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To investigate favorable single amino acid substitutions that improve antigen-antibody interactions, alanine (Ala) mutagenesis scanning of the interfacial residues of a cancer-targeted antibody, B5209B, was performed based on X-ray crystallography analysis. Two substitutions were shown to significantly enhance the binding affinity for the antigen, by up to 30-fold. One substitution improved the affinity by a gain of binding enthalpy, whereas the other substitution improved the affinity by a gain of binding entropy. Molecular dynamics simulations showed that the enthalpic improvement could be attributed to the stabilization of distant salt bridges located at the periphery of the antigen-antibody interface. The entropic improvement was due to the release of water molecules that were stably trapped in the antigen-antibody interface of the wild-type antibody. Importantly, these effects of the Ala substitutions were caused by subtle adjustments of the binding interface. These results will be helpful to design high-affinity antibodies with avoiding entropy-enthalpy compensation., (Copyright © 2018. Published by Elsevier Ltd.)

Keratin-associated protein 8.1 (KAP8.1) is a hair protein whose structure, biochemical roles, and protein distribution patterns have not been well characterized. In this study, we generated a monoclonal antibody against human KAP8.1 to analyze the protein's roles and distribution in the human hair shaft. Using this antibody, we revealed that KAP8.1 was predominantly expressed in discrete regions of the keratinizing zone of the hair shaft cortex. The protein expression patterns paralleled the distribution of KAP8.1 mRNA and suggested that KAP8.1 plays a role associated with cells to control hair curvature. Cross-reactivity among species and epitope analysis indicated that the monoclonal antibody recognized a linear epitope shared among human, mouse, and sheep KAP8.1. The antibody failed to interact with sheep KAP8.1 in native conformation, suggesting that structural features of KAP8.1 vary among species., (Copyright © 2018. Published by Elsevier Inc.)

Cadherin-17 (CDH17) is highly expressed in gastric cancer and is thus considered to be a good target for antibody therapy. CDH17 is classified as a nonclassical cadherin, in that it is composed of seven extracellular cadherin domains. We generated anti-CDH17 monoclonal antibodies (mAbs) which recognize the extracellular domain of CDH17. Competitive assay using AGS, a gastric cancer cell line, cells revealed that five selected anti-CDH17 mAbs recognize different epitopes on CDH17. As AGS cells were shown to exhibit broad expression pattern of CDH17 by flow cytometry, we separated three clones with a low (10,000/cell), medium (50,000/cell), and high (200,000/cell) expression level, designating them as AGS low , AGS med , and AGS high , respectively. The mAbs, coupled with saporin, exhibited effective cytotoxicity to AGS high , but poor cytotoxicity to AGS low . By contrast, the immunotoxin cocktail using the three clones D2101, D2005, and D2008, which recognize different epitopes, exhibited efficient cytotoxicity, even to the AGS low group. The effect of the immunotoxin cocktail is synergistic, as the combination index was demonstrated to be below 1.0, as calculated by the method of Chou and Talalay using CalcuSyn software. These results suggest that the immunotoxin cocktail targeted to multiple epitopes has synergistic effects on low expression level cells, which expand the applicable range of immunotoxin therapy for cancer.

Prostaglandin D2 (PGD2) is a lipid mediator involved in sleep regulation and inflammation. PGD2 interacts with 2 types of G protein-coupled receptors, DP1 and DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on T helper type 2 cells)/GPR44 to show a variety of biological effects. DP1 activation leads to Gs-mediated elevation of the intracellular cAMP level, whereas activation of DP2 decreases this level via the Gi pathway; and it also induces G protein-independent, arrestin-mediated cellular responses. Activation of DP2 by PGD2 causes the progression of inflammation via the recruitment of lymphocytes by enhancing the production of Th2-cytokines. Here we developed monoclonal antibodies (MAbs) against the extracellular domain of mouse DP2 by immunization of DP2-null mutant mice with DP2-overexpressing BAF3, murine interleukin-3 dependent pro-B cells, to reduce the generation of antibodies against the host cells by immunization of mice. Moreover, we immunized DP2-KO mice to prevent immunological tolerance to mDP2 protein. After cell ELISA, immunocytochemical, and Western blot analyses, we successfully obtained a novel monoclonal antibody, MAb-1D8, that specifically recognized native mouse DP2, but neither human DP2 nor denatured mouse DP2, by binding to a particular 3D receptor conformation formed by the N-terminus and extracellular loop 1, 2, and 3 of DP2. This antibody inhibited the binding of 0.5 nM [3H]PGD2 to mouse DP2 (IC50 = 46.3 ± 18.6 nM), showed antagonistic activity toward 15(R)-15-methyl PGD2-induced inhibition of 300 nM forskolin-activated cAMP production (IC50 = 16.9 ± 2.6 nM), and gave positive results for immunohistochemical staining of DP2-expressing CD4+ Th2 lymphocytes that had accumulated in the kidney of unilateral ureteral obstruction model mice. This monoclonal antibody will be very useful for in vitro and in vivo studies on DP2-mediated diseases.

The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (Kd, ∼10(-11) M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R(2) = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R(2) = 0.834). These results suggest that the BV-KinExA method described here provides a suitably accurate Kd evaluation of mAbs against proteins on the cell surface., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background and Purpose: Most of the cases of neuromyelitis optica (NMO) are characterized by the presence of an autoantibody, NMO-IgG, which recognizes the extracellular domains of the water channel, aquaporin-4. Binding of NMO-IgG to aquaporin-4 expressed in end-feet of astrocytes leads to complement-dependent disruption of astrocytes followed by demyelination. One therapeutic option for NMO is to prevent the binding of NMO-IgG to aquaporin-4, using high-avidity, non-pathogenic-chimeric, monoclonal antibodies to this water channel. We describe here the development of such antibodies., Experimental Approach: cDNAs encoding variable regions of heavy and light chains of monoclonal antibodies against the extracellular domains of human aquaporin-4 were cloned from hybridoma total RNA and fused to those encoding constant regions of human IgG1 and Igκ respectively. Then mammalian expression vectors were constructed to establish stable cell lines secreting mature chimeric antibodies., Key Results: Original monoclonal antibodies showed high avidity binding to human aquaporin-4, as determined by ELISA. Live imaging using Alexa-Fluor-555-labelled antibodies revealed that the antibody D15107 more rapidly bound to cells expressing human aquaporin-4 than others and strongly enhanced endocytosis of this water channel, while D12092 also bound rapidly to human aquaporin-4 but enhanced endocytosis to a lesser degree. Chimeric D15107 prevented complement-dependent cytotoxicity induced by NMO-IgG from patient sera in vitro., Conclusions and Implications: We have established non-pathogenic, high-avidity, chimeric antibodies against the extracellular domains of human aquaporin-4, which provide a novel therapeutic option for preventing the progress and recurrence of NMO/NMO spectrum disorders., (© 2015 The British Pharmacological Society.)

Introduction: ROBO1 is a membrane protein that contributes to tumor metastasis and angiogenesis. We previously reported that 90Y-labeled anti-ROBO1 monoclonal antibody (90Y-anti-ROBO1 IgG) showed an antitumor effect against ROBO1-positive tumors. In this study, we performed a biodistribution study and radioimmunotherapy (RIT) against ROBO1-positive small cell lung cancer (SCLC) models., Methods: For the biodistribution study, 111In-labeled anti-ROBO1 monoclonal antibody (111In-anti-ROBO1 IgG) was injected into ROBO1-positive SCLC xenograft mice via the tail vein. To evaluate antitumor effects, an RIT study was performed, and SCLC xenograft mice were treated with 90Y-anti-ROBO1 IgG. Tumor volume and body weight were periodically measured throughout the experiments. The tumors and organs of mice were then collected, and a pathological analysis was carried out., Results: As a result of the biodistribution study, we observed tumor uptake of 111In-anti-ROBO1 IgG. The liver, kidney, spleen, and lung showed comparably high accumulation of 111In-labeled anti-ROBO1. In the RIT study, 90Y-anti-ROBO1 IgG significantly reduced tumor volume compared with baseline. Pathological analyses of tumors revealed coagulation necrosis and fatal degeneration of tumor cells, significant reduction in the number of Ki-67-positive cells, and an increase in the number of apoptotic cells. A transient reduction of hematopoietic cells was observed in the spleen, sternum, and femur., Conclusions: These results suggest that RIT with 90Y-anti-ROBO1 IgG is a promising treatment for ROBO1-positive SCLC.

Aquaporin-4 (AQP4), the most abundant water channel in the brain, plays a central role in water homeostasis, neuronal activity, and migration of astrocytes in the central nervous system. Recent studies have demonstrated that AQP4 is a target of an autoantibody specifically detected in an autoimmune neurologic disease called neuromyelitis optica. Here we have generated a monoclonal antibody (MAb) against the C-terminal region of AQP4 using a baculovirus expressing mouse AQP4 as an immunogen. This antibody (clone E5206) recognized both human and mouse AQP4s in a denaturing condition and was able to precipitate AQP4 from cell lysates of CHO cells stably expressing AQP4. Western blot analysis using deletion mutants revealed that the epitope was located within a region between Asp(303) and Leu(320) in the C-terminal tail of AQP4. Although clone E5206 could not be used for immunostaining when cells or tissues were fixed with 4% paraformaldehyde or 10% formalin, it could be used when cells were fixed with 10% trichloroacetic acid and when a formalin-fixed tissue section was pretreated with antigen-retrieval reagents. This MAb can be a valuable tool for analysis of AQP4 in a variety of physiological and pathophysiological contexts, in human tissues and organs as well as in rodent models, both in vitro and in vivo.

Neuromyelitis optica is a demyelinating disease characterized by a disease-specific autoantibody designated as NMO-IgG that specifically recognizes aquaporin-4, and the binding of NMO-IgG to AQP4 causes the progress of the disease. Prevention of the binding of NMO-IgG, therefore, may alleviate the disease. Here we have developed monoclonal antibodies against AQP4 with a baculovirus display system in order to obtain high affinity monoclonal antibodies against the extracellular domains of AQP4. Our monoclonal antibodies can block the binding of NMO-IgG in spite of their heterogeneity. Taken together, we propose that our monoclonal antibodies can be applied in clinical therapy for NMO patients., (Copyright © 2013 Elsevier B.V. All rights reserved.)

L-Asparaginase (ASNase) is important for the treatment of childhood acute lymphoblastic leukemia. ASNase sensitivity has been shown to correlate with the asparagine synthetase (ASNS) protein content in acute lymphoblastic leukemia cell lines. However, there have been few studies to determine ASNS protein levels in human leukemias, since no appropriate monoclonal antibody is available for such quantitative analysis. In this study, we report the generation of anti-ASNS monoclonal antibodies, which are applicable to flow cytometry and enzyme-linked immunosorbent assay. These monoclonal antibodies should provide a valuable tool for the quantification of ASNS protein level and estimation of ASNase-resistance in leukemia cells.

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A(2A) adenosine receptor (A(2A)AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A(2A)AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A(2A)AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A(2A)AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active β(2)-adrenergic receptor structure, but locks A(2A)AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.

TEX101 was characterized as a unique germ cell marker molecule using the specific monoclonal antibody (mAb), TES101. Although this mAb has strong affinity/specificity for TEX101, TES101 mAb loses its reactivity under reducing conditions. In this study, we have generated new mAbs against TEX101 to compensate for the shortcomings of the TES101 mAb using different approaches. First, we immunized mice with the antigen on a baculovirus expression system and isolated new anti-TEX101 mAbs, 6002 and 6035. Second, we raised the mAb Ts4 from spleen cells of an immunologically naive old mouse. Western blot analysis revealed that the new mAbs possess immunoreactivity under reducing/non-reducing conditions. Immunopositive staining of the mAbs against Bouin-fixed sections was observed in spermatocytes, spermatids and testicular spermatozoa, but not in other cells, similar to paraformaldehyde (PFA)-fixed frozen sections stained with TES101 as previously reported. However, whereas the mAbs 6002/6035 mainly showed immunoreactivity only in spermatocytes in PFA-fixed frozen sections, the reactivity of the mAbs to spermatids and testicular spermatozoa was clearly recovered when the PFA-fixed sections were autoclaved or treated with SDS. Peptide mapping and deglycosylation analysis indicated that the epitopes for TES101, 6002 and 6035 are located within TEX101(25-94), whereas Ts4 recognized N-linked carbohydrate moieties on TEX101 in Triton X-100-soluble mouse testicular extracts but not in the extracellular or water-soluble fractions. These results suggest strongly that the molecular association or structure of N-linked carbohydrate moieties of TEX101 varies according to its subcellular localization within the seminiferous tubules. These new mAbs will be valuable tools for further analysis of TEX101, including its function(s).

We have been investigating the functional display of multipass membrane protein such as transporter or G-protein coupled receptor on the budded baculovirus (BV). We tested the use of a viral envelope protein gp64 transgenic mouse for the direct immunization of these membrane proteins displayed on BVs. The gp64 transgenic mice showed only a weak response to virus compared to wild type BALB/c mice. Immunizing gp64 transgenic mice with the BV expressing peptide transporter PepT1, we obtained 47 monoclonal antibodies (mAbs). These mAbs were specific to the PepT1 on the pancreatic cancer cells AsPC-1 by fluorocytometric analysis and exhibited antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity to AsPC-1. We also generated 7 mAbs by immunizing gp64 transgenic mice on a CCR2-deficient background with the BV expressing chemokine receptor CCR2 together with partially purified CCR2. These mAbs possessed specific binding to CCR2 in CHO cells on fluorocytometric analysis, and exhibited neutralizing activities for ligand-dependent inhibition of cyclic AMP production. This method provides a powerful tool for the generation of therapeutic/diagnostic mAbs against membrane proteins.

In hybridoma screening, quantitative kinetic evaluation is difficult since the concentration of each antibody in the hybridoma supernatant is unknown. From modeling calculations, we hypothesized that the ratio of two different antigen-antibody concentrations might allow discrimination of high-affinity monoclonal antibodies irrespective of the antibody concentration. Using anti-alpha-fetoprotein monoclonal antibodies of known affinity, we set the signal ratio of a time-resolved assay at >0.1, in which the antigen concentrations were 10 and 100 ng/mL. From anti-alpha-fetoprotein hybridoma screening with this assay, it was possible to effectively select high-affinity monoclonal antibodies with KD values below 1x10(-8) M. High-sensitivity sandwich enzyme-linked immunosorbent assay which detects domain III of alpha-fetoprotein has been established using selected high-affinity monoclonal antibodies. This screening method is useful for selection of high-affinity monoclonal antibodies of potential diagnostic value.

Objective: Liver X-activated receptor alpha (LXRalpha) regulates multiple genes controlling cholesterol metabolism and transport. To clarify its role in atherogenesis, we established a monoclonal antibody recognizing native human LXRalpha protein and studied the expression pattern in human atherosclerotic lesions., Methods and Results: A novel monoclonal antibody PPZ0412 was raised against the ligand-binding domain of LXRalpha, which can be used for immunostaining of human LXRalpha protein. LXRalpha protein was detected in the nucleus of macrophages in the liver, spleen, or lung and also in hepatocytes and adipocytes. In atherosclerotic lesions, the LXRalpha protein was detected in macrophages positive for scavenger receptor class A and/or CD68., Conclusions: In the human body, the LXRalpha protein is highly expressed in macrophage lineage cells and foam cells in atherosclerotic lesions and is identified as a target for intervention in atherosclerotic disease.

To obtain monoclonal antibodies (MAbs) that distinguish secretor and nonsecretor from their saliva in forensic casework, two (K7405 and K7422) and one (K7516) MAbs reacting to blood group A antigen were produced by immunization of mice with salivary mucin obtained from blood group A secretors and nonsecretors, respectively. K7405, produced by immunization with salivary mucin obtained from A secretor, reacted with the A substances bound to the carrier protein but not with the A substance separated from the carrier protein. On the other hand, the K7422 and K7516 were reactive to the A substance separated from carrier protein. From these results, we conclude that K7405 recognizes the A substances clustered on the carrier protein and K7422 and K7516 recognize the isolated A substance. In the forensic blood typing of body fluids, A secretors and A nonsecretors can be clearly discriminated by the combined application of two MAbs (K7405 and K7516), which react differently against saliva samples.

Monoclonal antibodies (Mabs) are valuable reagents for the purification, characterization and immunolocalization of proteins. In this study, we raised Mabs against human peroxisome proliferator-activated receptors (PPARs) using baculovirus particles displaying surface glycoprotein gp64-fusion proteins as the immunizing agent. In this system, to display fusion proteins on the viral surface, the amino terminal sequences of human PPARd and PPARg2 are inserted in-frame between the signal sequence and the mature domain of the gp64 nucleotide sequence.Mabs were raised by immunization with whole virus without a purification of the target antigens. The Mabs generated by this novel method were shown to recognize not only the gp64-PPARs fusion protein, but also mature, expressed proteins by a wide variety of techniques, including immunohistochemistry, immunoblotting, and electrophoretic mobility shift assays (EMSAs). Transfection of the transfer vector containing a nucleotide sequence encoding less than 30 amino acids along with linearized baculovirus DNA allows for the production of a high affinity antibody against the corresponding mature form. This method is of potential utility in that it allows the production of valuable antibodies without the requirement of a protein purification step.