C domain of FVIII. It is concluded that C domain of FVIII is one of the primary binding sites for the alloantibodies against FVIII in Chinese patients with hemophilia A.
Adolescent ; Adult ; Binding Sites, Antibody ; Case-Control Studies ; Child ; Child, Preschool ; Factor VIII ; immunology ; Hemophilia A ; blood ; immunology ; Humans ; Infant ; Isoantibodies ; blood ; Male ; Middle Aged ; Protein Interaction Domains and Motifs ; Young Adult

C4d is produced from the direct interaction between antibodies and tissue injury at an antibody binding site in a graft. C4d deposition along peritubular capillaries (PTCs) in a renal allograft is a characteristic finding of antibody-mediated rejection (AMR), and is a useful diagnostic tool of AMR. The C4d along PTCs is associated with poor graft survival. Therefore C4d is regarded as a biomarker of AMR and was included in the diagnosis criteria of AMR at 2007 Banff conference. However, although C4d assay is widely used, it has several limitations. ABO-incompatible transplantations develop C4d along the PTCs in the majority of grafts but this seems to be graft accommodation rather than AMR. Recent studies reported that more than half of renal allograft biopsies with chronic AMR were C4d-negative. Without treatment, the C4d-negative AMR can cause scarring within the graft, transplant glomerulopathy (TG) or even graft loss. C4d is not a certain indicator of antibody-mediated rejection and C4d staining is not always highly sensitive for detecting AMR. Measuring endothelial gene expression in kidney graft biopsies with alloantibody can be another sensitive and specific method to diagnose AMR and predict graft outcomes. Because of these complexities, at the 2011 Banff meeting, criteria for diagnosis of chronic AMR in the kidney were refined, and the need for inclusion of C4d-negative AMR in the Banff classification was investigated.
Antibodies ; Binding Sites, Antibody ; Biopsy ; Capillaries ; Cicatrix ; Factor IX ; Gene Expression ; Graft Survival ; Kidney ; Rejection (Psychology) ; Transplantation, Homologous ; Transplants

Protein A and protein G are two well-defined immunoglobulin (Ig)-binding proteins (IBPs), which show affinity for specific sites on Ig of mammalian hosts. Protein A and protein G contained several highly homologous IgG-binding domains which had been demonstrated to have function to bind to IgG. Whether combinations of Ig-binding domains of various IBPs could produce useful novel binding properties remains interesting. We constructed a combinatorial phage library which displayed randomly-rearranged A, B, C, D and E domains of protein A, B2 and B3 domains of protein G. Four rounds molecular evolution of this library directed by all four human IgG subclasses respectively generated a common arrangement of D-C respectively which didn't exist in SpA. The dynamic loss of control phages and increase of the phages displaying two or more binding domains, especially the selective enrichment of D-C and strict selection of its linking peptides demonstrated the efficient molecular evolutions and the significance of the selected D-C arrangement. The phage binding assays confirmed that D-C possessed a binding advantage with four human IgG subclasses compared to SpA. In this work, a novel combination of Ig-binding domains, D-C, was obtained and presented the novel Ig binding properties which provided a novel candidate molecule for the purification, production and detection of IgG antibodies and a new approach for the further study of structures and functions of IBPs.
Amino Acid Sequence ; Antibody Specificity ; Bacterial Proteins ; immunology ; metabolism ; Binding Sites ; Binding, Competitive ; Evolution, Molecular ; Immunoglobulin G ; immunology ; metabolism ; Molecular Sequence Data ; Peptide Library ; Sequence Alignment ; Staphylococcal Protein A ; immunology ; metabolism

The lower expression of CD20 antigen molecules on the B cell membrane is the primary characteristic of B-chronic lymphocytic leukemia (B-CLL). In this paper, we combined laser scanning confocal microscopy (LSCM) and quantum dots labeling to detect the expression and distribution of CD20 molecules on CD20+B lymphocyte surface. Simultaneously, we investigated the morphology and ultrastructure of the B lymphocytes that belonged to the normal persons and B-CLL patients through utilizing the atomic force microscope (AFM). In addition, we measured the force spectroscopy of CD20 antigen-antibody binding using the AFM tips modified with CD20 antibody. The fluorescent images indicated that the density of CD20 of normal CD20+B lymphocytes was much higher than that of B-CLL CD20+B cells. The AFM data show that ultrastructure of B-CLL CD20+B lymphocytes became more complicated. Moreover, the single molecular force spectroscopy data show that the special force of CD20 antigen-antibody was four times bigger than the nonspecific force between the naked AFM tip and cell surface. The force map showed that CD20 molecules distributed homogeneously on the normal CD20+B lymphocytes, whereas, the CD20 molecules distributed heterogenous on B-CLL CD20+B lymphocytes. Our data provide visualized evidence for the phenomenon of low-response to rituximab therapy on clinical. Meanwhile, AFM is possible to be a powerful tool for development and screening of drugs for pharmacology use.
Antigen-Antibody Reactions ; immunology ; Antigens, CD20 ; immunology ; B-Lymphocytes ; immunology ; ultrastructure ; Binding Sites, Antibody ; Cell Membrane ; immunology ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; immunology ; Microscopy, Atomic Force ; Microscopy, Confocal ; Quantum Dots

OBJECTIVETo identify the IgE-binding epitopes in the allergen Der p 1 of main house dust mites, which can be recognized by the specific IgE in the sera from allergic individuals, and obtain a hypoallergen derived from the T-B epitope fused peptide for potential use in specific immunotherapy (SIT).

METHODSThirty-one peptides containing 15 amino acids each, which covered the full 222 amino acids of Der p 1 protein sequence, were synthesized on the cellulous membrane by solid-phase peptide (SPOTs) synthesis, with 8 overlapping amino acids between every two neighboring peptides. The membrane bearing the spots of the synthesized peptides were incubated with the allergic serum pools consisting of the sera from 5 allergic individuals. The membrane was then probed with HRP-conjugated anti-human IgE, followed by enhanced chemiluminescence (ECL) for visualization and gray scale analysis of the positive peptide spots.

RESULTSThree strong IgE-binding epitopes were identified in the amino acid sequence of Der p 1 molecule, namely Ep1 (amino acids 85-99), Ep2 (amino acids 106-120) and Ep3 (amino acids 190-204).

CONCLUSIONThe 3 IgE-binding epitopes (B cell epitopes) identified in Der p 1 confirm the presence of linear epitopes in Der p 1, suggesting the possibility of constructing T/B epitope-fused hypoallergens.

Amino Acid Sequence ; Animals ; Antigens, Dermatophagoides ; immunology ; Arthropod Proteins ; immunology ; Binding Sites, Antibody ; Cysteine Endopeptidases ; immunology ; Epitopes ; immunology ; Immunoglobulin E ; immunology ; Lymphokines ; immunology ; Mites ; immunology ; Molecular Sequence Data

Binding sites of five monoclonal antibodies were obtained by reinforceable method of overlapping recombinant prion protein and synthetic peptide. Overlapping peptides of PrP core were expressed in Escherichia coli by insertion of serial PCR amplicons of ovine PrP gene fragments into pET32a. The expressed fusion peptides were then tested for the binding activity to PrP monoclonal antibodies in Western blotting. The binding sites of 5 monoclonal antibodies of ovine PrP were located respectively as follows: 2H3 in 199 aa-213 aa, 4C6, 5F11 and 7F11 in 139 aa-168 aa and 7F1 in 214 aa-227 aa. There oligo peptides were synthesized and used in ELISA test for more accurate localization of the binding sites. The binding sites of 4C6, 5F11 and 7F11 were further confirmed to be in 149 aa-158 aa. This conclusion may contribute to the research for pathogenesis and diagnostic method of scrapie and bovine transmissible spongiform encephalopathy.
Animals ; Antibodies, Monoclonal ; immunology ; metabolism ; Binding Sites, Antibody ; immunology ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Prion Diseases ; diagnosis ; Prions ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Scrapie ; diagnosis ; Sheep

We studied the immunogenicity of pseudorabies virus gC DNA vaccination by fusing the murine complement C3d receptor binding domain. First, pseudorabies virus gC gene was linked to four copies of C3d receptor binding domain (M284), and then cloned into the vector pcDNA3.1 to construct the recombinant plasmid sgC-M284. Through the experiment of immunized BALB/c mice, we found that the enzyme linked immunosorbent assay (ELISA) antibody titer for sgC-M284 was 17-fold higher than that for sgC alone, and protective rate of mice was augmented from 25% to 88% after lethal dose PrV (316 LD50) challenge. In addition, the IL-4 levels for sgC-M284 immunization approached that for the pseudorabies virus inactivated vaccine. In conclusion, we demonstrated murine C3d receptor binding domain fusion significantly increased Th2-biased immune response by inducing IL-4 production.
Adjuvants, Immunologic ; physiology ; Animals ; Antibody Formation ; immunology ; Binding Sites ; Cloning, Molecular ; Complement C3d ; genetics ; immunology ; Herpesvirus 1, Suid ; genetics ; immunology ; Interleukin-4 ; immunology ; Mice ; Mice, Inbred BALB C ; Pseudorabies Vaccines ; immunology ; Receptors, Complement 3d ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; pharmacology ; Viral Fusion Proteins ; immunology

Streptococcus suis (S. suis) IgG-binding protein (SPG) was present in all S. suis strains examined. It showed binding activities with IgG from various host species. Little was known about the biological role of this protein, but it was commonly believed that it acted as virulence factor. In this study, the genes encoding SPG were amplified respectively from the total DNA of the S. suis serotype 1/2, 1, 2 and 9 with PCR and expressed in Escherichia coli BL21 by plasmid pET28a as vector. The recombinant proteins were first purified with affinity chromatography (Ni-NTA), and further purified by sephadexG-200 gel chromatography. The recombinant SPG proteins were identified to have binding activities with IgG of different host species, and for human and porcine IgG they showed better binding activities. But the SPG from different serotypes of S. suis showed no great differences in their binding activities with IgG from the same host species.
Bacterial Proteins ; genetics ; metabolism ; Binding Sites, Antibody ; genetics ; Escherichia coli ; genetics ; metabolism ; Immunoglobulin G ; immunology ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Streptococcus suis ; immunology