Multiplexed detection of agents affecting erythropoiesis (AAEs) and overall strategy for optimizing initial testing procedure.

Erythropoietin receptor agonists (ERAs) are drugs acting on the early erythropoietic stages developed to treat anemia and other erythropoiesis disease and are prohibited by the World Anti-Doping Agency (WADA). As an alternative to ERAs, a new drug, belonging to the transforming growth factor-b inhibitors family, was recently developed to treat diseases linked to ineffective erythropoiesis. This drug, named as Luspatercept (Reblozyl®), is acting on the later stages of erythropoiesis to promote erythrocytes. This drug might be used by cheating athletes either independently or in combination with ERAs. Indeed, it was shown that Luspatercept and recombinant erythropoietin (rEPO) can act synergistically to increase red blood cells production, potentially allowing the use of lower doses for an efficient effect. Our aim was to find a way to combine the detection of ERAs and Luspatercept without impacting the sensitivity and specificity of ERAs detection from the current techniques implemented in antidoping laboratories and to reduce the time of analysis and total sample volume needed. Magnetic beads coated with antibodies were preferred for IP of samples for its potential multiplexing. Then, the following steps of the method were selected considering that SAR/SDS-PAGE are the electrophoretic methods authorized for initial testing procedure by WADA and that biotinylated primary antibodies used for the immunodetection results in the best sensitivity and specificity and is time saving. The method developed in this work for the combined detection of agents affecting erythropoiesis (AAEs) showed specificity, sensitivity, and robustness and is easily and quickly implementable to all antidoping laboratories.

High-resolution mass spectrometry confirms the presence of a hydroxyproline (Hyp) post-translational modification in the GGGGP linker of an Fc-fusion protein.

Flexible and protease resistant (G4S)n linkers are used extensively in protein engineering to connect various protein domains. Recently, several groups have observed xylose-based O-glycosylation at linker Ser residues that yield unwanted heterogeneity and may affect product quality. Because of this, an engineering effort was implemented to explore different linker sequence constructs. Here, we demonstrate the presence of an unexpected hydroxylation of a prolyl residue in the linker, made possible through the use of high-resolution mass spectrometry (HR-MS) and MSn. The discovery started with the detection of a poorly resolved ∼+17 Da mass addition at the reduced protein chain level of an Fc-fusion construct by liquid chromatography-MS. Upon further investigation at the peptide level using HR-MS, the mass increase was determined to be +15.99 Da and was localized to the linker peptide SLSLSPGGGGGPAR [210-223]. This peptide corresponds to the C-terminus of Fc [210-216], the G4P linker [217-221], and first 2 amino acids of a growth factor [222-223]. The linker peptide was first subjected to MS2 with collision-induced dissociation (CID) activation. The fragmentation profile localized the modification to the GGGPA [218-222] portion of the peptide. Accurate mass measurement indicated that the modification is an addition of an oxygen and cannot be CH4, thus eliminating several possibilities such as Pro→Leu. However, other possibilities cannot be ruled out. Higher-energy collision-induced dissociation (HCD)-MS2 and MS3 using CID/CID were both unable to differentiate between Ala222→ Ser222 or Pro221→ Hyp221. Finally, MS3 using high-resolution CID/HCD confirmed the mass increase to be a Pro221→Hyp221 post-translational modification.

Optimizing production of Fc-amidated peptides by Chinese hamster ovary cells.

BACKGROUND: Amidation of the carboxyl terminal of many peptides is essential for full biological potency, often increasing receptor binding and stability. The single enzyme responsible for this reaction is peptidylglycine α-amidating monooxygenase (PAM: EC 1.14.17.3), a copper- and ascorbate-dependent Type I membrane protein. METHODS: To make large amounts of high molecular weight amidated product, Chinese hamster ovary (CHO) cells were engineered to express exogenous PAM. To vary access of the enzyme to its substrate, exogenous PAM was targeted to the endoplasmic reticulum, trans-Golgi network, endosomes and lysosomes or to the lumen of the secretory pathway. RESULTS: PAM was equally active when targeted to each intracellular location and assayed in homogenates. Immunocytochemical analyses of CHO cells and a pituitary cell line demonstrated that targeting of exogenous PAM was partially successful. PAM substrates generated by expressing peptidylglycine substrates (glucagon-like peptide 1-Gly, peptide YY-Gly and neuromedin U-Gly) fused to the C-terminus of immunoglobulin Fc in CHO cell lines producing targeted PAM. The extent of amidation of the Fc-peptides was determined by mass spectrometry and amidation-specific enzyme immunoassays. Amidation was inhibited by copper chelation, but was not enhanced by the addition of additional copper or ascorbate. CONCLUSIONS: Peptide amidation was increased over endogenous levels by exogenous PAM, and targeting PAM to the endoplasmic reticulum or trans-Golgi network increased peptide amidation compared to endogenous CHO PAM.

The novel multispecies Fc-specific Pseudomonas exotoxin A fusion protein α-Fc-ETA' enables screening of antibodies for immunotoxin development.

Immunoconjugates that deliver cytotoxic payloads to cancer cells represent a promising class of therapeutic agents which are intensively investigated in various clinical applications. Prerequisites for the generation of effective immunoconjugates are antibodies which efficiently deliver the respective cytotoxic payload. To facilitate the selection of human or mouse antibodies that display favorable characteristics as immunotoxins, we developed a novel Pseudomonas exotoxin A (ETA)-based screening protein. The α-Fc-ETA' consists of a multispecies-specific Fc-binding domain antibody genetically fused to a truncated ETA version (ETA'). α-Fc-ETA' non-covalently bound to human and mouse antibodies but did not form immune complexes with bovine immunoglobulins. In combination with antibodies harboring human or mouse Fc domains α-Fc-ETA' inhibited proliferation of antigen-expressing tumor cells. The cytotoxic effects were strictly antibody dependent and were observed with low α-Fc-ETA' concentrations. Mouse antibodies directed against CD7 and CD317/HM1.24 that previously had been used for the generation of functional recombinant immunotoxins, also showed activity in combination with α-Fc-ETA' by inhibiting growth of antigen-positive myeloma and leukemia cell lines. In contrast, α-kappa-ETA', a similarly designed human kappa light chain-specific fusion protein, was only specifically active in combination with antibodies containing a human kappa light chain. Thus, the novel α-Fc-ETA' fusion protein is broadly applicable in screening antibodies and Fc-containing antibody derivatives from different species to select for candidates with favorable characteristics for immunotoxin development.

Human IgG Fc-glycosylation profiling reveals associations with age, sex, female sex hormones and thyroid cancer.

IgG functions rely on interactions of the Fc region with other proteins, which are optimized by tailoring of a conserved N-linked glycosylation at Asn-297. We conducted a study involving 735 control individuals and 138 thyroid cancer patients. Here we demonstrated that previously described age-related change in Fc-glycosylation was further characterized by definite sex specificity. In females, the incidences of most of glycosylated forms began to pose characteristic changes at ages of puberty or menopause. In addition, glycan-glycan relationships existed extensively within Fc glycosylation, which were characterized to be altered upon different states of subjects, such as age, sex and thyroid cancer. In thyroid cancer patients, detailed comparison of glycosylation incidences with control individuals yielded insight into aberrant change in IgG(1) Fc-glycosylation. This aberrant pattern was also featured by remarkable specificities of both age and sex. The receiver operating characteristic curve analysis was used to determine diagnostic values of Fc glycosylation. Finally, clinical measurement of two major female sex hormones estradiol and progesterone was conducted to determine potential associations of hormones with IgG Fc glycosylation. This study provided an important view to the associations of IgG Fc N-linked glycosylation with age, sex, female sex hormones and thyroid cancer. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Local tolerance and stability up to 24 months of a new 20% proline-stabilized polyclonal immunoglobulin for subcutaneous administration.

Subcutaneous administration of human IgG is an alternative to intravenous replacement therapy that is associated with more stable serum IgG levels and fewer systemic adverse events. Highly concentrated IgG solutions are most convenient to minimize infusion volume, but their preparation and stability presents substantial technical difficulties. We report on the stability and local tolerance of IgPro20, an l-proline-stabilized, 20% polyvalent human IgG developed for subcutaneous administration. Stability was tested according to ICH guidelines. Local tolerance and vasoactivity were examined in rabbit and rat models, respectively. The presence of l-proline in IgPro20 reduced viscosity and addition of Polysorbate 80 and inert gassing improved the appearance of the solution. After storage at 25 °C for 24 months, monomer + dimer content, aggregates, and fragments were within specification (≥ 90.0%, ≤ 4.0%, and ≤ 10.0%, respectively), and Fc function and antibody activities were maintained. In rats, intravenous injection of IgPro20 produced mild and transient hypotension comparable to that seen with intravenous IgG products. Local tolerance of IgPro20 in rabbits was comparable to that of a marketed subcutaneous IgG, Beriglobin P. Functionality and quality of IgPro20 are maintained during storage at 25 °C for at least 24 months. The product is well tolerated as assessed in animal models.

Identification and inhibition of drug target interference in immunogenicity assays.

A well-designed anti-drug antibody (ADA) immunoassay is critical for appropriately monitoring the immunogenicity profile of a therapeutic protein during its development. AMG 386 is a peptide-Fc fusion protein that inhibits angiogenesis by preventing the interaction of angiopoietins with the Tie2 receptor. In bridging immunoassays for ADA, interference by the drug target, present in the assay sample, can result in false positive antibody detection. We used a statistical design-of-experiments approach to identify angiopoietin interference in bridging immunoassays of anti-AMG 386 antibodies. We also demonstrated that a high-affinity monoclonal antibody, directed against an epitope on angiopoietin that competes with AMG 386 binding, could inhibit the angiopoietin interference while preserving the detection of ADA. This report describes the development and validation of methodologies for evaluating and addressing drug target interference in bioanalytical assays that involve interactions between drug, ADA, immune complexes, and drug target.

Immunoglobulin cleavage by the streptococcal cysteine protease IdeS can be detected using protein G capture and mass spectrometry.

The immunoglobulin degrading enzyme of Streptococcus pyogenes, IdeS, is an unusual cysteine protease produced by group A streptococci for which the only known substrate is immunoglobulin G (IgG). To date, IdeS has not been found to cleave any of the known synthetic substrates that other cysteine proteases hydrolyse, thus making the development of an IdeS detection assay difficult. Furthermore, at high doses of substrate, product generation is inhibited potentially due to the need for a dimeric enzyme complex with IgG. In this study we have developed a mass spectral assay for IdeS activity based on the detection of an Mr approximately 25,300 Fc fragment that retains the ability to bind streptococcal protein G. Using this assay procedure, evidence for a multimeric enzyme-substrate complex was obtained as well as identifying isolated heavy chains as a non-substrate inhibitor of IdeS activity. Under appropriate experimental conditions the assay could be used to detect IdeS activity in bacterial culture media or in human plasma without a requirement for purified reactants. The availability of a rapid and sensitive assay for IdeS should facilitate the detailed biochemical characterization of this unusual bacterial cysteine protease.

Fc function of a new intravenous immunoglobulin product: IGIV 10% triple virally inactivated solution.

BACKGROUND AND OBJECTIVES: Baxter AG has developed a new liquid intravenous immunoglobulin product [Immune Globulin Intravenous (IGIV) 10%] using a new manufacturing procedure. A modified Cohn fractionation and ion exchange chromatography is used to produce an IgG solution with no alterations to the Fc region. Three dedicated virus reduction steps are included: solvent-detergent treatment, nanofiltration, and incubation at low pH and elevated temperature in final formulation. We applied the reference method of the European Pharmacopoeia (EP) together with a flow-cytometric binding assay for the evaluation of the Fc function of the new product. MATERIALS AND METHODS: The EP reference method was done as described in the EP. The flow-cytometric method measured binding of IgG to Fc receptors of human monocytic THP-1 cells after exclusion of apoptotic cells. RESULTS: Sixteen lots of the new product expressed Fc functions between 84% and 110% when analysed with the EP reference method and Fc-binding activities between 82% and 121% when determined by the flow-cytometric method. CONCLUSION: All tested lots of the new product demonstrated a high level of Fc activity and met the requirements of the EP for Fc function.

Gene expression analysis of the CD4+ T-cell clones derived from gingival tissues of periodontitis patients.

The function of T cells infiltrating periodontitis lesions is complex and has not been fully elucidated. Here, we established T-cell clones from the gingival tissues of periodontitis patients and examined their gene expression. A total of 57 and 101 T-cell clones were established by means of immobilized anti-CD3 antibody and IL-2 from gingival tissues and peripheral blood, respectively. The gingival T-cell clones were derived from three patients, and the peripheral blood T-cell clones from two of these patients and a further patient whose gingival T-cell clones were not established. Gingival tissues were also obtained from a further 19 periodontitis patients. The expression of cytokines and molecules related to both regulatory function and tissue destruction were examined by means of reverse-transcription polymerase chain reaction. All the gingival T-cell clones expressed mRNA for TGF-beta1, CTLA-4, and CD25, and all the T-cell clones from peripheral blood expressed IFN-gamma and TGF-beta1 mRNAs. Most but not all the T-cell clones from gingival tissues and peripheral blood expressed mRNA for IFN-gamma and, CD25 and CTLA-4, respectively. The frequency of T-cell clones and gingival tissues expressing FOXP3, a possible master gene for mouse CD4(+)CD25(+) regulatory T cells, was very high (97%, 93%, and 100% for gingival T-cell clones, peripheral blood T-cell clones, and gingival tissues, respectively). Whereas the frequency of IL-4-expressing T-cell clones was lower for gingival T-cell clones (70% vs. 87%), the frequency of the gingival T-cell clones expressing IL-10 and IL-17 was higher than peripheral blood T-cell clones (75% vs. 62% for IL-10, 51% vs. 11% for IL-17). A similar expression profile was observed for gingival T-cell clones compared with gingival tissue samples with the exception of IL-4 expression, where the frequency of positive samples was lower in the gingival tissues (70% vs. 11%). These results suggest that the individual T cells infiltrating gingival lesions can express mRNA for both Th1 and Th2 cytokines as well as regulatory cytokines simultaneously.

High-level expression of single-chain Fv-Fc fusion protein in serum and egg white of genetically manipulated chickens by using a retroviral vector.

We report here the generation of transgenic chickens using a retroviral vector for the production of recombinant proteins. It was found that the transgene expression was suppressed when a Moloney murine leukemia virus-based retroviral vector was injected into chicken embryos at the blastodermal stage. When a concentrated viral solution was injected into the heart of developing embryos after 50 to 60 h of incubation, transgene expression was observed throughout the embryo, including the gonads. For practical production, a retroviral vector encoding an expression cassette of antiprion single-chain Fv fused with the Fc region of human immunoglobulin G1 (scFv-Fc) was injected into chicken embryos. The birds that hatched stably produced scFv-Fc in their serum and eggs at high levels (approximately 5.6 mg/ml). We obtained transgenic progeny from a transgenic chicken generated with this procedure. The transgene was stably integrated into the chromosomes of transgenic progeny. The transgenic progeny also expressed scFv-Fc in the serum and eggs.

B cells control the migration of a subset of dendritic cells into B cell follicles via CXC chemokine ligand 13 in a lymphotoxin-dependent fashion.

Certain classes of dendritic cells (DCs) meet rare cognate Ag-specific T and B cells inside primary B cell follicles for the development of germinal centers. However, the mechanisms underlying this coordination are still undefined. Cysteine-rich (CR) domain of the mannose receptor (CR-Fc)(+) DCs are a newly discovered subset of DCs that migrate rapidly into the primary lymphoid follicles from marginal zone after immunization. In this work, we uncover the key role of B cells in the establishment of a microenvironment that allows these DCs to be in the B cell area in a lymphotoxin (LT)-dependent fashion. CR-Fc(+) DCs are absent from the spleens of both LTbetaR- and LTalpha-deficient mice, suggesting that signaling by membrane LT is required for the presence of CR-Fc(+) DCs in the spleen. Interestingly, analysis of mutant mice that lack T, B, or NK cells demonstrates that B cell-derived membrane LT is essential for the unique localization of CR-Fc(+) DCs in the spleen. Using bone marrow transfer and ligand-blocking approaches, we provide evidence that B cell-derived LT acts indirectly on CR-Fc(+) DCs through LTbetaR(+) stromal cells. In analogous fashion to certain Ag-activated T and B cells, CR-Fc(+) DCs, expressing CXCR5, localize to primary lymphoid follicles in response to CXC ligand 13 (B lymphocyte chemoattractant). Together, we propose that B cells play a central role in establishing the chemotactic gradient that attracts not only Ag-activated T and B cells but also Ag-carrying CR-Fc(+) DCs. In turn, CR-Fc(+) DCs and T cells home to B cell follicles to interact with B cells in the developing germinal center.

Costimulatory molecules in human periodontal disease tissues.

An immunoperoxidase technique was used to examine CD28, CD152, CD80 and CD86 positive cells in gingival biopsies from 21 healthy/gingivitis and 26 periodontitis subjects. The samples were placed into 3 groups (small, intermediate, large) according to the size of the infiltrate. The percent CD28+ T cells in the connective tissue infiltrates was highly variable with no differences between the healthy/gingivitis and periodontitis groups. While there was an increase in positive cells in intermediate infiltrates from both healthy/gingivitis (28.5%) and periodontitis (21.4%) patients compared with small infiltrates (8.6% and 11.8%, respectively), this was not significant, although the percent CD28+ T cells did increase significantly in tissues with increased proportions of B cells relative to T cells (p=0.047). A mean of less than 5% infiltrating T cells were CD152+ which was significantly lower than the mean percent CD28+ T cells in intermediate healthy/gingivitis lesions (p = 0.021). The mean percent CD80+ and CD86+ B cells and macrophages was 1-7% and 8-16%, respectively, the difference being significant in intermediate healthy/gingivitis tissues (p = 0.012). Analysis of these cells in relation to increasing numbers of B cells in proportion to T cells and also to macrophages, suggested that CD80 was expressed predominantly by macrophages while CD86 was expressed by both macrophages and B cells. Few endothelial cells expressed CD80 or CD86. Keratinocytes displayed cytoplasmic staining of CD80 rather than CD86 although the numbers of positive specimens in the healthy/gingivitis and periodontitis groups reduced with increasing inflammation. In conclusion, percentages of CD28, CD152, CD80 and CD86 did not reflect differences in clinical status. However, the percent CD28+ T cells increased with increasing size of infiltrate and with increasing proportions of B cells suggesting increased T/B cell interactions with increasing inflammation. The percent CD152+ cells remained low indicating that CD152 may not be involved in negative regulation of T cells in periodontal disease. CD80 and CD86 have been reported to promote Th1 and Th2 responses, respectively, and the higher percent CD86+ cells suggests a predominance of Th2 responses in both healthy/gingivitis and periodontitis tissues. Nevertheless, other factors including cytokines themselves and chemokines which modulate T cell cytokine profiles must be monitored to determine the nature of Th1/Th2 responses in periodontal disease.

Chronic exposure to superantigen induces regulatory CD4(+) T cells with IL-10-mediated suppressive activity.

The repeated injection of bacterial superantigens (SAg), such as staphylococcus enterotoxin (SE) A or B, has been shown in mice to induce a state of unresponsiveness characterized by the lack of secretion of Th1 lymphokines, such as IL-2 and IFN-gamma, following subsequent SAg challenge. We made the observation, in vivo as well as in vitro, that unresponsiveness to SAg could be transferred from SEA- to SEB-reactive T cells (and reversibly from SEB- to SEA-specific T cells) in C57BL/6 mice but not in BALB/c mice. Since C57BL/6 mice, unlike BALB/c mice, possess TCR V(beta)3+ and V(beta)11+ T cells able to react with both SEA and SEB, we hypothesized that SAg-unresponsive V(beta)3(+) and V(beta)11+ T cells could mediate linked suppression of other SAg-reactive T cells. To analyze further this possibility, spleen cells from BALB/c mice made unresponsive to SEB were tested for their capacity to suppress the response of normal BALB/c cells to SEB. The production of both IFN-gamma and IL-2 following SEB stimulation was greatly impaired in co-cultures containing CD4(+) T cells, but not CD8(+) T cells, isolated from unresponsive animals. In vivo, the production of both IFN-gamma and IL-2 responses to SEB was dramatically reduced in animals adoptively transferred with unresponsive spleen cells. This suppression was abrogated in recipients injected with neutralizing anti-IL-10 antibodies. Moreover, in animals made unresponsive to SEB, SAg-reactive CD4(+) T cells were found to express high levels of CTLA-4, a molecule recently described to play an essential role in the suppressive function of regulatory T cells. Taken together these results demonstrate that the repetitive injection of SAg induces the differentiation of regulatory CD4(+) T cells capable of suppressing SAg-reactive naive T cells.

Expression of CD40 and its ligand, CD40L, in intestinal lesions of Crohn's disease.

OBJECTIVE: Selected mechanisms of the immune system participate in the development of inflammatory bowel disease. Recently, overexpression of the ligand for CD40 (CD40L), a lymphocyte costimulatory molecule, was shown to induce severe inflammatory bowel disease in transgenic mice. In the present study, we examined the expression of CD40 and CD40L on surgical specimens of ileum from 12 patients with Crohn's disease and 10 patients with diverticulitis. METHODS: Several CD40L+ cells were present in the affected tissue of patients with Crohn's disease, whereas few scattered CD40L+ cells were detected in sections of histologically normal ileum, resected distantly from the affected tissue, in patients with diverticulitis and in normal ileum portions obtained from colorectal cancer undergoing extensive surgery. The phenotype of CD40L+ cells was mainly CD4+. RESULTS: In patients with Crohn's disease, several CD40+ cells were detectable in the same areas of lymphocytes expressing CD40L, whereas in patients with diverticulitis, the number of CD40+ cells was significantly lower. Most of the CD40+ cells costained with CD20, thus showing to be B-lymphocytes, and only a few were CD14+ macrophages. Several von Willebrand-positive vessels were also positive for CD40. In addition, several infiltrating macrophages were found to express B7-1 and B7-2 molecules, the ligands of CD28 and CTLA-4, which cooperate with the CD40-CD40L pathway in lymphocyte activation. Staining of ileal lesions with anti-CTLA-4 antibodies resulted in detection of none or very few positive cells. In contrast, in patients with diverticulitis, an enhanced number of B7-1 and B7-2 and CTLA-4 was observed. CONCLUSION: The local accumulation of CD40L+ together with CD40+ cells within intestinal lesions of Crohn's disease suggests the involvement of this co-stimulatory pathway.

Effects of tumor necrosis factor and lymphotoxin on peripheral lymphoid tissue development.

Previously, we have reported that neutralization of surface lymphotoxin (LT-alphabeta) in mice which expressed an LT-beta receptor-Fc fusion protein, driven by the cytomegalovirus promoter, resulted in an array of anatomic abnormalities. We now report that mice which express a tumor necrosis factor (TNF) receptor p60-Fc fusion protein (which neutralizes TNF and soluble LT-alpha3 activity) develop unique lymphoid abnormalities. Our data demonstrate that some aspects of peripheral lymphoid organ development require both surface LT-alphabeta and TNF interacting with their specific receptors. However, these related cytokines are also capable of signaling distinct developmental events. Splenic MAdCAM-1 expression, follicular dendritic cell localization and normal Peyer's patch development all require both surface LT-alphabeta and TNF activity. Marginal zone formation and splenic B cell localization primarily require surface LT-alphabeta-LT-beta receptor interactions. Primary follicle formation was dependent upon TNF receptor(s) engagement. Interestingly spleen, lymph nodes and Peyer's patches from TNF receptor p60-Fc-expressing mice all develop different abnormalities, suggesting distinct pathways of development in these lymphoid organs. Thymus development appears to be independent of these signaling pathways. These results demonstrate that TNF and LT are crucial for normal peripheral, but not central lymphoid organ development.

Rapid detection of recombinant antibody fragments directed against cell-surface antigens by flow cytometry.

Cloning the correct genes coding for antibody variable domains (especially VL kappa) from hybridomas is often complicated by the presence of several immunoglobulin transcripts, some of them arising from the myeloma cell line. Indeed, four different VL genes were obtained after the amplification of immunoglobulin genes by PCR from the hybridoma HD37, which produces an antibody against the human CD19 B cell differentiation antigen. Most of the variants (eight out of 15) were derived from the kappa chain of the myeloma MOPC-21. For the rapid functional evaluation of recombinant antibody fragments against cell surface antigens, we established an efficient expression and detection system. First, deleted and mutated genes were eliminated by a colony screening procedure. Bacteria from picked colonies were then induced and grown in the presence of 0.4 M sucrose to increase the accumulation of soluble scFv in the periplasm (5-10 micrograms per ml of bacterial shake-tube culture). Finally, the cell-specific binding of scFv in crude periplasmic extracts was detected by flow cytometry. This procedure facilitated the efficient cloning of a functional anti-CD19 VH/VL combination from the hybridoma cDNA.

Mass spectrometry of the humanized monoclonal antibody CAMPATH 1H.

Two mass spectrometric techniques, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) have been used to study the intact humanized monoclonal antibody CAMPATH 1H, its fully and partially deglycosylated species, and 13 fragments prepared from it. The transformed ESI mass spectra of the glycosylated species gave complex patterns of molecular masses (M(r's). These have been substantially assigned to the presence of a mixture of glycoforms, each resulting from the combination of a single protein species with specific glycans of four distinct masses. The MALDI mass spectra of the glycosylated species, with the exception of that of the smallest fragment Fc/2, which indicated the presence of three of the glycans, gave single M(r) values comparable to the mean M(r) calculated from the ESI results. The M(r) values for the 10 prepared nonglycosylated species support the validity of the published amino acid sequence for the antibody and define the cleavage sites for the enzymic fragmentations. It is concluded that mass measurement of the Fc/2 fragment using ESI techniques provides a convenient means of preliminary assessment of the major glycosylated entities.

Thiophilic adsorption: rapid purification of F(ab)2 and Fc fragments of IgG1 antibodies from murine ascitic fluid.

A thiophilic adsorption method has been developed for rapid purification and separation of mouse F(ab)2 and Fc fragments obtained after proteolytic digestion of IgG1 monoclonal antibodies. Partially purified Mabs were digested with papain. Thiophilic chromatography was performed using stepwise elution with decreasing concentrations of ammonium sulphate. Most contaminating proteins did not react with the thiophilic adsorbent, and chromatography efficiently resolved the F(ab)2 and Fc fragments, as judged by electrophoresis. Fractions containing the F(ab)2 fragments retained about 90% of the total antibody activity loaded onto the column.

Purification and characterization of the Fas-ligand that induces apoptosis.

Fas is a 45-kD cell surface protein belonging to the tumor necrosis factor/nerve growth factor receptor family, and transduces the signal for apoptosis. The cytotoxic T lymphocyte (CTL) hybridoma, PC60-d10S requires the presence of Fas on target cells to induce cytolysis in target cells. This CTL cell line was weakly but specifically stained by a chimeric protein that consisted of the extracellular domain of mouse Fas and the Fc portion of human immunoglobulin G1 (mFas-Fc). Moreover, mFas-Fc inhibited the cytotoxic activity of PC60-d10S. Sublines of d10S that were stained intensively by mFas-Fc were isolated by repetitive fluorescence-activated cell sorter sorting. A cell-surface protein of about 40 kD was specifically precipitated by mFas-Fc from the lysates of these sublines. This protein was homogeneously purified by sequential affinity chromatographies using mFas-Fc and concanavalin A beads. The purified protein exhibited cytotoxic activity against cells expressing Fas but not to the cells which do not express Fas. These results indicated that the 40-kD membrane glycoprotein expressed on PC60-d10S cells is the Fas-ligand that induces the apoptotic signal by binding to Fas.