Soluble Antibody Research Articles

Chemical Modification of the Yeast Cell Surface Allows the Switch Between Display and Soluble Secretion of Full-Length Antibodies.

The classical yeast surface display (YSD)-based antibody hit discovery relies on the enrichment of candidates from large antibody fragment libraries by fluorescence-activated cell sorting (FACS), followed by sequencing of the remaining diversity. The sequences of hit candidates are then transferred into plasmids, which allow their expression as full-length IgG in mammalian cells. After production and downstream processing, antibodies are further qualified in biochemical and functional assays. This multistep process has been proven to be successful in academia and has also found its way to the biopharmaceutical industry. However, during the recent decade, several groups tried to simplify this process by enabling either switchable or simultaneous secretion and display of antibodies or antibody fragments by yeast cells. Therefore, functional testing of candidates can be accomplished immediately during the screening process, while eliminating the need for tedious sequence reformatting and production host change. In this protocol, we give guidance for the establishment of a full-length antibody display system on yeast cells, which permits a simple switch to soluble antibody secretion.

2499 Idiopathic Eosinophilic Syndrome With Hepatic Involvement

INTRODUCTION: Hepatic involvement in hypereosinophilic syndrome is rare. CASE DESCRIPTION/METHODS: A 66-year-old gentleman was referred for evaluation of persistent eosinophilia, cough, wheezing, and abnormal liver chemistries. No abdominal pain, fever, weight loss, and bowel habit changes. No traveling outside the U.S. On exam his vital signs were within normal range. Eyes, scleral icterus; Lungs; wheezing the bases; Abdomen, soft, nontender, nondistended, negative for, shifting dullness, no organomegaly, no distended veins on the abdominal wall; Extremities, no edema; Skin, no icterus no bruising. His initial laboratory findings were as follows, WBC 15.3 × 109/L, Hgb of 15.3 g/dL, platelets of 438 × 109/L, absolute neutrophil count of 6.7 × 109/L, absolute lymphocyte of 2.7 × 109/L, absolute eosinophil count of 4.5 × 109/L, alkaline phosphatase of 234 U/L, AST of 51 U/L, ALT of 94 U/L, total bilirubin of 0.4 mg/dL, albumin of 4.8 g/dL. With respect to the liver related laboratory findings: negative viral hepatitis panel, ANA of 1:160, total IgG of 1010 mg/dL, IgG1 of 659 mg/dL, IgG2 of 258 mg/dL, IgG3 of 12.9 mg/dL and IgG4 of 28 mg/dL; F-actin IgG antibody 23.3 units; negative anti-smooth muscle antibodies; soluble liver antibody <20 units; negative AMA, alpha-1 antitrypsin level of 167 mg/dL; and ceruloplasmin of 27 mg/dL. Comprehensive studies for stool ova and parasites was unremarkable. Peripheral blood smear showed eosinophilia. Bone marrow biopsy, with flow cytometric analysis revealing hypereosinophilia showing aberrant expression of CD 56. Abdominal MRI revealed a 5.8 cm hemangioma in the left lobe; no hepatic steatosis. Myeloproliferative neoplasms (MPN) and platelet-derived growth factor receptor α (PDGFRA A/B) workup was negative. Liver biopsy showed many eosinophils within portal areas with foci of sinusoidal involvement compatible with idiopathic eosinophilic syndrome; chronic hepatitis (grade 2/4), with portal and periportal fibrosis (stage II/IV); no steatosis. After starting prednisone 60 mg oral daily, tapered over 4 weeks, his cough, wheezing, hepatic enzymes' elevation and eosinophilia have resolved. DISCUSSION: Hypereosinophilic syndrome with hepatic involvement is rare, and should be in the differential diagnosis especially when hepatic enzymes' elevation is associated with eosinophilia.

Synchronous Germinal Center Onset Impacts the Efficiency of Antibody Responses.

The germinal center reaction is an important target for modulating antibody responses. Antibody production from germinal centers is regulated by a negative feedback mechanism termed antibody feedback. By imposing antibody feedback, germinal centers can interact and regulate the output of other germinal centers. Using an agent-based model of the germinal center reaction, we studied the impact of antibody feedback on kinetics and efficiency of a germinal center. Our simulations predict that high feedback of antibodies from germinal centers reduces the production of plasma cells and subsequently the efficiency of the germinal center reaction by promoting earlier termination. Affinity maturation is only weakly improved by increased antibody feedback and ultimately interrupted because of premature termination of the reaction. The model predicts that the asynchronous onset and changes in number of germinal centers could alter the efficiency of antibody response due to changes in feedback by soluble antibodies. Consequently, late initialized germinal centers have a compromised output due to higher antibody feedback from the germinal centers formed earlier. The results demonstrate potential effects of germinal center intercommunication and highlight the importance of understanding germinal center interactions for optimizing the antibody response, in particular, in the elderly and in the context of vaccination.

T-Cell Engagers Based Bioassay for Evaluation of PD-1/PD-L1 Inhibitors Activity.

PD-1/PD-L1-based therapy has been named a revolution in cancer treatment. By the end of 2018, more than 100 anti-PD-1 and anti-PD-L1 antibodies were in various stages of development, and more than 2000 clinical trials with their use have been registered. Characterization of such antibodies requires a bioassay to determine their biological activity. In this study, we developed a cell-based bioassay for analyzing the activity of anti-PD-1 and anti-PD-L1 antibodies. We chose reporter system consisting of two cell lines and compared several approaches for activation of effector cell line based on superantigens, soluble anti-CD3 antibodies, transmembrane anti-CD3 antibodies, chimeric antigenic receptors (CARs) and bispecific T-cell engager antibodies. The bispecific T-cell engager antibodies offer several advantages over the other approaches. We characterized the bioassay and demonstrated its applicability for analyzing the activity of anti-PD-1 and anti-PD-L1 antibodies. The proposed bioassay can be useful in the development of new therapeutic agents and methods for their characterization.

The phosphorylation kinetics of the key regulators in T cell receptor signaling pathway

Abstract T cell receptor (TCR) signaling pathway regulates T cell homeostasis and adaptive immune responses. Clinically, the activation of TCR is indispensable to the success of T cell-based immunotherapy. The engagement of TCR phosphorylates immunoreceptor tyrosine-based activation motifs (ITAM) on CD3, which is followed by a protein phosphorylation cascade. Protein phosphorylation, regulated by kinases and phosphatases, changes protein conformation and determines protein function. A better understanding of TCR signaling phosphorylation events is crucial to both decipher the T cell regulation mechanism and boost the efficacy of T cell immunotherapy. Here, we developed a panel of monoclonal antibodies that target several of the TCR signaling regulators and are applicable for both intracellular flow cytometry (ICFC) and western blot (WB). Using these antibodies, we performed a kinetic analysis of the TCR signaling pathway. Jurkat T cells were cross-linked by soluble anti-CD3 and anti-CD28 antibodies, and the phosphorylation kinetics of the key TCR signaling regulators, such as phospho-ZAP-70 and phospho-PLCg1, were evaluated by ICFC and WB. We show that TCR signaling activation induced by cross-linking CD3 and CD28 is a relatively transient event and the optimal activation of the key TCR signaling regulators varies from 1 to 15 minutes. Our data profiled the phosphorylation kinetics of the key regulators in TCR signaling pathway.

Role of the IL-33/ST2/p38 signaling pathway in the immune response of corneal epithelial cells to Aspergillus fumigatus infection.

To investigate the expression of interleukin (IL)-33 in the cornea and human corneal epithelial cells (HCECs) exposed to Aspergillus fumigatus (A. fumigatus), and to determine the function of IL-33/ST2/p38 signaling pathway in the immune response of corneal epithelial cells to A. fumigatus infection. The mRNA and protein expression of IL-33 in HCECs and mice corneas were examined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis, respectively. IL-33 expression was also detected in cornea samples from healthy donors and patients with fungal keratitis with immunohistochemistry. The cultured HCECs were treated with inactive A. fumigatus hyphae at various concentrations with or without recombinant human IL-33 protein, soluble recombinant ST2 protein, specific ST2 neutralizing antibody, or the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 for evaluation of the expression and activation of IL-33/ST2/p38 signaling in the regulation of proinflammatory cytokines. The production levels of IL-6 and IL-1β were determined by qRT-PCR and enzyme-linked immunosorbent assay (ELISA). The proliferation of HCECs was determined by a Cell Counting Kit-8 (CCK8) assay and cell count. IL-33 expression levels increased in the corneal tissues of patients with fungal keratitis and in mice corneas of experimental A. fumigatus infection, as well as in HCECs with infection of A. fumigatus. A. fumigatus strongly stimulated HCECs-generated proinflammatory cytokine (IL-6 and IL-1β) production at both the mRNA and protein levels. This production of pro-inflammatory mediators stimulated by A. fumigatus was further stimulated by IL-33 and was prevented by soluble ST2 protein or ST2 neutralizing antibody. Moreover, IL-33 naturally promoted the p38 phosphorylation induced by A. fumigatus, which was suppressed by soluble ST2 protein. The MAPK p38 inhibitor SB203580 also inhibited the A. fumigatus-induced proinflammatory cytokine production. IL-33 administration for 48h and 72h promoted the proliferation of HCECs, which was attenuated by treatment with soluble recombinant human ST2 protein. A. fumigatus elevates IL-33 expression in human and mice corneas and HCECs. Thus, IL-33/ST2/p38 signaling may play an important role in amplifying the immune response of corneal epithelial cells to A. fumigatus infection. Besides, IL-33 promotes the cell proliferation of HCECs via its receptor ST2. These findings suggest a novel autocrine mechanism of amplification of the fungal-induced inflammatory response in the corneal epithelium, highlighting a potential therapeutic target for fungal keratitis.

The soluble worm antigens-specific antibodies used as biomarkers of Schistosoma japonicum in a low prevalence and intensity endemic area of Hubei, China

The precise diagnosis of Schistosoma japonicum (S. japonicum) infection plays a critical role in achieving the ultimate goal of eliminating schistosomiasis in endemic regions. We evaluated the S. japonicum soluble worm antigen protein (SWAP) specific-IgG, IgG4 and IgE levels, and evaluated the association between S. japonicum infection and these antibodies in a sample of 837 residents from a S. japonicum-endemic area in Hubei province, China. The anticipants were divided into the Training Set (TS) and Validation Set (VS) based on the chronological order. Enzyme-linked immunosorbent assays were performed to detect the SWAP-specific antibodies. Three algorithms for identifying S. japonicum infection were generated in the TS and subsequently validated in the VS. The findings were further replicated in an independent cohort from an endemic area for Schistosoma mansoni (S. mansoni) in Brazil. Our results indicated for the first time that S. japonicum-infected individuals had higher levels of SWAP-specific IgG, IgG4 and IgE, and lower value of the IgE/IgG4 ratio than uninfected individuals in both the two sets (p < 0.01). Both the infected and uninfected individuals had a high prevalence of seropositivity for IgG. We further showed that the predictive model EGR (IgE/IgG4 ratio) score performed best in Chinese population (area under the receiver operating characteristic (AUROC) 0.905, sensitivity 82.7%, specificity 84.0% in the TS; AUROC 0.933, sensitivity 87.7%, specificity 89.1% in the VS). Nevertheless, the predictive model IgG4 score performed best in Brazilian cohort (AUROC 0.788, sensitivity 73.2%, specificity 73.3%). In summary, SWAP-specific IgG could be used as a biomarker for identifying individuals who have been previously exposed to S. japonicum, and furthermore the SWAP-specific IgE/IgG4 could be used as an immune biomarker for S. japonicum infection in particular in the endemic areas with low prevalence and intensity.

Effect of Molecular Chaperone on the Soluble Expression of Recombinant Fab Fragment in E. coli

Molecular chaperones are folding modulators that play a pivotal role in conformational quality control of proteome and assist proper protein folding upon translation. In this study, the effects of cytoplasmic chaperones on the production of anti-TNF-α Fab antibody in Escherichia coli were investigated. To increase the solubility of anti-TNF-α Fab, different chaperone plasmids including pG-KJE8 (GroELS and DnaKJE), pGro7 (GroELS), pKJE7 (DnaKJE), pG-Tf2 (GroELS and TF) and pTf16 (TF) co-expressed in BL21 cells containing pET-22b-Fab construct were used. The solubility of recombinant anti-TNF-α Fab was analyzed by SDS-PAGE. The reactivity of the recombinant protein was tested by ELISA and dot blot assays. SDS-PAGE analysis of the expressed Fab revealed that chaperone coexpression increased the solubility of the Fab antibody. Among the chaperone sets co expressed with Fab construct, the dnaK/dnaJ/grpE chaperones showed the highest effect on soluble expression with producing about 23.2% soluble Fab antibody. ELISA test results showed, the pKJE7 chaperone led to an approximately sevenfold increase in the activity of produced Fab fragment. Dot blot test results revealed reactivity of recombinant Fab to both transmembrane and soluble form of TNF-α. Our results demonstrated the significant advantage of molecular chaperones in improvement of the soluble expression of Fab antibody in E. coli.

Serum neopterin as well as ferritin, soluble interleukin-2 receptor, KL-6 and anti-MDA5 antibody titer provide markers of the response to therapy in patients with interstitial lung disease complicating anti-MDA5 antibody-positive dermatomyositis

Objective: This study identified biomarkers that can be used to assess disease activity and response to therapy in patients with interstitial lung disease complicating anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab)-positive clinically amyopathic dermatomyositis (CADM).Methods: In 15 patients with interstitial lung disease complicating anti-MDA5 Ab-positive CADM, anti-MDA5 Ab, neopterin, interleukin (IL)-18, ferritin, and soluble interleukin 2 receptor (sIL-2R) levels were measured in cryopreserved serum specimens before and at multiple times after remission induction therapy, and their correlations were assessed.Results: Anti-MDA5 Ab, neopterin, IL-18, ferritin, and sIL-2R levels did not differ significantly between patients who survived and those who succumbed to the disease. In many cases, serum anti-MDA5 Ab titers were over the upper limit (over 150 index value) before treatment in the usual measuring method, and gradually decreased to the normal range at stable phase. Meanwhile, serum neopterin levels (21.6 [15.3–48.3] nmol/L) were significantly elevated in newly diagnosed patients and fell to 6.8 (5–11.4) nmol/L at 6 months after treatment introduction.Conclusions: Elevated serum neopterin as well as ferritin, sIL-2R, KL-6, and anti-MDA5 Ab titer might help identify patients with interstitial lung disease complicated with DM and might be useful in monitoring response to therapy.

Cord blood CD4+/CD25+ T cells down-regulates NKG2D on cytokine-induced killer cells.

Studies have shown that CD4+ CD25+ regulatory T cells (Tregs) could inhibit cytokine-induced killer (CIK) cells against tumor cells, but minimal data have been reported on the underlying mechanisms. The purpose of this study was to investigate the potential suppressive mechanisms of cord blood Tregs on CIK cells in vivo and in vitro. The in vitro study demonstrated that Tregs were normally proliferated and had potent suppressive characteristics. CD4+ CD25+ LAP+ cells were highly expressed as part of activated Tregs, which limited CIK cell-mediated cytotoxicity and reduced the expression of the NKG2D receptor. Interestingly, the inhibitory ability of Tregs could be mimicked by soluble TGF-β1 and neutralizing TGF-β1 antibody could abrogate the inhibitory function of Tregs on CIK cells. In vivo results showed that adoptively transferred CIK cells could delay the tumor growth in nude mice. Moreover, depletion of CD4+ CD25+ Tregs in preculture or blockade of TGF-beta 1 strikingly enhanced CIK cells cytotoxicity. These data indicate that Tregs inhibit CIK cells cytotoxicity mainly by down regulating the expression of NKG2D receptor in a TGF-β dependent manner.

Development of new lateral-flow immunochromatographic strip using colloidal gold and mesoporous silica nanoparticles for rapid diagnosis of active schistosomiasis

Objective: To develop a new sandwich based lateral flow immunochromatographic strip for rapid detection of circulating Schistosoma mansoni antigen in serum and urine samples of patients with active schistosomiasis. Methods: This lateral flow immunochromatographic strip was prepared by using anti-Schistosoma mansoni soluble egg antigen monoclonal antibody conjugated gold nanoparticles (MAb-AuNPs) as antigen-detecting antibody, while crystalline material (MCM)-41-MAb bioconjugate was immobilized at the test line as antigen-capturing antibody. Both antigen capturing and detecting antibodies formed sandwich complexes with circulating Schistosoma mansoni antigen in the positive samples. Sandwich complexes immobilized at the test line gave distinct red color. The assay reliability was examined by using urine and serum samples of 60 Schistosoma mansoni infected patients, 20 patients infected with parasites other than Schistosoma, and 20 healthy individuals as negative controls. Results were compared with those obtained via sandwich enzyme linked immunosorbent assay (ELISA). Results: The detection limit of circulating Schistosoma mansoni antigen by lateral flow immunochromatographic strip was lower (3 ng/mL) than the detection limit by ELISA (30 ng/mL). The sensitivity and specificity of lateral flow immunochromatographic strip in urine samples were 98.3% and 97.5%, respectively compared to 93.5% and 90.0% by ELISA. In serum samples, they were 100.0% and 97.5%, respectively compared to 97.0% and 95.0% by ELISA. The strip test took approximately 10 min to complete. Conclusions: This new lateral flow immunochromatographic strip offers a sensitive, rapid, and field applicable technique for diagnosis of active schistosomiasis.

Expansion of Tumor-Infiltrating Lymphocytes from Melanoma Tumors.

Autologous, in vitro-expanded tumor-infiltrating lymphocytes (TIL) have been successfully used for treatment of melanoma patients. Expansion of TIL from tumors is usually performed in two steps. The details of the procedure differ between different laboratories, but the general concept remains the same. In the first step, small fragments of a tumor are placed in culture medium containing one or more T cell stimulating growth factors. Here the most common protocol using interleukin (IL)-2 is described. The length of the first step is flexible to allow generation of enough cell to start the second step of the procedure. The second step is a so-called rapid expansion protocol (REP) where harvested TIL from the first step are induced to massive proliferation via triggering of their T cell receptor (TCR) complex in presence of an excess of feeder cells and, again, T cell stimulating growth factors. This second expansion step is usually around 2weeks in length. Here will be described a REP that uses soluble anti-CD3 antibodies for TCR triggering, irradiated peripheral blood mononuclear cells (PBMC) as feeder cells, and IL-2 as the T cell growth factor. Furthermore, the described protocol utilizes gas-permeable cell culture flasks that yield large number of cells similar to conventional bioreactors but using standard laboratory equipment.

Parallelized Microscale Expression of Soluble scFv.

Antibody phage display is a key technology to generate recombinant, mainly human, antibodies for diagnostic and therapy, but also as tools for basic research. After antibody selection by "panning," a crucial step is the screening of monoclonal binders to isolate those which show antigen specificity. For this screening procedure, a highly parallelized approach to produce soluble antibody fragments in microtiter plates is essential. In this chapter, we give the protocol for the parallelized microscale production of scFvs for the screening procedure or further assays.

The adjuvant AlhydroGel elicits higher antibody titres than AddaVax when combined with HIV-1 subtype C gp140 from CAP256.

With the HIV-1 epidemic in southern Africa still rising, a prophylactic vaccine against the region’s most prolific subtype (subtype C) would be a significant step forward. In this paper we report on the effect of 2 different adjuvants, AddaVax and AlhydroGel, formulated with HIV-1 subtype C gp140, on the development of binding and neutralising antibody titres in rabbits. AddaVax is a squalene-based oil-in-water nano-emulsion (similar to MF59) which can enhance both cellular and humoral immune responses, whilst AlhydroGel (aluminium hydroxide gel) mainly drives a Th2 response. The gp140 gene tested was derived from the superinfecting virus (SU) from participant CAP256 in the CAPRISA 002 Acute infection cohort. The furin cleavage site of the Env protein was replaced with a flexible linker and an I559P mutation introduced. Lectin affinity purified soluble Env protein was mainly trimeric as judged by molecular weight using BN-PAGE and contained intact broadly neutralising epitopes for the V3-glycan supersite (monoclonal antibodies PGT128 and PGT135), the CD4 binding site (VRC01) and the V2-glycan (PG9) but not for the trimer-specific monoclonal antibodies PG16, PGT145 and CAP256-VRC26_08. When this soluble Env protein was tested in rabbits, AlhydroGel significantly enhanced soluble Env and V1V2 binding antibodies when compared to AddaVax. Finally, AlhydroGel resulted in significantly higher neutralization titres for a subtype C Tier 1A virus (MW965.26) and increased neutralization breadth to Tier 1A and 1B viruses. However, no autologous Tier 2 neutralisation was observed. These data suggest that adjuvant selection is critical for developing a successful vaccine and AlhydroGel should be further investigated. Additional purification of trimeric native-like CAP256 Env and/or priming with DNA or MVA might enhance the induction of neutralizing antibodies and possible Tier 2 HIV-1 neutralisation.

PD-1/PD-L Pathway Potentially Involved in ITP Immunopathogenesis

IntroductionImmune thrombocytopenia (ITP) is an autoimmune disease in which autoreactive T and B cells are activated by platelet autoantigens resulting in immune-mediated platelet destruction and/or suppression of platelet production. The binding of programmed death 1 (PD-1) to its ligands PD-L1 and PD-L2 on antigen-presenting cells turns off autoreactive T cells and induces peripheral tolerance. Aberrant PD-1/PD-L signalling could result in a breakdown of peripheral tolerance and lead to autoimmune diseases.MethodsThirty-four patients with primary active ITP who were diagnosed and/or followed up and 26 healthy controls were enrolled in this study. Platelet counts in all ITP patients were less than 30×109 /L at sampling. They had not been treated with any immunosuppressive agents for at least one week prior to sampling for this study.To determine the role of the PD-1/PD-L signalling pathway in ITP, we detected PD-1 expression on T cells and PD-L expression on dendritic cells (DCs) in both ITP patients with active disease and healthy controls by flow cytometry. To investigate the effects of PD-L1-Fc fusion protein (PD-L1-Fc) on T cells, PBMCs from ITP patients and healthy controls with autologous platelets were cultured with soluble anti-CD3 monoclonal antibody (mAb) and anti-CD28 mAb in the presence (PD-L1-Fc+ group) or absence (PD-L1-Fc- group) of the PD-L1-Fc (0.5 μg/mL) at 37 ˚C with 5% CO2 for 4 days. The cells were harvested and stained for flow cytometry to detect the apoptosis, activation and proliferation of T cells. IL-2 and IFN-γ levels in the co-culture supernatant were assayed by ELISA.ResultsEnhanced PD-1 expression on T cells and decreased PD-L1 expression on mDCs in ITP patientsWe found that PD-1 mean fluorescence intensities (MFIs) increased in CD4+ cells (P < 0.01) and in CD8+ cells (P < 0.01) from ITP patients compared with those from healthy controls. However, PD-L1 expression on monocyte-derived DCs was lower in patients with active ITP than in healthy controls (P < 0.01, Figure 1).PD-L1-Fc promoted T cell apoptosisAnnexin V-FITC and propidium iodide (PI) were used to detect T cell apoptosis. The percentage of apoptotic cells and dead cells were analysed to determine T cell apoptosis levels. In ITP patients, the percentage of apoptotic cells and dead cells were higher in PD-L1-Fc+ group than in the PD-L1-Fc- group (P < 0.01 for both CD4+ and CD8+ T cells). However, we found no significant difference in apoptosis between PD-L1-Fc- and PD-L1-Fc+ groups in healthy controls (Figure 2).PD-L1-Fc inhibited T cell activation and proliferationCD25 MFIs were analysed to determine the activation level of cocultured T lymphocytes. Compared with healthy controls, CD25 expression on CD4+ and CD8+ T cells was significantly increased in ITP patients (P < 0.05 for both CD4+ and CD8+ T cells). These results suggest that ITP patients had more activated CD4+ and CD8+ T cells than in healthy controls. PD-L1-Fc significantly inhibited T cell activation in ITP patients (P < 0.01, n=34) but not in healthy controls (P = 0.0834 in CD4+ T cells, P = 0.6834 in CD8+ T cells, n=26, Figure 3).To analyse proliferation, the frequency of divided cells was calculated according to the loss of CFSE fluorescence intensity. PD-L1-Fc inhibited proliferation of T cells from ITP patients (P < 0.01 in both CD4+ T and CD8+ T cells, n=34, Figure 4). We found no significant difference in proliferation in T cells from healthy controls (CD4+ T cells P = 0.9758, CD8+ T cells P = 0.5658, n=26).PD-L1-Fc inhibited IL-2 and IFN-γ productionIL-2 secretion was higher in ITP than in healthy controls in the absence of PD-L1-Fc (P = 0.0308, Figure 5). PD-L1-Fc significantly inhibited IL-2 and IFN-γ production in ITP patients. IL-2 and IFN-γ levels were lower in the PD-L1-Fc+ group than in the PD-L1-Fc- group in ITP (P < 0.01 for both IL-2 and IFN-γ, n=34). However, we did not detect a significant difference in secretion of IL-2 (P = 0.2016, n=26) or IFN-γ (P = 0.2989, n=26) in healthy controls.ConclusionsIn summary, our study suggests that the aberrant PD-1/PD-L negative costimulatory pathway may play a role in ITP. Enhancing PD-1/PD-L signalling might be a promising therapeutic approach for ITP by promoting T cell apoptosis, inhibiting T cell activation and proliferation, and reducing secretion of inflammatory factors. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Simplified Method for Transduction and Expansion of T Cells for Clinical Application

Introduction: Genetically modified T cells are being investigated to treat a variety of disorders and have been particularly successful in treating B cell cancers. As more effort is poured into new targets, molecular switches, and various other modifications, development of processes to quickly manufacture new products must keep up. Current manufacturing processes often require highly skilled operators and specialized equipment. Here, we demonstrate a simplified, novel method for transduction of T cells, followed by robust expansion in the G-Rex bioreactor with no need for intervention until harvest. A scaled-up, closed-version of the same process, including a closed harvest step with the GatheRex is currently under evaluation.Methods: Frozen peripheral blood mononuclear cells (PBMCs) from healthy donors were used as starting material. PBMCs were thawed, washed, and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags (32-C, Saint-Gobain Cell Therapy) or in G-Rex bioreactors (Wilson Wolf Corporation). Cells were cultured in TexMACS GMP medium (Miltenyi Biotec) with IL-7 and IL-15 throughout. For the transduction step, activated PMBCs and retroviral supernatant were incubated in cell culture bags coated with Retronectin (Takara) or in G-Rex bioreactors with vectofusin-1 (Miltenyi Biotec). Viral constructs contained either a CD34 or CD19 marker detectable by antibody staining. For transduction in the G-Rex, various cell densities, volumes, constructs, and multiplicity of infection (MOIs) were tested. Where applicable, the GatheRex device (Wilson Wolf Corporation) was used for volume reduction and harvest. Transduction efficiency and T cell phenotype were measured by flow cytometry. Cell count and viability were assessed with the NC-3000 (Chemometic). Glucose and lactate concentrations were checked daily for in-processing monitoring.Results: Overall transduction efficiency ranged from 30-90% depending on the experimental conditions. Incubating 1x107 activated PBMCs in 10 ml of medium in a 10-cm2 G-Rex (1.0 ml/cm2) with retrovirus at an MOI of 1 resulted in 3% transduced cells. Addition of vectofusin-1 to the same condition yielded transduction efficiency of 44%. Increasing the MOI to 10 lead to 86% transduced cells. Decreasing the transduction volume from 1.0 ml/cm2 to 0.4 ml/cm2 increased transduction efficiency from 34% to 55%. Reducing the volume further to 0.2 ml/cm2 did not improve efficiency, and rather had a negative impact compared to the 0.4 ml/cm2 condition (38%). 16-24 hrs after transduction, the volume of medium was increased to 10.0 ml/cm2 without a wash step. The dilution in place of a wash step had no negative impact on cell viability. 10.0 ml/cm2 medium supported high viability (>90%) and expansion (30-50 fold) over an additional 9 days without operator intervention. The phenotype of cells expanded in the G-Rex contained a mixed population of CD45RO+ and CD45RA+ cells, with a similar distribution of naive and memory cell subsets in G-Rex and bag cultures. Harvest of cells with the GatheRex was efficient; a 1L volume was reduced 10-fold in 5 minutes, and 95% of cells were recovered.Summary: T cells can be transduced with retroviral vectors in the G-Rex bioreactor. Clinically relevant levels of transgene expression can be achieved by combining reagents in the G-Rex, without complicated coating steps or time-consuming spinning steps. This simplified procedure reduces the hands-on time of the T cell transduction to minutes rather than hours. Transgenic cells can be expanded 30-50-fold in the G-Rex with limited operator intervention and without specialized equipment. DisclosuresKhalil:Bellicum Pharmaceuticals: Employment. Foster:Bellicum: Employment, Equity Ownership. Gagliardi:Bellicum Pharmaceuticals: Employment.

CD38-Specific Biparatopic Heavy Chain Antibodies Display Potent Complement-Dependent Cytotoxicity Against Multiple Myeloma Cells.

CD38 is overexpressed by multiple myeloma cells and has emerged as a target for therapeutic antibodies. Nanobodies are soluble single domain antibody fragments derived from the VHH variable domain of heavy chain antibodies naturally occurring in camelids. We previously identified distinct llama nanobodies that recognize three non-overlapping epitopes of the extracellular domain of CD38. Here, we fused these VHH domains to the hinge, CH2, and CH3 domains of human IgG1, yielding highly soluble chimeric llama/human heavy chain antibodies (hcAbs). We analyzed the capacity of these hcAbs to mediate complement-dependent cytotoxicity (CDC) to CD38-expressing human multiple myeloma and Burkitt lymphoma cell lines. Combinations of two hcAbs that recognize distinct, non-overlapping epitopes of CD38 mediated potent CDC, in contrast to the hcAb monotherapy with only weak CDC capacity. Similarly, combining daratumumab with a hcAb that recognizes a non-overlapping epitope resulted in dramatically enhanced CDC. Further, introducing the E345R HexaBody mutation into the CH3 domain strongly enhanced the CDC potency of hcAbs to CD38-expressing cells. Exploiting their high solubility, we genetically fused two distinct nanobodies into heteromeric dimers via a flexible peptide linker and then fused these nanobody dimers to the hinge, CH2 and CH3 domains of human IgG1, yielding highly soluble, biparatopic hcAbs. These biparatopic hcAbs elicited CDC toward CD38-expressing myeloma cells more effectively than daratumumab. Our results underscore the advantage of nanobodies vs. pairs of VH and VL domains for constructing bispecific antibodies. Moreover, the CD38-specific biparatopic heavy chain antibodies described here represent potential new powerful therapeutics for treatment of multiple myeloma.

Functional and immunogenic characterization of diverse HCV glycoprotein E2 variants

Induction of cross-reactive antibodies targeting conserved epitopes of the envelope proteins E1E2 is a key requirement for an hepatitis C virus vaccine. Conserved epitopes like the viral CD81-binding site are targeted by rare broadly neutralizing antibodies. However, these viral segments are occluded by variable regions and glycans. We aimed to identify antigens exposing conserved epitopes and to characterize their immunogenicity. We created hepatitis C virus variants with mutated glycosylation sites and/or hypervariable region 1 (HVR1). Exposure of the CD81 binding site and conserved epitopes was quantified by soluble CD81 and antibody interaction and neutralization assays. E2 or E1-E2 heterodimers with mutations causing epitope exposure were used to immunize mice. Vaccine-induced antibodies were examined and compared with patient-derived antibodies. Mutant viruses bound soluble CD81 and antibodies targeting the CD81 binding site with enhanced efficacy. Mice immunized with E2 or E1E2 heterodimers incorporating these modifications mounted strong, cross-binding, and non-interfering antibodies. E2-induced antibodies neutralized the autologous virus but they were not cross-neutralizing. Viruses lacking the HVR1 and selected glycosylation sites expose the CD81 binding site and cross-neutralization antibody epitopes. Recombinant E2 proteins carrying these modifications induce strong cross-binding but not cross-neutralizing antibodies. Conserved viral epitopes can be made considerably more accessible for binding of potently neutralizing antibodies by deletion of hypervariable region 1 and selected glycosylation sites. Recombinant E2 proteins carrying these mutations are unable to elicit cross-neutralizing antibodies suggesting that exposure of conserved epitopes is not sufficient to focus antibody responses on production of cross-neutralizing antibodies.

P3.CR-31 A Case of Thymoma with Autoimmune Hepatitis Following Preoperative Chemotherapy

We encountered a case of thymoma with autoimmune hepatitis. A 63-year-old woman was found to have an abnormal finding on chest radiography during a medical checkup, but did not seek further care. She was referred to our hospital one year later when a bilateral hilar abnormality was again found during a checkup. Chest computed tomography (CT) showed a 4.6 × 3.0 × 5.6 cm mass with calcifications in the anterior mediastinum. The tumor invaded the right lung and middle lobe, in association with a 2.0-cm daughter nodule in the head of the main tumor, with multiple pleural seeding lesions in the right thoracic cavity. Although she was asymptomatic, the anti-acetylcholine receptor antibody level was elevated to 33 nmol/L and the soluble IL-2 receptor antibody was elevated to 1,700 U/mL. The mass was diagnosed with CT-guided biopsy as a type-B2 thymic tumor (cT3N0M1a, c-Stage IV). She underwent induction chemotherapy (ADOC, 2 courses). After chemotherapy, abnormal liver enzyme levels (aspartate aminotransferase: 451 U/L, alanine aminotransferase: 529 U/L) were noted on preoperative examination. She was diagnosed with autoimmune hepatitis based on liver biopsy findings and treated with oral prednisolone 30 mg. Surgery was performed after prednisolone dose was gradually decreased to 20 mg. Extended thymectomy and pulmonary wedge resection using median sternotomy were performed. Pleural dissemination was resected using video-assisted thoracoscopic surgery. The pathological findings showed type-B3 thymic carcinoma (pT3N0M1a, p-Stage IV, Masaoka classification IVa). She was placed under observation without any additional treatment. Prednisolone dose has been gradually decreased to 15 mg without exacerbation of liver function. The anti-acetylcholine receptor antibody level decreased after surgery. Autoimmune diseases in association with thymoma are well known, but reports of autoimmune hepatitis are very rare. Autoimmune hepatitis must be considered when acute liver damage is observed.

Anti-CD40 Antibodies do not Cause Thromboembolism as Demonstrated by in Vitro and in Vivo Studies

Clinical trials with non-silenced anti-CD40L (anti-CD154) monoclonal antibodies (mAb) in patients with systemic lupus erythematosus (SLE) and idiopathic thrombocytopenic purpura (ITP) were halted after unexpected fatal thromboembolic events (TE). Previous results have shown that soluble CD40L/anti-CD40L mAb immune complexes (IC) can activate and induce pro-aggregatory effects on platelets in vitro via FcgRIIa. Pulmonary thrombi consisting of platelet aggregates and fibrin, and thrombocytopenia were found in humanized FcgRIIa transgenic mice after injection of pre-formed IC consisting of mouse soluble CD40L and anti-mouse CD40L mAb. To date, no such studies have been performed with IC of mouse sCD40 and anti-mouse CD40 mAbs. We therefore injected either single mouse proteins (soluble CD40L, soluble CD40) or antibodies (isotype controls, anti-mouse CD40L mAb, anti-mouse CD40 mAb), IC of soluble protein with the respective antibody into humanized FcgR transgenic mice. In addition, in vitro platelet aggregation was performed using blood from FcgR transgenic mice and human healthy donors. Whole blood samples from FcgR transgenic mice were treated with the same proteins and antibodies as in the in vivo study. For human whole blood, isotype controls, soluble CD40L, anti-CD40L mAb including IC in different ratios, soluble CD40, aCD40 mAbs (e.g. CFZ533) including IC in different ratios were used. We observed evidence of thromboembolism (thrombi formation in the lung shown by histopathology) and thrombocytopenia with the soluble CD40L/anti-CD40L mAb IC but not with the soluble proteins or antibodies alone, nor with the soluble CD40/anti-CD40 mAb IC. In vitro platelet aggregation assays performed using blood from FcgR transgenic mice and human healthy donors with recombinant proteins, mAbs and IC from respective species confirmed these findings. These data provide in vivo and in vitro evidence that all anti-CD40 mAbs, either alone or IC with soluble CD40 protein do not induce thromboembolism, indicating that the TE associated with anti-CD40L mAbs are target-related but not co-stimulation pathway specific. Combined with clinical evidence, these data further support the notion of minimal risk for patients developing life-threatening thromboembolic events following administration of a pathway blocking anti-CD40 mAb.