Specific Human Monoclonal Antibodies Research Articles

Preclinical studies of an innovative 5T4-ADC ACR246 with the potential to better treat 5T4-positive solid tumors.

e15005 Background: 5T4 oncofetal antigen shows limited expression in normal adult tissues but highly expressed by many different cancers. It drives spread of cancer cells through epithelial mesenchymal transition (EMT), potentiation of CXCL12/CXCR4 chemotaxis and favoring non-canonical Wnt pathway, leading to poor prognosis of cancer patients. Besides, 5T4 has a highly efficient endocytosis ability which enables it to be an ideal target for the development of ADC drugs. A handful of 5T4-targeted ADCs have been evaluated in early phase clinical trials for several years showing only preliminary anti-tumor activity. However, uncertainty of their safety profiles has also raised concerns due to high toxic payloads, MMAF and duocarmycin, being used. Thus, additional approaches to this target with the potential of improved efficacy and reduced toxicity are needed urgently. ACR246, an innovative 5T4-targeted ADC consisting of specific humanized monoclonal antibody site-specifically conjugated with a cell penetrating and moderate toxic payload (DNA topoisomerase I inhibitor, D2102) that has a robust bystander effect through a stable and cleavable linker, with a drug-to-antibody ratio (DAR) of 8, was designed to improve the safety and efficacy in treating tumor patients. Methods: The pharmacologic profiles of ACR246 were assessed in vitro and in vivo including efficacy, pharmacokinetics-pharmacodynamics (PK-PD), safety, and tissue distribution, in 5T4-positive cell lines, cell-derived xenograft (CDX) models and cynomolgus monkeys. Results: ACR246 bound specifically to 5T4 and was internalized into tumor cells followed by D2102 release, which induced DNA damage, cell cycle arrest and apoptosis in 5T4 positive tumor cells in vitro. In vivo low systemic exposure and tumor-specific accumulation of D2102 rendered ACR246 superior efficacy and safety property. Accordingly, ACR246 exhibited robust in vivo anti-tumor activity across various types of 5T4 positive CDX models in a dose-dependent manner, with DNA damage induced by the accumulated D2102 in tumor tissue. GLP toxicity studies indicated that ACR246 was well tolerated in cynomolgus monkeys. The estimated therapeutic index was 24. Conclusions: The overall preclinical profile marks ACR246 has the potential to become the best in class (BIC) 5T4-ADC, showing more favorable benefit-risk ratio in clinic.

Immunohistochemical Evaluation of NKP46 Receptor Expression and the Number of NK Cells in the Endometrium of Patients with Endometriosis.

Endometriosis is a medical condition that can cause infertility in women. Women with endometriosis experience a decrease in NK cell cytotoxic activity against endometrial cells, ultimately contributing to the spread of these cells. To assess the frequency of NK cells and the expression of the NKP46 receptor in endometrial tissue from patients with endometriosis using immunohistochemistry. 30 endometrial tissue specimens were collected from three groups of cases with mild (n=11), moderate (n=10), and severe endometriosis (n=9), respectively. Additionally, 20 normal endometrial tissue specimens were collected as the control group. Immunohistochemical staining was carried out using specific human monoclonal antibodies against CD56 and NKP46 molecules. Cases with severe endometriosis had a significantly higher number of CD56+ uterine NK cells (26.19±2.50) compared to fertile women (15.02±0.622) and women with mild to moderate endometriosis (p<0.001). However, there was no significant difference between the mild to moderate patients compared with the healthy women (p>0.05). Endometrial NKp46 expression was lower in women with severe endometriosis (0.447±0.0829) compared to fertile women (0.987±0.115, p=0.03). The NKp46+/CD56+ cell ratio was also lower in women with severe endometriosis (0.019±0.003) compared to fertile women (0.072±0.011, p=0.01). Women with severe endometriosis demonstrated an increased rate of infiltrated uterine NK cells and a significant decrease in NKP46 expression compared to fertile women. Therefore, NK cells and the NKp46 receptor may be involved in the development of endometriosis.

Abstract LB284: CLAUDIN-1 targeting antibody ALE.C04 drives single activity and restores anti-PD1 efficacy in solid tumors

Abstract Claudin 1 (CLDN1) is a protein confined within the normal epithelial tight junctions of different tissues. Upon malignant transformation, CLDN1 is overxpressed and epitopes become exposed outside the tight junctions (non-Junctional CLDN1). ALE.C04 is a highly specific humanized monoclonal antibody that recognizes a unique CLDN1 exposed epitope in different solid tumors. Herein we show that non-Junctional CLDN1 (NJ-CLDN1) is frequently overexpressed in solid tumors. Pre-clinically, ALE.C04 drives tumor growth inhibition in different CDXs and PDX in vivo tumor models. Notably, CLDN1 expression on tumor cells positively correlates with T cell exclusion, a mechanism described to hinder the efficacy of Checkpoint inhibitors (CPIs). On this line, the overexpression of Cldn1 in mouse tumor cells promoted T-cell exclusion and resistance to anti-PD1 treatment. Importantly, ALE.C04 restored both T-cell infiltration and anti-PD1 efficacy in tumors. Mechanistically, NJ-CLDN1 interacts with different components involved in extracellular matrix remodeling, thus establishing a physical barrier that exclude immune cells from the tumor nest. ALE.C04 perturbs the interface between CLDN1+ tumor cells and the stroma, thus restoring immune cell infiltration. Our pre-clinical data showed that: i) NJ-CLDN1 is a novel and druggable target in solid tumors ii) ALE.C04 is a selective anti-CLDN1 antibody capable of driving anti-tumor activity as both single agent and in combination with anti-PD1. Citation Format: Alberto Toso, Geoffrey Teixeira, Tina Zimmermann, Stefanie Gill Gill, Daniela Schmitter, Markus Meyer, Marion Muller, Laurent Mailly, Thomas Baumert, Luigi Manenti, Roberto Iacone. CLAUDIN-1 targeting antibody ALE.C04 drives single activity and restores anti-PD1 efficacy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB284.

Abstract 1758: Resistance to cancer therapy via upregulation of the NRP2/VEGF-C axis can be neutralized by ATYR2810

Abstract Introduction: Intrinsic or acquired resistance to cancer therapy is a major problem in the treatment of many cancers. Therapy-resistant cancer cells are able to evade the effect of cancer drugs, resulting in highly aggressive clones that can cause poor long-term prognosis. Neuropilin-2 (NRP2) is a cell surface receptor that acts as a co-receptor for vascular endothelial growth factor (VEGF) and has been shown to be associated with therapy resistance. The VEGF-C signaling pathway has also been linked to chemoresistance in several cancer types including breast, bladder, and gastric cancer. Previously we have shown that ATYR2810—a highly specific humanized monoclonal antibody that blocks NRP2/VEGF-C signaling—sensitizes triple negative breast cancer (TNBC), a highly aggressive and chemoresistant subtype of breast cancer, to chemotherapy and targeted anti-VEGF therapy both in vitro and in vivo. In this study we examined the role of enhanced VEGF-C signaling in tumor growth and therapeutic resistance, and the ability of ATYR2810 to neutralize these effects using in vivo models for lung and kidney cancers. Results: We generated a stable VEGF-C overexpressing non-small cell lung cancer (NSCLC) cell line and found that tumor growth was enhanced by increased VEGF-C signaling. Importantly, ATYR2810 monotherapy counteracted this VEGF-C-driven enhanced tumor growth as tumor growth inhibition was increased in the VEGF-C overexpressing tumors compared to control tumors. Further, VEGF-C overexpression was also found to enhance resistance to platinum-based chemotherapy in this model. However, sensitivity to chemotherapy was restored when ATYR2810 was used in combination with cisplatin. In clear cell renal cell carcinoma (ccRCC), the use of sunitinib—a small molecule inhibitor that inhibits the signaling of several receptor tyrosine kinases (RTKs), including vascular endothelial growth factor receptors (VEGFRs)—has reportedly been associated with the upregulation of VEGF-C and NRP2 expression as well as increased metastasis. In line with this, we found that the use of sunitinib leads to increased levels of circulating VEGF-C in the serum of ccRCC xenograft mice. The use of ATYR2810 in combination with sunitinib was found to act synergistically to inhibit tumor growth in ccRCC xenograft tumors leading to complete tumor regression in some animals. Conclusions: Here we demonstrate that high levels of VEGF-C expression increase tumor dependency on the VEGF-C/NRP2 signaling axis thereby promoting tumor growth and chemoresistance. ATYR2810 counteracts the aggressive traits of tumors expressing high levels of VEGF-C resulting in enhanced tumor growth inhibition and sensitivity to both chemotherapy and targeted therapy. Citation Format: Alison Barber, Zhiwen Xu, Lisa Eide, Clara Polizzi, Max Pastenes, Lauren Guy, Jasmine Stamps, Kristina Hamel, Zachary Fogassy, Sofia Klopp-Savino, Esther Chong, Yang Qing, Lara Glendening, Christoph Burkart, Leslie Nangle. Resistance to cancer therapy via upregulation of the NRP2/VEGF-C axis can be neutralized by ATYR2810 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1758.

193P CLAUDIN-1 targeting antibodies in solid tumors: From ALE.C04 to CLAUDIN-1 oncology platform

Claudin 1 (CLDN1) is a protein confined within the normal epithelial tight junctions of different tissues. Upon malignant transformation, CLDN1 is overxpressed and epitopes become exposed outside the tight junctions (non-Junctional CLDN1). ALE.C04 is a highly specific humanized monoclonal antibody that recognizes a unique CLDN1 exposed epitope in different solid tumors.

The Cellular Characterization of SARS-CoV-2 Spike Protein in Virus-Infected Cells Using the Receptor Binding Domain Binding Specific Human Monoclonal Antibodies.

ABSTRACTA human monoclonal antibody panel (PD4, PD5, PD7, SC23, and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2-infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 spike (S) protein, only PD5, PD7, and SC23 were able to bind to the receptor binding domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localized within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD-binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognized the uncleaved S protein, indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining were demonstrated for each isolate, SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection.IMPORTANCE The SARS-CoV-2 spike (S) protein receptor binding domain (RBD) mediates the attachment of SARS-CoV-2 to the host cell. This interaction plays an essential role in initiating virus infection, and the S protein RBD is therefore a focus of therapeutic and vaccine interventions. However, new virus variants have emerged with altered biological properties in the RBD that can potentially negate these interventions. Therefore, an improved understanding of the biological properties of the RBD in virus-infected cells may offer future therapeutic strategies to mitigate SARS- CoV-2 infection. We used physiologically relevant antibodies that were isolated from the B cells of convalescent COVID-19 patients to monitor the RBD in cells infected with SARS-CoV-2 clinical isolates. These immunological reagents specifically recognize the correctly folded RBD and were used to monitor the appearance of the RBD in SARS-CoV-2-infected cells and identified the site where the RBD first appears.

Abstract 311: Development of cludin-3 specific human monoclonal antibody(ABN501) as therapeutic anti-tumor agents

Abstract The tight junctions of epithelial tissues, that most malignant tumors are derived from, are associated in cell-cell interactions. Among the tight junction proteins, claudin-3 (CLDN3) is overexpressed in many types of solid cancers, such as breast, ovarian, colorectal, and gastric cancers. Although CLDN3 can be potential therapeutic target due to the overexpression in various types of cancers, high homology with other CLDN family members is a hurdle to develop antibodies specifically targeting CLDN3. In this study, human IgG1 monoclonal antibody (ABN501) against CLDN3 was developed by scFv phage display using CLDN3-overexpressing stable cells and CLDN3-embedded lipoparticles as antigens. It was confirmed that ABN501 specifically bound to human and mouse CLDN3 without cross-reactivity to other CLDN family members. Sub-nanomolar affinity in binding kinetics of ABN501 was measured in CLDN3 expressing cell lines. We observed the antibody-dependent cytotoxicity (ADCC) activity of ABN501 with human NK cells expressing CD16a (NK-92MI-CD16a), in various cancer cell lines according to CLDN3 expression levels. We also confirmed that ABN501 specifically targeted CLDN3-expressing tumors in biodistribution assay using fluorescence-conjugated ABN501. In addition, ABN501 showed anti-tumor effects when treated with NK-92MI-CD16a in xenograft mice bearing CLDN3 expressing tumors. Taken these results together, we suggest that ABN501, specifically recognizing to CLDN3, can be used as therapeutic agents for CLDN3 positive cancer, and developed in many different forms to treat cancers with its specificity for cancer diagnosis, antibody-drug conjugates, and chimeric antigen receptor (CAR) immunotherapy. Citation Format: Hong-Seok Choi, Euni Sim, Na Young Kim, Yong Jin Lee, Sae Hyung Lee, Hayeon Park, Hobin Yang, Jiwon Jo, Myeung-Ryun Seo, Heegeon Park, Ji-Hye Lee, Sungyoul Hong, Young Kee Shin, Jun-Young Choi. Development of cludin-3 specific human monoclonal antibody(ABN501) as therapeutic anti-tumor agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 311.

Abstract LB085: ATYR2810, a fully humanized monoclonal antibody targeting the VEGF-NRP2 pathway sensitizes highly aggressive and chemoresistant TNBC subtypes to chemotherapy

Abstract Introduction: Triple negative breast cancer (TNBC) represents 15% of all breast cancers, and is a notably aggressive subtype that is prone to early recurrence following initial diagnosis with a median survival of only 9 months following recurrence. One of the few treatment options for patients with advanced and metastatic TNBC is platinum-based chemotherapy, however not all tumors respond, and those that do often become refractory during the course of treatment. The cell surface receptor Neuropilin-2 (NRP2), which acts as a co-receptor for vascular endothelial growth factors (VEGFs), has been shown to be both highly expressed and associated with therapeutic resistance in TNBC. NRP2 also serves as a co-receptor for semaphorins, which may play tumor-suppressor functions in breast cancer. We have previously demonstrated that ATYR2810, a highly specific humanized monoclonal antibody which effectively blocks NRP2/VEGF signaling without disrupting NRP2/Semaphorin 3F signaling, down-regulated epithelial-mesenchymal transition (EMT) genes such as ZEB1 and sensitizes TNBC to chemotherapy both in vitro and in vivo. Results: In this study we have further characterized the breast cancer subtypes that are most responsive to ATYR2810 treatment. Using in vitro 3D colony formation assays (CFAs), we have interrogated ~15 breast cancer cell lines covering luminal, HER2+ and various TNBC subtypes, for responsiveness to ATYR2810 treatment in combination with chemotherapy. All responsive cell lines were associated with the more aggressive subtypes of TNBC such as BL2 (basal-like 2), ML (mesenchymal-like) and MSL (mesenchymal-stem like) subtypes. Additionally, we screened five patient derived xenografts (PDXs) and again found that the responsive PDXs were associated with the more aggressive and chemoresistant subtypes of TNBC. To gain insight into the potential mechanism for enhanced chemosensitivity by ATYR2810 treatment, we performed RNAseq on the PDX tumors. Interestingly, the chemokine receptor CXCR4, which is known to promote drug resistance and tumorigenic potential, was found to be significantly downregulated in responder PDX tumors treated with ATYR2810 in combination with cisplatin as compared to those treated with cisplatin alone. Further, gene set enrichment analysis (GSEA) showed that CXCR4 was found in numerous downregulated pathways enriched in the PDX responder tumors treated with ATYR2810/cisplatin, suggesting that CXCR4 may be driving the responder phenotype of the PDXs. Flow cytometry analysis of TNBC cells treated with ATYR2810 monotherapy in vitro resulted in a reduced frequency of cells expressing CXCR4. Furthermore, ATYR2810 monotherapy inhibited spontaneous lung metastasis in experimental models of mesenchymal TNBC. Conclusion: Taken together, our data suggest that ATYR2810 enhances chemosensitivity in highly aggressive TNBC subtypes, and that this response may be mediated through the downregulation of genes known to be associated with aggressive cancer states such as ZEB1 and CXCR4. ATYR2810 may therefore serve as a novel therapeutic agent for the treatment of advanced and metastatic TNBC and potentially other aggressive cancer types. Zhiwen Xu and Alison Barber are co-first authors of this abstract. Citation Format: Zhiwen Xu, Alison Barber, Christoph Burkart, Hira Lal Goel, Justin Rahman, Kristina Hamel, Zachary Fogassy, Lisa Eide, Clara Polizzi, Jasmine Stamps, Sofia Klopp Savino, Luke Burman, Esther Chong, Suzanne Paz, Arthur M. Mercurio, Leslie A. Nangle. ATYR2810, a fully humanized monoclonal antibody targeting the VEGF-NRP2 pathway sensitizes highly aggressive and chemoresistant TNBC subtypes to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB085.

HLA-DQ-Specific Recombinant Human Monoclonal Antibodies Allow for In-Depth Analysis of HLA-DQ Epitopes.

HLA-DQ donor-specific antibodies (DSA) are the most prevalent type of DSA after renal transplantation and have been associated with eplet mismatches between donor and recipient HLA. Eplets are theoretically defined configurations of surface exposed amino acids on HLA molecules that require verification to confirm that they can be recognized by alloantibodies and are therefore clinically relevant. In this study, we isolated HLA-DQ specific memory B cells from immunized individuals by using biotinylated HLA-DQ monomers to generate 15 recombinant human HLA-DQ specific monoclonal antibodies (mAb) with six distinct specificities. Single antigen bead reactivity patterns were analyzed with HLA-EMMA to identify amino acids that were uniquely shared by the reactive HLA alleles to define functional epitopes which were mapped to known eplets. The HLA-DQB1*03:01-specific mAb LB_DQB0301_A and the HLA-DQB1*03-specific mAb LB_DQB0303_C supported the antibody-verification of eplets 45EV and 55PP respectively, while mAbs LB_DQB0402_A and LB_DQB0602_B verified eplet 55R on HLA-DQB1*04/05/06. For three mAbs, multiple uniquely shared amino acid configurations were identified, warranting further studies to define the inducing functional epitope and corresponding eplet. Our unique set of HLA-DQ specific mAbs will be further expanded and will facilitate the in-depth analysis of HLA-DQ epitopes, which is relevant for further studies of HLA-DQ alloantibody pathogenicity in transplantation.

High affinity human Fc specific monoclonal antibodies for capture kinetic analyses of antibody-antigen interactions

We recently demonstrated that capturing human monoclonal antibodies (hmAbs) using high affinity anti-human Fc (AHC) antibodies allows reliable characterization of antibody-antigen interactions. Here, we characterized six human Fc specific mouse monoclonal antibodies (mAbs) and compared their binding profiles with three previously characterized goat AHC polyclonal antibodies (pAbs), exhibiting properties of a good capture reagent. All six mouse AHC mAbs specifically bound with high affinity to the Fc region of hIgG1, hIgG2, hIgG4 and to 43 different hIgG variants, containing substitutions and/or mutations in the hinge and/or Fc region, that have been reported to exhibit modified antibody effector function and/or pharmacokinetics. Biacore sensor surfaces individually derivatized with mouse AHC mAbs exhibited >2.5-fold higher hIgG binding capacity compared to the three goat AHC pAb surfaces and reproducibly captured hIgG over 300 capture-regeneration cycles. The results of the capture kinetic analyses performed on 31 antibody-antigen interactions using surfaces derivatized with either of the two highest affinity AHC mAbs (REGN7942 or REGN7943) were in concordance with those performed using goat AHC pAb surfaces. Our data demonstrate that AHC mAbs such as REGN7942 and REGN7943 that have properties superior than the three goat AHC pAbs are highly valuable research reagents, especially to perform capture kinetic analyses of antibody-antigen interactions on optical biosensors.

Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as a live virus vaccine candidate.

BackgroundDue to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus.MethodsHere, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type format or a membrane-anchored format lacking the polybasic cleavage site. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, the immunogenicity and protective efficacy of these NDV-based vaccines were investigated.FindingsWe report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly in mice. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs.InterpretationThe results suggested that the NDV vector expressing either the wild type S or membrane-anchored S without the polybasic cleavage site could be used as live vector vaccine against SARS-CoV-2.FundingThis work is supported by an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS) contract, the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract, philanthropic donations and NIH grants.

A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2.

An outbreak of coronavirus disease2019 (COVID-19)1-3, caused by severe acute respiratory syndrome coronavirus2 (SARS-CoV-2)4, has spread globally. Countermeasures are needed to treat and prevent further dissemination of the virus. Here we report the isolation of two specific human monoclonal antibodies (termed CA1 and CB6) from a patient convalescing from COVID-19. CA1 and CB6 demonstrated potent SARS-CoV-2-specific neutralization activity in vitro. In addition, CB6 inhibited infection with SARS-CoV-2 in rhesus monkeys in both prophylactic and treatment settings. We also performed structural studies, which revealed that CB6 recognizes an epitope that overlaps with angiotensin-converting enzyme2 (ACE2)-binding sites in the SARS-CoV-2 receptor-binding domain, and thereby interferes with virus-receptor interactions by both steric hindrance and direct competition for interface residues. Our results suggest that CB6 deserves further study as a candidate for translation to the clinic.

Development of an antibody cocktail for treatment of Sudan virus infection

Antibody-based therapies are a promising treatment option for managing ebolavirus infections. Several Ebola virus (EBOV)-specific and, more recently, pan-ebolavirus antibody cocktails have been described. Here, we report the development and assessment of a Sudan virus (SUDV)-specific antibody cocktail. We produced a panel of SUDV glycoprotein (GP)-specific human chimeric monoclonal antibodies (mAbs) using both plant and mammalian expression systems and completed head-to-head in vitro and in vivo evaluations. Neutralizing activity, competitive binding groups, and epitope specificity of SUDV mAbs were defined before assessing protective efficacy of individual mAbs using a mouse model of SUDV infection. Of the mAbs tested, GP base-binding mAbs were more potent neutralizers and more protective than glycan cap- or mucin-like domain-binding mAbs. No significant difference was observed between plant and mammalian mAbs in any of our in vitro or in vivo evaluations. Based on in vitro and rodent testing, a combination of two SUDV-specific mAbs, one base binding (16F6) and one glycan cap binding (X10H2), was down-selected for assessment in a macaque model of SUDV infection. This cocktail, RIID F6-H2, provided protection from SUDV infection in rhesus macaques when administered at 50 mg/kg on days 4 and 6 postinfection. RIID F6-H2 is an effective postexposure SUDV therapy and provides a potential treatment option for managing human SUDV infection.

Rituximab in dermatology: Revisited

Rituximab (RTX) is a specific mouse and human chimeric monoclonal antibody that has found to have numerous applications in dermatology. Though initially approved by the US FDA for treating rheumatoid arthritis and non-Hodgkin's lymphoma; currently its approval has extended to involve the pemphigus group of disorders; both as a first line drug as well as a therapeutic strategy for recalcitrant cases of pemphigus, unresponsive to conventional therapy. Apart from pemphigus, there have been a number of cutaneous disorders where rituximab has been found to be useful. This review will give a bird's eye view of the applications of rituximab in dermatology.

Ehrlichia chaffeensis Outer Membrane Protein 1-Specific Human Antibody-Mediated Immunity Is Defined by Intracellular TRIM21-Dependent Innate Immune Activation and Extracellular Neutralization.

Antibodies are essential for immunity against Ehrlichia chaffeensis, and protective mechanisms involve blocking of ehrlichial attachment or complement and Fcγ-receptor-dependent destruction. In this study, we determined that major outer membrane protein 1 (OMP-19) hypervariable region 1 (HVR1)-specific human monoclonal antibodies (huMAbs) are protective through conventional extracellular neutralization and, more significantly, through a novel intracellular TRIM21-mediated mechanism. Addition of OMP-1-specific huMAb EHRL-15 (IgG1) prevented infection by blocking attachment/entry, a mechanism previously reported; conversely, OMP-1-specific huMAb EHRL-4 (IgG3) engaged intracellular TRIM21 and initiated an immediate innate immune response and rapid intracellular degradation of ehrlichiae. EHRL-4-TRIM21-mediated inhibition was significantly impaired in TRIM21 knockout THP-1 cells. EHRL-4 interacted with cytosolic Fc receptor TRIM21, observed by confocal microscopy and confirmed by co-immunoprecipitation. E. chaffeensis-EHRL-4-TRIM21 complexes caused significant upregulation of proinflammatory cytokine/chemokine transcripts and resulted in rapid (<30 min) nuclear accumulation of NF-κB and TRIM21 and ehrlichial destruction. We investigated the role of TRIM21 in the autophagic clearance of ehrlichiae in the presence of EHRL-4. Colocalization between EHRL-4-opsonized ehrlichiae, polyubiquitinated TRIM21, autophagy regulators (ULK1 and beclin 1) and effectors (LC3 and p62), and lysosome-associated membrane protein 2 (LAMP2) was observed. Moreover, autophagic flux defined by conversion of LC3I to LC3II and accumulation and degradation of p62 was detected, and EHRL-4-mediated degradation of E. chaffeensis was abrogated by the autophagy inhibitor 3-methyladenine. Our results demonstrate that huMAbs are capable of inhibiting E. chaffeensis infection by distinct effector mechanisms: extracellularly by neutralization and intracellularly by engaging TRIM21, which mediates a rapid innate immune response that mobilizes the core autophagy components, triggering localized selective autophagic degradation of ehrlichiae.

Synergistic effect of IgG4 antibody and CTLs causes tissue inflammation in IgG4-related disease.

IgG4-related disease (IgG4-RD) is characterized by multi-organ irreversible damage resulting from tissue-specific infiltration of IgG4+ plasma cells and cytotoxic T lymphocytes (CTLs). However, whether IgG4 antibody contributes to the inflammation remains unclear. In this study, we established a mouse model that enabled us to evaluate the pathogenic function of IgG4 antibodies in response to a tissue-specific autoantigen using recombinant ovalbumin (OVA)-specific human IgG4 monoclonal antibody (rOVA-hIgG4 mAb) and the mice expressing OVA of the pancreatic islets (RIP-mOVA mice). We found no inflammatory effect of rOVA-hIgG4 mAb transfer alone; however, co-transfer with OVA-specific CD8 CTLs (OT-I T cells) induced tissue damage with dense lymphocytic inflammation in the pancreas of RIP-mOVA mice. rOVA-hIgG4 mAb caused accumulation of conventional DC1 cells (cDC1s) in the lymphoid tissues, and the dendritic cells (DCs) activated the OT-I T cells via cross-presentation. We also revealed that the synergistic effects of CTLs and antibodies were observed in the other subclasses including endogenous antibodies if they recognized the same antigen. The transfer of OVA-specific CD4 helper T cells (OT-II T cells) into RIP-mOVA mice induced the production of anti-OVA antibody, which had a synergistic effect, through acquisition of a T follicular helper (TFH) phenotype. Moreover, using OT-II T cells deficient in Bcl6 caused lower anti-OVA antibody production and inflammation with OT-I T cells. Our results indicated that autoreactive IgG4 antibodies play an important role of the tissue-specific CTL response in IgG4-RD.

Validation in China of a non‐invasive salivary pepsin biomarker containing two unique human pepsin monoclonal antibodies to diagnose gastroesophageal reflux disease

ObjectivesPeptest is a new non‐invasive reflux diagnostic test based on lateral flow technology that containing two highly specific human pepsin monoclonal antibodies for detecting pepsin, a biomarker for reflux disease. The primary aim of this multicenter clinical study was to validate the efficacy of Peptest in patients diagnosed with gastroesophageal reflux and healthy controls in China.MethodsPatients with suspected gastroesophageal reflux underwent an endoscopy and were classified into non‐erosive reflux disease and erosive esophagitis subgroups. A healthy control group was also recruited. All participants were given a reflux disease questionnaire—patients scoring greater than 12 and controls scoring zero. All participants provided a postprandial saliva sample and most patients gave an additional post‐symptom sample for pepsin analysis.ResultsAltogether 1032 participants aged between 19 and 78 years were recruited. They consisted of 488 patients with non‐erosive reflux disease, 221 with erosive esophagitis and 323 healthy controls. The number of postprandial and post‐symptom samples analyzed totaled 1031 and 692, respectively. The results across all centers showed an overall pepsin‐positive sensitivity of 85%, a specificity of 60%, a positive predictive value of 82%, a negative predictive value of 65% and a positive likelihood ratio of 2.12.ConclusionThe sensitivity of Peptest was high, but the specificity achieved in some centers was low, resulting overall in only a moderate specificity. Further diagnostic investigative studies are warranted.

In vitro effects of interleukin (IL)-1 beta inhibition on the epithelial-to-mesenchymal transition (EMT) of renal tubular and hepatic stellate cells

BackgroundThe epithelial to mesenchymal transition (EMT) is a multi-factorial biological mechanism involved in renal and hepatic fibrosis and the IL-1 beta has been assumed as a mediator of this process although data are not exhaustive. Therefore, the aim of our study was to evaluate the role of this cytokine in the EMT of renal proximal tubular epithelial cells (HK-2) and stellate cells (LX-2) and the protective/anti-fibrotic effect of its inhibition by Canakinumab (a specific human monoclonal antibody targeted against IL-1beta).MethodsBoth cell types were treated with IL-1 beta (10 ng/ml) for 6 and 24 h with and without Canakinumab (5 μg/ml). As control we used TGF-beta (10 ng/ml). Expression of EMT markers (vimentin, alpha-SMA, fibronectin) were evaluated through western blotting and immunofluorescence. Genes expression for matrix metalloproteinases (MMP)-2 was measured by Real-Time PCR and enzymatic activity by zymography. Cellular motility was assessed by scratch assay.ResultsIL-1 beta induced a significant up-regulation of EMT markers in both cell types and increased the MMP-2 protein expression and enzymatic activity, similarly to TGF-beta. Moreover, IL-1 beta induced a higher rate of motility in HK-2. Canakinumab prevented all these modifications in both cell types.ConclusionsOur results clearly demonstrate the role of IL-1 beta in the EMT of renal/stellate cells and it underlines, for the first time, the therapeutic potential of its specific inhibition on the prevention/minimization of organ fibrosis.

Abstract 3910: Characterization of BNC101 a human specific monoclonal antibody targeting the GPCR LGR5: First-in-human evidence of target engagement

Abstract At the base of normal intestinal crypts, stem cells maintain the highly regenerative gut epithelium. These intestinal stem cells are well characterized for their high expression of the G coupled protein receptor LGR5 (also known as GPR49). Together with R-spondins (potent Wnt signaling modulators) and stem cell growth factors, LGR5 forms part of a signaling cascade responsible for the regulation of cellular proliferation. Key mutations in the APC or BRAF pathways of intestinal stem cells lead to lesions and metastatic colorectal cancer if not diagnosed and resected at an early stage. The majority of primary and metastatic tumors arising from these mutations overexpress LGR5. It has been reported that metastatic colorectal cancer (CRC) patients expressing higher levels of LGR5 in tumor biopsies had increased rates of relapse. BNC101 is a first-in-class anti-LGR5 humanized monoclonal antibody. Biacore analysis of BNC101 has demonstrated a high affinity to LGR5 (Kd=16nM) with no cross-reactivity to LGR4 or LGR6 receptors. Immunoprecipitation studies have also shown that BNC101 has no off-target binding. The murine equivalent of BNC101 has demonstrated antitumor activity in multiple CRC patient-derived xenografts. Flow cytometry studies on CRC cell lines have revealed that LGR5 is located intracellularly with a small fraction present on the cell surface. To better understand its mechanism of action, BNC101 was conjugated to Alexa Fluor 647 and incubated with human CRC cell lines. It was shown that BNC101 interacts with membrane-bound LGR5 and is internalized within 5 minutes. Incubation of the cells for 24 hours at clinically relevant concentrations led to accumulation of fluorophore-conjugated BNC101 within the cell. This intracellular accumulation of BNC101 was further demonstrated with receptor recycling kinetic studies whereby only partial receptor recycling occurred, which may be attributed to the large intracellular LGR5 pool. CHO cell lines overexpressing LGR5 with a GFP tag were used to determine co-localization of BNC101 with its LGR5 ligand. BNC101 is currently in a safety and dose escalation phase I clinical trial in patients with recurrent metastatic CRC. We were able to demonstrate BNC101 target engagement with LGR5 for the first time in tumor biopsies following treatment. Tumor biopsies were analyzed by mass spectrometry together with Matrix Assisted Laser Desorption/Ionization; co-localization of BNC101 with LGR5 was observed, providing us with evidence for the first time that BNC101 infiltrates the patient tumor and engages with the overexpressed LGR5 receptor. Citation Format: Daniel J. Inglis, John Licari, Kristen R. Georgiou, Nicole L. Wittwer, Ross W. Hamilton, Donna M. Beaumont, Michaela A. Scherer, Tina C. Lavranos. Characterization of BNC101 a human specific monoclonal antibody targeting the GPCR LGR5: First-in-human evidence of target engagement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3910.

Intravenous Thrombolysis in Patients with Acute Ischemic Stroke after a Reversal of Dabigatran Anticoagulation with Idarucizumab: A Real-World Clinical Experience

Intravenous thrombolysis (IVT) is contraindicated in patients with acute ischemic stroke (AIS) using oral anticoagulants. A specific human monoclonal antibody was introduced to reverse immediately the anticoagulation effect of the direct inhibitor of thrombin, dabigatran. Until now, mostly individual cases presenting with successful IVT after a reversal of dabigatran anticoagulation in patients with AIS were published. Thus, we aimed to report real-world data from clinical practice. Patients with AIS on dabigatran treated with IVT after antidote reversal were enrolled in the retrospective nationwide study. Neurological deficit was scored using the National Institutes of Health Stroke Scale (NIHSS) and 90-day clinical outcome using modified Rankin scale (mRS) with a score 0-2 for a good outcome. Intracerebral hemorrhage (ICH) was defined as a presence of any sign of bleeding on control imaging after IVT, and symptomatic intracerebral hemorrhage (SICH) was assessed according to the Safe Implementation of Thrombolysis in Stroke-Monitoring Study (SITS-MOST) criteria. In total, 13 patients (7 men, mean age 70.0 ± 9.1 years) with a median NIHSS admission score of 7 points were analyzed. Of these patients, 61.5% used 2 × 150 mg of dabigatran daily. Antidote was administrated 427 ± 235 minutes after the last intake of dabigatran, with a mean activated prothrombin time of 38.1 ± 27.8 seconds and a mean thrombin time of 72.2 ± 56.1 seconds. Of the 13 patients, 2 had ICH and 1 had SICH, and no other bleeding complications were observed after IVT. Of the total number of patients, 76.9% had a good 3-month clinical outcome and 3 patients (23.1%) died. Recurrent ischemic stroke occurred in 2 patients (15.4%). The data presented in the study support the safety and efficacy of IVT after the reversal of the anticoagulation effect of dabigatran with antidote in a real-world clinical practice.