Recombinant Humanized Antibody Research Articles

Recombinant Humanized IgG1 Antibody Protects against oxLDL-Induced Oxidative Stress and Apoptosis in Human Monocyte/Macrophage THP-1 Cells by Upregulation of MSRA via Sirt1-FOXO1 Axis.

Oxidized low-density lipoprotein (oxLDL)-induced oxidative stress and apoptosis are considered as critical contributors to cardiovascular diseases. Methionine sulfoxide reductase A (MSRA) is a potent intracellular oxidoreductase and serves as an essential factor that protects cells against oxidative damage. Here, we firstly provide evidence that recombinant humanized IgG1 antibody treatment upregulated the expression of MSRA in THP-1 cells to defend against oxLDL-induced oxidative stress and apoptosis. It was also observed that the upregulation of MSRA is regulated by the forkhead box O transcription factor (FOXO1), and the acetylation of FOXO1 increased when exposed to oxLDL but declined when treated with recombinant humanized IgG1 antibody. In addition, we identified that silent information regulator 1 (SIRT1) suppresses FOXO1 acetylation. Importantly, SIRT1 or FOXO1 deficiency impaired the anti-oxidative stress and anti-apoptotic effect of recombinant humanized IgG1 antibody. Together, our results suggest that recombinant humanized IgG1 antibody exerts its anti-oxidative stress and anti-apoptotic function by upregulation of MSRA via the Sirt1-FOXO1 axis.

GMP Manufacturing and IND-Enabling Studies of a Recombinant Hyperimmune Globulin Targeting SARS-CoV-2.

Conventionally, hyperimmune globulin drugs manufactured from pooled immunoglobulins from vaccinated or convalescent donors have been used in treating infections where no treatment is available. This is especially important where multi-epitope neutralization is required to prevent the development of immune-evading viral mutants that can emerge upon treatment with monoclonal antibodies. Using microfluidics, flow sorting, and a targeted integration cell line, a first-in-class recombinant hyperimmune globulin therapeutic against SARS-CoV-2 (GIGA-2050) was generated. Using processes similar to conventional monoclonal antibody manufacturing, GIGA-2050, comprising 12,500 antibodies, was scaled-up for clinical manufacturing and multiple development/tox lots were assessed for consistency. Antibody sequence diversity, cell growth, productivity, and product quality were assessed across different manufacturing sites and production scales. GIGA-2050 was purified and tested for good laboratory procedures (GLP) toxicology, pharmacokinetics, and in vivo efficacy against natural SARS-CoV-2 infection in mice. The GIGA-2050 master cell bank was highly stable, producing material at consistent yield and product quality up to >70 generations. Good manufacturing practices (GMP) and development batches of GIGA-2050 showed consistent product quality, impurity clearance, potency, and protection in an in vivo efficacy model. Nonhuman primate toxicology and pharmacokinetics studies suggest that GIGA-2050 is safe and has a half-life similar to other recombinant human IgG1 antibodies. These results supported a successful investigational new drug application for GIGA-2050. This study demonstrates that a new class of drugs, recombinant hyperimmune globulins, can be manufactured consistently at the clinical scale and presents a new approach to treating infectious diseases that targets multiple epitopes of a virus.

Development of Human Recombinant Antibodies Against ROR1 Tumor Antigen.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal antigen expressed on many types of cancer cells, but not normal adult cells. ROR1 antigen contributes to cancer development and progression by several signaling pathways. ROR1 expression has been associated with tumor growth, survival, and metastasis. In this study specific human recombinant antibodies were selected against ROR1 antigen for their use in cancer immunotherapy. Phage display technology was used to produce phage antibody from a human scFv library. Phage concentration was determined to confirm the phage rescue process. Panning procedure was performed to isolate specific scFv clones against ROR1 epitope. Phage ELISA was done to evaluate the reactivity of the selected scFvs. Two specific human scFvs with frequencies of 20% and 25% were selected against ROR1 peptide. The antibodies showed specific reaction to the corresponding epitopes in phage ELISA. Cancer targeted therapy using human specific antibodies is a new strategy, which is used in cancer therapy. The selected specific scFvs that target ROR1 epitope are human antibodies that originated from a human library and have the potential to be used in clinic in cancer immunotherapy of ROR1 positive tumors without induction of human anti mouse antibody (HAMA) response.

Expanding the Malaria Antibody Toolkit: Development and Characterisation of Plasmodium falciparum RH5, CyRPA, and CSP Recombinant Human Monoclonal Antibodies.

Malaria, an infection caused by apicomplexan parasites of the genus Plasmodium, continues to exact a significant toll on public health with over 200 million cases world-wide, and annual deaths in excess of 600,000. Considerable progress has been made to reduce malaria burden in endemic countries in the last two decades. However, parasite and mosquito resistance to frontline chemotherapies and insecticides, respectively, highlights the continuing need for the development of safe and effective vaccines. Here we describe the development of recombinant human antibodies to three target proteins from Plasmodium falciparum: reticulocyte binding protein homologue 5 (PfRH5), cysteine-rich protective antigen (PfCyRPA), and circumsporozoite protein (PfCSP). All three proteins are key targets in the development of vaccines for blood-stage or pre-erythrocytic stage infections. We have developed potent anti-PfRH5, PfCyRPA and PfCSP monoclonal antibodies that will prove useful tools for the standardisation of assays in preclinical research and the assessment of these antigens in clinical trials. We have generated some very potent anti-PfRH5 and anti-PfCyRPA antibodies with some clones >200 times more potent than the polyclonal anti-AMA-1 antibodies used for the evaluation of blood stage antigens. While the monoclonal and polyclonal antibodies are not directly comparable, the data provide evidence that these new antibodies are very good at blocking invasion. These antibodies will therefore provide a valuable resource and have potential as biological standards to help harmonise pre-clinical malaria research.

A phase I study of TST001, a high affinity humanized anti-CLDN18.2 monoclonal antibody, in combination with capecitabine and oxaliplatin (CAPOX) as a first-line treatment of advanced G/GEJ cancer.

4062 Background: TST001 is a recombinant humanized IgG1 antibody specifically against human Claudin 18.2 (CLDN18.2) with high affinity and enhanced FcR engaging of NK cells, which can induce strong antibody-dependent cellular cytotoxicity (ADCC) activities. TST001 monotherapy dose-escalation study has been completed (IGCC2022-ABS-1152) and promising anti-tumor activities were observed in advanced gastric cancer patients with CLDN18.2 overexpression who had failed multiple lines of prior therapies. The current study is an open-label, multi-center, multiple cohorts, phase I clinical trial of TST001 monotherapy or in combination with standard treatments in Chinese patients with advanced or metastatic solid tumors (NCT04495296). Methods: This cohort is aimed to determine the recommended phase II dose, evaluate safety, tolerability and preliminary efficacy of TST001 in combination with CAPOX as a 1st line treatment of advanced G/GEJ cancer. A 3+3 design was utilized in the dose escalation phase with treatment naïve advanced G/GEJ cancer patients regardless of Claudin18.2 expression. Claudin 18.2 positive G/GEJ cancer patients were planned to be enrolled in the dose expansion phase in selected dose cohorts. Results: The TST001/CAPOX combination cohort was initiated in August 2021 and is ongoing in multiple sites in China with a data cutoff date of January 27, 2022. 12 subjects were dosed with TST001 with a dose range from 1 to 8mg/kg Q3W in combination with the standard dose of CAPOX in the dose escalation phase and one subject with central lab tested Claudin 18.2 overexpression was dosed in the expansion phase of TST001 at 6mg/kg Q3W with CAPOX. There was no subject experienced dose-limiting toxicity. Treatment-emergent adverse events (TEAEs) were mostly grade 1-2, including nausea (76.9%), anemia (69.2%), vomiting, AST increased (63.8% respectively), hypoalbuminemia, ALT increased (46.2%, respectively). Treatment related grade 3 AEs were hypertension （15.4%）, anemia, hypoproteinemia, WBC count decreased, hypocalcemia aggravated (7.7%, respectively). Among the eight patients from the dose escalation part (with no CLDN18.2 expression selection yet with measurable disease ) with at least one post-treatment tumor assessment, four had achieved partial response, three had achieved stable disease and one had progressive disease as the best tumor response per RECIST1.1. Conclusions: Conclusion: TST001 in combination with CAPOX in first line gastric cancer patients is safe and encouraging anti-tumor activities have been observed. Additional data from the 6 mg/kg dose expansion cohort will be updated at the meeting. Clinical trial information: NCT04495296.

A phase II study of KN046 monotherapy as 2nd line and above treatment for unresectable locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC).

e16305 Background: Pancreatic cancer is a malignancy with poor prognosis and high mortality ranking 7th worldwide and with a 5-year survival rate of 10.8%. More than 50% pts are diagnosed at the late stage. The monoclonal antibodies of immune checkpoint inhibitors are also being investigated but has very limit progression free survival (PFS) and overall survival (OS) benefit. KN046, a novel recombinant humanized bispecific antibody, can simultaneously block PD-1/PD-L1 and CTLA-4 pathways and restore T-cell immune response to tumor. KN046 plus nab-paclitaxel/gemcitabine as first-line treatment for unresectable locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC) in a phase 2 study arm 2 (NCT04324307) was reported before. Here, we report the efficacy and safety of KN046 as a monotherapy in the 2nd line and above setting (arm1). Methods: This ongoing phase 2 trial in China enrolled pts with histologically or cytologically confirmed unresectable locally advanced or metastatic PDAC who have ECOG PS of 0-1 and failed to at least one systemic anti-tumor treatment in arm 1. Single agent of KN046 (5mpk, Q2W) were administered until disease progression or intolerable toxicity. Tumor response was assessed according to RECIST 1.1 every 8 weeks. The primary endpoint is investigator-assessed objective response rate (ORR). Secondary endpoints including disease control rate (DCR), PFS, OS and safety, etc. Results: As of Jan 10, 2022, 21pts were enrolled, median (range) age was 57 (51-64) years, 14 (66.7%) pts ECOG 1, 16 (76.2%) pts had distant metastases, and 11 (52,4%) pts had received ≥2 lines systemic treatment. Median KN046 exposure time was 4.1(2.1,6,4) wks. 9 pts were included in the efficacy analysis with 1 PR and 3 SD. 21 pts in the safety analysis. Confirmed ORR was 11.1% (95% CI: 0.28,48.25), and DCR was 44.4% (95% CI: 13.7,78.8). Median PFS was 2.1 months (95% CI:1.61̃7.29), PFS rate in 6 and 9 months were 31.4% and 21%, respectively. Median OS was 7.5months (95% CI:3.02-NR), OS rate in 6 and 9 months were 61.3% and 49.5%, respectively. The incidence of KN046 related treatment-emergent adverse events (TRAE) was 71.4% (15/21), with 14.3% (3/21) ≥grade 3 TRAEs. Two pts experienced TRAE leading to discontinuation. The most common TRAEs (≥10%) were rash (n=6, 28.6%), alanine aminotransferase increased, chills, γ-glutamyl transferase increased and platelet count decreased (n = 3, 14.3%, respectively). No TRAE leading to death occurred. Conclusions: KN046, a bispecific antibody as 2nd line and above monotherapy for unresectable locally advanced or metastatic PDAC patients demonstrated a good PFS and OS benefit as well as acceptable safety profile, supporting KN046 in combination of chemotherapy as first line treatment. Clinical trial information: NCT04324307.

A multicenter, randomized, double-blind phase III clinical study to evaluate the efficacy and safety of KN046 combined with nab-paclitaxel and gemcitabine versus placebo combined with nab-paclitaxel and gemcitabine in patients with advanced pancreatic cancer (ENREACH-PDAC-01).

TPS4189 Background: Pancreatic cancer is biologically aggressive, and the majority of pts was diagnosed as locally advanced or metastatic disease. Nab-paclitaxel and gemcitabine is a frequently used regimen for advanced pancreatic cancer, but chemoresistance is unavoidable. The combination of immunotherapy with chemotherapy have shown efficacy for certain types of cancer, but no similar results have been obtained in pancreatic cancer. KN046, a novel recombinant humanized bispecific antibody, can simultaneously block PD-1/PD-L1 and CTLA-4 pathways and restore T-cell immune response to tumor. In a phase Ⅱ clinical trial (NCT04324307), as of August 10, 2021, 53 pts with unresectable advanced pancreatic cancer had received one cycle KN046 combined AG regimen treatment and 31 subjects had received post-baseline tumor assessment at least once. Objective response rate (ORR) was 45.2% (95% CI: 27.3%, 64.0%) and disease control rate (DCR) was 93.5% (95% CI: 78.6%, 99.2%)，excellent preliminary results have been achieved. Based on this, a phase III pivotal study (ENREACH-PDAC-01) is conducting now in China to verify the efficacy and safety of KN046 plus nab-paclitaxel and gemcitabine as first-line treatment for advanced pancreatic cancer (NCT05149326). Methods: This nationwide multicenter phase III clinical trial enrolled pts with histologically or cytologically confirmed unresectable locally advanced or metastatic pancreatic ductal adenocarcinomas with a WHO performance score of 0 or 1 and expected survival period of more than 3 months. Eligible pts will be randomized 1:1 to intervention group or control group and receive 4-6 cycles of KN046 (5mpk Q2W) or placebo combined with nab-paclitaxel (initial dose: 125mg/m2 D1,8,15 Q4W) and gemcitabine (initial dose: 1000mg/m2 D1,8,15 Q4W) followed by KN046 (5mpk Q2W) or placebo with gemcitabine（QW × 3, 1 week off） maintenance therapy. Treatment will continue until disease progression or intolerable toxicity, withdrawal of consent, loss to follow-up or death, end of study, whichever occurs first. The primary endpoint is overall survival (OS). Key secondary endpoints are ORR and progression free survival (PFS). PFS and ORR will be assessed independently per RECIST v1.1 at screening, every 8 weeks for 1 year and then every 12 weeks until disease progression. Tumor and blood samples will be collected at baseline and during study treatment for pharmacokinetic, immunogenicity and biomarker assessment. The first patient was enrolled in early February 2022. Clinical trial information: NCT05149326.

ED-B-Containing Isoform of Fibronectin in Tumor Microenvironment of Thymomas: A Target for a Theragnostic Approach.

Simple SummaryThe extra-domain B fibronectin (ED-B FN) is highly expressed in thymic epithelial tumors (TETs), as demonstrated by in vivo targeting using 131I-labeled L19 small immunoprotein (131I-L19-SIP) and immunohistochemistry with a predominant expression by stromal cells of a thymoma microenvironment rather than epithelial cells. Such high expression derived from the induction of stromal cells shifts FN production to the ED-B subtype. Our results suggest that Radretumab radioimmunotherapy (R-RIT) inefficacy is not related to low TET ED-B expression but to multifactorial aspects including patients’ inherent characteristics, the pattern expression of the target, the biological characteristics of the tumor, and the format of the target agent, which contribute to the resistance of tumor cells to treatment.Aim: to exploit tissue-specific interactions among thymic epithelial tumor (TETs) cells and extra-domain B fibronectin (ED-B FN). Material and methods: The stromal pattern of ED-B FN expression was investigated through tumor specimen collection and molecular profiling in 11 patients with recurrent TETs enrolled in prospective theragnostic phase I/II trials with Radretumab, an ED-B FN specific recombinant human antibody. Radretumab radioimmunotherapy (R-RIT) was offered to patients who exhibited the target expression. Experiments included immunochemical analysis (ICH), cell cultures, immunophenotypic analysis, Western blot, slot-blot assay, and quantitative RT-PCR of two primary thymoma cultures we obtained from patients’ samples and in the Ty82 cell line. Results: The in vivo scintigraphic demonstration of ED-B FN expression resulted in R-RIT eligibility in 8/11 patients, of which seven were treated. The best observed response was disease stabilization (n = 5/7) with a duration of 4.3 months (range 3–5 months). IHC data confirmed high ED-B FN expression in the peripherical microenvironment rather than in the center of the tumor, which was more abundant in B3 thymomas. Further, there was a predominant expression of ED-B FN by the stromal cells of the thymoma microenvironment rather than the epithelial cells. Conclusions: Our data support the hypothesis that thymomas induce stromal cells to shift FN production to the ED-B subtype, likely representing a favorable hallmark for tumor progression and metastasis. Collectively, results derived from clinical experience and molecular insights of the in vitro experiments suggested that R-RIT inefficacy is unlikely related to low target expression in TET, being the mechanism of R-RIT resistance eventually related to patients’ susceptibility (i.e., inherent characteristics), the pattern expression of the target (i.e., at periphery), the biological characteristics of the tumor (i.e., aggressive and resistant phenotypes), and/or to format of the target agent (i.e., 131I-L19-SIP).

CUB Domain-Containing Protein 1 (CDCP1) Is a Target for Radioligand Therapy in Castration-Resistant Prostate Cancer, including PSMA Null Disease.

With the improvement in overall survival with 177Lu-PSMA 617, radioligand therapy (RLT) is now a viable option for patients with metastatic castration-resistant prostate cancer (mCRPC). However, responses are variable, in part due to low PSMA expression in 30% of patients. Herein, we evaluated whether the cell surface protein CUB domain-containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, including in PSMA-low subsets. CDCP1 levels were evaluated using RNA sequencing from 119 mCRPC biopsies. CDCP1 levels were assessed in 17 post-enzalutamide- or abiraterone-treated mCRPC biopsies, 12 patient-derived xenografts (PDX), and prostate cancer cell lines. 4A06, a recombinant human antibody that targets the CDCP1 ectodomain, was labeled with Zr-89 or Lu-177 and tested in tumor-bearing mice. CDCP1 expression was observed in 90% of mCRPC biopsies, including small-cell neuroendocrine (SCNC) and adenocarcinomas with low FOLH1 (PSMA) levels. Fifteen of 17 evaluable mCRPC biopsies (85%) demonstrated membranous CDCP1 expression, and 4 of 17 (23%) had higher CDCP1 H-scores compared with PSMA. CDCP1 was expressed in 10 of 12 PDX samples. Bmax values of approximately 22,000, 6,200, and 2,800 fmol/mg were calculated for PC3, DU145, and C4-2B human prostate cancer cells, respectively. 89Zr-4A06 PET detected six human prostate cancer xenografts, including PSMA-low tumors. 177Lu-4A06 significantly suppressed growth of DU145 and C4-2B xenografts. The data provide the first evidence supporting CDCP1-directed RLT to treat mCRPC. Expanded studies are warranted to determine whether CDCP1 is a viable drug target for patients with mCPRC.

Monoclonal Antibodies Targeting Surface-Exposed Epitopes of Candida albicans Cell Wall Proteins Confer In Vivo Protection in an Infection Model.

ABSTRACTMonoclonal antibody (mAb)-based immunotherapies targeting systemic and deep-seated fungal infections are still in their early stages of development, with no licensed antifungal mAbs currently being available for patients at risk. The cell wall glycoproteins of Candida albicans are of particular interest as potential targets for therapeutic antibody generation due to their extracellular location and key involvement in fungal pathogenesis. Here, we describe the generation of recombinant human antibodies specifically targeting two key cell wall proteins (CWPs) in C. albicans: Utr2 and Pga31. These antibodies were isolated from a phage display antibody library using peptide antigens representing the surface-exposed regions of CWPs expressed at elevated levels during in vivo infection. Reformatted human-mouse chimeric mAbs preferentially recognized C. albicans hyphal forms compared to yeast cells, and increased binding was observed when the cells were grown in the presence of the antifungal agent caspofungin. In J774.1 macrophage interaction assays, mAb pretreatment resulted in the faster engulfment of C. albicans cells, suggesting a role of the CWP antibodies as opsonizing agents during phagocyte recruitment. Finally, in a series of clinically predictive mouse models of systemic candidiasis, our lead mAb achieved improved survival (83%) and a several-log reduction of the fungal burden in the kidneys, similar to the levels achieved for the fungicidal drug caspofungin and superior to the therapeutic efficacy of any anti-Candida mAb reported to date.

The pemphigus monoclonal antibody clone P5G6 (AX353) retains cell-surface staining by immunofluorescence and desmoglein binding by ELISA in an engineered scFv-Fc format

The recombinant human antibody clone P5G6 (AX353) in an engineered scFv-Fc format detects by immunofluorescence cell-surface proteins on cryosections of monkey esophagus. The same antibody detects by ELISA recombinant human desmoglein 3 and desmoglein 1 protein.

Antibodies against Anthrax Toxins: A Long Way from Benchlab to the Bedside.

Anthrax is an acute disease caused by the bacterium Bacillus anthracis, and is a potential biowarfare/bioterrorist agent. Its pulmonary form, caused by inhalation of the spores, is highly lethal and is mainly related to injury caused by the toxins secretion. Antibodies neutralizing the toxins of B. anthracis are regarded as promising therapeutic drugs, and two are already approved by the Federal Drug Administration. We developed a recombinant human-like humanized antibody, 35PA83 6.20, that binds the protective antigen and that neutralized anthrax toxins in-vivo in White New Zealand rabbits infected with the lethal 9602 strain by intranasal route. Considering these promising results, the preclinical and clinical phase one development was funded and a program was started. Unfortunately, after 5 years, the preclinical development was cancelled due to industrial and scientific issues. This shutdown underlined the difficulty particularly, but not only, for an academic laboratory to proceed to clinical development, despite the drug candidate being promising. Here, we review our strategy and some preliminary results, and we discuss the issues that led to the no-go decision of the pre-clinical development of 35PA83 6.20 mAb. Our review provides general information to the laboratories planning a (pre-)clinical development.

Fully human recombinant antibodies against EphA2 from a multi-tumor patient immune library suitable for tumor-targeted therapy

ABSTRACT Enhanced EphA2 expression is observed in a variety of epithelial-derived malignancies and is an important target for anti-tumor therapy. Currently, Therapeutic monoclonal antibodies against immune checkpoints have shown good efficacy for tumor treatment. In this study, we constructed an immune single-chain fragment variable (scFv) library using peripheral blood mononuclear cells (PBMCs) from 200 patients with a variety of malignant tumors. High affinity scFvs against EphA2 can be easily screened from the immune library using phage display technology. Anti-EphA2 scFvs can be modified into any form of recombinant antibody, including scFv-Fc and full-length IgG1 antibodies, and the recombinant antibody affinity was improved following modification. Among the modified anti-EphA2 antibodies the affinity of 77-IgG1 was significantly increased, reaching a pmol affinity level (10−12). We further demonstrated the binding activity of recombinant antibodies to the EphA2 protein, tumor cells, and tumor tissues using macromolecular interaction techniques, flow cytometry and immunohistochemistry. Most importantly, both the constructed scFvs-Fc, as well as the IgG1 antibodies against EphA2 were able to inhibit the growth of tumor cells to some extent. These results suggest that the immune libraries from patients with malignant tumors are more likely to screen for antibodies with high affinity and therapeutic effect. The constructed fully human scFv immune library has broad application prospects for specific antibody screening. The screened scFv-Fc and IgG1 antibodies against EphA2 can be used for the further study of tumor immunotherapy.

Preclinical Studies into Specific Toxicity of a Candidate Drug Based on Complement C3 neodeterminant Recombinant Humanized Antibody for Treating Traumatic Brain Injuries

This study aims to assess the specific toxicity (allergenicity, immunotoxicity and reproduction toxicity) of a dosage form of complement C3 neodeterminant recombinant humanized antibody. The reactions of general anaphylaxis, active cutaneous anaphylaxis and delayed-type reaction demonstrated no drug-related signs of experimental animal sensitization and immediate or delayed hypersensitivity in the treated animals. It was found that, in doses exceeding significantly the expected human therapeutic dose, the drug has no effect on humoral and cell-mediated immune responses. In addition, the drug does not suppress the phagocytic cell activity thereby indicating the absence of immunotoxic effect. Moreover, the drug has no adverse effects on male and female reproduction, progeny embryonal development, as well as prenatal and postnatal progeny development. The obtained data can be used in future clinical studies of the drug safety.

Reactivities of a Prostanoid EP2 Agonist, Omidenepag, Are Useful for Distinguishing between 3D Spheroids of Human Orbital Fibroblasts without or with Graves' Orbitopathy.

Background. To obtain new insights into the activation of the thyroid-stimulating hormone (TSH) and insulin-like growth factor 1 (IGF-1) receptors in human orbital fibroblasts (n-HOFs), the effects of the prostanoid EP2 agonist, omidenepag (OMD), and a rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) were evaluated using three-dimension (3D) n-HOFs spheroids in the absence and presence of the recombinant human TSH receptor antibodies, M22 and IGF-1. Methods. The effects of 100 nM OMD or 10 μM Rip on the physical properties, size, stiffness, and mRNA expression of several extracellular matrix (ECM) molecules, their regulator, inflammatory cytokines, and endoplasmic reticulum (ER) stress-related factors were examined and compared among 3D spheroids of n-HOFs, M22-/IGF-1-activated n-HOFs and GO-related human orbital fibroblasts (GHOFs). Results. The physical properties and mRNA expressions of several genes of the 3D n-HOFs spheroids were significantly and diversely modulated by the presence of OMD or Rip. The OMD-induced effects on M22-/IGF-1-activated n-HOFs were similar to the effects caused by GHOHs, but quite different from those of n-HOFs. Conclusions. The findings presented herein indicate that the changes induced by OMD may be useful in distinguishing between n-HOFs and GHOFs.

Characterization of an In Vivo Neutralizing Anti-Vaccinia Virus D8 Single-Chain Fragment Variable (scFv) from a Human Anti-Vaccinia Virus-Specific Recombinant Library.

A panel of potent neutralizing antibodies are protective against orthopoxvirus (OPXV) infections. For the development of OPXV-specific recombinant human single-chain antibodies (scFvs), the IgG repertoire of four vaccinated donors was amplified from peripheral B-lymphocytes. The resulting library consisted of ≥4 × 108 independent colonies. The immuno-screening against vaccinia virus (VACV) Elstree revealed a predominant selection of scFv clones specifically binding to the D8 protein. The scFv-1.2.2.H9 was engineered into larger human scFv-Fc-1.2.2.H9 and IgG1-1.2.2.H9 formats to improve the binding affinity and to add effector functions within the human immune response. Similar binding kinetics were calculated for scFv-1.2.2.H9 and scFv-Fc-1.2.2.H9 (1.61 nM and 7.685 nM, respectively), whereas, for IgG1-1.2.2.H9, the Michaelis-Menten kinetics revealed an increased affinity of 43.8 pM. None of the purified recombinant 1.2.2.H9 formats were able to neutralize VACV Elstree in vitro. After addition of 1% human complement, the neutralization of ≥50% of VACV Elstree was achieved with 0.0776 µM scFv-Fc-1.2.2.H9 and 0.01324 µM IgG1-1.2.2.H9, respectively. In an in vivo passive immunization NMRI mouse model, 100 µg purified scFv-1.2.2.H9 and the IgG1-1.2.2.H9 partially protected against the challenge with 4 LD50 VACV Munich 1, as 3/6 mice survived. In contrast, in the scFv-Fc-1.2.2.H9 group, only one mouse survived the challenge.

CD200 Expression on Multiple Myeloma Cells Induces Attenuation of T Cell-Mediated Cytotoxicity Via DOK2

IntroductionThe CD200/CD200 receptor (CD200R) axis is known to exert immunoregulatory effects in myeloid-derived cells and constitutes a putative immune checkpoint in hematological malignancies, in which CD200 expression is associated with poor prognosis. In multiple myeloma (MM), CD200 is expressed in the majority of patient-derived primary cells. However, its functional importance as well as the related downstream mechanism upon CD200 ligand binding to its CD200R on T cells are not well understood. In this study, we analyze the functional role of CD200 as a potential immune checkpoint in MM and decipher the mechanism of CD200-mediated immune escape.MethodsPrimary MM cells and MM cell lines were analyzed for CD200 surface expression by flow cytometry. To overexpress CD200 on non-expressing MM cell lines we used a Sleeping Beauty transposon vector system. CD200+/- MM cell lines L363, U266 and MM.1s were co-cultured with CD3/CD28-activated healthy donor T cells. T cell-mediated cytotoxicity in these co-cultures was assessed with flow cytometry and/or luciferase assay. Moreover, to analyze the effects of CD200R activation on downstream signaling pathways, activated T cells were treated with recombinant human CD200 (rhCD200) and/or anti-CD200 blocking antibody and subsequently, Western blotting was performed.ResultsOf n=120 primary MM samples (n=120) analyzed, CD200 protein expression could be detected in ca. 75 % of the cases. In contrast, all n=9 MM cell lines tested neither displayed surface nor cytoplasmic CD200 expression. Therefore, using a Sleeping Beauty transposon vector system we stably expressed CD200 on MM cell lines for further analyses. In the presence of CD200-expressing MM cells up to 50% decrease in CD3+ T cell-mediated cytotoxicity against MM cells was observed in flow cytometry and luciferase assay. Proliferation rates of MM cell lines remained unchanged regardless of the level of CD200 overexpression as determined by Alamar blue assays. In myeloid-derived cells, CD200R directly interacts with docking protein-2 (DOK2). In activated T cells, we observed DOK2 phosphorylation upon CD200 binding when treated with rhCD200 in a time- and concentration-dependent manner. Applying an anti-CD200 blocking antibody, this effect could be reversed, thus revealing a possible mechanism for the observed attenuation of T cell function.ConclusionOur study shows that anti-MM cytotoxicity from primary healthy donor CD3+ T cells is attenuated by CD200 expression on MM cells. We also demonstrate that this inhibitory mechanism in CD3+ T cells is mediated via DOK2, providing a potential target for immunotherapeutic approaches in MM. DisclosuresEinsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding.

OAB-017: Attenuation of T cell cytotoxicity mediated by CD200 expression on multiple myeloma cells

<h3>Background</h3> The CD200/CD200 receptor (CD200R) axis is known to entail an immunoregulatory function in myeloid derived cells. The ligand, CD200, is implicated in solid and hematological malignancies to be associated with poor prognosis. Multiple myeloma (MM), a plasma cell malignancy, shows strong expression of CD200 in the majority of patient derived primary cells. However, the downstream mechanism upon CD200 ligand binding to the CD200R in T cells is not well understood. In this study, we evaluate the role of CD200 as a potential immune checkpoint in MM and seek to unravel the mechanism of immune escape mediated by CD200. <h3>Methods</h3> CD200 expression on patient-derived primary MM cells and MM cell lines was tested using flow cytometry. A Sleeping Beauty Transposon vector system was used to overexpress CD200 on MM cell lines. CD3/CD28-activated healthy donor T cells were co-cultured with CD200+/- MM cell lines L363, U266 and MM.1s. Using flow cytometry or luciferase assay, the co-culture was analyzed to assess cytotoxicity. To study downstream signaling effects of CD200R activation, CD3/CD28-activated T cells were treated with recombinant human CD200 (rhCD200) and/or anti-CD200 blocking antibody. Western blotting was performed to analyze potential downstream effector signaling. <h3>Results</h3> Approximately three-quarters of patient-derived primary MM cells (n=120) expressed CD200. In MM cell lines (n=9), no surface or cytoplasmic expression of CD200 was observed. Hence, we stably expressed CD200 on MM cell lines using a Sleeping Beauty transposon vector system. In both, flow cytometric analysis and luciferase assay, we observed up to 50% decrease in CD3+ T cell-mediated cytotoxicity in the presence of CD200-expressing MM cells. Possible differences in proliferation rates of MM cell lines due to CD200 overexpression were ruled out by performing an Alamar blue assay. In myeloid derived cells, docking protein-2 (DOK2) is known to directly interact with CD200R. DOK2 was phosphorylated upon CD200 binding in activated T cells when treated with rhCD200 in a time and concentration-dependent manner. This effect could be reversed with an anti-CD200 blocking antibody, providing a mechanistic explanation for the observed attenuation of T cell function. <h3>Conclusions</h3> CD200 expression on MM cell lines leads to attenuated cytotoxicity from primary healthy donor CD3+ T cells. Here, we provide evidence that this inhibitory mechanism in CD3+ T cells is mediated via DOK2.

Clinical management of snakebite envenoming: Future perspectives.

Snakebite envenoming is a major cause of morbidity and mortality in rural communities throughout the tropics. Generally, the main clinical features of snakebites are local swelling, tissue necrosis, shock, spontaneous systemic hemorrhage, incoagulable blood, paralysis, rhabdomyolysis, and acute kidney injury. These clinical manifestations result from complex biochemical venom constituents comprising of cytotoxins, hemotoxins, neurotoxins, myotoxins, and other substances. Timely diagnosis of envenoming and identification of the responsible snake species is clinically challenging in many parts of the world and necessitates prompt and thorough clinical assessment, which could be supported by the development of reliable, affordable, widely-accessible, point-of-care tests. Conventional antivenoms based on polyclonal antibodies derived from animals remain the mainstay of therapy along with supportive medical and surgical care. However, while antivenoms save countless lives, they are associated with adverse reactions, limited potency, and are relatively inefficacious against presynaptic neurotoxicity and in preventing necrosis. Nevertheless, major scientific and technological advances are facilitating the development of new molecular and immunologic diagnostic tests, as well as a new generation of antivenoms comprising human monoclonal antibodies with broader and more potent neutralization capacity and less immunogenicity. Repurposed pharmaceuticals based on small molecule inhibitors (e.g., marimastat and varespladib) used alone and in combination against enzymatic toxins, such as metalloproteases and phospholipase A2s, have shown promise in animal studies. These orally bioavailable molecules could serve as early interventions in the out-of-hospital setting if confirmed to be safe and efficacious in clinical studies. Antivenom access can be improved by the usage of drones and ensuring constant antivenom supply in remote endemic rural areas. Overall, the improvement of clinical management of snakebite envenoming requires sustained, coordinated, and multifaceted efforts involving basic and applied sciences, new technology, product development, effective clinical training, implementation of existing guidelines and therapeutic approaches, supported by improved supply of existing antivenoms.

Measuring the Anticoagulation Effects of Exosite I Thrombin Inhibitors: Utility and Limitations of Clotting Assays, Thromboelastography, and Calibrated Automated Thrombography

The ability to anticoagulate patients at elevated risk for thrombosis without inducing a bleeding diathesis remains an unmet need of patient care both in a hospital setting and at home. Traditional anticoagulants including warfarin and heparin effectively inhibit clotting, but incur a bleeding risk and require continued monitoring after a stable profile has been established. Current generation direct oral anticoagulants (DOACs) including dabigatran, rivaroxaban, and apixaban overcome many of the liabilities of warfarin and heparin (e.g. lack of target specificity and need for monitoring), but are still susceptible to bleeding diathesis. Thus there remains a need for novel anticoagulants that offer an improved therapeutic index. The recombinant human antibody JNJ-64179375 (JNJ-9375) is a novel thrombin inhibitor that binds to the exosite I region with high affinity and selectivity. In contrast to DOACs that inhibit the protease active site, JNJ-9375 inhibits the interaction of thrombin with its substrates including fibrinogen and PAR-1 while retaining active site and exosite II mediated interactions. This targeted and distinct mechanism of action which modulates the actions of thrombin offers the potential for efficacious anticoagulation with the potential for reduced risk of bleeding compared to active site-specific DOACs.To assess the ability to measure exosite I-specific thrombin inhibitors effects in blood, we compared JNJ- 9375 to the active site ligand dabigatran in clotting assays, thromboelastography (TEG), and calibrated automated thrombography (CAT) assays. We focused on clinically relevant concentrations of these inhibitors based on the efficacious doses reported in the RE-LY study for dabigatran (NCT00262600) and a Phase 1 study for JNJ-9375 (NCT02949206). In conventional clotting assays (prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT)) dabigatran and JNJ-9375 each prolonged clotting times in a dose-dependent manner. TT assays were the most sensitive to dabigatran and JNJ-9375, with 80 nM dabigatran prolonging the clotting time by 5-fold and 100 nM JNJ-9375 prolonging the clot time 4-fold over vehicle control. PT and aPTT assays were less sensitive than the TT assay, requiring ≥ 300 nM dabigatran and ≥ 1 µM JNJ- 9375 to double the PT or aPTT clotting times. In TEG assays both JNJ-9375 and dabigatran prolonged the reaction time (R), with 200 nM JNJ-9375 prolonging R by 1.3- to 1.5-fold and 100 nM dabigatran prolonging R from 1.6- to 1.8-fold over buffer control. In CAT assays initiated with 5 pM tissue factor (PPP Reagent, Stago), dabigatran inhibited peak thrombin and Endogenous thrombin potential (ETP) with an approximate IC50 of 1 µM. In contrast, titration of JNJ-9375 up to 1 µM increased both the Peak thrombin and ETP ≥ 20% above buffer control. This increase in peak thrombin and ETP with JNJ-9375 was also observed with other exosite I-specific ligands such as hirugen, suggesting a general effect of exosite I binding on the primary endpoints of the CAT assay. This insensitivity of the CAT assay to exosite I ligands likely reflects the inability of these molecules to inhibit proteolysis of the peptidyl substrate z-Gly-Gly-Arg-AMC, which directly binds to the active site and is used as the readout of thrombin activity.In summary, of the assays tested the TT clotting assay offers the greatest sensitivity to detect multiple classes of direct thrombin inhibitors in plasma, while TEG can detect direct thrombin inhibitors in whole blood. In contrast, the CAT assay is insensitive to exosite I ligands such as JNJ-9375. These results highlight the importance of the relationship between the endpoint of a given coagulation assay and the mechanism of action of the anticoagulant being tested. DisclosuresBunce:Janssen Research & Development: Employment. Huang Devine:Janssen Research & Development: Employment. Chintala:Janssen Research & Development: Employment.