Epitope Mapping of Monoclonal Antibody PMab-233 Against Tasmanian Devil Podoplanin.

The highly O-glycosylated membrane glycoprotein podoplanin (PDPN) is frequently overexpressed in several malignant cancers, such as oral cancer, lung cancer, germinal neoplasia, mesothelioma, and brain tumor. The expression of PDPN is strongly associated with cancer progression and poor prognosis. PDPN possesses three tandem repeats of platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) and PLAG-like domain (PLD), and binds to C-type lectin-like receptor 2 (CLEC-2) on platelets, followed by PDPN-mediated platelet aggregation. We have previously established a novel anti-Tasmanian devil PDPN (tasPDPN) monoclonal antibody (mAb), PMab-233, which specifically detects tasPDPN using flow cytometry, Western blot, and immunohistochemical analyses. However, the specific binding epitope of tasPDPN for PMab-233 remains to be clarified. Herein, a series of deletion or point mutants of tasPDPN were utilized for investigating the binding epitopes of PMab-233 using flow cytometry. The findings of this study demonstrated that Asp30, Thr33, and Thr34 of tasPDPN, which are located in PLAG1, are responsible for the binding of PMab-233 to tasPDPN.

Epitope Mapping of PMab-225 an Anti-Alpaca Podoplanin Monoclonal Antibody Using Flow Cytometry.

Podoplanin (PDPN) is a mucin-type membrane glycoprotein, and possesses three platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 at the N-terminus of PDPN, and one or two PLAG-like domains (PLDs) in the middle of PDPN. PDPN is expressed on normal tissues, such as podocytes of the kidney and type I alveolar cells of the lung, and is also overexpressed in numerous malignant cancers. Previously, we reported a novel anti-alpaca podoplanin (aPDPN) monoclonal antibody (mAb), PMab-225, using Cell-Based Immunization and Screening (CBIS) method. PMab-225 specifically detected aPDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/aPDPN) cells using flow cytometry and western blotting, and strongly stained alpaca tissues such as lung type I alveolar cells by immunohistochemistry. However, the specific binding epitope of aPDPN for PMab-225 remains unclear. Thus, in this study, a series of deletion or point mutations of aPDPN were utilized for investigating the binding epitope of PMab-225 using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-225 epitope might exist between 80 amino acid (aa) and 85 aa of aPDPN. Furthermore, the analysis of point mutants demonstrated that Thr84 of aPDPN, which exists in PLD, could be included in the critical epitope of PMab-225.

Identification of Fc Gamma Receptor Glycoforms That Produce Differential Binding Kinetics for Rituximab.

Fc gamma receptors (FcγR) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of FcγRs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the FcγR glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fcγ receptors RI (CD64), RIIaArg131/His131 (CD32a), RIIb (CD32b) and RIIIaPhe158/Val158 (CD16a) and analyze the role of the glycans in the binding mechanism with IgG. The interactions of the monoclonal antibody rituximab with each FcγR were characterized and we discuss the CHO-FcγRIIIaPhe158/Val158 and CHO-FcγRI interactions and compare them to the equivalent interactions with human (HEK293) and murine (NS0) produced receptors. Our results reveal clear differences in the binding profiles of rituximab, which we attribute in each case to the differences in host cell-dependent FcγR glycosylation. The glycan profiles of CHO expressed FcγRI and FcγRIIIaPhe158/Val158 were compared with the glycan profiles of the receptors expressed in NS0 and HEK293 cells and we show that the glycan type and abundance differs significantly between the receptors and that these glycan differences lead to the observed differences in the respective FcγR binding patterns with rituximab. Oligomannose structures are prevalent on FcγRI from each source and likely contribute to the high affinity rituximab interaction through a stabilization effect. On FcγRI and FcγRIIIa large and sialylated glycans have a negative impact on rituximab binding, likely through destabilization of the interaction. In conclusion, the data show that the IgG1-FcγR binding kinetics differ depending on the glycosylation of the FcγR and further support a stabilizing role of FcγR glycans in the antibody binding interaction.

Effect of temperature and food restriction on immune function in striped hamsters (Cricetulus barabensis).

Small mammals in temperate areas face seasonal fluctuations of temperature and food availability, both of which may influence their immune responses, which are critical to survival. In the present study, we tested the hypothesis that low temperature and food restriction suppress immune function in striped hamsters (Cricetulus barabensis). Thirty-seven adult male hamsters were randomly assigned to warm (23±1°C) and cold (5±1°C) treatment groups, which were further divided into fed and food-restricted groups. Body mass was not affected by cold stress, food restriction or the interaction cold stress×food restriction. Cold stress decreased total body fat mass, haematological parameters including white blood cells, lymphocytes and neutrophilic granulocytes, and immunoglobin (Ig) M titres 5 days after injecting keyhole limpet haemocyanin (KLH). However, cold temperature increased bacterial killing capacity, indicative of innate immunity, and did not affect the mass of the thymus and spleen, intermediate granulocytes, the phytohaemagglutinin (PHA) response and the levels of blood glucose and serum leptin. Corticosterone concentration was affected significantly by the interaction cold stress×food restriction but not by cold stress or food restriction alone. Food restriction reduced thymus mass, but other immunological parameters including body fat mass, spleen mass, haematological parameters, innate immunity, PHA response, the titres of IgM and IgG, and the levels of blood glucose and serum leptin were all not affected by food restriction or the interaction cold stress×food restriction. Innate immunity was positively correlated with leptin levels, whereas no significant correlations were observed in the levels of blood glucose, serum leptin, corticosterone and all the detected immune parameters. Our results show that cold stress suppressed humoral immunity but enhanced innate immunity and did not affect cellular immunity in striped hamsters. Most immunological indices were not influenced by food restriction. Blood glucose, leptin and corticosterone could not explain the changes of innate, cellular and humoral immunity upon cold stress or food restriction in striped hamsters.

Male-specific submaxillary gland protein, a lipocalin allergen of the golden hamster, differs from the lipocalin allergens of Siberian and Roborovski dwarf hamsters.

BACKGROUND: An increasing number of asthma cases upon exposure to hamsters and anaphylactic reactions following hamster bites are being reported, but the allergens responsible are still poorly characterized. In the Golden hamster, male-specific submaxillary gland protein (MSP), a lipocalin expressed in a sex- and tissue-specific manner in the submaxillary and lacrimal glands, is secreted in the saliva, tears and urine. The purpose of this study was to determine if MSP is an allergen, to identify IgE-reactive proteins of different hamster species and to analyse potential cross-reactivities. METHODS: Fur extracts were prepared from four hamster species. Hamster-allergic patients were selected based on a history of positive IgE-test to hamster epithelium. The IgE-reactivity of patients' sera was investigated by means of immunoblot and ELISA. IgE-reactive proteins in fur extracts and the submaxillary gland were identified using anti-MSP antibodies, Edman sequencing or mass spectrometry. MSP was purified from Golden hamster and recombinant MSP was expressed in E. coli. RESULTS: Four patients had IgE-antibodies against 20.5-kDa and 24-kDa proteins of Golden hamster fur extract, which were identified as MSP. IgE-reactive MSP-like proteins were detected in European hamster fur extract. Three patient sera showed IgE-reactive bands at 17-21 kDa in Siberian and Roborovski hamster fur extracts. These proteins were identified as two closely related lipocalins. Immunoblot inhibition experiments showed that they are cross-reactive and are different from MSP. CONCLUSION: MSP lipocalin of the Golden hamster was identified as an allergen, and it is different from the cross-reactive lipocalin allergens of Siberian and Roborovski hamsters. Our findings highlight the need for specific tools for the in vitro and in vivo diagnosis of allergy to different hamster species.

Prevalence and isotypic complexity of the anti-Chinese hamster ovary host cell protein antibodies in normal human serum.

Host cell-derived protein impurities may be present at low levels in biopharmaceutical products. Antibodies to host cell proteins are present in individuals with no known exposure to these products. In this study, antibodies to drug process-specific Chinese hamster ovary host cell-derived proteins (CHO-HCP) were measured in unexposed individuals using a validated enzyme-linked immunosorbent assay. Samples that tested positive for anti-CHO-HCP reactivity were further characterized to determine the isotypes and IgG subclasses expressed in each positive individual. The specificity of the detected anti-CHO-HCP antibody isotypes was confirmed by the competitive inhibition assay and the uncoated plate specificity testing. These antibody characterization experiments revealed that the prevalent anti-CHO-HCP antibody subclasses were predominantly IgG1 (present in 66.6% of individuals) and IgG2 (60%), with 33.3% positive for IgG3 while IgG4 was detected in low abundance. Forty percent (40%) of the individuals with IgG type reactivity were also identified as IgM-positive. Anti-CHO-HCP-specific IgE was not detected in the assays used in this study. Some individuals exhibited single isotype anti-CHO-HCP reactivity; others contained a combination of multiple antibody isotypes. Data presented in this study demonstrate the isotypic complexity and the high prevalence of anti-CHO-HCP antibodies in human serum with no known exposure to CHO cell-derived therapeutic biologics.

Blockade of the PD-1/PD-1L pathway reverses the protective effect of anti-CD40L therapy in a rat to mouse concordant islet xenotransplantation model.

BACKGROUND: We have previously demonstrated that costimulatory blockade with anti-CD40L monoclonal antibody (mAb) prolongs the survival of non-vascularized concordant rat to mouse islet xenografts. Here, we examine whether signaling through the PD-1/PD-1L pathway is required for the anti-CD40L therapy to prolong concordant islet graft survival using a novel anti-murine PD-1 mAb (clone 4F10). METHODS: C57BL/6 mice received a cellular concordant islet xenograft under the left kidney capsule and four experimental groups were prepared. Group I: untreated control; group II: recipient mice were treated with three doses of 0.5 mg of anti-CD40L mAb (clone MR1) on days 0, 2 and 4; group III: mice were treated with 0.5 mg of anti-PD-1 (CD279) mAb (clone 4F10) every other day for 8 days; and finally group IV: mice received the combined treatment that consisted of anti-CD40L plus anti-PD-1 mAb. RESULTS: Concordant islet xenografts transplanted in control untreated mice showed a median survival time (MST) of 17 +/- 7.43 days, whereas anti-CD40L treatment led to a significant prolongation of graft survival (MST: 154 +/- 65.56, P < 0.0001). The administration of anti-PD-1 alone significantly accelerated graft rejection compared to non-treated controls (MST: 10 +/- 2.24 vs. MST: 17 +/- 7.43, P < 0.0004). Remarkably, the combined administration of anti-CD40L and anti-PD-1 reversed the protective effect obtained with anti-CD40L alone (anti-CD40L, MST: 154 +/- 65.56 vs. anti-CD40L plus anti-PD-1, MST: 10 +/- 7.72, P < 0.0002). CONCLUSION: Overall, our data indicate that the PD-1/PD-1L pathway is required for the achievement of prolonged graft survival in anti-CD40L-treated mice in a setting of rat to mouse concordant islet xenotransplantation.

Molecular cloning and sequences of interleukin-10 in the Djungarian (Phodopus sungorus), Chinese (Cricetulus griseus), and Syrian (Mesocricetus auratus) hamster.

Interleukin 10 (IL-10) genes of Djungarian, Chinese, and Syrian hamsters were cloned. The clones of IL-10 consisted of 537 bp nucleotides and 178 amino acids in full length, and the nucleotide and amino acid sequences exhibited a high degree of homology with those of the mouse and human. Since the number and position of signal sequences, N-glycosylations and cysteine sites in the IL-10 amino acid sequences of the hamsters were the same as those of the mouse, we suggest that the IL-10 molecular structures of the hamster are closer to that of the mouse than human.

Food restriction in pregnant rat-like hamsters (Cricetulus triton) affects endocrine, immune function and odor attractiveness of male offspring.

We studied the effect of intrauterine food restriction (FR) on the immune function, endocrine status and attractiveness of scents of male rat-like hamsters, Cricetulus triton. Work was conducted on field-caught parents from the North China plain and their laboratory-born progeny. Restricted pregnant dams were fed 70% of the mean daily intake of hamsters with free access to food. FR caused a marked and protracted weight reduction of the body, adrenal, testes and epididymides in males. During the refeeding period, the spleen and thymus, but not the adrenal weight of the malnourished offspring caught up with that of the control after about 60 days. The present results demonstrated that estrous females preferred the odors of control males to that of FR males. Males whose mothers were food restricted during gestation had lower testosterone concentrations, immune responses and reproductive organ mass but had higher circulating cortisol than did the males in the control group. Thus, the effect of maternal FR may be an important cause in population regulation in the rat-like hamster. The testosterone level was positively correlated with immune function in rat-like hamsters, but the lower immunity was not suppressed by higher level of testosterone, as previously suggested. We also found a negative relationship between cortisol and immune function in the rat-like hamster.

A ligand for the murine NK activation receptor Ly-49D: activation of tolerized NK cells from beta 2-microglobulin-deficient mice.

Mouse NK cells express inhibitory NK receptors that recognize target cell MHC class I molecules and activation receptors that are less well defined. The Ly-49D activation receptor on C57BL/6 NK cells recognizes Chinese hamster ovary cells and triggers natural killing. In this study, we demonstrate that a Chinese hamster classical MHC class I molecule is the ligand for Ly-49D in a reporter gene assay system as well as in NK cell killing assays. Ly-49D recognizes the Chinese hamster class I molecule better when it is expressed with Chinese hamster beta(2)-microglobulin (beta(2)m) than murine beta(2)m. However, it is still controversial that Ly-49D recognizes H-2D(d), as we were unable to demonstrate the specificity previously reported. Using this one ligand-one receptor recognition system, function of an NK activation receptor was, for the first time, investigated in NK cells that are tolerized in beta(2)m-deficient mice. Surprisingly, Ly-49D-killing activity against ligand-expressing targets was observed with beta(2)m-deficient mouse NK cells, albeit reduced, even though "tolerized" function of Ly-49D was expected. These results indicate that Ly-49D specifically recognizes the Chinese hamster MHC class I molecule associated with Chinese hamster beta(2)m, and indicate that the Ly-49D NK cell activation receptor is not tolerized in beta(2)m deficiency.

Antibodies to heterologous proteins in hemophilia A patients receiving recombinant factor VIII (Recombinate).

As a consequence of the manufacturing process, trace quantities of Chinese hamster ovary cell protein, bovine serum albumin and murine immunoglobulin G are present in Recombinate recombinant human factor VIII (rhFVIII). The development of antibodies (Abs) to these heterologous proteins was evaluated during long-term rhFVIII therapy of hemophilia A in 68 previously treated and 73 previously untreated patients. Ab prevalence was also assessed in 157 non-hemophilic subjects. Abs against heterologous proteins could be detected in varying percentages of patients and non-hemophilic subjects. Abs arose in patients sporadically, and levels were typically low. There were no adverse events associated with development or presence of anti-heterologous protein Abs. These data indicate that sustained immune responses to trace levels of heterologous proteins are very infrequent during long-term rhFVIII therapy.

Recognition of CHO cells by inhibitory and activating Ly-49 receptors.

Upon ligand recognition, members of the murine Ly-49 receptor family can transmit inhibitory or activating signals that regulate NK cell function. Ly-49A, G, and D have been shown to recognize the murine class I molecule H-2Dd as a potential ligand. Recent studies also have demonstrated also that Ly-49D+ NK cells can lyse CHO cells, although the ligand responsible for this recognition was not identified. Because allorecognition by NK cells may be important in bone-marrow transplantation and because of the overlapping class I recognition by these receptors, recognition of CHO cells by Ly-49G and A was investigated. Our data suggest that Ly-49G and probably A transmit inhibitory signals in response to CHO cells. Receptor inhibition was assessed by examining NK lytic function, IFN-gamma secretion, and DAP12 phosphorylation in response to CHO cells by sorted subsets of Ly-49D vs. G B6 NK cells. Our results suggest that CHO cells may express a common ligand(s) that is capable of engaging Ly-49D, G, and possibly A in C576BL/6 NK cells. In addition to our findings that Ly-49 inhibitory receptors also recognize CHO cells, activating receptors other than Ly-49D are present in B6 mice that can lyse CHO cells.

Different membrane cofactor protein (CD46) isoforms protect transfected cells against antibody and complement mediated lysis.

The need for organ transplantation, especially of kidneys, exceeds the availability of human donors and the possibility of xenotransplantation from suitable animals is now being addressed. The immediate barrier to success is hyperacute graft rejection, resulting from naturally occurring xenoreactive antibodies and the activation of complement. It is proposed that the intensity of the hyperacute response can be reduced by providing additional regulatory molecules to limit activation of the complement cascade, initially as transfected gene products in cultured cells as an in vitro model and eventually as a transgene in potential donor animals, such as pigs. Limiting the activity of C3b reduces the production of the C3a, C4a and C5a anaphylotoxins, thus curtailing not only the immediate C3b-mediated lytic pathway but also the later effects of a cellular inflammatory response including endothelial and platelet cell activation. To develop and assess the first part of this strategy, we have transfected several cDNA's encoding isoforms of CD46 (membrane cofactor protein). At least four different CD46 isoforms are commonly expressed in almost all human cells, and we have compared two of these and a third form to determine if they mediate different functions. After transfection, CD46-expressing CHO-K1 cells were selected with methionine sulphoximine and identified using monoclonal antibodies. Transfectants with suitable CD46 expression were assayed for primary CD46 function using a lysis assay dependent on the reaction of antibody and complement. In this in vitro model of hyperacute rejection, normal human sera containing natural xenoreactive antibodies were shown to lyse CHO cells, but only in the presence of complement.(ABSTRACT TRUNCATED AT 250 WORDS)

The identification of mammalian centrosomal antigens using human autoimmune anticentrosome antisera.

Human autoimmune sera were screened for the presence of anticentrosome autoantibodies. Two high titer sera were identified that reacted with HeLa, CHO, and PtK2 centrosomes by immunofluorescence, although the fluorescent patterns that were obtained using the two antisera were separate and distinct. Serum obtained from patient IJ contained antibodies that reacted with epitopes present only in mitotic centrosomes; staining of interphase centrosomes was never detected uing IJ antiserum. Immunoblot analysis demonstrated that antibodies present in IJ antiserum reacted with a 190 kD spindle pole antigen. Immunofluorescent staining of cultured mammalian cells demonstrated that antibodies present in serum obtained from patient SPJ reacted with both interphase and mitotic centrosomes. Characterization of SPJ antiserum by immunoblotting demonstrated that antibodies present in the SPJ serum recognized proteins of Mrs of 39, 185, and 220 kD, although the possibility that the 185 kD polypeptide was a proteolytic breakdown product of the 220 kD protein has not been eliminated. Neither antiserum was able to inhibit microtubule nucleation from centrosomes in a lysed cell system in which pure 6S tubulin was added to permeabilized cells following pretreatment of the cells with either SPJ or IJ antiserum. These antisera should be useful probes for studying the biochemistry of the mammalian centrosome.

Production of Chinese hamster monoclonal antibody to a human cell-surface antigen using a hamster-human somatic cell hybrid as antigen.

Hybridomas producing monoclonal antibody (MAb) of predefined specificity were isolated from a Chinese hamster that had received injections of a Chinese hamster-human somatic cell hybrid. A standard method for the production of murine hybridomas was used to produce Chinese spleen lymphocyte X murine plasmacytoma hybridomas, and they were screened for complement-mediated cytotoxicity against a panel of Chinese hamster-human somatic cell hybrids and agglutination of human erythrocytes. Two hybridomas were established in tissue culture following limiting dilution cloning, and their reactivity with a panel of Chinese hamster-human somatic cell hybrids indicates that they are specific for the previously identified human cell-surface antigen a1. The Chinese hamster appears to respond preferentially to human antigens of Chinese hamster-human somatic cell hybrids, and it will serve as a useful tool for the production of MAb specific for human cell-surface antigens expressed in the many well-characterized Chinese hamster-human somatic cell hybrids available.