Human Lambda Research Articles

Structural modification of 2-phenylquinoline-4-carboxylic acid containing SIRT3 inhibitors for the cancer differentiation therapy.

Inhibition of SIRT3 exhibited potency in triggering leukemic cell differentiation. In discovery of potent SIRT3 inhibitors for cancer differentiation therapy, structural modification was performed on the previously developed lead compound P6. A total of 33 compounds were designed and synthesized. In the enzyme inhibitory assay, several molecules S18, S26, S27 and T5 showed potent SIRT3 inhibitory activity with IC50 value of 0.53, 1.86, 5.06, and 2.88 μM, respectively. Moreover, the tested compounds exhibited SIRT3 inhibitory selectivity over SIRT1 and SIRT2. Compounds S27 and T5 were potent in inhibition the growth of MM1.S and RPMI-8226 cells in the invitro antiproliferative test. Significantly, representative compounds, especially S27 and T5, promoted differentiation of tested MM cells in the cellular morphological evaluation, accompanied by increasing the expression of differentiation antigen CD49e and human immunoglobulin light chain lambda and kappa. Additionally, molecule S18 without antiproliferative potency itself, showed significant inhibitory activity against growth factor IL-6 induced RPMI-8226 cell proliferation. Collectively, potent SIRT3 selective inhibitors with MM cell differentiation potency were developed for further discovery of anticancer drugs.

Resolving haplotype variation and complex genetic architecture in the human immunoglobulin kappa and lambda chain loci in individuals of diverse ancestry

Abstract Immunoglobulins (IGs), critical components of the human immune system, are composed of heavy and light protein chains encoded at three genomic loci. The IG Kappa (IGK) chain locus consists of two large, inverted segmental duplications. The complexity of IG loci has hindered effective use of standard high-throughput methods for characterizing genetic variation within these regions. To overcome these limitations, we leverage long-read sequencing to create haplotype-resolved IGK assemblies in an ancestrally diverse cohort (n=36), representing the first comprehensive description of IGK haplotype variation. We identify extensive locus polymorphism, including novel single nucleotide variants (SNVs) and a common novel ∼24.7 Kbp structural variant harboring a functional IGKV gene. Among 47 functional IGKV genes, we identify 141 alleles, 64 (45.4%) of which were not previously curated. We report inter-population differences in allele frequencies for 14 IGKV genes, including alleles unique to specific populations within this dataset. In addition, we identify haplotypes carrying signatures of gene conversion that associate with enrichment of SNVs in the IGK distal region. Finally, we identify extensive polymorphism in the IG Lambda (IGL) locus, including copy number variation for genes encoding IGLC and IGLJ genes. These data lay a foundation for advancing our understanding of how genetic variation in IG loci impacts expressed antibody repertoires in human populations.

P807 HLA type impact on immunization and efficacy of ustekinumab in IBD patients: a multicenter study

Abstract Background About 30% of patients treated with ustekinumab will present primary or secondary failure. Outcomes and frequency of ustekinumab immunization are still controversial. Some HLA types (especially DQA1*05) appear to be predictive factors of anti TNFα immunization. This study aimed to assess the impact of HLA type on the risk of immunization and on the efficacy of ustekinumab. Methods IBD patients treatede with ustekinumab in Saint-Etienne and Lyon hospitals were included between 2017 and 2023. Ustekinulab through level was measured by ELISA and HLA type (DQA1, DQB1, DRB1) determined by PCR. Anti-drug antibodies (ADA) detection was done using both a "drug sensitive" assay (ELISA) and an innovative "drug tolerant" method based on anti human lambda chain. Data about clinical, biological (CRP, fecal calprotectin) and endoscopic responses were retrospectively collected. Results Among the 115 patients included (43% males, mean age of 40 years old), 98 had Crohn disease. Nighty-eight percent of the patients were previously treated with at least one anti-TNFα and 33% of them were immunized. In this study, 43 patients were immunized against ustekinumab with the "drug tolerant" assay and only 2 with the "drug sensitive" method. Immunization tended to reduce (not significatively) clinical, biological and endoscopic responses. Ustekinumab through level was significately higher for patients with good clinical (p = 0.009) and biological (p = 0.036). HLA DQA1*05 or DRB1*03 carriage was associated with higher risk of immunization (OR = 5.13, p = 0.048 / OR = 5.36, p = 0.006) and with a trend to decrease clinico-biological response. Other HLA types, notable DQA1*01 tended to be associated with a lower immunization (OR = 0.7, p=0.3) and a better response (OR = 2.55, p = 0.018). A small group involved (26 patients), combination therapy at induction had no significant benefit in terms of immunization prevention or therapeutic response. Sub-groups analysis seemed to confirm some HLA types involvement, by breaking free of imbalances due to haplotype transmission. Conclusion Our study suggests the usefulness of ustekinumab monitoring and a more common immunization than previously described. HLA type seem to influence immunization and ustekinumab response. Some specific HLA types and patients profiles tend to be associated with a higher risk of immunization not only to anti-TNFα but also to ustekinumab.

Saccharomyces cerevisiae DNA polymerase IV overcomes Rad51 inhibition of DNA polymerase δ in Rad52-mediated direct-repeat recombination.

Saccharomyces cerevisiae DNA polymerase IV (Pol4) like its homolog, human DNA polymerase lambda (Polλ), is involved in Non-Homologous End-Joining and Microhomology-Mediated Repair. Using genetic analysis, we identified an additional role of Pol4 also in homology-directed DNA repair, specifically in Rad52-dependent/Rad51-independent direct-repeat recombination. Our results reveal that the requirement for Pol4 in repeat recombination was suppressed by the absence of Rad51, suggesting that Pol4 counteracts the Rad51 inhibition of Rad52-mediated repeat recombination events. Using purified proteins and model substrates, we reconstituted in vitro reactions emulating DNA synthesis during direct-repeat recombination and show that Rad51 directly inhibits Polδ DNA synthesis. Interestingly, although Pol4 was not capable of performing extensive DNA synthesis by itself, it aided Polδ in overcoming the DNA synthesis inhibition by Rad51. In addition, Pol4 dependency and stimulation of Polδ DNA synthesis in the presence of Rad51 occurred in reactions containing Rad52 and RPA where DNA strand-annealing was necessary. Mechanistically, yeast Pol4 displaces Rad51 from ssDNA independent of DNA synthesis. Together our in vitro and in vivo data suggest that Rad51 suppresses Rad52-dependent/Rad51-independent direct-repeat recombination by binding to the primer-template and that Rad51 removal by Pol4 is critical for strand-annealing dependent DNA synthesis.

A Novel Inactive Isoform with a Restored Reading Frame Is Expressed from the Human Interferon Lambda 4 TT Allele at rs368234815.

The TT allele of the dinucleotide variant rs368234815 (TT/ΔG) abolishes the open reading frame (ORF) created by the ancestral ΔG allele of the human interferon lambda 4 (IFNL4) gene, thus preventing the expression of a functional IFN-λ4 protein. While probing the expression of IFN-λ4 in human peripheral blood mononuclear cells (PBMCs), using a monoclonal antibody that binds to the C-terminus of IFN-λ4, surprisingly, we observed that PBMCs obtained from TT/TT genotype individuals could also express proteins that reacted with the IFN-λ4-specific antibody. We confirmed that these products did not emanate from the IFNL4 paralog, IF1IC2 gene. Using cell lines and overexpressing human IFNL4 gene constructs, we obtained evidence from Western blots to show that the TT allele could express a protein that reacted with the IFN-λ4 C-terminal-specific antibody. It had a molecular weight similar if not identical to IFN-λ4 expressed from the ΔG allele. Furthermore, the same start and stop codons used by the ΔG allele were used to express the novel isoform from the TT allele suggesting that a restoration of the ORF had occurred in the body of the mRNA. However, this TT allele isoform did not induce any IFN-stimulated gene expression. Our data do not support a ribosomal frameshift that leads to the expression of this new isoform, implying that an alternate splicing event may be responsible. An N-terminal-specific monoclonal antibody did not react with the novel protein isoform suggesting that the alternate splicing event likely occurs beyond exon 2. The new isoform is glycosylated similar to the functional IFN-λ4 and is also secreted. Furthermore, we show that the ΔG allele can also potentially express a similarly frameshifted isoform. The splicing event that leads to the generation of these novel isoforms and their functional significance remains to be elucidated.

Avelumab use in Merkel cell carcinoma treatment

Avelumab is a programmed death-ligand 1 (PD-L1) blocking human IgG1 lambda monoclonal antibody. It was the first immunotherapy to be approved for the treatment of MCC. In March 2017, the FDA granted accelerated approval to avelumab for the treatment of adults and pediatric patients 12 years and older with metastatic Merkel cell carcinoma (MCC) –irrespective of prior therapy. In July 2017, the European Medicines Agency (EMA) recommended the approval of avelumab as a monotherapy for the treatment of adult patients with metastatic Merkel cell carcinoma (mMCC). Approvals were based on the efficacy and safety demonstrated in JAVELIN Merkel 200 (NCT02155647), a multi-center, open-label, single-arm, phase II clinical trial [1]. Part A of the study consisted of patients treated in the second line with metastatic, chemotherapy-refractory MCC. Part B consisted of systemic treatment-naive patients who received avelumab as a first-line treatment for metastatic or distally recurrent MCC. In the first line the ORR is 39.7%. Durable responses lasting at least 6 months were observed and the majority of responses are observed early with the median time to response of 6.1 week. PFS rate at 6 and 12 months are 41% and 31%, respectively. Median OS is 20.3 months. The OS rate at 1 year is 60%.

Tubulointerstitial nephritis in cases of lupus nephritis: An experience from a tertiary care referral center of Eastern India

Context: Up to 60% of patients with systemic lupus erythematosus develop lupus nephritis (LN). Tubulointerstitial nephritis (TIN) includes interstitial inflammation, tubulitis, tubular atrophy (TA), and interstitial fibrosis. These are considered independent risk factors for renal outcome. Aims: To evaluate the occurrence of TIN in LN and to correlate it with clinical and histopathological variables and renal outcome. Settings and Design: It was a prospective, single-center study. Subjects and Methods: One hundred and thirty-two LN cases were evaluated. Light microscopic scoring of interstitial inflammation, fibrosis, and TA was done as follows: 0 (nil); 1+ (mild - <25% of the area of observed cortex); 2+ (moderate - >25% to 50% of the area of observed cortex); 3+ (severe - >50% of the area of observed cortex). For direct immunofluorescence study, fluorescein isothiocyanate-conjugated polyclonal rabbit antisera against human IgG, IgA, IgM, C3c, C1q, kappa, and lambda antibodies (DAKO, Germany) were used. Statistical Analysis Used: Statistical software GraphPad Prism version 6.1. Results: Significant TIN was present in 6% of cases associated with high National Institutes of Health activity and chronicity indices irrespective of the modified International Society of Nephrology and Renal Pathology Society class of LN. In the cases where inflammation and fibrosis are marked, significantly raised serum creatinine, low estimated glomerular filtration rate, high 24-h urinary protein excretion, and reduced survival without any complete remission were seen. Severe interstitial and tubular inflammations without chronicity were also associated with low survival rate due to frequent relapse and significant hypertension. Conclusions: Activity and chronicity indices describing TIN components become essential to predict the survival, therapeutic response, and disease prognosis in LN.

Serum Free Light Chain Quantification Testing: Comparison of Two Methods for Disease Monitoring.

Levels of free immunoglobulin light chains in serum and urine are a sensitive measure of dysregulated immunoglobulin synthesis. The development of an assay for free light chains in serum was a major advance in laboratory testing for monoclonal gammopathies. The original assay by The Binding Site, called Freelite®, has been in common use in laboratory monitoring of monoclonal gammopathies. Two clinical entities, myeloma-defining condition and light chain-predominant multiple myeloma, rely on quantitative measurements of serum free light chains. Using polyclonal antisera specific to free light chains, Diazyme Laboratories developed a latex immunoturbidimetric assay for quantification of human kappa and lambda serum free light chains. We evaluated the Diazyme assay by comparing the results of kappa and lambda free light chain quantification, and kappa/lambda ratio with the results on the same specimens by the Freelite method. We also compared the correlation of the 2 methods to evaluate response to treatment and to changes in clinical status of patients with multiple myeloma. The results of Freelite and Diazyme methods are comparable. There was no statistically significant difference in the performance of the 2 assays for quantification of light chains, kappa/lambda ratio, or correlation of clinical parameters from patients with multiple myeloma at various stages of monitoring the disease in 2 geographically diverse laboratory and clinical environments. The Diazyme method is comparable to Freelite and provides an opportunity to add the test to front-end automation and improvement in efficiency of the assay.

Immunoglobulin peptide against melanoma

Was investigated a peptide named L12P obtained from human immunoglobulin Lambda joining 1 (IGLJ1) before recombination. The peptide previously showed activity against fungi, which motivated us to investigate the antitumoral activity against murine B16F10 melanoma tumor cells. To test the biological activity of the peptide against tumor cells different tumor phenotypes were initially assayed in vitro, such as migration, adherence and cell cycle. Hence, the selected peptide displayed significant action in vitro against melanoma cells. In conclusion the present work may suggest a functional molecules derived from immunoglobulin with future potential therapeutic.

A real‐life experience as a proof of Guselkumab effectiveness and safety in patients with moderate to severe psoriasis

Psoriasis is a skin disorder characterized by chronic inflammation driven by different immunologic pathways, among which the IL-23/Th17 axis plays a pivotal role. For this reason, the use of IL23p19 inhibitors in psoriasis treatment has been evaluated over the years. Guselkumab, a totally human IgG1 lambda monoclonal antibody, that selectively blocks the p 19 subunit of IL- 23 has demonstrated high efficacy and safety throughout several, randomized, double-blind phase III trials (VOYAGE 1 and 2, NAVIGATE and ECLIPSE). We designed a single-center retrospective cohort study in a population consisting of 46 patients followed from December 2018 to April 2021. After a diagnosis of moderate to severe psoriasis, all the patients were considered suitable to receive treatment with Guselkumab. In our population, among those who achieved clinical improvement in terms of Psoriasis Area Severity Index (PASI), PASI 75, 90, and 100 were achieved on average on weeks 14, 19, 21 respectively. We then analyzed a subgroup of our population, consisting of 35 patients, who had an identical follow-up time of 28 weeks, thus observing the trend in mean PASI at subsequent assessments and the number of patients who had reached PASI 75, PASI 90, and PASI 100 at week 4 (10; 3; 1), week 12 (12; 13; 11), week 20 (7; 6; 2), and week 28 (1; 4; 6), respectively. The results obtained are in line with those obtained from previous studies, thus confirming that Guselkumab is an excellent choice in terms of security, long-term efficacy, and overall tolerance.

POS-490 GLOMERULAR MICROANGIOPATHY ASSOCOATED WITH PROLONGED USE OF BEVACIZUMAB IN A PATIENT WITH RENAL-CELL CARCINOMA - CASE REPORT

Drug-induced thrombotic microangiopaty (TMA) known to be associated with the usage of the vascular endothelial growth factor inhibitors; since 2007, several cases of TMA, associated with bevacizumab, had been reported. In 2019, Person et al. described specific pattern of injury in 17 patients receiving bevacizumab, and introduced the term bevacizumab-associated glomerular microangiopathy instead of TMA; none on these patients had renal-cell carcinoma We present a case of glomerular microangiopathy, associated with prolonged bevacizumab treatment in a patient with renal-cell carcinoma 61 years old Caucasian female was referred for kidney biopsy. She underwent resection of her right kidney for the renal-cell carcinoma twice, and received bevacizumab for 8 years; 18 months before admission she developed new-onset arterial hypertension and nephrotic syndrome. At admission, she had general edema, her blood pressure was 200/100 mm Hg; the rest of her physical examination was unremarkable. Her urine protein excretion was 6400mg/24 hours with blunt urine sediment; hemoglobin was 10.4g/dL, platelet count was 322x109/L, ESR 59mL/min, serum creatinine 120µmol/L, total protein 5.4g/dL, serum albumin 2.4g/dL, LDH 224U/L. Other blood chemistry parameters, immunoglobulins, C3, C4 and CRP levels were normal. ECG, chest CT, kidneys and abdomen ultrasound showed nothing but right kidney postoperative changes and mild bilateral hydrothorax. Kidney biopsy light microscopy on paraffin-imbedded sections stained with haematoxylin-eosin, periodic acid-Schiff (PAS), Masson’s trichrome, and Jones silver found 10 sclerosed glomeruli, the rest 26 were enlarged, with lobular appearance, without hypercellularity or crescents. Most capillary lumens were widened, with single-contour capillary walls, lacking endothelial layer, and occupied with homogenous acellular mild eosinophilic, PAS negative, non-fuchsinophilic, Jones-negative coarse pseudothrombi (Figures 1, 2). Diffuse acute tubular epithelial injury with brush-border loss, without casts or interstitial fibrosis/tubular atrophy, and mild thickening of arterioles and small arteries due to muscular layer hypertrophy was found. Immunofluorescence on formalin fixed paraffin-embedded tissue with FITC-conjugated antibodies to human IgA, IgG, IgM, C1q, C3, fibrinogen, kappa and lambda light chains showed diffuse homogenous IgA, IgG, IgM, C1q, C3, fibrinogen, kappa and lambda +/++ expression in pseudothrombi. Based on the presence of pseudothrombi and absence of the TMA clinical features we diagnosed bevacizumab-associated glomerular microangiopathy, discontinued bevacizumab, and started ACE-inhibitors and aspirin with the outpatient follow-up. A month later, her edema resolved, blood pressure was 150/90 mm Hg, urine protein excretion was 780mg/24 hours, serum albumin 3.1g/dL, and serum creatinine 112µmol/L. A year later, she was doing well, without edema; her blood pressure was 130/80 mm Hg, hemoglobin 13.0g/dL, total protein 7.3g/dL, serum albumin 4.2g/dL, and serum creatinine 88µmol/LView Large Image Figure ViewerDownload Hi-res image Download (PPT) The patient treated with bevacizumab for 8 years developed bevacizumab-associated glomerular microangiopathy, which is an extremely rare complication in a renal-cell carcinoma setting. Withdrawn of bevacizumab lead to the resolution of clinical symptoms and restoration of kidney function

Investigation of the antiviral activity of the recombinant human interferon lambda 1 in human conjunctiva cell culture

The aim of the study was to determine the efficacy of recombinant human interferon lambda 1 (IFN-λ1) against human adenovirus serotype 5 in a culture of human conjunctival cells Chang conjunctiva clone 1-5c-4. Material and methods. The study design consisted of three experimental schemes, reflecting a prophylactic and two options for a therapeutic and prophylactic treatment regimen (with the constant presence of the virus in the culture medium and with its removal after adsorption). The antiviral activity of IFN-λ1 was determined by the number of viable cells after exposure to the virus (MTT test). Results and discussion. It has been established that IFN-λ1 has antiviral activity against human adenovirus in vitro under a prophylactic and therapeutic-prophylactic scheme of administration at an infection dose of 1 and 10 TCID50 (50% tissue culture infectious dose), but not at an infection dose of 100 TCID50. The antiviral effect of the use of IFN-λ1 in a therapeutic and prophylactic regimen at an infection dose of 1 TCID50 was comparable to that of IFN-α. At the same time, both interferons did not have a toxic effect on the cell culture even at a concentration of 84 and 58 μg/ml, respectively. The antiviral activity and the absence of cytotoxic action provide the basis for further study of the possibility of development of based on IFN-λ1 drug for eye conjunctiva viral diseases treatment.

The Novel Subtype-Selective Histone Deacetylase (HDAC) Inhibitor, Chidamide, Exerts Dual Anti-Myeloma and Bone Protective Effect in Vitro and In Vivo

Background and Purpose: Despite the advance in multiple myeloma treatment, drug resistance is still inevitable and the prognosis of most patients remains poor. Chidamide, a new subtype-selective histone deacetylase (HDAC) inhibitor, which is produced independently in China, is designed to inhibit the activity of HDAC 1, 2, 3 and 10. In the present work, we investigated the effect of chidamide on multiple myeloma and its associated osteolytic bone disease with different models.Experiment Design: In vitro cytotoxicity of chidamide was tested on myeloma cell lines (MM.1s, CAG, ARP-1, 8226, U266, H929, LP-1, ANBL-6, OPM2) with CCK-8 and flow cytometry. Synergistic combinations with standard drugs in 8226 and CAG cell lines were analyzed by compusyn software. The effect on osteoclast formation was assessed in osteoclastic cultures. The mechanisms of chidamide were explored by western blot and immunofluorescence. Murine models were employed for in vivo works, IVIS in vivo imaging system, ELISA (human Ig lambda, CTX-I, PINP) and micro-CT were used to surveillance the tumor burden and examine the bone destruction.Results: The cytotoxic effect of chidamide on myeloma cells is due to induction of cell apoptosis and cell cycle disruption (G1 phase arrest) in a dose dependent manner (IC50 varies from 2 to 5 uM in different cell lines) which mediated by increasing Cleaved Caspase-3, Caspase-7, Caspase-8, Caspase-9, p21 and p27, decreasing C-Myc, Mcl-1, Bcl-xL, CDK4 and CDK6. SensitiveSOCS3/JAK2/STAT3 cell signaling was involved in regulating these proteins by increasing the expression of SOCS3, reducing the expression of phospho-JAK2 and phospho-STAT3, thus inhibiting the proliferation and inducing cell cycle disruption. After treating with chidamide, the expression levels of HDAC1, 2, 3, 10 were not changed significantly, which indicated chidamide suppressed the function of these targets while not affected their expression. Notably, chidamide exerted synergistic anti-myeloma effect with dexamethasone, carfilzomib or pomalidomide (combination index < 1). Also, chidamide showed effective cytotoxicity in the co-culture of myeloma cells with bone marrow stromal cells and osteoclasts. Importantly, chidamide suppressed in vitro osteoclast formation and resorption, inhibited the expression of the key molecules (Cathepsin K, c-fos, NFATc1) and de-phosphorylation of p-AKT and p-JNK, and disrupted the F-acting ring formation. Finally, chidamide significantly reduced the tumor volume in the subcutaneous xenografts models, also the level of monoclonal protein Ig lambda secreted by myeloma cells decreased in disseminated myeloma murine models after treatment. Moreover, chidamide not only prevented tumor-associated bone loss in the disseminated murine model which was partially related with reducing the tumor burden but also had a direct bone protective effect on RANKL-induced rapid bone loss in non-tumor bearing mouse model with serum PINP (bone formation) and trabecular numbers increased while serum CTX-I (bone resorption) and trabecular space diminished compared with the vehicle group.Conclusion: Our work demonstrates dual anti-myeloma and bone protective effect of chidamide in vitro and in vivo, and shows its synergistic anti-myeloma effect with dexamethasone, carfilzomib and pomalidomide. These findings strongly suggest the potential clinical use of this drug in multiple myeloma in the near future. DisclosuresNo relevant conflicts of interest to declare.

Interferon Lambda Delays the Emergence of Influenza Virus Resistance to Oseltamivir.

Influenza viruses are a leading cause of morbidity and mortality worldwide. These air-borne pathogens are able to cross the species barrier, leading to regular seasonal epidemics and sporadic pandemics. Influenza viruses also possess a high genetic variability, which allows for the acquisition of resistance mutations to antivirals. Combination therapies with two or more drugs targeting different mechanisms of viral replication have been considered an advantageous option to not only enhance the effectiveness of the individual treatments, but also reduce the likelihood of resistance emergence. Using an in vitro infection model, we assessed the barrier to viral resistance of a combination therapy with the neuraminidase inhibitor oseltamivir and human interferon lambda against the pandemic H1N1 A/Netherlands/602/2009 (H1N1pdm09) virus. We serially passaged the virus in a cell line derived from human bronchial epithelial cells in the presence or absence of increasing concentrations of oseltamivir alone or oseltamivir plus interferon lambda. While the treatment with oseltamivir alone quickly induced the emergence of antiviral resistance through a single mutation in the neuraminidase gene, the co-administration of interferon lambda delayed the emergence of drug-resistant influenza virus variants. Our results suggest a possible clinical application of interferon lambda in combination with oseltamivir to treat influenza.

Pharmacokinetic-Pharmacodynamic Modelling of Systemic IL13 Blockade by Monoclonal Antibody Therapy: A Free Assay Disguised as Total.

A sequential pharmacokinetic (PK) and pharmacodynamic (PD) model was built with Nonlinear Mixed Effects Modelling based on data from a first-in-human trial of a novel biologic, MEDI7836. MEDI7836 is a human immunoglobulin G1 lambda (IgG1λ-YTE) monoclonal antibody, with an Fc modification to reduce metabolic clearance. MEDI7836 specifically binds to, and functionally neutralizes interleukin-13. Thirty-two healthy male adults were enrolled into a dose-escalation clinical trial. Four active doses were tested (30, 105, 300, and 600 mg) with 6 volunteers enrolled per cohort. Eight volunteers received placebo as control. Following single subcutaneous administration (SC), individual time courses of serum MEDI7836 concentrations, and the resulting serum IL13 modulation in vivo, were quantified. A binding pharmacokinetic-pharmacodynamic (PK-PD) indirect response model was built to characterize the exposure-driven modulation of the target over time by MEDI7836. While the validated bioanalytical assay specification quantified the level of free target (i.e., a free IL13 assay), emerging clinical data suggested dose-dependent increase in systemic IL13 concentration over time, indicative of a total IL13 assay. The target time course was modelled as a linear combination of free target and a percentage of the drug-target complex to fit the clinical data. This novel PK-PD modelling approach integrates independent knowledge about the assay characteristics to successfully elucidate apparently complex observations.

Identification and Targeting of Thomsen-Friedenreich and IL1RAP Antigens on Chronic Myeloid Leukemia Stem Cells Using Bi-Specific Antibodies.

IntroductionQuiescent leukemia stem cells (LSCs) play a major role in therapeutic resistance and disease progression of chronic myeloid leukemia (CML). LSCs belong to the primitive population; CD34+CD38-Lin-, which does not distinguish normal hematopoietic stem cells (HSC) from CML LSCs. Because Thomsen–Friedenreich/CD176 antigen is expressed on CD34+ HSC and IL1RAP is tightly correlated to BCR-ABL expression, we sought to increase the specificity towards LSC by using additional biomarkers.MethodsWe evaluated the co-expression of both antigens on CD34+ peripheral blood mononuclear cells (PBMCs) from both healthy volunteers and CML patients, using flow cytometry. Then, we used site-directed mutagenesis to induce knob-in-hole mutations in the human IgG heavy chain and the human lambda light chain to generate the bi-specific antibody (Bis-Ab) TF/RAP that binds both antigens simultaneously. We measured complement-directed cytotoxicity (CDC) in CML samples with the Bis-Ab by flow cytometry.ResultsIn contrast to healthy volunteers, CML samples displayed a highly significant co-expression of CD176 and IL1RAP. When either a double-positive cell line or CML samples were treated with increasing doses of Bis-Ab, increased binding and CDC was observed indicating co-operative binding of the Bis-Ab as compared to monoclonal antibodies.DiscussionThese results show that the bi-specific antibody is capable of targeting IL1RAP+ and CD176+ cell population among CML PBMCs, but not corresponding normal cells in CDC assay. We hereby offer a novel strategy for the depletion of CML stem cells from the bulk population in clinical hematopoietic stem cell transplantation.

The importance of serum immunoglobulin free light chain assessment for predicting outcome in patients with newly diagnosed multiple myeloma in real clinical practice

Background. The prognosis of patients with multiple myeloma (MM) is significantly different depending on the biological characteristics of the tumor substrate, the microenvironment of the bone marrow, as well as factors associated with the patient’s body. Therefore, the search for new reliable and easily identifiable prognostic markers is relevant for the effective management of patients with this disease.The objective of the study was to assess the prognostic value of the study of serum free light chains (FLC) of immunoglobulins κ and λ and their ratio κ / λ FLC in the blood serum of patients with newly diagnosed MM in real clinical practice.Materials and methods. 369 patients with first diagnosed MM (134 men and 235 women) were examined who were hospitalized in the hematology department of the City Clinical Hospital No. 2 Novosibirsk in the period since January 2012 to December 2017. The median age of the patients was 67 (32–82) years. All patients received induction courses of chemotherapy based on bortezomib. The control group consisted of 56 conditionally healthy individuals: 34 women (60.7 %) and 22 (39.3 %) men with a median age of 62 (40–68) years. The concentration of FLC-κ and FLC-λ (mg / L) in blood serum was determined by immunoturbidimetric method on a Hitachi 911 automated biochemical analyzer using the Freelite Human Lambda and Freelite Human Kappa reagent kits (Binding Site, Great Britain).Results. It was found that in patients with MM, the concentration of serum FLC-κ or FLC-λ was statistically significantly higher compared to the control group and varied depending on the type of MM (p &lt;0.001). The diagnostic sensitivity of the quantitative determination of FLC and their ratio for MM was 98.64 %, compared with 94.04 % in a standard immunochemical study. The values of the ratio κ / λ FLC &lt;0.04 or&gt; 65, as well as the concentration of FLC-κ and FLC-λ are higher than the median obtained in the whole group (FLC-κ ≥702 mg / L and FLC-λ ≥493.2 mg / L), correlate with known factors of poor prognosis for MM (with a high concentration of β2‑microglobulin (&gt;3.5 mg / L) (r = 0.461; p &lt;0.001), plasma cell bone marrow infiltration &gt;60 % (r = 0.420; p &lt;0.001), renal failure (creatinine &gt;177 μmol / L) (r = 0.380; p = 0.002), and also with high lactate dehydrogenase activity (&gt;450 U / L) (r = 0.520; p &lt;0.001) and is associated with poor outcomes. The median overall survival in the group of patients with κ / λ FLC &lt;0.04 or &gt;65 was 49 months compared to 76 months in the group with κ / λ FLC 0.04–65 (log-rank p = 0.012).Conclusion. The determination of free FLC in the blood serum of patients with MM can be used to assess the prognosis of their survival. The value of the κ / λ FLC ratio &lt;0.04 or &gt;65 allows us to divide patients with MM into risk groups with significantly different outcomes and can be used to identify patients at high risk who need more aggressive therapy and more detailed monitoring of the response.

Abstract LB-089: Targeting transferrin receptor 1 (TfR1) with the ch128.1/IgG1 antibody inhibits Epstein-Barr virus (EBV) driven lymphoproliferative growth and lymphomagenesis in immunosuppressed mice bearing human B cells

Abstract Epstein-Barr virus (EBV) is a human herpesvirus that is associated with malignancies of both B lymphocyte and epithelial cell origin. EBV is associated with the development of approximately 1.5% of all cancers worldwide, including AIDS-related non-Hodgkin lymphoma (AIDS-NHL), Hodgkin lymphoma (HL), and post-transplant lymphoproliferative disease (PTLD). Primary infection of B cells with EBV results in their polyclonal activation and immortalization. The transferrin receptor 1 (TfR1), also known as CD71, is important for iron uptake and regulation of cellular proliferation. The TfR1 is expressed at high levels in proliferating cells including activated lymphocytes and malignant cells. We have developed a mouse/human chimeric antibody targeting TfR1 (ch128.1/IgG1) that has shown significant anti-tumor activity in immunosuppressed mouse models bearing human malignant B cells, including multiple myeloma and AIDS-NHL cells. In this study, we examined the effect of targeting TfR1 to inhibit EBV driven transformation of human B cells in vivo using an immunodeficient NSG mouse model. T cell-depleted human peripheral blood mononuclear cells were infected by exposure to EBV-containing supernatants from a B-lymphoblastoid cell line (B95-8). Mice were inoculated with these cells immediately following infection with EBV (day 0), or after cells were cultured for 7 days post infection prior to injection (day 7), and treated with ch128.1/IgG1 or an IgG1 isotype control antibody at 2 and 28 days post-inoculation. We monitored survival, serum levels of human cytokines, and characterized tumor-like growths in spleen and liver by assessing expression of human CD19 (marker for human B cells), EBV latent membrane protein 1 (LMP1; a marker for EBV infection), and human immunoglobulin light chain kappa or lambda (markers for monoclonality) by immunohistochemistry. Treatment with ch128.1/IgG1 significantly enhanced survival, compared to mice treated with the isotype control IgG1. Lymphoma-like cells of human B cell origin that were EBV-LMP1-positive, and often monoclonal, developed in most mice injected with EBV-infected cells (day 0 and 7) treated with the IgG1 control antibody, but only rarely in ch128.1/IgG1 treated animals. In addition, serum levels of human IL-6, IL-8, IFN-γ, CXCL13, sCD25, and sCD27, indicators of human B cell activation and proliferation, were lower in animals treated with ch128.1/IgG1. Taken together, these results show that ch128.1/IgG1 inhibited EBV driven lymphomagenesis using the NSG mouse model newly adapted to study EBV infection/transformation, indicating that there is significant potential for agents targeting TfR1 as therapeutic strategies to prevent EBV-associated B cell malignancies, including AIDS-NHL, HL, and PTLD. Citation Format: Laura E. Martinez, Tracy R. Daniels-Wells, Yu Guo, Larry I. Magpantay, Pierre V. Candelaria, Manuel L. Penichet, Otoniel Martinez-Maza, Marta Epeldegui. Targeting transferrin receptor 1 (TfR1) with the ch128.1/IgG1 antibody inhibits Epstein-Barr virus (EBV) driven lymphoproliferative growth and lymphomagenesis in immunosuppressed mice bearing human B cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-089.

RanBP2-Mediated SUMOylation Promotes Human DNA Polymerase Lambda Nuclear Localization and DNA Repair

Cellular DNA is under constant attack by a wide variety of agents, both endogenous and exogenous. To counteract DNA damage, human cells have a large collection of DNA repair factors. Among them, DNA polymerase lambda (Polλ) stands out for its versatility, as it participates in different DNA repair and damage tolerance pathways in which gap-filling DNA synthesis is required. In this work, we show that human Polλ is conjugated with Small Ubiquitin-like MOdifier (SUMO) proteins both in vitro and in vivo, with Lys27 being the main target of this covalent modification. Polλ SUMOylation takes place in the nuclear pore complex and is mediated by the E3 ligase RanBP2. This post-translational modification promotes Polλ entry into the nucleus, which is required for its recruitment to DNA lesions and stimulated by DNA damage induction. Our work represents an advance in the knowledge of molecular pathways that regulate cellular localization of human Polλ, which are essential to be able to perform its functions during repair of nuclear DNA, and that might constitute an important point for the modulation of its activity in human cells.

Expression of human lambda expands the repertoire of OmniChickens.

Most of the approved monoclonal antibodies used in the clinic were initially discovered in mice. However, many targets of therapeutic interest are highly conserved proteins that do not elicit a robust immune response in mice. There is a need for non-mammalian antibody discovery platforms which would allow researchers to access epitopes that are not recognized in mammalian hosts. Recently, we introduced the OmniChicken®, a transgenic animal carrying human VH3-23 and VK3-15 at its immunoglobulin loci. Here, we describe a new version of the OmniChicken which carries VH3-23 and either VL1-44 or VL3-19 at its heavy and light chain loci, respectively. The Vλ-expressing birds showed normal B and T populations in the periphery. A panel of monoclonal antibodies demonstrated comparable epitope coverage of a model antigen compared to both wild-type and Vκ-expressing OmniChickens. Kinetic analysis identified binders in the picomolar range. The Vλ-expressing bird increases the antibody diversity available in the OmniChicken platform, further enabling discovery of therapeutic leads.