scholarly journals 891 S-531011, a novel anti-human CCR8 antibody: antibody screening and evaluation of biological profiles

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A934-A934
Author(s):  
Mai Yoshikawa ◽  
Yoji Nagira ◽  
Morio Nagira ◽  
Tetsuya Yoshida ◽  
Shinpei Yoshida ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Animal Studies ◽  
Treg Cells ◽  
Immunological Tolerance ◽  
Negative Control ◽  
Affinity Maturation ◽  
University Hospital ◽  
Hybridoma Cells ◽  
Humanized Antibody

BackgroundRegulatory T cells (Tregs) are involved in tumor progression and inhibition of anti-tumor immune responses by promotion of immunological tolerance in the tumor microenvironment. On the other hand, Treg cells in peripheral blood are also essential role in preventing autoimmunity and uncontrolled inflammation. So, selective control of tumor infiltrating Treg cells might be an attractive approach of immune-oncology therapies without disrupting their systemic anti-inflammatory functions. Here, we focused on CCR8 (C-C motif chemokine receptor 8) as a target molecule which was selectively and highly expressed on tumor-infiltrating Tregs and developed a novel anti-human CCR8 specific antibody.MethodsWe immunized mice with human CCR8 by our original immunization method which could strongly induce antibodies for membrane proteins, and then constructed hybridoma cells. Anti-human CCR8 (human CCR4 as a negative control) binding assay and human CCL1-CCR8 neutralizing assay were simultaneously performed by using supernatants of hybridoma cells to isolate human CCR8 specific strong-neutralizing antibodies. After humanization and affinity maturation of some selected clones, we selected our lead antibody by binding specificity, neutralizing activity, antibody dependent cellular cytotoxicity (ADCC) and thermodynamic stability as index.ResultsWe rapidly induced human CCR8 specific antibodies in mouse with our unique immunization methods and constructed thousands of hybridomas secreting anti-human CCR8 antibodies. We also successfully humanized some of lead antibodies which show high affinity and specificity and isolated novel anti-human CCR8 specific humanized antibody S-531011 as our development antibody after affinity maturation. S-531011 selectively recognizes human CCR8 on the surface of tumor-infiltrating Tregs and shows strong ADCC. While human CCL1 is known as a dominant ligand of CCR8 which binds extracellular loop2 and N-terminal of CCR8, S-531011 recognizes similar epitopes and effectively neutralizes CCL1-CCR8 signaling. Furthermore, S-531011 also shows favorable blood kinetics in vivo and potently inhibits tumor growth in tumor bearing human CCR8 knock-in mouse model.ConclusionsWe develop S-531011, a novel anti-human CCR8 humanized antibody which could selectively recognize and deplete tumor infiltrating Tregs. Based on our pre-clinical data, S-531011 has strong anti-tumor effect and we expect that it might be a potent novel tumor immuno-therapeutic agent with fewer side effect.Ethics ApprovalThe present study was approved by the Institutional Ethics Committee of Osaka University Hospital (approved number: 13266-15). Animal studies were approved by the Institutional Animal Care and Use Committee (approved number: S20192D, S20197D and S20198D).

scholarly journals Protection against Autoimmune Diabetes by Silkworm-Produced GFP-Tagged CTB-Insulin Fusion Protein

2011 ◽  
Vol 2011 ◽  
pp. 1-14 ◽  
Author(s):  
Qiaohong Meng ◽  
Wenfeng Wang ◽  
Xiaowen Shi ◽  
Yongfeng Jin ◽  
Yaozhou Zhang
Keyword(s):  
Fusion Protein ◽  
Oral Administration ◽  
Fluorescent Protein ◽  
Autoimmune Diabetes ◽  
Nod Mice ◽  
Treg Cells ◽  
Immunological Tolerance ◽  
Protein Vaccine ◽  
Green Fluorescent

In animals, oral administration of the cholera toxin B (CTB) subunit conjugated to the autoantigen insulin enhances the specific immune-unresponsive state. This is called oral tolerance and is capable of suppressing autoimmune type 1 diabetes (T1D). However, the process by which the CTB-insulin (CTB-INS) protein works as a therapy for T1Din vivoremains unclear. Here, we successfully expressed a green fluorescent protein- (GFP-) tagged CTB-Ins (CTB-Ins-GFP) fusion protein in silkworms in a pentameric form that retained the native ability to activate the mechanism. Oral administration of the CTB-Ins-GFP protein induced special tolerance, delayed the development of diabetic symptoms, and suppressed T1D onset in nonobese diabetic (NOD) mice. Moreover, it increased the numbers of CD4+CD25+Foxp3+T regulatory (Treg) cells in peripheral lymph tissues and affected the biological activity of spleen cells. This study demonstrated that the CTB-Ins-GFP protein produced in silkworms acted as an oral protein vaccine, inducing immunological tolerance involving CD4+CD25+Foxp3+Treg cells in treating T1D.

A novel rationally designed agonist antibody fragment that functionally mimics thrombopoietin

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 2541-2541
Author(s):  
M. Renshaw ◽  
S. Frederickson ◽  
B. Lin ◽  
X. Su ◽  
Y. Wang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Rational Design ◽  
Antibody Fragment ◽  
Clinical Problem ◽  
Negative Control ◽  
Platelet Production ◽  
Platelet Counts ◽  
Low Platelet Count

2541 Background: Thrombocytopenia, or low platelet count, is a significant clinical problem associated with ITP, cancer chemotherapy, or other clinical settings. To avoid transfusions, agents that stimulate platelet production are in development. Clinical trials with recombinant versions of thrombopoietin (TPO), which stimulates platelet production in a lineage specific manner by binding to cMpl receptor (cMpl-R) on megakaryocytic progenitors, were demonstrated to increase platelet counts in humans. However, the possible generation of an anti-TPO immune response that cross-reacts with and impairs the function of the endogenous cytokine is a significant disadvantage of this treatment approach. Alternate thrombopoietic agents that lack native TPO primary sequences have been developed to address this concern. Methods: Using rational design, antibody fragments (Fabs) that mimic TPO were created. A peptide with cMpl-R binding capability was grafted into different CDRs of a fully human Fab scaffold. Functional presentation of the peptide was optimized using phage display and cell-based panning. Select antibodies and fragments containing two grafted peptides were assayed for their ability to stimulate cMpl-R in vitro. In vivo stimulation of platelet production was tested in normal mice injected daily for five days with either rhTPO (90 μg/kg) or Fab59 (0.2 mg/kg, 2 mg/kg or 5 mg/kg) or negative control Fab (5mg/kg). Results: Several candidates demonstrated agonist activity in an in vitro cMpl-R signaling reporter assay, including Fab59 which was estimated to be equipotent to TPO. In vivo, a rise in peripheral platelet counts comparable to rhTPO was seen with Fab59 at 2 mg/kg and 5 mg/kg. Serum antibodies generated in response to dosing with Fab59 did not cross-react with murine or human TPO. Conclusion: These rationally-designed mimetic Fabs may provide a therapeutic intervention for thrombocytopenia while avoiding the potential generation of neutralizing antibodies to endogenous TPO. [Table: see text]

scholarly journals Cas9-directed immune tolerance in humans—a model to evaluate regulatory T cells in gene therapy?

Gene Therapy ◽  
2021 ◽  
Author(s):  
Dimitrios Laurin Wagner ◽  
Lena Peter ◽  
Michael Schmueck-Henneresse
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Regulatory T Cells ◽  
Immune Tolerance ◽  
Neutralizing Antibodies ◽  
Treg Cells ◽  
Gene Therapies ◽  
Early Results ◽  
Cell Immunity

AbstractThe dichotomic nature of the adaptive immune response governs the outcome of clinical gene therapy. On the one hand, neutralizing antibodies and cytotoxic T cells can have a dramatic impact on the efficacy and safety of human gene therapies. On the other hand, regulatory T cells (Treg) can promote tolerance toward transgenes thereby enabling long-term benefits of in vivo gene therapy after a single administration. Pre-existing antibodies and T cell immunity has been a major obstacle for in vivo gene therapies with viral vectors. As CRISPR-Cas9 gene editing advances toward the clinics, the technology’s inherent immunogenicity must be addressed in order to guide clinical treatment decisions. This review summarizes the recent evidence on Cas9-specific immunity in humans—including early results from clinical trials—and discusses the risks for in vivo gene therapies. Finally, we focus on solutions and highlight the potential role of Cas9-specific Treg cells to promote immune tolerance. As a “beneficial alliance” beyond Cas9-immunity, antigen-specific Treg cells may serve as a living and targeted immunosuppressant to increase safety and efficacy of gene therapy.

scholarly journals An affinity-matured human monoclonal antibody targeting fusion loop epitope of dengue virus with in vivo therapeutic potency

2020 ◽  
Author(s):  
Tomohiro Kotaki ◽  
Takeshi Kurosu ◽  
Ariadna Grinyo ◽  
Edgar Davidson ◽  
Siti Churrotin ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Neutralizing Antibodies ◽  
Human Monoclonal Antibody ◽  
Variable Region ◽  
Cross Reactivity ◽  
Affinity Maturation ◽  
Interferon Α ◽  
Fusion Loop

AbstractDengue virus (DENV), from the genus flavivirus of the family flaviviridae, causes serious health problems globally. Human monoclonal antibodies (HuMAb) can be used to elucidate the mechanisms of neutralization and antibody-dependent enhancement (ADE) of DENV infections, leading to the development of a vaccine or therapeutic antibodies. Here, we generated eight HuMAb clones from an Indonesian patient infected with DENV. These HuMAbs exhibited the typical characteristics of weak neutralizing antibodies including high cross-reactivity with other flaviviruses and targeting of the fusion loop epitope (FLE). However, one of the HuMAbs, 3G9, exhibited strong neutralization ability (NT50 < 0.1 µg/ml) and possessed a high somatic hyper-mutation rate of the variable region, indicating affinity-maturation. Administration of this antibody significantly improved the survival rate of interferon-α/β/γ receptor knockout C57BL/6 mice after a lethal DENV challenge. Additionally, Fc-modified 3G9 molecules that had lost their in vitro ADE activity showed significantly enhanced therapeutic potency in vivo and competed strongly with an ADE-prone antibody in vitro. Taken together, the affinity-matured FLE-targeting antibody 3G9 exhibits several promising features for therapeutic application including a low NT50 value, potential for pan-flavivirus infection treatment, and suppression of ADE. This study demonstrates the therapeutic potency of affinity-matured FLE-targeting antibodies.

scholarly journals Potential for Antigen-Specific Tolerizing Immunotherapy in Systematic Lupus Erythematosus

2021 ◽  
Vol 12 ◽  
Author(s):  
Sean Robinson ◽  
Ranjeny Thomas
Keyword(s):  
Lupus Erythematosus ◽  
Survival Rates ◽  
Clinical Manifestations ◽  
Treg Cells ◽  
Immunological Tolerance ◽  
Immunosuppressive Drugs ◽  
Systemic Autoimmune Disease ◽  
Systematic Lupus Erythematosus ◽  
Curative Therapy

Systemic lupus erythematosus (SLE) is a chronic complex systemic autoimmune disease characterized by multiple autoantibodies and clinical manifestations, with the potential to affect nearly every organ. SLE treatments, including corticosteroids and immunosuppressive drugs, have greatly increased survival rates, but there is no curative therapy and SLE management is limited by drug complications and toxicities. There is an obvious clinical need for safe, effective SLE treatments. A promising treatment avenue is to restore immunological tolerance to reduce inflammatory clinical manifestations of SLE. Indeed, recent clinical trials of low-dose IL-2 supplementation in SLE patients showed that in vivo expansion of regulatory T cells (Treg cells) is associated with dramatic but transient improvement in SLE disease markers and clinical manifestations. However, the Treg cells that expanded were short-lived and unstable. Alternatively, antigen-specific tolerance (ASIT) approaches that establish long-lived immunological tolerance could be deployed in the context of SLE. In this review, we discuss the potential benefits and challenges of nanoparticle ASIT approaches to induce prolonged immunological tolerance in SLE.

scholarly journals Construction of polycistronic baculovirus surface display vectors to express the PCV2 Cap(d41) protein and analysis of its immunogenicity in mice and swine

2020 ◽  
Vol 51 (1) ◽  
Author(s):  
Ya-Yi Chen ◽  
Wei-Chen Yang ◽  
Yu-Kang Chang ◽  
Chi-Young Wang ◽  
Wei-Ru Huang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Surface Display ◽  
Recombinant Baculovirus ◽  
Negative Control Group ◽  
Negative Control ◽  
Control Group ◽  
Challenge Study ◽  
Ifn Γ ◽  
Cellular Immune

Abstract To increase expression levels of the PCV2 Cap(d41) protein, novel baculovirus surface display vectors with multiple expression cassettes were constructed to create recombinant baculoviruses BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41). Our results reveal that the recombinant baculovirus BacDD-4Cap(d41) was able to express the highest levels of Cap(d41) protein. Optimum conditions for expressing the PCV2 Cap(d41) protein were determined, and our results show that 107 of Sf-9 infected with the recombinant baculovirus BacDD-4Cap(d41) at an MOI of 5 for 3 days showed the highest level of protein expression. Mice immunized with the 4Cap(d41) vaccine which was prepared from the recombinant baculovirus-infected cells (107) elicited higher ELISA titers compared to the Cap (d41) vaccine. The 4Cap(d41) vaccine could elicit anti-PCV2 neutralizing antibodies and IFN-γ in mice, as confirmed by virus neutralization test and IFN-γ ELISA. Moreover, the swine lymphocyte proliferative responses indicated that the 4Cap(d41) vaccine was able to induce a clear cellular immune response. Flow cytometry analysis showed that the percentage of CD4+ T cells and CD4+/CD8+ ratio was increased significantly in SPF pigs immunized with the 4Cap(d41) vaccine. Importantly, the 4Cap(d41) vaccine induced an IFN-γ response, further confirming that its effect is through cellular immunity in SPF pigs. An in vivo challenge study revealed that the 4Cap(d41) and the commercial vaccine groups significantly reduce the viral load of vaccinated pigs as compared with the CE negative control group. Taken together, we have successfully developed a 4Cap(d41) vaccine that may be a potential subunit vaccine for preventing the disease associated with PCV2 infections.

scholarly journals An affinity-matured human monoclonal antibody targeting fusion loop epitope of dengue virus with in vivo therapeutic potency

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomohiro Kotaki ◽  
Takeshi Kurosu ◽  
Ariadna Grinyo-Escuer ◽  
Edgar Davidson ◽  
Siti Churrotin ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Neutralizing Antibodies ◽  
Human Monoclonal Antibody ◽  
Variable Region ◽  
Cross Reactivity ◽  
Affinity Maturation ◽  
Interferon Α ◽  
Fusion Loop

AbstractDengue virus (DENV), from the genus flavivirus of the family flaviviridae, causes serious health problems globally. Human monoclonal antibodies (HuMAb) can be used to elucidate the mechanisms of neutralization and antibody-dependent enhancement (ADE) of DENV infections, leading to the development of a vaccine or therapeutic antibodies. Here, we generated eight HuMAb clones from an Indonesian patient infected with DENV. These HuMAbs exhibited the typical characteristics of weak neutralizing antibodies including high cross-reactivity with other flaviviruses and targeting of the fusion loop epitope (FLE). However, one of the HuMAbs, 3G9, exhibited strong neutralization (NT50 < 0.1 μg/ml) and possessed a high somatic hyper-mutation rate of the variable region, indicating affinity-maturation. Administration of this antibody significantly prolonged the survival of interferon-α/β/γ receptor knockout C57BL/6 mice after a lethal DENV challenge. Additionally, Fc-modified 3G9 that had lost their in vitro ADE activity showed enhanced therapeutic potency in vivo and competed strongly with an ADE-prone antibody in vitro. Taken together, the affinity-matured FLE-targeting antibody 3G9 exhibits promising features for therapeutic application including a low NT50 value, potential for treatment of various kinds of mosquito-borne flavivirus infection, and suppression of ADE. This study demonstrates the therapeutic potency of affinity-matured FLE-targeting antibodies.

scholarly journals Protective Effect of the α7 Nicotinic Receptor Agonist PNU-282987 on Dopaminergic Neurons Against 6-Hydroxydopamine, Regulating Anti-neuroinflammatory and the Immune Balance Pathways in Rat

2021 ◽  
Vol 12 ◽  
Author(s):  
Ying Jiang ◽  
Huizi Ma ◽  
Xuemei Wang ◽  
Zhan Wang ◽  
Yaqin Yang ◽  
...  
Keyword(s):  
T Cells ◽  
Immunohistochemical Staining ◽  
Treg Cells ◽  
Immunological Tolerance ◽  
Motor Deficits ◽  
Inflammatory Reactions ◽  
Protein Levels ◽  
Immune Balance ◽  
6 Hydroxydopamine

Neuroinflammation and inner immune dysfunction are increasingly accepted as important components of the etiopathogenesis of Parkinson’s disease (PD). According to emerging evidence, a7 nicotinic acetylcholine receptor (α7nAChR), a ligand-gated ion channel, plays an important role in inflammatory reactions and is also expressed on the surface of T cells. In particular, regulatory T cells (Tregs) are critical for the maintenance of immunological tolerance. In the present study, we investigated the roles of α7nAChR in inhibiting inflammation and maintaining the immune balance in rats with 6-hydroxydopamine (6-OHDA)-induced lesions and the possible mechanisms regulating the proportion of Tregs in vivo. Adult male Wistar rats (n = 90) were subjected to a unilateral injection of 6-OHDA into the left medial forebrain bundle, and PNU-282987, an α7nAChR agonist, was intraperitoneally injected 2 h prior to the induction of lesions by 6-OHDA and again at days 1, 7, and 13 postlesion. Behavioral tests and immunohistochemical staining to detect the expression of tyrosine hydroxylase (TH) in the bilateral substantial nigra (SN) were performed. Subsequently, CD4+ T lymphocytes and the expression of forkhead/winged helix transcription factor p3 (Foxp3, which is a marker of Treg cells) in the SN were also assessed using immunofluorescence staining. The expression of glial fibrillary acidic protein (GFAP) in the SN was determined by performing immunohistochemical staining. Additionally, the protein levels of α7nAChR, extracellular signal-regulated kinase (Erk) phosphorylated-Erk (p-Erk) and Foxp3 in the ventral midbrain were determined using Western blotting, and the relative expression of the TNF-α, IL-1β, and IL-10 mRNAs were detected using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). We found that PNU-282987 significantly improved the motor deficits induced by 6-OHDA, reduced the loss of TH in the SN, suppressed the overactivation of GFAP+ cells and expression of related inflammatory cytokines, and increased the number of Foxp3+ cells. In addition, we also showed that PNU-282987 significantly increased the protein expression of the a7nAchR, p-Erk, and Foxp3 in 6-OHDA-lesioned rats (p &lt; 0.05). These results indicated that α7nAChR activation could exert an anti-inflammatory effect and participate in the process of modulating the immune balance during 6-OHDA-induced injury, potentially through the α7nAChR/p-Erk/Foxp3 signaling pathway.

Decellularized bone extracellular matrix in skeletal tissue engineering

2020 ◽  
Vol 48 (3) ◽  
pp. 755-764
Author(s):  
Benjamin B. Rothrauff ◽  
Rocky S. Tuan
Keyword(s):  
Tissue Engineering ◽  
Bone Regeneration ◽  
Tissue Regeneration ◽  
Animal Studies ◽  
Tumor Resection ◽  
Regenerative Capacity ◽  
Skeletal Tissue ◽  
Large Bone

Bone possesses an intrinsic regenerative capacity, which can be compromised by aging, disease, trauma, and iatrogenesis (e.g. tumor resection, pharmacological). At present, autografts and allografts are the principal biological treatments available to replace large bone segments, but both entail several limitations that reduce wider use and consistent success. The use of decellularized extracellular matrices (ECM), often derived from xenogeneic sources, has been shown to favorably influence the immune response to injury and promote site-appropriate tissue regeneration. Decellularized bone ECM (dbECM), utilized in several forms — whole organ, particles, hydrogels — has shown promise in both in vitro and in vivo animal studies to promote osteogenic differentiation of stem/progenitor cells and enhance bone regeneration. However, dbECM has yet to be investigated in clinical studies, which are needed to determine the relative efficacy of this emerging biomaterial as compared with established treatments. This mini-review highlights the recent exploration of dbECM as a biomaterial for skeletal tissue engineering and considers modifications on its future use to more consistently promote bone regeneration.

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