Murine IgG Research Articles

Screening of Neutralizing Antibodies against FaeG Protein of Enterotoxigenic Escherichia coli.

The misuse of antibiotics in veterinary medicine presents significant challenges, highlighting the need for alternative therapeutic approaches such as antibody drugs. Therefore, it is necessary to explore the application of antibody drugs in veterinary settings to reduce economic losses and health risks. This study focused on targeting the F4ac subtype of the FaeG protein, a key adhesion factor in enterotoxigenic Escherichia coli (ETEC) infections in piglets. By utilizing formaldehyde-inactivated ETEC and a soluble recombinant FaeG (rFaeG) protein, an antibody library against the FaeG protein was established. The integration of fluorescence-activated cell sorting (FACS) and a eukaryotic expression vector containing murine IgG Fc fragments facilitated the screening of anti-rFaeG IgG monoclonal antibodies (mAbs). The results demonstrate that the variable regions of the screened antibodies could inhibit K88-type ETEC adhesion to IPEC-J2 cells. Furthermore, in vivo neutralization assays in mice showed a significant increase in survival rates and a reduction in intestinal inflammation. This research underscores the potential of antibody-based interventions in veterinary medicine, emphasizing the importance of further exploration in this field to address antibiotic resistance and improve animal health outcomes.

Combined treatment with cetuximab and STA9090 has synergistic anticancer effects on human non-small cell lung cancer.

Cetuximab (CET), a human murine chimeric IgG monoclonal antibody and an inhibitor of epidermal growth factor receptor (EGFR), has been shown to be effective in treating various types of cancer. However, its use is hindered by limitations such as resistance development, variability in patient response, side effects, and challenges in biomarker identification. Therefore, CET is often combined with other targeted therapies or chemotherapies to enhance its effectiveness. In this study, we investigate the anticancer effects and underlying mechanisms of the combination of CET, an EGFR inhibitor, and STA9090, an inhibitor of heat shock protein 90 (Hsp90), in both in vitro and in vivo models of non-small cell lung cancer (NSCLC). The results demonstrate significantly stronger effects on NSCLC cells in response to combination therapy than to treatment with either agent alone, indicating that the combination of CET and STA9090 has potential synergistic effects. Additionally, the combination therapy inhibits tumor growth in a xenograft nude mouse model more effectively than treatment with either agent alone, suggesting improved efficacy when used together. Furthermore, the synergistic effects of the combination therapy are likely due to inactivation of the receptor tyrosine kinase (RTK) pathway, which is overly activated in cancer and contributes to tumor growth, angiogenesis, and metastasis. Consequently, our findings suggest that STA9090 has potent direct antitumor activity and synergizes with CET against NSCLC tumors. It is highly likely that these synergistic effects are mediated through RTK pathway inactivation caused by the combination. Therefore, our findings strongly and consistently support the potential synergistic effect of STA9090, an RTK inhibitor, in combination with EGFR-targeting agents.

Glycoprotein VI Platelet Receptor Blockade Attenuates Progression of Established Abdominal Aortic Aneurysms in Murine Models

Objective: Abdominal aortic aneurysms (AAA) frequently feature the formation of a nonocclusive intraluminal thrombus (ILT) along the site of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation via the glycoprotein VI receptor pathway impacts the pathogenesis of AAA. Methods and Results: RNA sequencing was performed on age-matched control human aortic tissue, AAA aortic tissue, and AAA ILT. Platelet transcripts comprised 25% of significantly enriched genes in the comparison of AAA thrombus to AAA tissue and control aortic wall, with GPVI increased by 9.1 Log2-FC (P = 3.21E−08). Circulating GPVI was significantly elevated in platelets isolated from AAA patients versus age-matched controls. Soluble GPVI (sGPVI—indicator of platelet activation), was significantly increased in the plasma of patients with fast-growing AAAs compared to slow-growing AAAs and healthy control subjects. To determine if GPVI blockade was protective from AAA progression, mice underwent either the angiotensin II (AngII) infusion or the topical elastase models of aneurysm formation. Following model initiation, aortic diameter was monitored by weekly ultrasounds. After two weeks, mice which developed an aneurysm (defined as an aortic diameter of >1.2mm) were randomized into control (IgG—50μg) or treatment (JAQ1—50μg, monoclonal GPVI antibody) groups. As expected, platelets isolated from JAQ1 treated mice did not activate in response to the GPVI specific agonist convulxin. Furthermore, JAQ1 treatment attenuated aneurysm progression, increased type I collagen deposition, attenuated macrophage accumulation, and increased survival in both murine AAA models versus IgG control treated mice. Conclusions: Platelets play a critical role in AAA pathophysiology and specific blockade of the GPVI-mediated activation pathway attenuates progression of established aneurysms in two independent murine models. Thus, our work demonstrates that specific targeting of GPVI to blunt platelet activation in AAA could be a potential therapeutic for a pathology with no current pharmacological treatments. R01HL147171-04 and R01HL158801-02. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract LB355: Cryo-immune vaccination (CIV) by SYNC-T therapy: Preclinical modeling of a novel device-multidrug immunotherapeutic approach to eradicate advanced metastatic cancers

Abstract A major challenge to further development of immune checkpoint therapies (ICT) for advanced cancer is the inherent toxicity of higher-order multidrug combinations likely needed to eradicate metastatic disease. Recently, early clinical studies have investigated a new general approach to address this challenge, termed cryo-immune vaccination (CIV). The CIV approach by SYNC-T therapy involves cycles of local cryolysis of a solid tumor followed by infusion of the same lesion with a low-dose multi-API formulation. CIV has never been explored methodically in preclinical or clinical settings, so the basis for systemic responses that have been observed in early clinical testing are not fully understood. Accordingly, as trials of the safety and efficacy of CIV proceed, there is now a need of preclinical model systems to help illuminate the mechanistic foundations of the approach, investigate safety and efficacy, and learn where and how it might be best leveraged. To develop the conceptual framework of CIV, we adapted the orthotopic metastatic 4T1 breast cancer model used extensively in our laboratory as a foundation to study CIV antitumor responses and mechanisms of action. Briefly, breast tumors were initiated in naïve murine subjects by orthotopic injection of 1x104 cells into the mammary fad pads of immunocompetent BALB/c hosts. When tumors reached ~5-7 mm, a time when occult pulmonary metastases have seeded, primary tumors were exposed surgically on skin flaps and contacted directly with a liquid nitrogen-cooled pointed steel rod for 15-30 sec. The rod was then withdrawn and tumor tissue allowed to thaw fully before injection into the cryolysed tumor of a selected ICT multidrug cocktail (or negative control murine IgG) in 25-50 µl total volume. The selected multidrug cocktail was modeled on SYNC-T combination therapy currently under study in a Phase 1 clinical trial (Syncromune, Inc.) Briefly, the ICT drug cocktails were variably composed of murine-reactive antibodies against CTLA4, PD-1 or OX40 (agonist), CD40L (agonist) and CpG oligonucleotide. As a starting point, allometric scaling was used to model dosing analogous to the proprietary ICT drug formulations SV-101 and SV-102 in clinical testing (Syncromune, Inc.). For exploratory toxicology, a standard panel of blood-borne liver and kidney toxicity biomarkers was assessed. For exploratory pharmacology, a ‘super mouse’ design was employed with ELISA-based determinations to measure antibody distribution in blood serum. To gain initial mechanistic insights, responses will be compared in host mice that are genetically deficient in selected components of the adaptive or innate immune systems. Progress in methodological and mechanistic studies will be presented. Citation Format: James B. DuHadaway, Alexander J. Muller, Lisa D. Laury-Kleintop, U. Margaretha Wallon, Susan K. Gilmour, Marie Webster, Jason R. Williams, Gabriela R. Rossi, Mario R. Mautino, Jonathan Lewis, Charles J. Link, George C. Prendergast. Cryo-immune vaccination (CIV) by SYNC-T therapy: Preclinical modeling of a novel device-multidrug immunotherapeutic approach to eradicate advanced metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB355.

Abstract 5901: A targetable secreted protein drives pancreatic cancer metastatic colonization and HIF1a nuclear retention

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an increasingly diagnosed cancer that kills 90% of afflicted patients. Thus, effective systemic therapy to PDAC remains a significant unmet medical need. NPTX1 is over-expressed in in vivoselected highly metastatic pancreatic cancer cells and promotes liver metastasis: Ex vivo mRNAseq of highly metastatic human and murine PDAC liver metastatic tumors identified neuronal pentraxin 1 (NPTX1) as a cancer secreted protein that becomes over-expressed during PDAC metastatic progression. CRISPRi-based silencing of NPTX1 reduced liver metastatic tumor burden by 15-fold in the highly metastatic clones. NPTX1 is over-expressed in PDAC liver metastases and predicts survival of PDAC patients: To assess NPTX1 protein expression in situ, two independent board-certified pathologists developed an immunohistochemical (IHC) scoring system and blindly scored NPTX1 expression in a large cohort of 125 primary and 47 liver metastatic PDAC tumors obtained through the rapid-autopsy program at MSKCC. Liver metastatic samples exhibited significantly higher IHC scores than primary PDAC samples (>2-fold increase). NPTX1 upregulation promotes cell growth under hypoxia: NPTX1 depletion suppressed growth of PDAC cells under hypoxia but not normoxia. Extracellular supplementation of recombinant NPTX1 was sufficient to rescue cell growth of NPTX1 depleted cells supporting the critical role of secreted NPTX1 in promoting cell growth under hypoxia. AMIGO2 is a cell surface NPTX1 receptor and mediates HIF1a nuclear retention via specific HIF1a residues: We next sought to identify the cell surface receptor for NPTX1. We reasoned that a receptor for NPTX1 may become upregulated as a feedback response to depletion of its ligand. mRNA sequencing of ex vivo NPTX1-depleted tumors identified AMIGO2. AMIGO2 promoted nuclear retention of HIF1a and conversely, its depletion drove the degradation of HIF1a. HIF1a/hypoxia reporter assay revealed that AMIGO2 depletion led to 300-fold reduction of HIF1a activity. Overexpression of a phosphomimetic construct at HIF1a serine 641 and 643, not a full length HIF1a ORF rescued AMIGO2 mediated HIF1a reporter signal reduction. Therapeutic targeting of NPTX1-AMIGO2 axis in primary and metastatic PDAC: Through phage display and mouse immunization campaigns, we identified 6 human IgG1 and 1 murine IgG1 antibodies that exhibited high affinity binding to human NPTX1. We identified anti- NPTX1 murine IgG1 antibody 31B01(31B01) as a lead antibody that substantially and significantly suppressed liver metastatic colonization by >80% outperforming gemcitabine. We also tested the impact of 31B01 on the pancreas orthotopic tumor growth. 31B01 suppressed orthotopic patient derived organoid (PDO) growth (>70% reduction), caused a regression response and dramatically extended the overall survival of treated mice harboring PDAC PDO. Citation Format: Norihiro Yamaguchi, Gloria Wu, Ethan Ravetch, Mai Takahashi, Abdul G. Khan, Akimasa Hayashi, Wenbin Mei, Dennis Hsu, Elisa de Stanchina, Ivo C. Lorenz, Christine Iacobuzio-Donahue, Sohail F. Tavazoie. A targetable secreted protein drives pancreatic cancer metastatic colonization and HIF1a nuclear retention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5901.

A Syngeneic Murine Model of Antiphospholipid Syndrome (APS) Recapitulates Aspects of Human APS

Background Antiphospholipid syndrome (APS) is characterized by arterial or venous thrombosis, or recurrent pregnancy loss, in the presence of persistently positive tests for antiphospholipid antibodies (aPL). aPL include lupus anticoagulants (LAC), anti-β2-glycoprotein 1 (β2GPI) and anti-cardiolipin antibodies (aCL). Among them, antibodies directed toward β2GPI have been best characterized and shown in most studies to have a significant association with thrombosis. Most murine models of APS are generated by infusion of either polyclonal IgG isolated from APS patients, or monoclonal IgG directed against human or murine β2GPI. However, one or more of the following limitations may limit their clinical relevance: 1) Unlike chronically elevated autologous aPL in APS patients, cross-species IgG infused into mice circulate for only limited periods of time and may provoke allogeneic responses; 2) Monoclonal human or murine anti-β2GPI IgG, unlike polyclonal aPL from patients, tend to be high affinity, monospecific, and may recognize epitopes different than those recognized by clinical aPL. Hence, we sought to develop a relevant syngeneic murine model of APS that employed polyclonal murine anti-β2GPI IgG that would circulate in mice for extended periods, and not induce allogeneic inflammatory responses. Methods β2GPI-deficient ( apoh -/-) mice were previously developed in our lab on a C57/BL6 background using CRISPR/cas9. IgG isolated from apoh -/- mice immunized with human plasma β2GPI was used as a source of polyclonal murine aPL IgG, while IgG isolated from apoh -/- mice treated with adjuvant alone served as control IgG in all experiments. Wild-type (WT) C57/BL6 mice (two groups of 8 mice each) were injected intravenously with 20 µg of either murine polyclonal aPL or control IgG, and after 72 hours were evaluated for venous thrombosis using the IVC stasis model, with thrombi harvested and measured 48 h after IVC ligation. In parallel studies, we injected mice with aPL or control IgG and evaluated 1) activation of washed platelets, 2) aortic wall gene expression, and 3) levels of circulating neutrophil extracellular traps (NETs) using blood and tissue samples isolated at identical time points as IVC ligation studies (mice in these studies did not undergo IVC ligation). Platelet activation was assessed by measuring the binding of JON/A and expression of platelet P-selectin by flow-cytometry. Total RNA isolated from aortic walls of 3 mice each treated with murine aPL or control IgG was analyzed by RNA sequencing. Results WT C57/BL6 mice that received 20 µg of polyclonal murine aPL sustained plasma anti-β2GPI IgG levels >500 ng/mL for more than 120 h after IgG injection. In the IVC stasis model, six out of eight mice receiving aPL IgG and four out of eight mice receiving control IgG developed thrombi (lack of thrombi in all mice was likely due to bleeding of extensive IVC back branches in C57/BL6 mice). Thrombi weight in aPL-treated mice were significantly larger than those from mice that received control IgG (19.7 ± 1.7 mg vs 13.5 ± 1.0 mg, P = 0.015). Thrombin (0.1 U/ml)-induced platelet activation was significantly greater for platelets isolated from aPL-administered mice compared to those from control mice 72h after IgG injection as determined by binding of JON/A (1246.7 ± 55.3 MFI vs 879 ± 37.9 MFI, P = 0.005) and expression of P-selectin (5729.7 ± 553.9 MFI vs 3777.3 ± 402.5 MFI, P=0.046). Gene enrichment analysis of mRNA from aortas demonstrated significant upregulation of gene pathways related to complement and coagulation cascades, blood coagulation, and oxidoreductase activity in the aPL-treated mice. Individually upregulated genes included C8, C9 fga and fgb. Elevated levels of circulating nucleosomes were also observed in aPL treated mice but did not reach significance due to small numbers. Conclusion These results demonstrate that anti-β2GPI polyclonal aPL raised in apoh-/- mice circulate for extended periods when reinfused into WT mice, enhance thrombosis, induce platelet hyperreactivity, and cause changes in mRNA expression in the vascular wall. Further characterization of anti-β2GPI IgG for LAC activity, anti-PS/PT reactivity and β2GPI domain 1 reactivity is in progress. While all animal models of APS have weaknesses, this syngeneic model provides an additional approach that recapitulates many aspects of vascular inflammation seen in human APS.

Features and protective efficacy of human monoclonal antibodies targeting Mycobacterium tuberculosis arabinomannan.

A better understanding of the epitopes most relevant for antibody-mediated protection against tuberculosis (TB) remains a major knowledge gap. We have shown that human polyclonal IgG to the Mycobacterium tuberculosis (Mtb) surface glycan arabinomannan (AM) and related lipoarabinomannan (LAM) is protective against TB. To investigate the impact of AM epitope recognition and Fc-gamma receptor (FcgR)-binding on antibody functions against Mtb, we isolated a high-affinity human monoclonal antibody (mAb; P1AM25) to AM and show its binding to oligosaccharide (OS) motifs we previously found to be associated with in vitro functions of human polyclonal anti-AM IgG. Human IgG1 P1AM25, but not two other high-affinity human IgG1 anti-AM mAbs reactive with different AM OS motifs, enhanced Mtb phagocytosis by macrophages and reduced intracellular growth in an FcgR-dependent manner. P1AM25 in murine IgG2a, but neither murine IgG1 nor a non-FcgR-binding IgG, given intraperitoneally prior to and after aerosolized Mtb infection was protective in C57BL/6 mice. Moreover, we demonstrate the protective efficacy of human IgG1 P1AM25 in passive transfer with Mtb-infected FcgR-humanized mice. These data enhance our knowledge of the important interplay between both antibody epitope specificity and Fc effector functions in the defense against Mtb and could inform development strategies of vaccines against TB.

Evaluation of murine OX40L-murine IgG1(MM1) fusion protein on immunogenicity against L. mexicana infection in BALB/c mice

The majority of OX40L is found on professional antigen-presenting cells (APC), the potency of OX40L to enhance the immunogenicity of potential vaccines against leishmania is not yet fully investigated. There is no report of administration of OX40L on cutaneous leishmaniasis either in therapy or prophylactic immunisation and the present study for the first time reports the effect of OX40L on L. mexicana infection. In this study, B9B8E2 cells were transfected with the murine OX40L and IgG1 plasmids, were used to produce the mOX40-mIgG1 (MM1). The therapeutic effects of MM1(mOX40L-mIgG1) was tested in a challenge experiment using L. mexicana infected BALB/c mice. Mice received two doses of MM1, on day 3 and 7 after the infection. Mice receiving MM1 generated an inflammatory reaction a few days after the injection of the OX40L, which was gradually dampened and finally disappeared 3 weeks later. There was a significant delay in the growth of developing lesions in mice receiving OX40L compared to controls injected with PBS and the size of lesions in the group receiving MM1 was significantly smaller than that of injected with either PBS. 40% of mice given MM1 remained lesion free for two months, when experiments were terminated. The results clearly indicate the high therapeutic effect of mOX40L-mIgG1 fusion protein in L. mexicana infection. The effect of OX40L on the enhancement of immunisation, needs to be further investigated for developing new vaccine strategies.

Anti-complement factor H (CFH) autoantibodies could delay pristane-induced lupus nephritis

PurposeAnti-complement factor H (CFH) autoantibodies could be detected in lupus and its significance remained to be elucidated. Herein, we aimed to explore the roles of anti-CFH autoantibodies based on pristane-induced lupus mice.MethodsTwenty-four female Balb/c mice were randomly divided into four groups, with one group injected with pristane (pristane group), one group with pristane and then human CFH (hCFH) (pristane-CFH group) 3 times, and the other two as vertical controls, PBS group and PBS-CFH group. Histopathological analysis was performed six months after pristane administration. Levels of hCFH, anti-CFH autoantibodies and anti-dsDNA antibody were detected. Murine IgG (mIgG) were purified and cross-reactivity, epitopes, subclasses and functional analysis were further evaluated in vitro.ResultsImmunization with hCFH and subsequent development of anti-CFH autoantibodies significantly attenuated nephritis of pristane-induced lupus, including lower levels of urinary protein and serum creatinine, decreased levels of serum anti-dsDNA antibody, greatly ameliorated renal histopathologic damage, decreased IgG, complements (C1q, C3) deposits and lower inflammatory factor (IL-6) expression in glomerulus. Furthermore, the purified mIgG (contained anti-CFH autoantibodies) could recognize both hCFH and murine CFH, and the epitopes were predominantly located in hCFH short consensus repeats (SCRs) 1–4, 7 and 11–14. The IgG subclasses were predominant IgG1. The autoantibodies could enhance the binding between hCFH and C3b, and increase factor I mediated-C3b lysis in vitro.ConclusionOur results suggested that anti-CFH autoantibodies could attenuate pristane-induced lupus nephritis by increasing bio-functions of CFH on regulating complement activation and controlling inflammation.

Siglec15 promotes the migration of thyroid carcinoma cells by enhancing the EGFR protein stability.

Sialic acid-bound immunoglobulin-like lectin 15 (Siglec15) has emerged as a novel therapeutic target in tumor immunotherapy. This study is designed to investigate the function and mechanism of Siglec15 in thyroid carcinoma (THCA). The information on patients with THCA from TGCA and GEO database were used to analyze the expression of Siglec15 in THCA. THCA cells were treated with Siglec15-mFc, a recombinant fusion protein consisting of the extracellular domain of human Siglec15 and murine IgG Fc. THP-1 cells expressing human Siglec15 and its mutant were co-cultured with THCA cells to mimic the contact between Siglec15-expressing tumor-associated macrophages and THCA cells. Wound-healing assay and transwell migration assay were used to examine the migration abilities of BCPAP and C643 cells. Pull-down assay was performed to examine the interaction between Siglec15 and epidermal growth factor receptor (EGFR) on the cancer cells. Cycloheximide (CHX) assay was used to evaluate the stability of the protein. The expression of Siglec15 in thyroid carcinoma tissues is higher than in normal tissues. Siglec15 promotes the migration of THCA cells by binding to EGFR in a sialic acid-dependent manner and increases EGFR protein expression. Inhibition of the EGFR pathway blocks the effect of Siglec15 on the migration of THCA cells. Our findings reveals that Siglec15 promotes the migration of thyroid carcinoma cells by enhancing the EGFR protein stability.

Dsg3 epitope-specific signalling in pemphigus.

Pemphigus is an autoantibody driven disease that impairs the barrier function of the skin and mucosa by disrupting desmosomes and thereby impeding cellular cohesion. It is known that the different clinical phenotypes of pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are dependent on the autoantibody profile and target antigens that, amongst others, are primarily desmoglein (Dsg)1 and/or Dsg3 for PV and Dsg1 for PF. However, it was reported that autoantibodiesagainst different epitopes of Dsg1 and Dsg3 can be pathogenic or not. The underlying mechanisms are very complex and involve both direct inhibition of Dsg interactions and downstream signalling. The aim of this study was to find out whether there is target-epitope-specific Dsg3 signalling by comparing the effects of the two pathogenic murine IgGs, 2G4 and AK23. Dispase-based dissociation assay, Western Blot analysis, Stimulated emission depletion microscopy, Fura-based Ca2+ flux measurements, Rho/Rac G-Protein-linked immunosorbent assay, Enzyme-linked immunosorbent assay. The IgGs are directed against the EC5 and EC1 domain of Dsg3, respectively. The data show that 2G4 was less effective in causing loss of cell adhesion, compared to AK23. STED imaging revealed that both autoantibodies had similar effects on keratin retraction and reduction of desmosome number whereas only AK23 induced Dsg3 depletion. Moreover, both antibodies induced phosphorylation of p38MAPK and Akt whereas Src was phosphorylated upon treatment with AK23 only. Interestingly, Src and Akt activation were p38MAPK-dependent. All pathogenic effects were rescued by p38MAPK inhibition and AK23-mediated effects were also ameliorated by Src inhibition. The results give first insights into pemphigus autoantibody-induced Dsg3 epitope-specific signalling which is involved in pathogenic events such as Dsg3 depletion.

A recombinant 9E1 monoclonal antibody binds membrane and soluble channel catfish immunoglobulin M

The development and validation of the recombinant 9E1 monoclonal antibody against channel catfish IgM is described. The variable heavy and light chain domains of the 9E1 hybridoma were cloned into murine IgG1 and IgK expression vectors. These expression plasmids were co-transfected into 293F cells and mature IgG was purified from culture supernatant. It is demonstrated that the recombinant 9E1 monoclonal antibody binds to soluble IgM in ELISA and ELISPOT assays and to membrane-bound IgM by immunofluorescence with different B-cell types. The recombinant 9E1 monoclonal antibody will be a valuable tool in the continued examination of the channel catfish adaptive immune system

Combination CD200R/PD-1 blockade in a humanised mouse model

There is an increasing number of immune-checkpoint inhibitors being developed and approved for cancer immunotherapy. Most of the new therapies aim to reactivate tumour-infiltrating T cells, which are responsible for tumour killing. However, in many tumours, the most abundant infiltrating immune cells are macrophages and myeloid cells, which can be tumour-promoting as well as tumouricidal. CD200R was initially identified as a myeloid-restricted, inhibitory immune receptor, but was subsequently also found to be expressed within the lymphoid lineage. Using a mouse model humanised for CD200R and PD-1, we investigated the potential of a combination therapy comprising nivolumab, a clinically approved PD-1 blocking antibody, and OX108, a CD200R antagonist. We produced nivolumab as a murine IgG1 antibody and validated its binding activity in vitro as well as ex vivo. We then tested the combination therapy in the immunogenic colorectal cancer model MC38 as well as the PD-1 blockade-resistant lung cancer model LLC1, which is characterised by a large number of infiltrating myeloid cells, making it an attractive target for CD200R blockade. No significant improvement of overall survival was found in either model, compared to nivolumab mIgG1 monotherapy. There was a trend for more complete responses in the MC38 model, but investigation of the infiltrating immune cells failed to account for this. Importantly, MC38 cells expressed low levels of CD200, whereas LLC1 cells were CD200-negative. Further investigation of CD200R-blocking antibodies in tumours expressing high levels of CD200 could be warranted.

In Vivo Screening of Immunotherapy Resistant Head and Neck Squamous Cell Carcinoma Identifies B7-H3 Inhibition as a Potential Radiosensitizer

<h3>Purpose/Objective(s)</h3> Patients with locally advanced head and neck squamous cell carcinoma (HNSCC) have poor outcomes following conventionally fractionated radiation (conRT) alone or in combination with chemotherapy. Unfortunately, the addition of anti-PD-1/L1 therapy to conRT has not been found to improve patient outcome. To identify other, more active conRT and immunotherapy combinations, we performed an <i>in vivo</i> shRNA screen of immune-related and other "druggable" genes in an immunotherapy and comparative conRT resistant murine HNSCC model (MOC2) to identify potential radiosensitizers. <h3>Materials/Methods</h3> The <i>in vivo</i> screen utilized shRNA libraries focused on immune-related and other "druggable" targets (n=242) in murine immunoevasive HNSCC tumor cells (MOC2), which were implanted into C57BL/6 flanks and treated with conRT (2Gy x 5) or sham. For validation, the same MOC2 tumor model was treated with murine isotype IgG or anti-B7H3 antibody with conRT. Flow cytometry was performed on single tumor cells approximately 10 days following the last conRT treatment using a murine specific adaptive immunotherapy panel. Data from the HNSCC Cancer Genome Atlas (TCGA) cohort was used for expression of B7-H3 in HPV-negative (-) and HPV-positive (+) HNSCC tumors. A subset of these patients with HPV (-) tumors (n=94) treated uniformly with surgery and post-operative radiation were analyzed for locoregional recurrence (LRR). Statistical analysis was performed in a scientific 2-D graphing and statistics software. <h3>Results</h3> Our <i>in vivo</i> shRNA screen identified several immune-related genes associated with conRT sensitization, including the under-studied CD276 (gene encoding immune checkpoint molecule B7-H3) (RSA log p-value -2.8). Notably, PD-L1 was predicted to have a relatively poor response in combination with conRT (RSA log p-value -0.38). Although ongoing at the time of writing, the validation study showed that conRT + anti-B7-H3 treatment led to a median survival of at least 28.3 days, significantly greater than RT + IgG of 22.3 days (p=3.5e⁻³), IgG (p=4.4e⁻⁶) or anti-B7-H3 alone (p=7e⁻⁶) (both 15.5 days). On initial flow cytometry, RT + anti-B7H3 had a potential increase in NK cells (1.55) compared to no RT + anti-B7H3 (0.22) or no RT + IgG (1.00). B7-H3 was expressed at a significantly higher level in HPV (-) versus HPV (+) HNSCC tumors from the TCGA (p=1.37e-13). In a subset of HPV (-) tumors from the HNSCC TCGA cohort treated with surgery and conRT, B7-H3 expression was significantly associated with increased LRR (p=0.035). <h3>Conclusion</h3> Our immunocompetent <i>in vivo</i> screen identified B7H3 inhibition as a potential novel radiosensitizer in HNSCC. In an immunocompetent syngeneic model, conRT + anti-B7H3 treatment demonstrated improved survival. As B7-H3 is targetable clinically, it provides an exciting and rational target for combinational therapy with RT.

Podocyte expression of human phospholipase A2 receptor 1 causes immune-mediated membranous nephropathy in mice

Antibody-mediated autoimmune pathologies like membranous nephropathy are difficult to model, particularly in the absence of local target antigen expression in model organisms such as mice and rats; as is the case for phospholipase A2 receptor 1 (PLA2R1), the major autoantigen in membranous nephropathy. Here, we generated a transgenic mouse line expressing the full-length human PLA2R1 in podocytes, which has no kidney impairment after birth. Beginning from the age of three weeks, these mice spontaneously developed anti-human PLA2R1 antibodies, a nephrotic syndrome with progressive albuminuria and hyperlipidemia, and the typical morphological signs of membranous nephropathy with granular glomerular deposition of murine IgG in immunofluorescence and subepithelial electron-dense deposits by electron microscopy. Importantly, human PLA2R1-expressing Rag2-/- mice, which lack mature and functioning B and T lymphocytes, developed neither anti-PLA2R1 antibodies nor proteinuria. Thus, our work demonstrates that podocyte expression of human PLA2R1 can induce membranous nephropathy with an underlying antibody-mediated pathogenesis in mice. Importantly, this antibody-mediated model enables proof-of-concept evaluations of antigen-specific treatment strategies, e.g., targeting autoantibodies or autoantibody-producing cells, and may further help understand the autoimmune pathogenesis of membranous nephropathy.

IgG against the Membrane-Proximal Portion of the Desmoglein 3 Ectodomain Induces Loss of Keratinocyte Adhesion, a Hallmark in Pemphigus Vulgaris

Pemphigus vulgaris is a severe autoimmune blistering disease characterized by IgG autoantibodies (auto-abs) against the desmosomal adhesion molecules desmoglein (DSG) 3 and DSG1. Underlying mechanisms leading to blister formation upon binding of DSG-specific IgG auto-abs are not fully understood. Numerous studies showed the pathogenicity of IgG auto-ab binding to the aminoterminal region 1 (EC1) of the DSG3 ectodomain. However, auto-abs in pemphigus vulgaris are polyclonal, including IgG against both aminoterminal- and membrane-proximal epitopes of the DSG3 ectodomain. In this study, the pathogenicity of a previously uncharacterized murine monoclonal IgG antibody, 2G4, directed against the membrane-proximal region (EC5) of the DSG3 ectodomain was characterized and tested in various specificity and functionality assays. The results clearly show that 2G4 is capable of inhibiting intercellular keratinocyte adhesion and of inducing cellular DSG3 redistribution by activation of the p38MAPK signal transduction pathway. In this study, we provide evidence that an IgG auto-abs directed against the membrane-proximal region EC5 of DSG3 induces acantholysis, the hallmark in pemphigus vulgaris. These findings challenge the current concept that IgG auto-abs targeting the NH2-terminal portion of the DSG3 ectodomain are pathogenic only. Our study provides further aspects for a deeper understanding of desmosomal keratinocyte adhesion and improves our insight into the complex auto-ab‒induced blister formation in pemphigus vulgaris.

Comparison of CD3e Antibody and CD3e-sZAP Immunotoxin Treatment in Mice Identifies sZAP as the Main Driver of Vascular Leakage.

Anti-CD3-epsilon (CD3e) monoclonal antibodies (mAbs) and CD3e immunotoxins (ITs) are promising targeted therapy options for various T-cell disorders. Despite significant advances in mAb and IT engineering, vascular leakage syndrome (VLS) remains a major dose-limiting toxicity for ITs and has been poorly characterized for recent “engineered” mAbs. This study undertakes a direct comparison of non-mitogenic CD3e-mAb (145-2C11 with Fc-silentTM murine IgG1: S-CD3e-mAb) and a new murine-version CD3e-IT (saporin–streptavidin (sZAP) conjugated with S-CD3e-mAb: S-CD3e-IT) and identifies their distinct toxicity profiles in mice. As expected, the two agents showed different modes of action on T cells, with S-CD3e-mAb inducing nearly complete modulation of CD3e on the cell surface, while S-CD3e-IT depleted the cells. S-CD3e-IT significantly increased the infiltration of polymorphonuclear leukocytes (PMNs) into the tissue parenchyma of the spleen and lungs, a sign of increased vascular permeability. By contrast, S-CD3e-mAbs-treated mice showed no notable signs of vascular leakage. Treatment with control ITs (sZAP conjugated with Fc-silent isotype antibodies) induced significant vascular leakage without causing T-cell deaths. These results demonstrate that the toxin portion of S-CD3e-IT, not the CD3e-binding portion (S-CD3e-mAb), is the main driver of vascular leakage, thus clarifying the molecular target for improving safety profiles in CD3e-IT therapy.

Development of an immunoassay for aglycosylated murine IgG1 in mouse serum via generation of a specific tool antibody.

Aim: To develop a method for the quantitation of effector functionless mouse surrogate IgG1 drug molecules in mouse matrices. Materials & methods: A panel of antibodies that bound specifically to N297G mutation-containing mouse IgG molecules was generated in rats. The panel was screened to identify an antibody that could be used as both the capture and detection reagent in an electrochemiluminescent immunoassay. Results & conclusion: The quantitative assay developed with the N297G-specific antibody passed acceptance criteria across multiple IgG1 fragment crystallizable (Fc)-containing protein formats and provides accurate quantitation of the total levels of mouse surrogate protein Fc present in in vivo mouse serum samples. These results are useful in understanding drug integrity and the development of precise pharmacokinetic/pharmacodynamic relationships.

IgG Binds Escherichia coli Serine Protease EspP and Protects Mice From E. coli O157:H7 Infection.

Shiga toxin-producing Escherichia coli O157:H7 is a virulent strain causing severe gastrointestinal infection, hemolytic uremic syndrome and death. To date there are no specific therapies to reduce progression of disease. Here we investigated the effect of pooled immunoglobulins (IgG) on the course of disease in a mouse model of intragastric E. coli O157:H7 inoculation. Intraperitoneal administration of murine IgG on day 3, or both on day 3 and 6, post-inoculation improved survival and decreased intestinal and renal pathology. When given on both day 3 and 6 post-inoculation IgG treatment also improved kidney function in infected mice. Murine and human commercially available IgG preparations bound to proteins in culture filtrates from E. coli O157:H7. Bound proteins were extracted from membranes and peptide sequences were identified by mass spectrometry. The findings showed that murine and human IgG bound to E. coli extracellular serine protease P (EspP) in the culture filtrate, via the IgG Fc domain. These results were confirmed using purified recombinant EspP and comparing culture filtrates from the wild-type E. coli O157:H7 strain to a deletion mutant lacking espP. Culture filtrates from wild-type E. coli O157:H7 exhibited enzymatic activity, specifically associated with the presence of EspP and demonstrated as pepsin cleavage, which was reduced in the presence of murine and human IgG. EspP is a virulence factor previously shown to promote colonic cell injury and the uptake of Shiga toxin by intestinal cells. The results presented here suggest that IgG binds to EspP, blocks its enzymatic activity, and protects the host from E. coli O157:H7 infection, even when given post-inoculation.

Expression and characterization of a recombinant broadly-reactive monoclonal antibody against group 1 and 2 influenza viruses

Production of broadly-reactive antibodies is critical for universal immunodiagnosis of rapidly-evolving influenza viruses. Most monoclonal antibodies (mAbs) are generated in mice using the hybridoma technology which involves labor- and time-consuming screening and low yield issues. In this study, a recombinant antibody based on a broadly-reactive mAb against the hemagglutinin (HA) stalk of H7N9 avian influenza virus was expressed in CHO cells and its biological characteristics, cross-reactivity and epitope recognition were identified. The variable genes of the parental antibody were amplified and cloned into the antibody-expressing plasmids containing the constant genes of murine IgG1. The recombinant antibody was expressed in high yield and purity in CHO cells and showed similar features to the parental antibody, including negative hemagglutination inhibition activity against H7N9 virus and high binding activity with the H7N9 HA protein. Notably, the recombinant antibody exhibited a broad reactivity with different influenza subtypes belonging to group 1 and group 2, which was associated with its recognition of a highly-conserved epitope in the stalk, as observed for the parental antibody. Our results suggest that cell-based antibody expression system can be utilized as an important alternative to the hybridoma technology for antibody production for influenza virus diagnostics.