IgG Heavy Chain Research Articles

A fully caninised anti-NGF monoclonal antibody for pain relief in dogs

BackgroundMonoclonal antibodies are a major class of biological therapies in human medicine but have not yet been successfully applied to veterinary species. We have developed a novel approach, PETisation, to rapidly convert antibodies for use in veterinary species. As an example, anti-nerve growth factor (anti-NGF) monoclonal antibodies (mAbs) which are effective in reducing acute and chronic pain in rodents and man are potentially useful for treating pain in dogs but a fully caninised mAb is required in order to avoid an immune response. The aim of this study was to determine the optimal properties of a caninised anti-NGF mAb for safe, repeated administration to dogs, to determine its pharmacokinetic properties and to evaluate its efficacy in a model of inflammatory pain in vivo.ResultsStarting with a rat anti-NGF mAb, we used a novel algorithm based on expressed canine immunoglobulin sequences to design and characterise recombinant caninised anti-NGF mAbs. Construction with only 2 of the 4 canine IgG heavy chain isotypes (A and D) resulted in stable antibodies which bound and inhibited NGF with high-affinity and potency but did not bind complement C1q or the high-affinity Fc receptor gamma R1 (CD64). One of the mAbs (NV-01) was selected for scale-up manufacture, purification and pre-clinical evaluation. When administered to dogs, NV-01 was well tolerated, had a long serum half-life of 9 days, was not overtly immunogenic following repeated dosing in the dog and reduced signs of lameness in a kaolin model of inflammatory pain.ConclusionsThe combination of stability, high affinity and potency, no effector activity and long half-life, combined with safety and activity in the model of inflammatory pain in vivo suggests that further development of the caninised anti-NGF mAb NV-01 as a therapeutic agent for the treatment of chronic pain in dogs is warranted.

LIM Kinase 1 (LIMK1) Interacts with Tropomyosin-related Kinase B (TrkB) and Mediates Brain-derived Neurotrophic Factor (BDNF)-induced Axonal Elongation

BDNF/TrkB signaling plays critical roles in axonal outgrowth of neurons, the process of which requires the remodeling of the cytoskeleton structure, including microtubules and filamentous actin. However, the mechanism by which BDNF/TrkB signaling regulates cytoskeleton reorganization is still unclear. Here, we identified a novel interaction between LIMK1 and TrkB, which is required for the BDNF-induced axonal elongation. We demonstrated that BDNF-induced TrkB dimerization led to LIMK1 dimerization and transphosphorylation independent of TrkB kinase activity, which could further enhance the activation and stabilization of LIMK1. Moreover, activated LIMK1 translocated to the membrane fraction and phosphorylated its substrate cofilin, thus promoting actin polymerization and axonal elongation. Our findings provided evidence of a novel mechanism for the BDNF-mediated signal transduction leading to axonal elongation.

B cell exchange across the blood-brain barrier in multiple sclerosis

In multiple sclerosis (MS) pathogenic B cells likely act on both sides of the blood-brain barrier (BBB). However, it is unclear whether antigen-experienced B cells are shared between the CNS and the peripheral blood (PB) compartments. We applied deep repertoire sequencing of IgG heavy chain variable region genes (IgG-VH) in paired cerebrospinal fluid and PB samples from patients with MS and other neurological diseases to identify related B cells that are common to both compartments. For the first time to our knowledge, we found that a restricted pool of clonally related B cells participated in robust bidirectional exchange across the BBB. Some clusters of related IgG-VH appeared to have undergone active diversification primarily in the CNS, while others have undergone active diversification in the periphery or in both compartments in parallel. B cells are strong candidates for autoimmune effector cells in MS, and these findings suggest that CNS-directed autoimmunity may be triggered and supported on both sides of the BBB. These data also provide a powerful approach to identify and monitor B cells in the PB that correspond to clonally amplified populations in the CNS in MS and other inflammatory states.

Cleavage of IgG1 in gingival crevicular fluid is associated with the presence of Porphyromonas gingivalis

Immunoglobulin (Ig) G1 plays an important role in the adaptive immune response. Kgp, a lysine-specific cysteine protease from Porphyromonas gingivalis, specifically hydrolyses IgG1 heavy chains. The purpose of this study was to examine whether cleavage of IgG1 occurs in gingival crevicular fluid (GCF) in vivo, and whether there is any association with the presence of Porphyromonas gingivalis and other periodontopathogens. GCF was obtained from nine patients with aggressive periodontitis, nine with chronic periodontitis and five periodontally healthy individuals. The bacterial loads of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia were analysed by real-time polymerase chain reaction, and the presence and cleavage of IgG1 and IgG2 were determined using Western blotting. Kgp levels were measured by ELISA. Cleaved IgG1 was identified in the GCF from 67% of patients with aggressive periodontitis and in 44% of patients with chronic periodontitis. By contrast, no cleaved IgG1 was detectable in healthy controls. No degradation of IgG2 was detected in any of the samples, regardless of health status. Porphyromonas gingivalis was found in high numbers in all samples in which cleavage of IgG1 was detected (P < 0.001 compared with samples with no IgG cleavage). Furthermore, high numbers of Tannerella forsythia and Prevotella intermedia were also present in these samples. The level of Kgp in the GCF correlated with the load of Porphyromonas gingivalis (r = 0.425, P < 0.01). The presence of Kgp (range 0.07-10.98 ng/mL) was associated with proteolytic fragments of IgG1 (P < 0.001). However, cleaved IgG1 was also detected in samples with no detectable Kgp. In patients with periodontitis, cleavage of IgG1 occurs in vivo and may suppress antibody-dependent antibacterial activity in subgingival biofilms especially those colonized by Porphyromonas gingivalis.

The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus

In addition to sculpting eukaryotic transcripts by removing introns, pre-mRNA splicing greatly impacts protein composition of the emerging mRNP. The exon junction complex (EJC), deposited upstream of exon-exon junctions after splicing, is a major constituent of spliced mRNPs. Here, we report comprehensive analysis of the endogenous human EJC protein and RNA interactomes. We confirm that the major "canonical" EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions and that the majority of exon junctions carry an EJC. Unexpectedly, we find that endogenous EJCs multimerize with one another and with numerous SR proteins to form megadalton sized complexes in which SR proteins are super-stoichiometric to EJC core factors. This tight physical association may explain known functional parallels between EJCs and SR proteins. Further, their protection of long mRNA stretches from nuclease digestion suggests that endogenous EJCs and SR proteins cooperate to promote mRNA packaging and compaction.

Engineering of Sialylated Mucin-type O-Glycosylation in Plants

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.

A simple novel measure of passive transfer of maternal immunoglobulin is predictive of preweaning mortality in piglets

Two factors that contribute to preweaning mortality in piglets are the initiation of lactation by sows and their ability to nurse their piglets. The objective of this study was to determine if the quantification of the transfer of immunoglobulins (Igs) from sow to piglet could act as a measure of these sow factors in terms of their influence on preweaning mortality. To measure passive transfer, a simple, rapid Ig immunocrit method was developed. For validation, the smallest piglets from 204 gilts were sacrificed on day 1 after birth and blood was collected. Piglet serum Ig concentrations were measured three ways: (1) by protein A sepharose precipitation, SDS-PAGE, and densitometry of the IgG heavy chain; (2) by precipitation of Ig with (NH(4))(2)SO(4) followed by spectrophotometric measurement; and (3) by precipitation of Ig with (NH(4))(2)SO(4) and measurement of the ratio of precipitate to sample volume using a hematocrit microcapillary (the Ig immunocrit method). Results from the (NH(4))(2)SO(4) methods correlated (r=0.86) with those obtained using SDS-PAGE. Day 1 weights and immunocrit ratios and preweaning survival data were then collected from every piglet from first (n=90), and second (n=145) parity sows. Bodyweight and immunocrit ratios accounted for 4.8% and 16.7% (P<0.01) of the variation in preweaning mortality, respectively. Litter average immunocrit ratios were not correlated with preweaning mortality. In conclusion, the Ig immunocrit method is a simple, rapid measure of passive transfer from sow to piglet, and is useful in assessing the initiation of colostrum and the nursing ability of sows, and the effect of these events on preweaning piglet mortality.

A human-derived protein SBP (HBsAg-binding protein) can bind to hepatitis B virus surface antigen (HBsAg) and enhance the immune response to hepatitis B virus (HBV) vaccine

A high titer of antibody to HBsAg (Hepatitis B virus surface antigen) (anti-HBs) is a requisite for the prevention of HB (Hepatitis B), and adjuvants generally play a great role in eliciting special anti-HBs to HB vaccine. However, adjuvants still need to be improved because of their shortages such as unremarkable efficacy, undesirable side effect or poor security. In this study, we used HBsAg separated from HB patient sera to screen a human liver cDNA expression library, and found a novel HBsAg-binding protein (SBP), which is located at the human chromosome 14q32.33 and is similar to human IgG heavy chain in structure. Western blot demonstrated that SBP existed in both healthy human sera and HB patient sera. Furthermore, SBP could bind to HBsAg by its N-terminal domain. Notably, we confirmed that SBP could promote dendritic cells (DC) to phagocytize HBsAg more effectively and enhance the immunogenicity of HB vaccine, when SBP was mixed proportionally with HBsAg and the resulting mixture was infused into mice. These results suggest that SBP could be developed into a safe and promising adjuvant of HB vaccine.

Construction and development of a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma

We present a detailed method for constructing a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma. Two novel mammalian library vectors pcDNA3-CHm and pcDNA3-CLm were constructed that contained restriction enzyme sites NheI, ClaI and antibody constant domain. Mammalian expression vector pcDNA3-CHm contains IgG heavy-chain (HC) constant region and glycosylphosphatidylinositol anchor (GPI) that could be anchored full-length antibodies on the surface of mammalian cells. GOLPH2 prokaryotic expression vector was carried out in Escherichia coli and purified by immobilized metal affinity chromatography. Variable domain of heavy-chain and variable domain of light-chain genes were respectively inserted into the vector pcDNA3-CHm and pcDNA3-CLm by ligation, and antibody libraries are displayed as whole IgG molecules on the cell surface by co-transfecting this HC-GPI with a light chain. By screening the cell library using magnetic beads and cell ELISA, the cell clone that displayed GOLPH2-specific antibodies on cell surfaces was identified. The mammalian cell-based antibody display library is a great potential application for displaying full-length functional antibodies of targeting hepatocellular carcinoma on the surface of mammalian cells. Anti-GOLPH2 display antibody was successfully isolated from the library.

Acute phase protein profile in agoutis (Dasyprocta azarae; Lichtenstein, 1823) in captivity, determined by sodium dodecyl sulfate polyacrilamyde gel electrophoresis

The acute phase response refers to a nonspecific and complex systemic reaction of the organism that occurs shortly after any tissue injury. The acute phase response is considered a part of the innate host defense system, which is responsible for the survival of the host during the critical early stages of attack, and in evolutionary terms, it precedes the acquired immune response. The purpose of this study was to determine serum protein concentrations, including the acute phase protein profile in agoutis (Dasyprocta azarae) in captivity, by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Blood samples from 11 adult healthy animals (nine females and two males) were obtained. The serum proteinogram had 21 proteins with molecular weights ranging from 15 to 240 kD. The acute phase proteins identified were: ceruloplasmin, transferrin, albumin, haptoglobin, α-1-acid glycoprotein, and hemoglobin. IgA, IgG heavy and light chains, and nonnominal identified proteins of 240, 210, 140, 98, 78, 48, 35, 31, 23, and 15 kD were also identified. The determination of the acute phase protein concentrations is a useful method for the early detection of subclinical disease or changes in the healthy animal, with predictive information on the development of disease in the future. It is possible to standardize the reference values of the serum protein profile of agoutis, which can be used for diagnosis and prognosis, treatment and clinical follow-up of nutritional disorders, and immune-mediated inflammatory diseases that may affect these animals.

Serum IgG-Like Glycoferroprotein: Identifi cation of Its Final Dissociation Form of Thermostable Protein Coupled with Albumin

Human serum IgG-like glycoferroprotein identical to ascitic IgG-like glycoferroprotein that binds labeled monoclonal antibodies to CA125 is a complex consisting of three proteins: IgG, human serum albumin, and unidentified thermostable protein. Final dissociation form of serum IgG-like glycoferroprotein also appears as a complex of three nonidentical polypeptides with a molecular weight of 55 kDa (PC55) migrating in the albumin zone of thermostable protein coupled with albumin and structures chemically identical to human serum albumin and IgG heavy chains. Under denaturing conditions of electrophoresis in polyacrylamide gel, IgG-like glycoferroprotein and PC55 have the same molecular weight (about 55 kDa), while under reducing conditions their weight is about 75 kDa. Transition form (form the lower to the higher molecular weight) appears as an oblique (at about ≈ 30°) protein band creating a ladder string effect. Ladder string effect was reproduced with thermostable protein coupled with albumin, PC55, IgG-like glycoferroprotein, with all commercially available human and bovine albumins, rat albumin as well as with heated and renatured albumins and can serve as electrophoretic identification sign for thermostable protein coupled with albumin. Renatured after boiling (100°C for 15 min) bovine albumin under reducing conditions appeared as bow string twisted in helix, that raises molecule in 2.5 turns from ≈ 2 to ≈ 75 kDa. These data attest to the existence of an albumin double and to its possible double structure.

Abstract 1566: Inhibition of TGF-β in tumor-draining lymph nodes can enhance the potency of dendritic cell-based vaccine immunotherapy

Abstract Transforming growth factor-β (TGF-β), an immunosuppressive cytokine, produced in the tumor microenvironment will flow into tumor-draining lymph nodes where are primary priming sites for generation of anti-tumor immune responses, and play a critical role in suppressing anti-tumor immune responses. We focused on TGF-β-mediated immunosuppression in tumor draining lymph nodes, and examined whether local inhibition of TGF-β there could enhance the potency of dendritic cell (DC) -based vaccine immunotherapy in tumor-bearing mice. C57BL/6 mice bearing Lewis lung carcinoma (LLC1) subcutaneously were vaccinated with DCs loaded with LLC1-derived antigen. In order to inhibit TGF-β in tumor-draining lymph nodes, those mice were administered the plasmid DNA encoding the extracellular domain of TGF-β type II receptor fused to the human IgG heavy chain (TGFR DNA) near the established tumor intramuscularly. As results, the level of TGF-β in tumor-draining lymph nodes was decreased by administrations of TGFR DNA near the established tumor. In tumor-draining lymph nodes, the proliferative activity of FoxP3+ regulatory T cells was eliminated, whereas tumor antigen-specific CD8+ cells producing interferon-α were increased as compared with those in mice administered DC vaccine alone. In spleen, tumor antigen-specific cytotoxic as well as natural killer activity were augmented in mice administered DC vaccine with TGFR DNA. Subsequently, growth of the established tumor was suppressed effectively. In conclusion, inhibition of TGF-β-mediated immunosuppression in tumor-draining lymph nodes can significantly enhance the potency of DC vaccine. This animal model provides for a novel rationale with DC vaccine immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1566. doi:1538-7445.AM2012-1566

Abstract 1538: Enhanced antitumor effects of intratumoral (IT) hu14.18-IL2 immunocytokine (IC) compared to intravenous (IV) hu14.18-IL2 are distinguished by increased activated tumor infiltrating lymphocytes (TILs) and IC retention

Abstract Introduction: Hu14.18-IL2 (APN301, Apeiron Biologics) is an immunocytokine (IC) consisting of human IL2 linked to each IgG heavy chain of the hu14.18 mAb, which recognizes the GD2 disialoganglioside. Phase 2 clinical trials of IV hu14.18-IL2 IC in neuroblastoma and melanoma are underway, with activity already demonstrated in neuroblastoma. We have previously shown that intratumoral IC treatment (IT-IC) results in enhanced anti-tumor activity in mouse models. These studies were designed to determine the mechanisms involved in the enhanced activity and to provide justification for future clinical testing of localized IT administration of this and other immunocytokines. Methods: We characterize tumor growth, survival outcomes, histology and phenotype of tumor infiltrating lymphocytes (TILs) by flow cytometry of IT-hu.14.18-IL2 treatment of A/J mice bearing subcutaneous NXS2 neuroblastomas. Looking at these parameters, IT-IC treated mice are compared to IV-IC treated, IT PBS treated and untreated mice. Data: Mice receiving IT-IC show significant increases [by immunohistochemistry (IHC) and by flow cytometry] of NKp46+ Natural Killer (NK) cells and CD8a+ cytotoxic T cells in TIL populations compared to control tumor-bearing mice. Improved survival and inhibition of tumor growth are observed in mice receiving IT-IC vs. untreated mice or mice receiving IV-IC. Comparisons within treatment groups, or independent of treatment groups, show that the number of NK or CD8 T cells in the tumor correlates inversely with change in tumor size. Analyses by IHC and flow-cytometry show greater IC detection in tumor after IT-IC vs. IV-IC. Moreover, IT-IC results in improved IC retention in tumors and increased NKG2D effector receptors on intratumoral NKG2A/C/E cells and on CD8 T cells when compared to control mice or mice receiving IV-IC. The augmented NKG2D seen in TILs was not seen in spleen cells, supporting the localized nature of the intratumoral changes induced by this treatment. Conclusions: In this murine neuroblastoma model, enhanced antitumor effects of IT hu14.18-IL2 compared to IV hu14.18-IL2 are distinguished by increased activated TILs and IC retention, improved survival and inhibition of tumor growth. These observations suggest that localized administration of immunocytokines in human patients may show analogous advantages over IV administration. In accordance, we have designed a phase I/II clinical trial protocol investigating the maximum tolerated dose (MTD) and efficacy endpoints of intratumorally administered hu14.18-IL2 in stage III/IV melanoma patients with recurrent or refractory disease, and are working towards approval for its activation. Supported by R01-CA-32685-27. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1538. doi:1538-7445.AM2012-1538

Mass spectrometric detection of antigen-specific immunoglobulin peptides in paraneoplastic patient sera

Paraneoplastic neurological syndromes (PNS) are severe immune mediated effects of cancer. The presence of IgG autoantibodies against onconeural antigens in serum is a hallmark of the disease. Multiple paraneoplastic antibodies have been described, including antibodies against HuD, Yo, amphiphysin and CV2. In this study, we test the hypothesis that primary amino-acid structures of the antigen binding part of antibodies from various individuals share common sequences that are specific for each auto-antigen.We selected 60 patients with PNS, associated with antibodies against HuD, Yo, Amp or CV2. Affinity purified IgG was separated using SDS-PAGE and IgG heavy chains were excised, trypsinized and subjected to tandem mass spectrometry. We selected masses that uniquely identified a PNS autoantibody group, and used MS/MS fragmentation spectra to obtain information on peptide sequences. Out of 19,173 unique masses, 28 immunoglobulin-derived peptides were found exclusively in samples from a single autoantibody defined PNS group.Our results confirm that specific peptide structures exist in the antigen binding site of IgG that are shared between individuals harboring autoantibodies against the same onconeural antigen. Thus, the immune response in these patients followed converging paths during the rearrangement, selection and maturation of immunoglobulin sequences. The identified peptides can be applied in the diagnosis of PNS, but these data also indicate that a similar approach in a variety of other diseases involving an immune response would have an appealing outlook.

Induction of IgA Deposits and Glomerulonephritis by IgA Rheumatoid Factor

Induction of IgA Deposits and Glomerulonephritis by IgA Rheumatoid Factor

Plasma Half-life Extension of Small Recombinant Antibodies by Fusion to Immunoglobulin-binding Domains

Many therapeutic proteins possessing a small size are rapidly cleared from circulation. Half-life extension strategies have therefore become increasingly important to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Here, we performed a comparative analysis of the half-life extension properties of various bacterial immunoglobulin-binding domains (IgBDs) derived from Staphylococcus protein A (SpA), Streptococcus protein G (SpG), and Finegoldia (formerly Peptostreptococcus) protein L (PpL). These domains, composed of 50-60 amino acid residues, were fused to the C terminus of a single-chain Fv and a bispecific single-chain diabody, respectively. All fusion proteins were produced in mammalian cells and retained their antigen-binding properties. The half-lives of the antibody molecules were prolonged to varying extents for the different IgBDs. The strongest effects in mice were observed for domain C3 of SpG (SpG(C3)) followed by domains B and D of SpA, suggesting that SpG(C3) is particularly useful to extend the plasma half-life of small proteins.

Hydrolysis of Secreted Sialoglycoprotein Immunoglobulin A (IgA) in ex Vivo and Biochemical Models of Bacterial Vaginosis

Bacterial vaginosis (BV) is a common polymicrobial imbalance of the vaginal flora associated with a wide variety of obstetric and gynecologic complications including serious infections and preterm birth. As evidenced by high recurrence rates following treatment, interventions for BV are still lacking. Several hydrolytic activities, including glycosidases and proteases, have been previously correlated with BV and have been hypothesized to degrade host sialoglycoproteins that participate in mucosal immune functions. Sialidase activity is most predictive of BV status and correlates strongly with adverse health outcomes. Here we combine clinical specimens with biochemical approaches to investigate secretory immunoglobulin A (SIgA) as a substrate of BV-associated glycosidases and proteases. We show that BV clinical specimens hydrolyze sialic acid from SIgA, but not in the presence of the sialidase inhibitor dehydro-deoxy-sialic acid. The collective action of BV-associated glycosidases exposes underlying mannose residues of SIgA, most apparent on the heavily N-glycosylated secretory component of the antibody. Terminal sialic acid residues on SIgA protect underlying carbohydrate residues from exposure and hydrolysis by exoglycosidases (galactosidase and hexosaminidase). It is known that both IgG and SIgA are present in the human reproductive tract. We show that the IgG heavy chain is more susceptible to proteolysis than its IgA counterpart. Gentle partial deglycosylation of the SIgA secretory component enhanced susceptibility to proteolysis. Together, these data support a model of BV in which SIgA is subject to stepwise exodeglycosylation and enhanced proteolysis, likely compromising the ability of the reproductive mucosa to neutralize and eliminate pathogens.

Transient gene expression with CHO cells in conditioned medium: a study using TubeSpin®bioreactors

Background Transient gene expression (TGE) allows rapid protein production in mammalian cells and has become an important tool in the pharmaceutical product development pipeline [1]. Polyethylenimine (PEI)-mediated, high-density transfection allowed to express recombinant proteins at yields exceeding 1 g/L in only a few weeks [2]. Although highly efficient protocols are available, volumetric scale-up of TGE is still a challenge. A major issue is the need to perform the transfection in fresh medium rather than in conditioned (spent) medium. This implies a medium exchange step just before transfection. In CHO-DG44 [3] cells we observed up to a 100-fold decrease in volumetric protein production if transfections were performed in conditioned medium, compared to fresh medium. The reasons for such a negative effect of conditioned medium on transfectability and/or protein production expression are not yet known. To study this problem we transfected CHO cells at small-scale in TubeSpin bioreactor 50 tubes using 41 different commercially available serum-free media formulations in combination with different transfection parameters and culture conditions. By comparing the transient production of a recombinant IgG antibody among the different media, we observed variation of up to 400-fold when transfecting in fresh media and up to 20-fold when using conditioned medium. The optimization of the PEI:DNA ratio allowed a significant improvement in yields of transfection in conditioned medium. Methods Suspension-adapted CHO-DG44 cells [3] were routinely cultivated in TubeSpin bioreactor 50 tubes (TPP, Trasadingen, Switzerland) in ProCHO5 medium (Lonza, Vervier, Belgium) supplemented with 13.6 mg/L hypoxanthine, 3.9 mg/L thymidine, and 4 mM glutamine. The 38 media samples for transfection were provided by Excellgene SA. Conditioned medium is defined as a cell culture medium where cells have been growing for more than two days up to a density between 4-5 million cells/mL. Cell growth was accessed with the Packed Cell Volume method. The dual expression vector pXLGCHO-A3, containing the cDNAs coding for human anti-Rhesus D IgG1 heavy and light chain cloned in separate expression cassettes in a head-to-head orientation, was kindly provided by Excellgene SA [4]. Transfections are performed at a cell density of 5.5 million cells/mL at a volume of 5 mL by the direct addition of 15 μg of pDNA and 76 μg of linear 25 kDa Polyethylenimine (PEI, Polysciences, Eppenheim, Germany)

Extensive Bone Marrow Infiltration and Abnormal Free Light Chain Ratio Identifies Patients with Smoldering Myeloma At High Risk for Progression to Symptomatic Disease,

Abstract 3926Asymptomatic/smoldering multiple myeloma (SMM) is a proliferative plasma cell disorder characterized by a substantial risk of progression to symptomatic myeloma. According to current recommendations, patients with SMM should be followed without treatment until they develop symptomatic disease. However, the risk of progression to symptomatic myeloma varies between different series and for individual patients; thus, significant effort is needed in order to identify factors that could discriminate those who are at high risk for progression. Such patients should be followed closer and should be considered candidates for clinical trials. In order to evaluate previously recognized risk factors and study patterns of progression we analyzed our series of patients with SMM, who have been diagnosed and followed in the Department of Clinical Therapeutics in Athens, Greece.SMM was defined as serum monoclonal (M) protein (IgG or IgA) level of ≥3 g/dL and/or bone marrow plasma cells ≥10%, absence of end-organ damage, such as lytic bone lesions, anemia, hypercalcemia, or renal failure, that can attributed to a plasma cell proliferative disorder (IMWG criteria, Br J Haematol 2003;121:749–57). Progression to symptomatic myeloma was defined as per the IMWG proposed criteria.We analyzed 95 patients with SMM, 53% of whom were females, 70% had IgG heavy chain, 22% had IgA, 5% had a biclonal SMM and 3% had light chain only SMM, while 65% had a kappa light chain and 35% a lambda light chain. Median infiltration by clonal plasma cells in BM trephine biopsy was 20% (range 10–90%), 10% of patients had ≥60% clonal plasma cells in BM biopsy. Fifty patients had MRI of the spine at the time of diagnosis of SMM and 19.5% had an abnormal pattern of BM infiltration (diffuse, focal or variegated pattern). In patients with available bone marrow immunohistochemistry data, 61% had clonal plasma positive for CD56, 17% for CD20 and 19% for cyclin D1.The median follow up of the cohort was 27 months (range 1–253 months) and 23 (24%) patients have progressed to symptomatic myeloma. The one-year, 2-year and 3-year cumulative probability of progression was 7%, 12% and 20% respectively. Nine patients (9.5%) progressed within the first two years from the diagnosis of SMM. All these patients had an M-protein of ≥1 g/dl (10 g/L), 67% had bone marrow plasma cells >60% and 80% had an abnormal MRI pattern of BM infiltration. The 3-year probability of progression to symptomatic myeloma was 4%, 18% and 87% for patients with <20%, 20–59% and ≥60% clonal plasma cells in bone marrow biopsy (P<0.001). The 2-year probability of progression to symptomatic myeloma was 0%, 13% and 60% for patients with <20%, 20–59% and ≥60% clonal plasma cells in BM biopsy (P<0.001). Patients with significantly abnormal free light chain ratio (either kappa/lambda ≥8 or kappa/lambda ≤0.125, according to Dispenzieri et al, Blood 2008;111:785–9) had a 3-year probability of progression to symptomatic MM of 41% vs. 15% (p=0.07). There was no significant difference in the risk of progression to symptomatic MM for patients with IgA vs. IgG myeloma. In multivariate analysis, abnormal FLC ratio less than 0.125 or more than 8 (HR: 6.4, 95% CI 1.3–34.5 p=0.032) and BM clonal plasma cells infiltration ≥60% (HR: 23, 95% CI 5–125, p<0.001) were independent risk factors for progression to symptomatic myeloma.Progression to symptomatic MM was manifested by the development of anemia in 52% of patients who progressed to symptomatic MM, development of lytic bone lesions or pathologic fracture in 48%, an increase of serum creatinine to ≥2 mg/dl in 13%, development of a soft tissue plasmacytoma in 4% and development of hypercalcemia in 4%.In conclusion, in our series of patients the 3-year probability of progression to symptomatic myeloma is about 20%, but there is a subgroup of patients with extensive bone marrow infiltration (≥60%) and highly abnormal FLC ratio, who have a substantial risk of progression to symptomatic disease within the first two years from the diagnosis of SMM. These high risk patients may also have other features such as abnormal MRI of the spine. Patients at high risk for progression should be considered for clinical trials evaluating the role of treatment before the development of symptomatic disease, which in most cases is manifested with anemia and/or lytic bone disease or pathologic fractures. Disclosures:No relevant conflicts of interest to declare.

Proteasome Activity Not M-Protein Production Influences Multiple Myeloma (MM) Responsiveness to Bortezomib (BZB): The Key Role of PSMB5,

Abstract 3491M-protein load may sensitize MM cells to BZB but models thus far have not directly tested that hypothesis, in one case using a subclone of an IgGκ-producing human MM cell line differing 5X from the parent line with respect to intracellular M-protein concentration (Ca Res 2007;67:1783), and in another using MM cell lines differing in proteasome activity (Blood 2009;113:3040). In other models proteasome activity has been increased by exposure to low doses of bortezomib or by over-expression of PSMB5, the gene for the β5c subunit, reducing sensitivity to BZB (Blood 2008;112:2489). To examine more stringently these aspects of the load-capacity hypothesis with respect to responsiveness to BZB, we used ALMC1, MM1S and KMS12-BM MM cells. ALMC1 cells produce an intact IgGλ and excess amyloid-forming λ light chains and provided a target for M-protein IgGλ gene knockdown (KD). All 3 cell lines were used to evaluate the impact of KD of PSMB5 and PSMB8 expression on proteasome-directed lys48-ubiquitinated proteins, proteasome activity, caspase activation and dose-response to BZB.We achieved gene silencing with no apoptosis or loss of viability with optimized use of streptolysin-O permeabilization (J Immun Meth 2008;333:147). To decrease intracellular M-protein, we targeted the ALMC1 IgG heavy chain+λ light chain genes (IgGλ) and to reduce proteasome activity the PSMB5 or PSMB8 (β5i) genes (4 siRNA per gene with scr control; Dharmacon). To test for efficacy of KD we used RT-qPCR for IgGλ, PSMB5 and PSMB8. We optimized flow cytometry assays to test for intracellular M-protein levels with IgG Fc-receptor and λ light chain specific monoclonal antibodies, and to test for proteasome-directed ubiquitinated proteins with anti-ubiquitin lysine48-specific monoclonal antibodies. To test for proteasome activity we used the site-specific luminogenic peptide substrates Suc-LLVY-aminoluciferin (AML) (β5), Z-LRR-AML (β2) and Z-nLPnLD -AML (β1), and for caspase-3/7 activation a similar substrate containing the tetrapeptide DEVD, reagents optimized for cell lysis, protease or caspase and luciferase activity and read-out in relative luminescent units (RLU) (Promega). In 24-hour culture with BZB (0, 1.56, 3.125, 6.25, 12.5 and 25nM), we tested for viability and proliferation by MTT assay (Invitrogen). All KD experiments, and all luciferase and MTT assays (with 104 and 2.5×104 cells/well in triplicate for each situation), were conducted on at least 6 and 3 separate days respectively. Statistical analyses were performed with GraphPad PRISM.Twenty-four hours after KD, IgGλ, PSMB5 and PSMB8 mRNA were decreased by > 90% compared to scr controls. With KD of IgGλ the intracellular M-protein was reduced 10X but with KD of PSMB5 or PSMB8 the M-protein levels were unchanged as was β5 activity. KD of IgGλ, PSMB5 or PSMB8 had no impact on lys48-specific protein levels, or on proteasome or caspase 3/7 activity. Dose-response to BZB after KD of IgGλ or PSMB8 was no different than scr controls but after KD of PSMB5 the BZB dose-response curve was shifted significantly to the left (Figure 1). Among the 3 cell lines there were distinctive differences in baseline proteasome activity (KMS12-BM>ALMC1>MM1S), sensitivity to BZB (MM1S>ALMC1>KMS12-BM), and lys48-specific protein levels (MM1S>ALMC1>KMS12-BM). Results with KD of PSMB5 and PSMB8 in KMS12-BM and MM1S cells showed a similar left-shift of the BZB dose-response curves with KD of PSMB5 but not PSMB8. In addition, β5 and β1 activity was significantly reduced in all 3 cell lines when exposed to BZB after KD of PSMB5 but not PSMB8 (β1 activity is shown in Figure 2). [Display omitted] [Display omitted] These data indicate that proteasome activity, both baseline and post-BZB exposure, influence sensitivity to BZB and that acute reductions in intracellular M-protein levels do not. Results with KD of PSMB5, encoding the β5c subunit, highlight the key role of β5c particularly with respect to control of β1 activity either directly through a role in proteasome assembly and turnover or indirectly as a result of β5c substrates binding non-specifically within the proteasome and blocking the β1 site. Modulation of PSMB5 expression during BZB exposure, perhaps via interference with the TCF11 or Nrf2 feedback loops, may enhance MM cell sensitivity to BZB. Disclosures:No relevant conflicts of interest to declare.