Antigen–Antibody Binding

Recognition of poly(C) DNA and RNA sequences in mammalian cells is achieved by a subfamily of the KH (hnRNP K homology) domain-containing proteins known as poly(C)-binding proteins (PCBPs). To reveal the molecular basis of poly(C) sequence recognition, we have determined the crystal structure, at 1.7-A resolution, of PCBP2 KH1 in complex with a 7-nucleotide DNA sequence (5'-AACCCTA-3') corresponding to one repeat of the human C-rich strand telomeric DNA. The protein-DNA interaction is mediated by the combination of several stabilizing forces including hydrogen bonding, electrostatic interactions, van der Waals contacts, and shape complementarities. Specific recognition of the three cytosine residues is realized by a dense network of hydrogen bonds involving the side chains of two conserved lysines and one glutamic acid. The co-crystal structure also reveals a protein-protein dimerization interface of PCBP2 KH1 located on the opposite side of the protein from the DNA binding groove. Numerous stabilizing protein-protein interactions, including hydrophobic contacts, stacking of aromatic side chains, and a large number of hydrogen bonds, indicate that the protein-protein interaction interface is most likely genuine. Interaction of PCBP2 KH1 with the C-rich strand of human telomeric DNA suggests that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.

The tumor-associated glycoprotein (TAG)-72 is expressed in the majority of human adenocarcinomas but is rarely expressed in most normal tissues, which makes it a potential target for the diagnosis and therapy of a variety of human cancers. Here we describe the construction, affinity maturation, and biological characterization of an anti-TAG-72 humanized antibody with minimum potential immunogenicity. The humanized antibody was constructed by grafting only the specificity-determining residues (SDRs) within the complementarity-determining regions (CDRs) onto homologous human immunoglobulin germ line segments while retaining two mouse heavy chain framework residues that support the conformation of the CDRs. The resulting humanized antibody (AKA) showed only about 2-fold lower affinity compared with the original murine monoclonal antibody CC49 and 27-fold lower reactivity to patient serum compared with the humanized antibody HuCC49 that was constructed by CDR grafting. The affinity of AKA was improved by random mutagenesis of the heavy chain CDR3 (HCDR3). The highest affinity variant (3E8) showed 22-fold higher affinity compared with AKA and retained the original epitope specificity. Mutational analysis of the HCDR3 residues revealed that the replacement of Asn(97) by isoleucine or valine was critical for the affinity maturation. The 3E8 labeled with (125)I or (131)I showed efficient tumor targeting or therapeutic effects, respectively, in athymic mice with human colon carcinoma xenografts, suggesting that 3E8 may be beneficial for the diagnosis and therapy of tumors expressing TAG-72.

Highly pathogenic avian influenza (HPAI) H5N1 virus infection is still a potential threat to public health worldwide. While vaccines and antiviral drugs are currently under development, neutralizing antibodies could offer an alternative strategy to prevent and treat H5N1 virus infection. In the present study, we had developed a humanized antibody against H5N1 viruses from mouse-derived hybridoma in order to minimize its immunogenicity for potential clinical application. The humanized antibody hH5M9 was generated by transferring the mouse complementarity determining region (CDR) residues together with four key framework region (FR) residues onto the FR of the human antibody. This humanized antibody exhibited high affinity and specificity comparable to the parental mouse or chimeric counterpart with broad and strong neutralization activity against all H5N1 clades and subclades except for Egypt clades investigated. Furthermore, through epitope mapping we identified a linear epitope on the top region of hemagglutinin (HA) that was H5N1 specific and conserved. Our results for the first time reported a humanized antibody against H5N1 viruses by CDR grafting method. With the expected lower immunogenicity, this humanized antibody was expected to be more efficacious than murine or human-mouse chimeric antibodies for future application in humans.

Designed ankyrin repeat proteins (DARPins) are a novel class of binding proteins that bind their target protein with high affinity and specificity and have very favorable expression and stability properties. We describe here the in vitro selection of DARPins against human epidermal growth factor receptor 2 (Her2), an important target for cancer therapy and diagnosis. Several DARPins bind to the same epitope as trastuzumab (Herceptin), but none were selected that bind to the epitope of pertuzumab (Omnitarg). Some of the selected DARPins bind with low nanomolar affinity (Kd=7.3 nm) to the target. Further analysis revealed that all DARPins are highly specific and do not cross-react with epidermal growth factor receptor I (EGFR1) or any other investigated protein. The selected DARPins specifically bind to strongly Her2-overexpressing cell lines such as SKBR-3 but also recognize small amounts of Her2 on weakly expressing cell lines such as MCF-7. Furthermore, the DARPins also lead to a highly specific and strong staining of plasma membranes of paraffinated sections of human mamma-carcinoma tissue. Thus, the selected DARPins might be used for the development of diagnostic tests for the status of Her2 overexpression in different adenocarcinomas, and they may be further evaluated for their potential in targeted therapy since their favorable expression properties make the construction of fusion proteins very convenient.

Monoclonal antibodies have recently started to deliver on their promise as highly specific and active drugs; however, a more effective, knowledge-based approach to the selection, design, and optimization of potential therapeutic antibodies is currently limited by the surprising lack of detailed structural information for complexes formed with target proteins. Here we show that complexes formed with minimal antigen binding single chain variable fragments (scFv) reliably reflect all the features of the binding interface present in larger Fab fragments, which are commonly used as therapeutics, and report the development of a robust, reliable, and relatively rapid approach to the determination of high resolution models for scFv-target protein complexes. This NMR spectroscopy-based approach combines experimental determination of the interaction surfaces and relative orientations of the scFv and target protein, with NMR restraint-driven, semiflexible docking of the proteins to produce a reliable and highly informative model of the complex. Experience with scFvs and Fabs targeted at a number of secreted regulatory proteins suggests that the approach will be applicable to many therapeutic antibodies targeted at proteins, and its application is illustrated for a potential therapeutic antibody targeted at the cytokine IL-1beta. The detailed structural information that can be obtained by this approach has the potential to have a major impact on the rational design and development of an increasingly important class of biological pharmaceuticals.

Antibody therapeutics are a promising drug class due to their high specificity and favorable pharmacokinetics. While there are many methods for the development of antibodies specific to disease associated antigens, selecting antibodies against functional epitopes with high specificity and affinity can be difficult for certain epitopes. We describe a generalizable method for synthesizing antibody mimetics by site specifically conjugating small molecules (with high affinity and specificity to disease associated antigens) to an Fc fragment to develop drugs with the benefits of an antibody. As a proof of concept, an E269pAcPhe Fc antibody Fc fragment was produced and subsequently site-specifically labeled with a linker-modified folic acid compound to generate an Fc-folic acid antibody-mimetic. This was chosen as the model system because the high-affinity folate receptor FR-α is highly expressed in a number of cancer types including breast and ovarian cancer. The specificity of the Fc-folic acid conjugate was assessed via flowcytometry with the folate-receptor positive breast cancer cell line MDA-MB-231 by measuring Fc-folic acid binding in both the absence and presence of an excess of folic acid. Fc-small molecule conjugates could be developed into a unique class of antibody-like therapeutics.

Cytochrome P450 46A1 (CYP46A1) initiates the major pathway of cholesterol elimination from the brain and thereby controls cholesterol turnover in this organ. We determined x-ray crystal structures of CYP46A1 in complex with four structurally distinct pharmaceuticals; antidepressant tranylcypromine (2.15 Å), anticonvulsant thioperamide (1.65 Å), antifungal voriconazole (2.35 Å), and antifungal clotrimazole (2.50 Å). All four drugs are nitrogen-containing compounds that have nanomolar affinity for CYP46A1 in vitro yet differ in size, shape, hydrophobicity, and type of the nitrogen ligand. Structures of the co-complexes demonstrate that each drug binds in a single orientation to the active site with tranylcypromine, thioperamide, and voriconazole coordinating the heme iron via their nitrogen atoms and clotrimazole being at a 4 Å distance from the heme iron. We show here that clotrimazole is also a substrate for CYP46A1. High affinity for CYP46A1 is determined by a set of specific interactions, some of which were further investigated by solution studies using structural analogs of the drugs and the T306A CYP46A1 mutant. Collectively, our results reveal how diverse inhibitors can be accommodated in the CYP46A1 active site and provide an explanation for the observed differences in the drug-induced spectral response. Co-complexes with tranylcypromine, thioperamide, and voriconazole represent the first structural characterization of the drug binding to a P450 enzyme.

The release of ubiquitin from attachment to other proteins and adducts is critical for ubiquitin biosynthesis, proteasomal degradation and other cellular processes. De-ubiquitination is accomplished in part by members of the UCH (ubiquitin C-terminal hydrolase) family of enzymes. We have determined the 2.25 A resolution crystal structure of the yeast UCH, Yuh1, in a complex with the inhibitor ubiquitin aldehyde (Ubal). The structure mimics the tetrahedral intermediate in the reaction pathway and explains the very high enzyme specificity. Comparison with a related, unliganded UCH structure indicates that ubiquitin binding is coupled to rearrangements which block the active-site cleft in the absence of authentic substrate. Remarkably, a 21-residue loop that becomes ordered upon binding Ubal lies directly over the active site. Efficiently processed substrates apparently pass through this loop, and constraints on the loop conformation probably function to control UCH specificity.

The antigenic binding site(s) of antibodies to factor XII associated with the antiphospholipid syndrome

The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the membrane of the parasitized erythrocyte which provide the parasite with nutrients, adjust the erythrocyte electrolyte composition to the needs of the parasite, and dispose of metabolic waste products and osmolytes. Patch-clamp recordings identified inwardly and outwardly rectifying (OR) anion conductances in the host erythrocyte membrane as electrophysiological correlate of the NPP. The OR conductance is regulated by serum. Here we show that serum albumin (SA) stimulated OR-generated Cl^- and lactate outward currents with an EC₅₀ of approximately 100 nM while other proteins such as ovalbumin or casein did not. The stimulatory efficacy did not differ between fatty acid free bovine SA and recombinant human SA and disruption of the SA tertiary structure abolished the effect suggesting that intact SA protein and not other bound factors interact with the erythrocyte membrane. Taken together, the data indicate a high affinity and specificity interaction of native SA with the parasitized erythrocytes which might underlie the observed dependence of P. falciparum growth in vitro on SA.

Murine hybridomas were generated by immunizing mice with purified alpha-fetoprotein (AFP) to determine the cross-reactivity of AFP antibodies with human serum albumin (HSA). Eighteen out of 23 hybridoma products were cross-reactive with HSA. All 23 monoclonal antibodies (MAbs) could be subgrouped into 3 groups by their binding affinity to AFP and HSA, and one MAb from each group, IIB3, IIIB1 and IIIC6, were chosen for this study. The affinity to AFP was highest for IIB3 followed by IIIC6 and IIIB1, and that to HSA was in the order IIIB1 greater than IIB3. Immunohistochemical staining revealed no cross-reactions of these antibodies with HSA at the tissue level. IIB3 was the most efficient in the quantitative assays, indicating high affinity and specificity (non-cross-reactivity) of the antibodies, both of which are prerequisites for in vitro assays. Scans with good tumor localization were obtained from mice which received 131I-labeled IIB3 or IIIC6 after xenografting of the human hepatocellular carcinoma. The images obtained with IIB3 were inhibited in the presence of either AFP or HSA in the circulation. These findings indicated that the use of cross-reactive and/or high-affinity antibodies against AFP provides an inhibitory factor for tumor localization. MAb IIIC6, because of its high specificity and moderate affinity to AFP, seems to be the best candidate for immunoscintigraphy of the tumors in future studies.

We have studied the role of lymphoproliferative tumors in the pathogenesis of autoimmune and the origin of the autoantibodies in PNP. Objectives of this study is to understand the characteristics of immunoglobulin genes of the B-cells isolated from the tumor which can produce auto-antibody. Total RNA of the tumor cells was then isolated and the mRNA was reversely transcribed into cDNA. V(H) and V(L) genes were amplified and cloned and their sequences were analyzed. 49 V(H) genes (527 to 577 bp) and 36 V(L) genes (445 to 454 bp) sequences were cloned from five tumors. All of the IgV(L) were mostly homologous to IGKV4-01 germ-line gene. The frequency of IgV(H) germ-line gene usage in a decreasing order was IGHV3-23 > IGHV3-9 > IGHV4-31 > IGHV4-59. There was more nucleotide changes occurred in complementary determining regions (CDR) than those in the framework regions (FR) in the V(H) and V(L) of B cell clones. In both V(H) and V(L), the probability (P) that the number of observed replacement mutations (R) in the CDRs would occur by random chance was low. The clones of B-lymphocytes in the tumors carrying possibly functional rearranged immunoglobulin heavy and light chain genes were isolated and there were high incidences of somatic mutations in CDRs and relatively conserved sequences in framework region.

Desired drug candidates should have both a high potential binding chance and high specificity. Recently, many drug screening strategies have been developed to screen compounds with high possible binding chances or high binding affinity. However, there is still no good solution to detect whether those selected compounds possess high specificity. Here, we developed a reverse DFCNN (Dense Fully Connected Neural Network) and a reverse docking protocol to check a given compound’s ability to bind diversified targets and estimate its specificity with homemade formulas. We used the RNA-dependent RNA polymerase (RdRp) target as a proof-of-concept example to identify drug candidates with high selectivity and high specificity. We first used a previously developed hybrid screening method to find drug candidates from an 8888-size compound database. The hybrid screening method takes advantage of the deep learning-based method, traditional molecular docking, molecular dynamics simulation, and binding free energy calculated by metadynamics, which should be powerful in selecting high binding affinity candidates. Also, we integrated the reverse DFCNN and reversed docking against a diversified 102 proteins to the pipeline for assessing the specificity of those selected candidates, and finally got compounds that have both predicted selectivity and specificity. Among the eight selected candidates, Platycodin D and Tubeimoside III were confirmed to effectively inhibit SARS-CoV-2 replication in vitro with EC50 values of 619.5 and 265.5 nM, respectively. Our study discovered that Tubeimoside III could inhibit SARS-CoV-2 replication potently for the first time. Furthermore, the underlying mechanisms of Platycodin D and Tubeimoside III inhibiting SARS-CoV-2 are highly possible by blocking the RdRp cavity according to our screening procedure. In addition, the careful analysis predicted common critical residues involved in the binding with active inhibitors Platycodin D and Tubeimoside III, Azithromycin, and Pralatrexate, which hopefully promote the development of non-covalent binding inhibitors against RdRp.

Molecular Recognition by a Binary Code

Synthetic Fab fragments that bind the HIV-1 gp41 heptad repeat regions