IgG Heavy Chain Research Articles

Characteristics Peripheral Blood IgG and IgM Heavy Chain Complementarity Determining Region 3 Repertoire before and after Immunization with Recombinant HBV Vaccine.

Immunization with recombinant HBV vaccine induces specific immune responses in human causing B lymphocytes to produce protective HBsAb, and to form memory B lymphocytes, thereby facilitating HBV immunity in the body. However, B lymphocytes heterogeneity and characteristics are not fully elucidated. In this study, we conducted high-throughput sequencing of BCR heavy chain CDR3 repertoires in 3 healthy volunteers before and after the third immunization with recombinant HBV vaccine. We used Roche 454 Genome Sequencer FLX system to perform a comparative analysis of IgM and IgG H chain CDR3 repertoires. First, we found that the diversity of IgG H chain CDR3 repertoires was 1/6 of IgM on average. Moreover, after the third immunization with HBV vaccine, the diversity of IgG H chain CDR3 repertoires was 1/26 of IgM on average. Second, we detected relatively high levels of HBsAbs in all the healthy volunteers after immunization with HBV vaccine. The volunteers shared a small number of CDR3 sequences before and after immunization, and among each other. However, we did not find completely identical BCR H chain CDR3 amino acid sequences in these volunteers. Lastly, after immunization with recombinant HBV vaccine, the volunteers showed high frequency of IgG H chain CDR3 amino acid sequences mostly resulting from rearrangements of IGHV, IGHD and IGHJ, suggesting that the mechanism of high frequency CDR3 generation might be associated with the maturation of IgG affinity (somatic hypermutation) during the recombinant HBV vaccine-induced B lymphocyte responses. This study identified the characteristics and changes of BCR CDR3 repertoires before and after immunization with HBV vaccine, and evaluated the performance of the sequencing technology for this application. Our findings provide a basis for further research in B lymphocyte generated HBsAb heterogeneity and monitoring the maintenance of memory B lymphocytes.

A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis.

Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15–120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

Perfil eletroforético do colostro de ovelhas suplementadas com propileno glicol e cobalto associado à vitamina B12 no final da gestação

Resumo: Este trabalho teve por objetivo avaliar o proteinograma do colostro de ovelhas submetidas a administração de propileno glicol e de cobalto associado à vitamina B12 no final da gestação. Dezoito ovelhas da raça Santa Inês, prenhas e com idade variando entre 18 meses a cinco anos foram distribuídas, por amostragem probabilística em três grupos experimentais, aproximadamente 30 dias antes da data prevista para o parto. No Grupo 1 (G1/n=6) foram administrados 30mL de propileno glicol P.A. via oral diariamente; no Grupo 2 (G2/n=6) foi administrado 1mg de cloreto de cobalto em solução a 1% via oral diariamente e 2mg de vitamina B12, via intramuscular semanalmente e no Grupo 3 (G3/n=6): grupo controle. Logo após o parto procedeu-se a colheita de 30mL de colostro, que foram acondicionados em recipientes apropriados e encaminhados ao laboratório. Após homogeneização, adicionou-se a cada 1.000μL de colostro, 75μL de solução de renina, que foi mantido em banho-maria a 37ºC por aproximadamente 20 minutos e centrifugado a 21.000G durante dez minutos em centrífuga refrigerada. Posteriormente, a fração intermediária, correspondente ao soro do colostro, foi aliquotada e mantida em ultrafreezer a -80oC para posterior determinação das proteínas. A determinação da proteína total do soro colostral foi realizada empregando-se reagente comercial. A separação das proteínas foi realizada utilizando-se a técnica de eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE). Foram identificadas as proteínas IgA, lactoferrina, albumina, IgG de cadeia pesada (IgGCP), β-caseína, IgG de cadeia leve (IgGCL), β-lactoglobulina and α-lactoalbumina, não havendo influência da administração dos suplementos na fase final da gestação sobre as concentrações protéicas do colostro.

IgG Subclass Staining in Routine Renal Biopsy Material.

Immunofluorescence staining plays a vital role in nephropathology, but the panel of antibodies used has not changed for decades. Further classification of immunoglobulin (Ig)G-containing immune-type deposits with IgG subclass staining (IgG1, IgG2, IgG3, and IgG4) has been shown to be of diagnostic utility in glomerular diseases, but their value in the evaluation of renal biopsies has not been addressed systematically in large renal biopsy material. Between January 2007 and June 2014, using direct immunofluorescence, we stained every renal biopsy for the IgG subclasses if there was moderate to prominent glomerular IgG staining and/or IgG-predominant or IgG-codominant glomerular staining. The total number of biopsies stained was 1084, which included 367 cases of membranous glomerulonephritis, 307 cases of lupus nephritis, 74 cases of fibrillary glomerulonephritis, 53 cases of proliferative glomerulonephritis with monoclonal IgG deposits, and 25 cases of antiglomerular basement membrane disease, among others. We found that monoclonality of IgG deposits cannot always be reliably determined on the basis of kappa and lambda light chain staining alone, particularly if concomitant (frequently nonspecific) IgM staining is present. In IgG heavy and heavy and light chain deposition disease (3 cases), subclass staining is very helpful, and in proliferative glomerulonephritis with monoclonal IgG deposits subclass staining is necessary. IgG subclass staining is useful in differentiating primary from secondary membranous glomerulonephritis. In proliferative glomerulonephritis with polyclonal IgG deposition, IgG1 dominance/codominance with concomitant IgG3 and IgG2 but weak or absent IgG4 staining favors an underlying autoimmune disease. IgG subclass staining is a very useful diagnostic method in a selected cohort of renal biopsies, particularly in biopsies with glomerulonephritis with monoclonal IgG deposits.

Generation and Characterization of a Human/Mouse Chimeric GD2-Mimicking Anti-Idiotype Antibody Ganglidiximab for Active Immunotherapy against Neuroblastoma.

Vaccination with proteins mimicking GD2 that is highly expressed on neuroblastoma (NB) cells is a promising strategy in treatment of NB, a pediatric malignancy with poor prognosis. We previously showed efficacy of ganglidiomab in vivo, a murine anti-idiotype (anti-Id) IgG1. In order to tailor immune responses to variable regions, we generated a new human/mouse chimeric anti-Id antibody (Ab) ganglidiximab by replacing murine constant fragments with corresponding human IgG1 regions. DNA sequences encoding for variable regions of heavy (VH) and light chains (VL) were synthesized by RT-PCR from total RNA of ganglidiomab-producing hybridoma cells and further ligated into mammalian expression plasmids with coding sequences for constant regions of human IgG1 heavy and light chains, respectively. We established a stable production cell line using Chinese hamster ovarian (CHO) cells co-transfected with two expression plasmids driving the expression of either ganglidiximab heavy or light chain. After purification from supernatants, anti-idiotypic characteristics of ganglidiximab were demonstrated. Binding of ganglidiximab to anti-GD2 Abs of the 14.18 family as well as to NK-92tr cells expressing a GD2-specific chimeric antigen receptor (scFv(ch14.18)-zeta) was shown using standard ELISA and flow cytometry analysis, respectively. Ganglidiximab binding affinities to anti-GD2 Abs were further determined by surface plasmon resonance technique. Moreover, binding of anti-GD2 Abs to the nominal antigen GD2 as well as GD2-specific Ab-mediated cytotoxicity (ADCC, CDC) was competitively inhibited by ganglidiximab. Finally, ganglidiximab was successfully used as a protein vaccine in vivo to induce a GD2-specific humoral immune response. In summary, we report generation and characterization of a new human/mouse chimeric anti-Id Ab ganglidiximab for active immunotherapy against NB. This Ab may be useful to tailor immune responses to the paratope regions mimicking GD2 overexpressed in NB.

Glycosylation status of serum immunoglobulin G in patients with prostate diseases.

Occurrences of high values in patients with benign prostate disease and low values in patients with highly suspicious cancer have diminished the trustworthiness of prostate‐specific antigen as an early diagnostic marker of prostate cancer. In the search for other complimentary markers, we focused on serum IgG from patients with prostate diseases as well as normal subjects. IgG purified from the sera of normal control subjects and patients with prostate diseases, was digested with peptide N‐glycanase. Released glycans were quantified using MALDI‐time of flight mass spectrometry. We report that N‐linked (N‐acetylhexosamine)2(deoxyhexose)(mannose)3(N‐acetylglucosamine)2 was significantly increased in the IgG heavy chains of patients with prostate cancer compared with that of either benign prostatic disease patients or healthy subjects, whereas (hexose)(N‐acetylhexosamine)2(deoxyhexose)(mannose)3 (N‐acetylglucosamine)2 was more abundant in the heavy chains of healthy subjects and benign prostatic disease patients. Thus, an absence of the terminal hexose of N‐linked glycans has been closely connected to the progression of prostate cancer. Furthermore, surface plasmon resonance analyses have revealed that IgG from patients with prostate cancer has a decreased binding for Sambucus nigra lectin, compared with that from the benign prostatic disease patients or from normal subjects, suggesting lower levels of (N‐acetylneuraminic acid)(α2‐6)galactose/N‐acetylgalactosamine groups in the N‐linked glycans of patient IgG. Meanwhile, wheat germ agglutinin binding to IgG of the cancer group was significantly larger than that for the benign prostatic disease group but smaller than that for normal subjects. Our study indicates that the glycosylation changes in IgG can become useful diagnostic parameters for prostate cancer.

Reformatting Rituximab into Human IgG2 and IgG4 Isotypes Dramatically Improves Apoptosis Induction In Vitro.

The direct induction of cell death, or apoptosis, in target cells is one of the effector mechanisms for the anti CD20 antibody Rituximab. Here we provide evidence that Rituximab’s apoptotic ability is linked to the antibody IgG isotype. Reformatting Rituximab from the standard human IgG1 heavy chain into IgG2 or IgG4 boosted in vitro apoptosis induction in the Burkitt’s lymphoma B cell line Ramos five and four-fold respectively. The determinants for this behavior are located in the hinge region and CH1 domain of the heavy chain. By transplanting individual IgG2 or IgG4 specific amino acid residues onto otherwise IgG1 like backbones, thereby creating hybrid antibodies, the same enhancement of apoptosis induction could be achieved. The cysteines at position 131 of the CH1 domain and 219 in the hinge region, involved in IgG2 and IgG4 disulfide formation, were found to be of particular structural importance. Our data indicates that the hybrid antibodies possess a different CD20 binding mode than standard Rituximab, which appears to be key in enhancing apoptotic ability. The presented work opens up an interesting engineering route for enhancing the direct cytotoxic ability of therapeutic antibodies.

Chronic bacterial infection activates autoreactive B cells and induces isotype switching and autoantigen-driven mutations.

The links between infections and the development of B-cell-mediated autoimmune diseases are still unclear. In particular, it has been suggested that infection-induced stimulation of innate immune sensors can engage low affinity autoreactive B lymphocytes to mature and produce mutated IgG pathogenic autoantibodies. To test this hypothesis, we established a new knock-in mouse model in which autoreactive B cells could be committed to an affinity maturation process. We show that a chronic bacterial infection allows the activation of such B cells and the production of nonmutated IgM autoantibodies. Moreover, in the constitutive presence of their soluble antigen, some autoreactive clones are able to acquire a germinal center phenotype, to induce Aicda gene expression and to introduce somatic mutations in the IgG heavy chain variable region on amino acids forming direct contacts with the autoantigen. Paradoxically, only lower affinity variants are detected, which strongly suggests that higher affinity autoantibodies secreting B cells are counterselected. For the first time, we demonstrate in vivo that a noncross-reactive infectious agent can activate and induce autoreactive B cells to isotype switching and autoantigen-driven mutations, but on a nonautoimmune background, tolerance mechanisms prevent the formation of consequently dangerous autoimmunity.

IgG is involved in the migration and invasion of clear cell renal cell carcinoma

OBJECTIVE:To investigate if IgG can be expressed in clear cell renal cell carcinoma (cRCC) , and the expression of IgG is involved in the cancer progression. If IgG expression can...

An Improved Homologous Recombination Method for Rapid Cloning of the Antibody Heavy Chain Gene and Its Comparison with the Homologous Recombination and Traditional Cloning Methods

Background: The homologous recombination (HR) is one of the conventional cloning methods for the production of recombinant DNA. It is a quick method for in vivo DNA cloning without using the high cost restriction enzymes. A few modifications in the cloning procedure can increase the efficiency of this method. M aterials and Methods: In this study, effect of heating on the rate of the IgG1 heavy chain gene cloning was investigated in the HR method and then it was compared with HR method without heating and traditional cloning method. For doing this comparison, three cloning methods including HR, HR with the heat treatment, and traditional cloning were used to clone the human IgG1 heavy chain into the pcDNA3.1(+) plasmid. R es ults: The results showed that adding heat in the HR method converts insert and vector from the double strand DNA to the single strand DNA. This allows them to copulate with each other better and faster than the two other methods. The heat addition also helps the cell enzyme system for a faster and easier recombination and moreover it increases the cloning efficiency of the HR method in case of in vitro processing. Conclusion: The results showed that giving heat in the HR method increases cloning rate 7.5% and this increase reaches 10% in comparison with the traditional method.

Urine proteome analysis to evaluate protein biomarkers in children with autism

BackgroundAutism is a complex developmental disability for which no specific diagnostic markers have been identified so far. The present study aimed to evaluate whether there is any abnormal protein(s) excreted in the urine of autistic children by proteome analysis which may act as diagnostic marker. MethodsUrine proteome analysis was carried out in first void urine samples of autistic and normal children (n=30) in the age group of 4–12years by 2D-PAGE followed by MALDI–TOF–MS analysis. ResultsComparison of 2D-PAGE gels revealed that many urinary proteins are expressed differentially in autistic children. Total numbers of spots observed were 250 and 159 in autism and normal samples respectively, out of which 95 matches were observed. In addition, 3 spots of abnormally expressed peptides were selected, excised and analyzed. Peptide sequence with significant match score was for kininogen-1 (KNG-1)–50 (spot-1), IgG1 heavy chain variable region-35(spot-2) and mannan-binding lectin serine protease-2 isoform-2 precursor-45(spot-3). All the autistic children showed significant increase (p<0.001) in urinary kininogen level measured quantitatively by ELISA, when compared to normal children. ConclusionIncreased urinary kininogen-1 level in all the autistic children and the possibility of this protein as a diagnostic marker need further investigation.

Abstract SY35-01: The next generation of ADCs

Abstract The concept of an antibody-drug conjugate (ADC) is deceptively simple: a monoclonal antibody against a tumor-specific antigen delivers a potent cytotoxic payload directly to the malignant cells, resulting in targeted killing. However, this apparent simplicity belies a complex product with multiple interacting components. Working to optimize these components, we have identified three important concepts that impact the therapeutic potential of ADCs. First, we have learned that stability of an ADC can vary according to the specific conjugation site on the antibody. During the development of engineered cysteine antibodies (EC-mAbs), we evaluated various cysteine substitutions and found that S239C on the IgG heavy chain is particularly stable when conjugated using standard maleimide chemistry. Alternatively, we have devised a method for producing stable conjugation to native cysteine residues by designing maleimides that catalyze their own ring-opening after coupling to the antibody. These modifications in ADC stability appear to result in both improved activity and decreased toxicity in preclinical models. Second, we have observed that modulation of ADC hydrophobicity results in profound changes in both biodistribution and antitumor activity. When utilizing typical drug-linkers, ADCs with a high drug-to-antibody ratio (DAR &amp;gt;4) appear to be cleared from circulation more rapidly by reticuloendothelial cells. By reducing drug-linker hydrophobicity, we can circumvent this clearance pathway and it becomes possible to increase the DAR, resulting in more potent ADCs that retain desirable pharmacokinetic properties. We have achieved a similar result with the use of short, discrete PEG oligomers to mask the drug linker, thereby reducing the apparent hydrophobicity of the ADC. Finally, the chemical properties of the released payload have a major impact on both efficacy and toxicity. The difference between polar and nonpolar payloads will be discussed, including bystander activity, potential mechanisms of resistance, and adverse events observed in clinical trials. A new class of DNA crosslinking payloads is represented by the pyrrolobenzodiazepines (PBD) dimer, which is several logs more potent than auristatins and has the ability to overcome multi-drug resistance phenotypes. These examples demonstrate how rapidly the ADC field is evolving and provide hope that the next generation of ADCs will be even more effective and well-tolerated in the fight against cancer. Citation Format: Jonathan G. Drachman. The next generation of ADCs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY35-01. doi:10.1158/1538-7445.AM2015-SY35-01

Analysis of the Effect of β-Endorphin-Like Peptide Pentarphin on Intracellular Ca²⁺ Concentration in Early Mouse Embryos Using Fluorescent Probes.

At the early stages of mammalian embryo development, Ca(2+) influences the processes of cleavage and differentiation. An important role in these processes is played by peptide growth factors. One of them, pentarphin, is a peptide growth factor similar to the central part of β-endorphin molecule and a fragment 369-373 of IgG heavy chain. We used fluorescent Ca(2+) indicators Fluo-3 AM and Fura Red AM. Pentarphin added to embryos in a concentration of 0.1 μM 1.5-2 fold increased intercellular Ca(2+) level in comparison with the initial level.

Functional assessment of antibody oxidation by native mass spectrometry

Oxidation of methionine (Met) residues is one of several chemical degradation pathways for recombinant IgG1 antibodies. Studies using several methodologies have indicated that Met oxidation in the constant IgG1 domains affects in vitro interaction with human neonatal Fc (huFcRn) receptor, which is important for antibody half-life. Here, a completely new approach to investigating the effect of oxidative stress conditions has been applied. Quantitative ultra-performance liquid chromatography mass spectrometry (MS) peptide mapping, classical surface plasmon resonance and the recently developed FcRn column chromatography were combined with the new fast-growing approach of native MS as a near native state protein complex analysis in solution. Optimized mass spectrometric voltage and pressure conditions were applied to stabilize antibody/huFcRn receptor complexes in the gas phase for subsequent native MS experiments with oxidized IgG1 material. This approach demonstrated a linear correlation between quantitative native MS and IgG-FcRn functional analysis.In our study, oxidation of the heavy chain Met-265 resulted in a stepwise reduction of mAb3/huFcRn receptor complex formation. Remarkably, a quantitative effect of the heavy chain Met-265 oxidation on relative binding capacity was only detected for doubly oxidized IgG1, whereas IgG1 with only one oxidized heavy chain Met-265 was not found to significantly affect IgG1 binding to huFcRn. Thus, mono-oxidized IgG1 heavy chain Met-265 most likely does not represent a critical quality attribute for pharmacokinetics.

Effects of intramammary infection on whey proteinograms of sheep during lactation

The study aimed to identify potential biomarkers of mammary gland infection in Santa Inês sheep. Commercial flocks of sheep provided the same hygiene, sanitary, and nutritional management under semi-intensive production systems were monitored during the lactation stage-and assessed 15, 30, 60, and 90 days after delivery (through the end of lactation and weaning). The California Mastitis Test (CMT) was performed on the mammary glands. Milk was collected for bacterial examination and protein analysis. Bacterial culture and biochemical characterization of the samples were performed. Forty-two milk samples from healthy glands (negative CMT and bacterial testing) and 43 milk samples from infected glands (positive CMT and bacterial testing) taken at the predefined time points were assessed. A rennin solution was used to obtain the whey. The proteins analysis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which allowed for the quantification of nine whey proteins produced in healthy glands: serum albumin, lactoferrin, IgA, IgG heavy-chain (IgG HC), IgG light-chain (IgG LC), total IgG (IgG HC + IgG LC), α-lactalbumin, β-lactoglobulin, protein with MW 15.000 Da, protein with MW 29.000 Da and eleven whey proteins secreted by infected glands, including haptoglobin and α-1-acid glycoprotein. A comparison of whey proteins between healthy and infected glands showed increases (P&lt;0.05) in the secreted and total contents of all proteins, except for IgG LC and α-lactoalbumin. The most significant changes were observed in α-1-acid glycoprotein, lactoferrin and haptoglobin, which showed three-, five-, and seven-fold increases in secretion, respectively. This study showed that haptoglobin, α-1-acid glycoprotein, lactoferrin, albumin, and the IgA and IgG immunoglobulins may serve as potential biomarkers for mammary gland infection in sheep.

Rare Case of a Patient Presenting with Parathyroid Hormone-Related Peptide Mediated Hypercalcemia and IgG Heavy Chain Disease: Case Report and Review of Literature

IgG Heavy Chain Disease (γHCD) is a rare plasma cell disorder. Hypercalcemia related to plasma cell dyscrasias is related to non-PTHrP related mechanisms. Here we describe the first case of a patient with γHCD and PTHrP related hypercalcemia. Methods: Patient case derived from chart review from 2011 to 2015. Literature review performed searching PubMed 1968-current. Results: The patient was diagnosed with hypercalcemia with elevated PTHrP and exclusion of other etiologies of hypercalcemia. She was diagnosed with (γHCD) by M-spike 0.64 g/dL, IFE showing a broad band of IgG heavy chain, without associated light chains and severe depression of the non-mono-clonal IgG. Serum immunoglobulins demonstrated elevated IgG (2110 mg/dL), normal IgA (46 mg/dL) and decreased IgM (<21 mg/dL). Bone marrow biopsy showed 5% PCs, non-clonal by kappa/lambda, but exclusive for IgG by IHC, without any staining for IgA or IgM. The patient was started on therapy with improved hypercalcemia and PTHrP levels. Conclusions: This is the first reported case of γHCD presenting with PTHrP related hypercalcemia. Given that skeletal involvement is uncommon in γHCD, hypercalcemia secondary to γHCD may at times be a PTHrP driven phenomenon and we recommend that this test be ordered in such cases.

Proteomic Investigation on Grp94-IgG Complexes Circulating in Plasma of Type 1 Diabetic Subjects.

The glucose-regulated protein94 (Grp94) has been found in complexes with IgG in plasma of Type 1 (T1) diabetic subjects; however, the pathogenetic meaning of Grp94-IgG complexes has not yet been elucidated. To shed light on the nature and structure of these complexes in vivo, we conducted a proteomic analysis on plasma of both T1 diabetic subjects and healthy control subjects. IgG purified from plasma was submitted to 2D PAGE followed by Western blotting and mass analysis. Grp94 was detected in plasma of all diabetic but not control subjects and found linked with its N-terminus to the IgG heavy chain. Mass analysis of heavy chain of IgG that binds Grp94 also in vitro, forming stable complexes with characteristics similar to those of native ones, permitted identifying CH2 and CH3 regions as those involved in binding Grp94. At the electron microscopy, IgG from diabetic plasma appeared as fibrils of various lengthes and dimensions, suggestive of elevated aggregating tendency conferred to IgG by Grp94. The nonimmune nature of complexes turned out to be responsible for the particular stability and structure adopted by complexes in plasma of diabetic subjects. Results are of relevance to understanding the pathogenetic mechanisms underlying diabetes and its complications.

Pomalidomide and Dexamethasone Is an Effective Regimen for Advanced-Stage Relapsed/Refractory Multiple Myeloma: Experience of a Single Center

Introduction Almost all patients with MM eventually relapse, and the remission duration in relapsed MM decreases with each regimen. Pomalidomide is an IMiD with antiproliferative, anti-inflammatory and anti-angiogenic effects that was recently approved for the use of relapsed MM failing lenalidomide and bortezomib. Based on these findings, we aimed to evaluate the efficacy of pomalidomide and dexamethasone (PD) for heavily-pretreated relapsed or refractory MM (RRMM) at our Institution. Methods We retrospectively reviewed the records of all patients with RRMM treated with PD at Tom Baker Cancer Center between 01/10 and 06/14. Eligible patients were age 18 years or older; had RRMM after two or more prior therapies (including lenalidomide, bortezomib or thalidomide); and had an Eastern Cooperative Group performance status of 0 to 2. Patients received oral pomalidomide 2-4 mg/d on days 1-21, and dexamethasone 20 mg or 40 mg on a weekly basis. Definitions of response and progression were used according to the EBMT modified criteria. The primary endpoint of the study was to assess the efficacy and feasibility of PD in this group of patients. All analyses were performed using the SPSS 20.0 software and all p-values were 2-sided and statistically significant if <0.05 Results Between 01/10 and 06/14, 31 patients were identified for the study. Clinical and laboratory characteristics are listed in Table 1.The median age for this cohort of patients was 64 years (46-81). Seventy-one percent of patients had IgG isotype, 19.4% had IgA, and 9.7% had light chain only disease. The median number of therapies prior to PD was 4 (2-11). All patients received lenalidomide and bortezomib prior to PD; 11 patients had bortezomib, 8 had lenalidomide and 7 had both prior to PD. Six patients out of 11 receiving bortezomib prior to PD responded (54%) versus 12.5% (1/8), 14% (1/7), and 20% (1/5) for those receiving lenalidomide, lenalidomide/bortezomib and other regimens respectively prior to PD. (p=0.07) Two patients received pomalidomide at a dose of 3 mg, 1 at 2 mg and 28 at 4 mg. After a median of 5 cycles, the ORR was 29% (9/31) with 1 patient achieving nCR and 8 patients (25.8%) PR. (Table 2 ) The median time to first response was 8 weeks with majority of cases achieving at least PR after 2 cycles of therapy. Stable disease was seen in 22.6% and progression in 29% of cases. FISH cytogenetics at relapse were available in 24 patients with 6 cases exhibiting high risk disease (25%). At a median follow-up of 20 months, 16 patients (51.6%) are alive and 21 (67.7%) had already progressed. Median OS was 19.1 months and median PFS 5.6 months. Median PFS was 6.5 months in the group with SR cytogenetics compared to 3.1 months for the HR group. (p0.2) Median OS was similar between SR and HR (19.4 vs 23 months) (p=0.14) With regards to toxicity, 9 patients experienced grade 3/4 hematological toxicity. Eight patients required blood transfusion and one patient discontinued therapy due to grade 4 thrombocytopenia. In conclusion, Pomalidomide is an efficacious drug for the treatment of RRMM. The current report confirms the ORR seen in previous studies and the benefit in HR disease. Further combinations with pomalidomide in the setting of HR disease and for those patients who immediately progressed on IMiD therapies should be considered.Table 1Clinical and Laboratory characteristics for RRMM patients treated with Pomalidomide and DexamethasoneCharacteristicNMedianRange%Age (years)316446-81Gender-Male -Female14 1745.2% 54.8%ISS StageI II III5 14 1216.1% 45.2% 38.7%Heavy chainIgG IgA Free light chain only Light chainKappa Lambda22 6 3 25 671% 19.4% 9.7% <![if !supportEmptyParas]> <![endif]>80.6% 19.4%Hemoglobin (g/L)3111375-142Creatinine (µmol/L)318650-290Calcium (µmol/L)312.32.06-2.64LDH (IU/L)3118984-489B2-microglobulin (mg/L)314.21.36-16.3Albumin (g/L)313321-42BMPC (%)31308-98FISH CytogeneticsStandard risk High risk24 18 675% 25%Prior TherapiesASCT Thalidomide Lenalidomide Bortezomib Carfilzomib21 13 31 31 667.7% 41.9% 100% 100% 19.4%Table 2Response rates for RRMM patients treated with Pomalidomide and DexamethasoneCharacteristicMedian (Range)N%Number of cycles5 (1-31)Overall Response rate9/3129%Near Complete Response Complete Response1 03.2% 0%Very Good Partial Response00%Partial Response825.8%Stable Disease722.6%Minimal Response619.4%Progression929%Alive1651.6% DisclosuresJimenez Zepeda:Janssen Ortho: Honoraria. Bahlis:Celgene: Honoraria, Research Funding.

Overexpression of Serpinb1 in Chinese hamster ovary cells increases recombinant IgG productivity

We report the discovery and validation of a novel CHO cell engineering target for improving IgG expression, serpin peptidase inhibitor, clade B, member 1 (Serpinb1). Transcriptomic studies using microarrays revealed that Serpinb1 was up-regulated in cultures with IgG heavy and light chain transcription transiently repressed compared with cultures treated with non-targeting siRNA. As proof of concept, a lentiviral vector was employed to overexpress the Chinese Hamster Serpinb1 in a CHOZN® Glutamine Synthetase (−/−) recombinant IgG producing CHO line. The lentiviral stable pool demonstrated 4.2-fold SERPINB1 overexpression compared with the non-transduced control. The peak viable cell density (VCD) and peak IgG volumetric productivity of the lentiviral stable pool increased 1.3 and 2.0 fold, respectively, compared with the non-transduced control. For host cell engineering, a plasmid encoding SERPINB1 was transfected into the CHOZN® GS (−/−) host cell line to create several stable pools. Single-cell clones isolated from the pools were characterized for their SERPINB1 expression levels and growth. The clone (SERPINB1_OE_27) with the highest SERPINB1 expression had decreased peak viable cell density and exponential phase growth rate. Selected SERPINB1 OE clones were subsequently evaluated for their IgG expression capabilities using GS selection. Clone SERPINB1_OE_42 with moderate SERPINB1 overexpression demonstrated increased IgG productivity in “bulk” selection. We conclude that manipulating Serpinb1 expression can lead to increased recombinant IgG productivity, but the effect in host cell lines may vary by clone and by overexpression level. This work represents the ongoing effort in applying “-omics” findings to novel CHO host cell line engineering.

Expression and distribution of immunoglobulin G in the normal liver, hepatocarcinoma and postpartial hepatectomy liver

The liver has the extraordinary properties of regeneration and immune tolerance; however, the mechanisms governing these abilities are poorly understood. To address these questions, we examined the possible expression of immunoglobulins in the human and rat liver and the relationship of IgG expression to hepatocyte proliferation, metastasis, apoptosis and immune tolerance. Immunohistochemistry, in situ hybridization, laser-guided microdissection and reverse transcription-PCR were performed to examine the expression of IgG in normal human and rat liver, severe combined immunodeficient mouse (SCID) liver and human liver cancers and corresponding cell lines. Small interfering RNA (siRNA) was transfected into cultured hepatocarcinoma cells to downregulate the expression of IgG heavy chain genes. Cell proliferation and apoptosis were assayed with flow cytometry. Cell metastasis was assayed with a Transwell cell assay. Partial hepatectomy (70%) was performed in rats to examine the relationship between hepatocyte IgG and hepatocyte proliferation. IgG, together with essential enzymes for its synthesis, were expressed in the cytoplasm of hepatocytes of normal adult human and hepatoma patients and rat livers, SCID mouse liver and BRL-3A, L-02 and HepG-2 cell lines. Downregulation of IgG inhibited cell proliferation and metastasis and promoted apoptosis. Postsurgery livers expressed significantly more IgG than the livers before surgery and decreased to the original levels when hepatocytes stopped regeneration. IgA and IgM but not IgD and IgE were also positive in hepatocytes. Our findings demonstrate that normal and malignant hepatocytes are capable of synthesizing immunoglobulin, which has important roles in hepatocyte proliferation, apoptosis and cancer growth with profound clinical implications.