IgG Heavy Chain Research Articles

Metaphylactic effect of minerals on the immune response, biochemical variables and antioxidant status of newborn calves

The aim of this study was to evaluate whether injectable zinc and copper affect host immune responses and antioxidant status of newborn calves. For this study, 19 newborn calves were divided into two groups. The control group consisted of 10 animals; and the treated group consisted of nine animals that received copper edetate (Cu-ed) and zinc edetate (Zn-ed) subcutaneously at the first day of life at doses of 0.3mg/kg and 1.0mg/kg respectively. Blood and faecal samples were collected for laboratory analyses (seric biochemistry, proteinogram, antioxidant enzymes and parasitological examination) on days 10, 20 and 30 after birth. On day 10, treated animals showed increased levels of total proteins, as well as increased globulin levels compared to the control group, a finding probably related to the increase in ceruloplasmin and IgG heavy chain. Thirty days after mineral metaphylactic administration, IgG light chain and acid glycoprotein levels significantly increased in treated animals (p<.05). There were no significant differences between groups regarding the biochemical analyses (triglycerides, cholesterol, glucose and urea). On the other hand, the superoxide dismutase and catalase activities increased on day 10 after treatment. In the control group, eight animals showed severe diarrhoea and one died 8days after birth. Two animals from this group showed mild diarrhoea. Only three treated animals had severe diarrhoea, and six showed signs of mild diarrhoea. All animals that showed severe diarrhoea (control=8; treated=3) had hyperthermia (over 39.5°C), and therefore, antibiotic therapy was administered (sulfadiazine and trimethoprim) for five consecutive days. In summary, Zn-ed and Cu-ed decreased the frequency and intensity of diarrhoea, modulated superoxide dismutase (SOD) and catalase (CAT) enzymes and also heightened the immune responsiveness of newborn calves, suggesting a new approach to improve cattle performance and minimize the occurrence of diarrhoea.

Expression of immunoglobulin G in human podocytes, and its role in cell viability and adhesion.

Podocyte injury occurs during the initiation and development of numerous forms of glomerular disease, and antibodies targeting podocytes have become a biomarker for diagnosis and monitoring treatment response. Accumulating evidence has suggested that immunoglobulin (Ig) is expressed in non-B lineage cells, including epithelial cancer cells, myeloid cells and several types of normal cells. The main aim of the present study was to ascertain the expression of IgG in human podocytes and to determine its potential role in cellular bioactivity. The present study detected positive staining for IgG heavy chain (Igγ) and its subtype γ4, and the light chains κ and λ in the cytoplasm or on the membrane by immunofluorescence. In addition, positive bands were detected for Igγ, γ1, γ3, γ4, κ and λ in the lysates of a podocyte cell line by western blotting. Mass spectrometry confirmed IgG1 as an intact tetramer in the culture supernatant. Constant region transcripts of Igγ, γ1, γ3, γ4, κ and λ were identified by reverse transcription-polymerase chain reaction, and DNA sequencing of these transcripts revealed 96–99% similarity with Ig mRNAs in the National Center for Biotechnology Information database. Compared with the diverse gene rearrangements from B cell-derived Ig, podocyte-derived Ig exhibited conservative V(D)J patterns in the variable regions of Igγ and κ chains. Furthermore, the present study investigated the mechanism underlying IgG production in these cells by examining the expression of recombination activating gene (RAG)1, RAG2 and activation-induced cytidine deaminase. The expression levels of these proteins suggested that podocyte-derived Ig and traditional Ig may be generated in a similar manner. Furthermore, small interfering RNA-mediated downregulation of IgG expression reduced podocyte viability and adhesive capabilities. These findings suggested that IgG is expressed in podocytes and that this expression may be associated with podocyte function. Due to its potential biological and clinical significance, this phenomenon warrants further investigation.

An update on medium- and low-abundant blood plasma proteome of horse

The main objectives of the study were to: (1) deeply analyse the serum protein composition of Equus caballus, (2) assess the effectiveness of the high-abundant protein depletion and improve the concentration of medium- and low-abundant proteins. The analysis were performed on the blood plasma of three healthy part-Arabian mares. The implementation of two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation – time of flight mass spectrometry allowed us to establish a horse plasma proteome map. Serum proteins were resolved at pH 4 to 7, followed by 12% SDS-PAGE. As a result 136 spots were successfully identified, representing the products of 46 unique genes. Of these, 22 gene products have not been previously identified in horse serum/plasma samples using proteomic tools. Gene ontology analysis showed that almost 30% of all identified gene products belong to the coagulation and complement cascades. These results can undoubtedly serve as a useful and prospective prerequisite for the future analysis of horse plasma proteome changes in different physiological and pathophysiological conditions. The use of the medium- and low-abundant protein enrichment tool increased their abundance and allowed us to identify a higher number of protein gene products. The highest depletion efficiency was observed for the most abundant plasma proteins, that is albumin, IgG heavy chains and serotransferrin.

Protein-Protein Interaction Network Analysis of Salivary Proteomic Data in Oral Cancer Cases

Background:Oral cancer is a frequently encountered neoplasm of the head and neck region, being the eight most common type of human malignancy worldwide. Despite improvement in its control, morbidity and mortality rates have improved little in the past decades. Therefore, prevention and/or early detection are a high priority. Proteomics with network analysis have emerged as a powerful tool to identify important proteins associated with cancer development and progression that can be potential targets for early diagnosis. In the present study, network- based protein- protein interactions (PPI) for oral cancer were identified and then analyzed for use as key proteins/potential biomarkers.Material and Methods:Gene expression data in articles which focused on saliva proteomics of oral cancer were collected and 74 candidate genes or proteins were extracted. Related protein networks of differentially expressed proteins were explored and visualized using cytoscape software. Further PPI analysis was performed by Molecular Complex Detection (MCODE) and BiNGO methods.Results:Network analysis of genes/proteins related to oral cancer identified kininogen-1, angiotensinogen, annexin A1, IL-8, IgG heavy variable and constant chains, CRP, collagen alpha-1 and fibronectin as 9 hub-bottleneck proteins. In addition, based on clustering with the MCODE tool, vitronectin, collagen alpha-2, IL-8 and integrin alpha-v were established as 5 distinct seed proteins.Conclusion:A hub-bottleneck protein panel may offer a potential /candidate biomarker pattern for diagnosis and treatment of oral cancer disease. Further investigation and validation of these proteins are warranted.

Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein.

PurposeThe goal of this study was to purify and characterize Ebola virus glycoprotein (GP)-specific IgG antibodies from hybridoma clones.Materials and MethodsFor hybridoma production, mice were injected by intramuscular-electroporation with GP DNA vaccines, and boosted with GP vaccines. The spleen cells were used for producing GP-specific hybridoma. Enzyme-linked immunosorbent assay, Western blot assay, flow cytometry, and virus-neutralizing assay were used to test the ability of monoclonal IgG antibodies to recognize GP and neutralize Ebola virus.ResultsTwelve hybridomas, the cell supernatants of which displayed GP-binding activity by enzyme-linked immunosorbent assay and the presence of both IgG heavy and light chains by Western blot assay, were chosen as a possible IgG producer. Among these, five clones (C36-1, D11-3, D12-1, D34-2, and E140-2) were identified to secrete monoclonal IgG antibodies. When the monoclonal IgG antibodies from the 5 clones were tested for their antigen specificity, they recognized GP in an antigen-specific and IgG dose-dependent manner. They remained reactive to GP at the lowest tested concentrations (1.953–7.8 ng/mL). In particular, IgG antibodies from clones D11-3, D12-1, and E140-2 recognized the native forms of GP expressed on the cell surface. These antibodies were identified as IgG1, IgG2a, or IgG2b kappa types and appeared to recognize the native forms of GP, but not the denatured forms of GP, as determined by Western blot assay. Despite their GP-binding activity, none of the IgG antibodies neutralized Ebola virus infection in vitro, suggesting that these antibodies are unable to neutralize Ebola virus infection.ConclusionThis study shows that the purified IgG antibodies from 5 clones (C36-1, D11-3, D12-1, D34-2, and E140-2) possess GP-binding activity but not Ebola virus-neutralizing activity.

Glycobiology of Aging.

Glycosylation is one of the most frequent post-translational modification of proteins. Many membrane and secreted proteins are decorated by sugar chains mainly linked to asparagine (N-linked) or to serine or threonine (O-linked). The biosynthesis of the sugar chains is mainly controlled by the activity of their biosynthetic enzymes: the glycosyltransferases. Glycosylation plays multiple roles, including the fine regulation of the biological activity of glycoproteins. Inflammaging is a chronic low grade inflammatory status associated with aging, probably caused by the continuous exposure of the immune system to inflammatory stimuli of endogenous and exogenous origin. The aging-associated glycosylation changes often resemble those observed in inflammatory conditions. One of the most reproducible markers of calendar and biological aging is the presence of N-glycans lacking terminal galactose residues linked to Asn297 of IgG heavy chains (IgG-G0). Although the mechanism(s) generating IgG-G0 remain unclear, their presence in a variety of inflammatory conditions suggests a link with inflammaging. In addition, these aberrantly glycosylated IgG can exert a pro-inflammatory effect through different mechanisms, triggering a self-fueling inflammatory loop. A strong association with aging has been documented also for the plasmatic forms of glycosyltrasferases B4GALT1 and ST6GAL1, although their role in the extracellular glycosylation of antibodies does not appear likely. Siglecs, are a group of sialic acid binding mammalian lectins which mainly act as inhibitory receptors on the surface of immune cells. In general activity of Siglecs appears to be associated with long life, probably because of their ability to restrain aging-associated inflammation.

A Case of Immunotactoid Glomerulopathy (ITG) Associated with Monoclonal B-Cell Lymphocytosis (MBL) with Chronic Lymphocytic Leukemia (CLL) Phenotype Treated with Obinutuzumab

Abstract Introduction: Although 44 to 90% of CLL cases have kidney infiltration on autopsy, renal involvement generally goes undetected and renal failure is unusual (Da'As N et al, Eur J Haematol 2001). There is no standard therapy for nephrotic syndrome (NS) in CLL. Treatment of CLL itself may result in regression of NS in most patients and treatment with alkylating agents, cyclosporine A, fludarabine and even splenectomy have been shown to be effective. Controversy exists regarding effectiveness of rituximab in CLL-associated nephrotic syndrome. (Yahata et al, Am J Nephrol 2000). Here we report the first case, to our knowledge, of MBL/CLL-associated NS successfully treated with obinutuzumab. Case Report: 71 years old female presented to ER with hypertensive urgency and anasarca. Past medical history was relevant for progressive hypertension (HTN) for 1 year prior. Creatinine (Cr) was 1.9mg/dL, BUN 45mg/dL, urine protein was >300mg/dl on urinalysis and subsequent 24hr urine protein was 9824mg/24hrs. Patient (pt) had renal ultrasound revealing morphologically normal kidneys without evidence of renal artery stenosis. Echocardiogram was normal. CBC revealed anemia (Hgb 9.2mg/dl) and mild thrombocytopenia (plt 121), WBC count was 7.6 x 10e3/mcl with relative lymphocytosis (47.46 %, ALC 3.594 x10e3 /mcl). On left kidney core biopsy 25 glomeruli were seen by light microscopy, 1 of which was globally sclerotic. The remaining glomeruli showed diffuse duplication of basement membranes with cellular interposition and mild to moderate intracapillary hypercellularity with endothelial cell swelling and/or leukocyte accumulation (Figure 1). Proximal tubules displayed patchy degenerative and regenerative changes including loss of apical brush border, nuclear enlargement and prominent nucleoli, and few interstitial foam cells (Figure 2). By immunofluorescence microscopy, glomeruli stained for IgG, C3, C1 and kappa light chain. There was no staining for lambda light chain. Stains for IgG heavy chain subtypes were positive for IgG1 only (Figure 3), consistent with monoclonal IgG1-kappa deposition. Electron microscopy revealed electron dense deposits in the mesangium, also in subendothelial and subepithelial locations in the capillary walls. On high-power magnification, these deposits revealed a microtubular substructure (i.e., with a central lumen) (Figure 4). These pathologic findings were consistent with ITG. Findings of ITG with IgG1 deposit as well as bicytopenia with relative lymphocytosis warranted further workup for gammopathy. SPEP revealed nonselective protein loss (total protein was 4.5g/dL, Albumin 2.9g/dL, total globulin 1.6g/dL, Albumin/Globulin ratio 1.8) with non-detectable M spike. UPEP was negative. IgG was 203mg/dL, IgA 45mg/dL, IgM 50mg/dL. Serum free kappa light chain was 35.79mg/L with normal Lambda light chain level of 20.08 mg/L with Kappa/Lambda ratio 1.78. Beta-2 microglobulin level was 6.4mg/L. Peripheral blood flow cytometry was ordered, which revealed 20% monoclonal B-cells (CD5+/CD23+) with CLL phenotype. Pt was started on prednisone 60mg daily for 7 days for ITG and decision was made to start CLL-targeted treatment with Obinutuzumab for 6 cycles, with the intent of improving the glomerulopathy, as pt did not meet IWCLL-2008 criteria for initiation of treatment for CLL. Obinutuzumab was given in the usual schedule of 100 mg on day 1, 900 mg on day 2 and 1000 mg on day 8 and 15 of first month, followed by 1000 mg every 28 days for 5 additional cycles. Repeat flow cytometry in 6 months after diagnosis showed complete response of monoclonal lymphocytosis (minimal residual disease negative). There was complete normalization of kidney function (Cr 0.73mg/dl and glomerular filtration rate 78ml/min) and no further evidence of proteinuria or edema. Hypertension has fully resolved, without additional need for anti-hypertensives. 30 months post treatment pt remains in remission for both MBL and nephrotic syndrome. Discussion: Treatment with obinutuzumab may lead to complete resolution and sustained remission of nephrotic syndrome associated with CLL. The more efficient B-cell depletion achieved with obinutuzumab may make this a better treatment choice for CLL associated NS than rituximab. Further evaluation with clinical trials is warranted. Download : Download high-res image (128KB) Download : Download full-size image Disclosures Skarbnik: Novartis: Speakers Bureau; Abbvie: Other: Ad board, Speakers Bureau; Genentech: Speakers Bureau; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau.

Generation and characterization of cross neutralizing human monoclonal antibody against 4 serotypes of dengue virus without enhancing activity.

BackgroundDengue disease is a leading cause of illness and death in the tropics and subtropics. Most severe cases occur among patients secondarily infected with a different dengue virus (DENV) serotype compared with that from the first infection, resulting in antibody-dependent enhancement activity (ADE). Our previous study generated the neutralizing human monoclonal antibody, D23-1B3B9 (B3B9), targeting the first domain II of E protein, which showed strong neutralizing activity (NT) against all four DENV serotypes. However, at sub-neutralizing concentrations, it showed ADE activity in vitro.MethodsIn this study, we constructed a new expression plasmid using the existing IgG heavy chain plasmid as a template for Fc modification at position N297Q by site-directed mutagenesis. The resulting plasmid was then co-transfected with a light chain plasmid to produce full recombinant IgG (rIgG) in mammalian cells (N297Q-B3B9). This rIgG was characterized for neutralizing and enhancing activity by using different FcγR bearing cells. To produce sufficient quantities of B3B9 rIgG for further characterization, CHO-K1 cells stably secreting N297Q-B3B9 rIgG were then established.ResultsThe generated N297Q-B3B9 rIgG which targets the conserved N-terminal fusion loop of DENV envelope protein showed the same cross-neutralizing activity to all four DENV serotypes as those of wild type rIgG. In both FcγRI- and RII-bearing THP-1 cells and FcγRII-bearing K562 cells, N297Q-B3B9 rIgG lacked ADE activity against all DENV serotypes at sub-neutralizing concentrations. Fortunately, the N297Q-B3B9 rIgG secreted from stable cells showed the same patterns of NT and ADE activities as those of the N297Q-B3B9 rIgG obtained from transient expression against DENV2. Thus, the CHO-K1 stably expressing N297Q-B3B9 HuMAb can be developed as high producer stable cells and used to produce sufficient amounts of antibody for further characterization as a promising dengue therapeutic candidate.DiscussionHuman monoclonal antibody, targeted to fusion loop of envelope domainII (EDII), was generated and showed cross-neutralizing activity to 4 serotypes of DENV, but did not cause any viral enhancement activity in vitro. This HuMAb could be further developed as therapeutic candidates.

Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

Proteomic analysis of influenza haemagglutinin-specific antibodies following vaccination reveals convergent immunoglobulin variable region signatures.

Analysis of the anti-haemagglutinin serum antibody proteome from six H1N1pdm09 influenza A vaccinated subjects demonstrated restricted IgG1 heavy chain species encoded by IGHV5-51 and IGHV3-7 gene families in 2 subjects and either IGHV5-51 or IGHV3-7 in 4 individuals. All subjects exhibited a dominant IGKV3-20 light chain, however 5 subjects also exhibited IGKV3-11 and IGKV4-1 families. Sequences were closely aligned with the matched germline sequence, with few shared mutations. This study illustrates the feasibility of using a proteomic approach to determine the expressed V region signatures of serum antibodies induced by vaccination.

Identification and verification of hybridoma-derived monoclonal antibody variable region sequences using recombinant DNA technology and mass spectrometry

Antibody engineering requires the identification of antigen binding domains or variable regions (VR) unique to each antibody. It is the VR that define the unique antigen binding properties and proper sequence identification is essential for functional evaluation and performance of recombinant antibodies (rAb). This determination can be achieved by sequence analysis of immunoglobulin (Ig) transcripts obtained from a monoclonal antibody (MAb) producing hybridoma and subsequent expression of a rAb. However the polyploidy nature of a hybridoma cell often results in the added expression of aberrant immunoglobulin-like transcripts or even production of anomalous antibodies which can confound production of rAb. An incorrect VR sequence will result in a non-functional rAb and de novo assembly of Ig primary structure without a sequence map is challenging. To address these problems, we have developed a methodology which combines: 1) selective PCR amplification of VR from both the heavy and light chain IgG from hybridoma, 2) molecular cloning and DNA sequence analysis and 3) tandem mass spectrometry (MS/MS) on enzyme digests obtained from the purified IgG. Peptide analysis proceeds by evaluating coverage of the predicted primary protein sequence provided by the initial DNA maps for the VR. This methodology serves to both identify and verify the primary structure of the MAb VR for production as rAb.

Probing Transient Release of Membrane-Sequestered Tyrosine-Based Signaling Motif by Solution NMR Spectroscopy.

Membrane sequestration of tyrosine-based signaling motifs of antigen receptors effectively restricts the signaling activities in resting lymphocytes. However, low level of basal signaling in resting cells is required for lymphocyte survival and antigen responsiveness, of which the molecular mechanism remains obscure. Here we probe the transient release of the cytoplasmic domain of the membrane-bound IgG heavy chain (mIgG-tail) by hydrogen exchange NMR spectroscopy, illustrating a dynamic molecular basis for its basal signaling activity. To solve the severe resonance overlap problem in the 2D spectra of mIgG-tail, a non-uniformly sampled pseudo-4D hydrogen exchange NMR experiment has been exploited to quantitatively measure site-specific hydrogen exchange rates. Our solution NMR study reveals transient solvent exposure of the ITT signaling motif that can be further enhanced by calcium ion, and provides insight into the mechanism of lymphocyte basal signaling.

Antibody and Antibody Mimetic Immunotherapeutics

Antibody and Antibody Mimetic Immunotherapeutics

Computational design of a specific heavy chain/κ light chain interface for expressing fully IgG bispecific antibodies.

The use of bispecific antibodies (BsAbs) to treat human diseases is on the rise. Increasingly complex and powerful therapeutic mechanisms made possible by BsAbs are spurring innovation of novel BsAb formats and methods for their production. The long-lived in vivo pharmacokinetics, optimal biophysical properties and potential effector functions of natural IgG monoclonal (and monospecific) antibodies has resulted in a push to generate fully IgG BsAb formats with the same quaternary structure as monoclonal IgGs. The production of fully IgG BsAbs is challenging because of the highly heterogeneous pairing of heavy chains (HCs) and light chains (LCs) when produced in mammalian cells with two IgG HCs and two LCs. A solution to the HC heterodimerization aspect of IgG BsAb production was first discovered two decades ago; however, addressing the LC mispairing issue has remained intractable until recently. Here, we use computational and rational engineering to develop novel designs to the HC/LC pairing issue, and particularly for κ LCs. Crystal structures of these designs highlight the interactions that provide HC/LC specificity. We produce and characterize multiple fully IgG BsAbs using these novel designs. We demonstrate the importance of specificity engineering in both the variable and constant domains to achieve robust HC/LC specificity within all the BsAbs. These solutions facilitate the production of fully IgG BsAbs for clinical use.

Abstract 1319: Epigenome-wide association study reveals differential DNA methylation consistent with progression of multiple myeloma

Abstract Purpose: Multiple myeloma (MM) is the second most common hematological malignancy in the US. It is characterized by a clonal expansion of plasma cells in the bone marrow and extramedullary sites and is preceded by two precursor conditions including monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM). Strong evidence suggests a germline and environmental etiology. However, efforts to characterize heritable changes in gene activity, such as DNA methylation, have not been widely reported. Methods: We examined epigenome-wide DNA methylation as markers of MGUS, SMM and MM in peripheral blood obtained from treatment-naïve European American cases with heavy-chain IgG or IgA MGUS (n=60), SMM (n=31) and MM (n=54) and age- and sex-matched controls (n=79) included from the University of Alabama at Birmingham, University of Chicago and the Mayo Clinic, Rochester [54.5% males; mean age, 64 years (range, 36 to 86)]. We quantified DNA methylation of over 450,000 CpG and non-CpG loci using the Infinium HumanMethylation450 array (Illumina). Differentially methylated positions were calculated using a general linear model framework adjusted for confounders and cellular heterogeneity. Results: A total of 6 CpGs were differentially methylated in MM cases compared to controls at a level of genome-wide statistical significance. MM was associated with hypomethylation at differentially methylated positions inside SBNO2 (P=3.37x10-10), WIZ (P=1.12x10-8), CA6 (P=4.29x10-8) and ADORA1 (P=3.68x10-8) as well as intergenic positions proximal to TNFRSF8 (Chr 1p36.22; P=2.24x10-9) and ENDOV (Chr 17q25.3; P=2.74x10-8). Each of these loci, with the exception of CA6 and ADORA1, were hypomethylated in each of the 3 plasma cell dyscrasia phenotypes including MGUS and SMM and MM cases compared to controls (P&amp;lt;0.03), albeit not at a level of genome-wide statistical significance. Conclusions: These preliminary findings suggest that differences in DNA methylation may contribute to altered risk of MM, as well as its precursor conditions, and may play a role in plasma cell dyscrasia progression as a consequence of heritable changes in gene activity due to past exposures. Replication in a large yet similarly well-characterized population is warranted. Citation Format: Stephen D. Gragg, Devin Absher, Xiangqin Cui, Christina Pillion, Richard Myers, Shaji Kumar, Luciano Costa, Brian Chiu, Celine Vachon, Elizabeth Brown. Epigenome-wide association study reveals differential DNA methylation consistent with progression of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1319. doi:10.1158/1538-7445.AM2017-1319

Karakterisasi Antibodi Poliklonal terhadap Aflatoksin M1

The aim of the research was characterized the polyclonal antibody at AFM for diagnostic reagens on track 1 and measure the levels of AFM in milk. The series of experiments are given a polyclonal antibody testing 1 dimetilaminobenzidin (DAB) by dot blot immunoassay (DBIA) so it will look brown color display on the positive control sample; purification and estimation antibody of AFM concentration with ammonium sulfate; 1 dialysis; fractionation using HiTrap Protein A HP column, measured using a spectrophotometer at a wavelength of 280 nm; and characterized of IgG using SDS-PAGE. DBIA test results showed a typical reaction betweenantigen AFM1-BSA with AFM1-BSA antibody to rabbit serum in the form of brown dots after addition of DAB substrate. The results of spectrophotometric against rabbit serum fractionation showed the type of IgG heavy chain.

An Efficient Transient Expression System for Enhancing the Generation of Monoclonal Antibodies in 293 Suspension Cells.

Recombinant monoclonal antibodies (mAbs) are useful in research, diagnosis, and therapy. The increased demands of recombinant mAbs require efficient production systems. A variety of expression vectors have been developed for stable or transient production of mAbs in mammalian cells. Although a few commercial expression systems of mAbs can be listed, the high expense often impedes academic research. In this study, we described the development of a transient mammalian system based on a bicistronic vector, which contained an internal ribosome entry site (IRES) and enhancer elements to express the IgG1 light chain (LC) and heavy chain (HC) in one transcript. Optimization of all components of the expression system, including gene transfer methods and regulatory elements, yielded maximal expression levels in serum-free 293 suspension cells (Freestyle 293-F). This method enabled consistent production of the anti-programmed cell death protein 1 (PD-1) mAb up to 300 mg/L in less than one week under transiently transfected conditions. Furthermore, purified anti-PD-1 IgGs showed specific affinity to the target antigen human PD-1 and PHA-stimulated human T cells. The simplicity of the procedure made it suitable for the fast and high-yield production of IgG antibodies in small scales, which expedited the screening of a large number of recombinant candidates.

Novel antibody-like single-chain TCR antibody Fc fusion protein

Abstract We have previously reported the construction of a fusion protein composed of a soluble single-chain T cell receptor genetically linked to the constant domain of the human IgG1 heavy chain (TCR-Ig). The antigen recognition portion of the protein binds to an unmutated peptide derived from human p53 (amino acids 264–272) presented in the context of HLA-A2.1, whereas the IgG1 Fc provides effector functions. The protein is capable of forming dimers, specifically staining tumor cells, and promoting target and effector cell conjugation. The protein also has potent antitumor effects against p53+/HLA-A2.1+ human tumor xenografts in athymic nude mice and can mediate cell killing by antibody-dependent cellular cytotoxicity. Therefore, TCR-Ig behaves like an antibody, but possesses the ability to recognize antigens derived from intracellular targets. To test TCR-Ig in fully immunocompetent models, we have generated murine tumor cells stably expressing the human p53 epitope by constructing a single chain trimer composed of the human p53 peptide genetically linked to murine beta 2 microglobulin genetically linked to HLA-A2.1, with the inclusion of a disulfide trap to enhance stability. Utilizing haNK (NK-92 cells stably expressing high affinity Fc receptor and IL-2) as effector cells in the presence of TCR-Ig we demonstrated specific killing of murine single chain trimer expressing tumor cells. We are currently evaluating the antitumor activities and vaccinal effects of TCR-Ig in an HLA-A2.1 transgenic mouse model. TCR-Ig may represent a novel group of immunotherapeutics that has the potential to expand the range of tumors available for targeted therapies beyond those currently addressed by conventional antibody-based approaches.

The Challenges of Recombinant Endostatin in Clinical Application: Focus on the Different Expression Systems and Molecular Bioengineering

Angiogenesis plays an essential role in rapid growing and metastasis of the tumors. Inhibition of angiogenesis is a putative strategy for cancer therapy. Endostatin (Es) is an attractive anti-angiogenesis protein with some clinical application challenges including; short half-life, instability in serum and requirement to high dosage. Therefore, production of recombinant endostatin (rEs) is necessary in large scale. The production of rEs is difficult because of its structural properties and is high-cost. Therefore, this review focused on the different expression systems that involved in rEs production including; mammalian, baculovirus, yeast, and Escherichia coli (E. coli) expression systems. The evaluating of the results of different expression systems declared that none of the mentioned systems can be considered to be generally superior to the other. Meanwhile with considering the advantages and disadvantage of E. coli expression system compared with other systems beside the molecular properties of Es, E. coli expression system can be a preferred expression system for expressing of the Es in large scale. Also, the molecular bioengineering and sustained release formulations that lead to improving of its stability and bioactivity will be discussed. Point mutation (P125A) of Es, addition of RGD moiety or an additional zinc biding site to N-terminal of Es , fusing of Es to anti-HER2 IgG or heavy-chain of IgG, and finally loading of the endostar by PLGA and PEG- PLGA nanoparticles and gold nano-shell particles are the effective bioengineering methods to overcome to clinical changes of endostatin.

Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG2a, lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG1) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.