Antibody-drug conjugates targeting CD248+ myofibroblasts effectively alleviate renal fibrosis in mice.

Myofibroblasts, or activated fibroblasts, play a critical role in the process of renal fibrosis. Targeting myofibroblasts to inhibit their activation or induce specific cell death has been considered to be an effective strategy to attenuate renal fibrosis. However, specific biomarkers for myofibroblasts are needed to ensure the efficacy of these strategies. Here, we verified that CD248 was mainly expressed in myofibroblasts in patients with chronic kidney disease, which was inversely correlated with renal function. The same result was also confirmed in renal fibrotic mice induced by unilateral ureteral obstruction and aristolochic acid nephropathy. By using an antibody-drug conjugate (ADC) named IgG78-DM1, in which maytansinoid (DM1) was linked to a fully human antibody IgG78 through an uncleavable SMCC linker, we demonstrated that it could effectively bind with and kill CD248+ fibroblasts in vitro and alleviate renal fibrosis in mice models. Besides, we confirmed that IgG78-DM1 had qualified biosafety in vivo. Our results confirmed that CD248 can be used as a specific marker for myofibroblasts, and specific killing of CD248+ myofibroblasts by IgG78-DM1 has excellent anti-fibrotic effect in renal fibrotic mice. Our study expanded the application of ADC and provided a novel strategy for the treatment of renal fibrosis.

Antibody-drug conjugates targeting CD248 inhibits liver fibrosis through specific killing on myofibroblasts.

BACKGROUND: Chronic liver injury induces pathological repair, resulting in fibrosis, during which hepatic stellate cells (HSCs) are activated and transform into myofibroblasts. CD248 is mainly expressed on myofibroblasts and was considered as a promising target to treat fibrosis. The primary aim of this study was to generate a CD248 specific antibody-drug conjugate (ADC) and evaluate its therapeutic efficacy for liver fibrosis and its safety in vivo. METHODS: CD248 expression was examined in patients with liver cirrhosis and in mice with CCl4-induced liver fibrosis. The ADC IgG78-DM1, which targets CD248, was prepared and its bioactivity on activated primary HSCs was studied. The anti-fibrotic effects of IgG78-DM1 on liver fibrosis were evaluated in CCl4-induced mice. The reproductive safety and biosafety of IgG78-DM1 were also evaluated in vivo. RESULTS: CD248 expression was upregulated in patients with liver cirrhosis and in CCl4-induced mice, and was mainly expressed on alpha smooth muscle actin (α-SMA)+ myofibroblasts. IgG78-DM1 was successfully generated, which could effectively bind with and kill CD248+ activated HSCs in vitro and inhibit liver fibrosis in vivo. In addition, IgG78-DM1 was demonstrated to have qualified biosafety and reproductive safety in vivo. CONCLUSIONS: Our study demonstrated that CD248 could be an ideal target for myofibroblasts in liver fibrosis, and CD248-targeting IgG78-DM1 had excellent anti-fibrotic effects in mice with liver fibrosis. Our study provided a novel strategy to treat liver fibrosis and expanded the application of ADCs beyond tumors.

Correction to: Characterization of the first fully human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy.

Tumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors. We previously isolated a novel single-chain variable fragment (scFv) 78 against TEM1 from a yeast display scFv library. Here we evaluated the potential applications of scFv78 as a tool for tumor molecular imaging, immunotoxin-based therapy and nanotherapy. Epitope mapping, three-dimensional (3D) structure docking and affinity measurements indicated that scFv78 could bind to both human and murine TEM1, with equivalent affinity, at a well-conserved conformational epitope. The rapid internalization of scFv78 and scFv78-labeled nanoparticles was triggered after specific TEM1 binding. The scFv78-saporin immunoconjugate also exerted dose-dependent cytotoxicity with high specificity to TEM1-positive cells in vitro. Finally, specific and sensitive tumor localization of scFv78 was confirmed with optical imaging in a mouse tumor model that has highly endogenous mTEM1 expression in the vasculature. Our data indicate that scFv78, the first fully human anti-TEM1 recombinant antibody, recognizes both human and mouse TEM1 and has unique and favorable features that are advantageous for the development of imaging probes or antibody-toxin conjugates for a large spectrum of human TEM1-positive solid tumors.

Characterization of the first fully human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy.

Tumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors. We previously isolated a novel single-chain variable fragment (scFv) 78 against TEM1 from a yeast display scFv library. Here, we evaluated the potential applications of scFv78 as a tool for tumor molecular imaging, immunotoxin-based therapy and nanotherapy. Epitope mapping, three-dimensional structure docking and affinity measurements indicated that scFv78 could bind to both human and murine TEM1, with equivalent affinity, at a well-conserved conformational epitope. The rapid internalization of scFv78 and scFv78-labeled nanoparticles was triggered after specific TEM1 binding. The scFv78-saporin immunoconjugate also exerted dose-dependent cytotoxicity with high specificity to TEM1-positive cells in vitro. Finally, specific and sensitive tumor localization of scFv78 was confirmed with optical imaging in a tumor mouse model that has highly endogenous mTEM1 expression in the vasculature. Our data indicated that scFv78, the first fully human anti-TEM1 recombinant antibody, recognizes both human and mouse TEM1 and has unique and favorable features that are advantageous for the development of imaging probes or antibody-toxin conjugates for a large spectrum of human TEM1-positive solid tumors.

Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy.

Phage display technology (PDT), a combinatorial screening approach, provides a molecular diversity tool for creating libraries of peptides/proteins and discovery of new recombinant therapeutics. Expression of proteins such as monoclonal antibodies (mAbs) on the surface of filamentous phage can permit the selection of high affinity and specificity therapeutic mAbs against virtually any target antigen. Using a number of diverse selection platforms (e.g. solid phase, solution phase, whole cell and in vivo biopannings), phage antibody libraries (PALs) from the start point provides great potential for the isolation of functional mAb fragments with diagnostic and/or therapeutic purposes. Given the pivotal role of PDT in the discovery of novel therapeutic/diagnostic mAbs, in the current review, we provide an overview on PALs and discuss their impact in the advancement of engineered mAbs.

Efficient construct of a large and functional scFv yeast display library derived from the ascites B cells of ovarian cancer patients by three-fragment transformation-associated recombination.

Over the past decade, yeast display technology has emerged as a powerful tool for the isolation of high-affinity immunoglobulin fragments with potential utility as clinical diagnostic and therapeutic reagents. Despite significant refinement of the various methodologies underpinning library construction and selections, certain aspects remain challenging and process limiting. We have sought to significantly improve the robustness of the single-chain Fv (scFv) library construction step by overcoming the technical inefficiencies frequently encountered during the PCR-mediated assembly of scFvs from the discrete heavy and light V-domain repertoires. Using a novel primer set designed to provide maximum amplification coverage of the known germ-line V-domain repertoire, we have exploited the potential of the in vivo homologous gap-repair apparatus of Saccharomyces cerevisiae to assemble intact scFvs directly from co-transformed PBMC-derived VH, VL, and linearized vector component fragments. We have successfully applied this three-fragment assembly strategy to construct a large (>10(9)) scFv yeast display library from the ascites immune repertoire of ovarian cancer patients and validated the approach by applying FACS-based sorting to readily isolate scFvs that recognize various tumor marker antigens (TMAs). It is expected that this simplified construction method may find general utility, both for de novo scFv library construction and for subsequent combinatorial affinity maturation manipulations that require more than two fragments.

Gi-biased ß2AR signaling links GRK2 upregulation to heart failure.

RATIONALE: Phosphorylation of ß(2)-adrenergic receptor (ß(2)AR) by a family of serine/threonine kinases known as G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) is a critical determinant of cardiac function. Upregulation of G protein-coupled receptor kinase 2 (GRK2) is a well-established causal factor of heart failure, but the underlying mechanism is poorly understood. OBJECTIVE: We sought to determine the relative contribution of PKA- and GRK-mediated phosphorylation of ß(2)AR to the receptor coupling to G(i) signaling that attenuates cardiac reserve and contributes to the pathogenesis of heart failure in response to pressure overload. METHODS AND RESULTS: Overexpression of GRK2 led to a G(i)-dependent decrease of contractile response to ßAR stimulation in cultured mouse cardiomyocytes and in vivo. Importantly, cardiac-specific transgenic overexpression of a mutant ß(2)AR lacking PKA phosphorylation sites (PKA-TG) but not the wild-type ß(2)AR (WT-TG) or a mutant ß(2)AR lacking GRK sites (GRK-TG) led to exaggerated cardiac response to pressure overload, as manifested by markedly exacerbated cardiac maladaptive remodeling and failure and early mortality. Furthermore, inhibition of G(i) signaling with pertussis toxin restores cardiac function in heart failure associated with increased ß(2)AR to G(i) coupling induced by removing PKA phosphorylation of the receptor and in GRK2 transgenic mice, indicating that enhanced phosphorylation of ß(2)AR by GRK and resultant increase in G(i)-biased ß(2)AR signaling play an important role in the development of heart failure. CONCLUSIONS: Our data show that enhanced ß(2)AR phosphorylation by GRK, in addition to PKA, leads the receptor to G(i)-biased signaling, which, in turn, contributes to the pathogenesis of heart failure, marking G(i)-biased ß(2)AR signaling as a primary event linking upregulation of GRK to cardiac maladaptive remodeling, failure and cardiodepression.

Modulation of immune and inflammatory responses on experimental arthritis following intraarticular gene transfer of tumor necrosis factor receptor-immunoglobulin Fc.

In spite of popularity of TNF-α antagonist in the treatment of rheumatoid arthritis (RA), their modes of action are not fully understood. In the present study, we further explore the effects of gene transfer route of a TNF-α antagonist on arthritis. Recombinant adeno-associated virus 2 (rAAV2) encoding rat TNF receptor-immunoglobulin Fc (ratTNFR:Fc) fusion gene was injected intraarticularly in rats with collagen-induced arthritis (CIA). As revealed by examination of the clinical, radiographical, and histological aspects, local gene transfer of rAAV2/ratTNFR:Fc ameliorated the arthritis symptoms and inhibited the development of CIA. Compared with the vector control group, expressions of TNF-α, IL-1, and IFN-γ were down-regulated, and IL-10 release was up-regulated in the rAAV2/ratTNFR:Fc-treated group. Furthermore, administration of rAAV2/ratTNFR:Fc ameliorated the enlargement of spleen and significantly reduced spleen cell proliferation. Low level of nitric oxide (NO) in spleen was observed in CIA rats following the delivery of rAAV2/ratTNFR:Fc when compared to the vector control group. This study provides the evidence that intraarticular delivery of rAAV2/ratTNFR:Fc suppress the progression of arthritis by restoring the balance between pro-inflammatory and anti-inflammatory cytokines and inhibiting spleen cell proliferation. Our findings also implicate that the down-regulation of NO release on arthritis is involved in the anti-inflammatory mechanisms of TNF-α antagonist.

Interaction with CD68 and Regulation of GAS6 Expression by Endosialin in Fibroblasts Drives Recruitment and Polarization of Macrophages in Hepatocellular Carcinoma.

Fibroblasts and macrophages play key roles in the development of hepatocellular carcinoma (HCC). However, cross-talk between these two kinds of cells has not been well studied. Endosialin (CD248/TEM1) is a transmembrane glycoprotein that is expressed in certain cancer cells, tumor stromal cells, and pericytes. In this study, we found that endosialin is mainly expressed in cancer-associated fibroblasts (CAF) in HCC and its expression inversely correlates with patient prognosis. Endosialin interacted with CD68 to recruit macrophages and regulated expression of GAS6 in CAFs to mediate M2 polarization of macrophages. The fully human antibody IgG78 bound glycosylated endosialin and induced its internalization in CAFs, thus weakening the cross-talk between CAFs and macrophages. In subcutaneous and orthotopic xenograft models of HCC in nude mice, treatment with IgG78 significantly inhibited tumor growth. These results indicate that endosialin-positive CAFs promote HCC progression and highlight IgG78 as a promising therapeutic candidate for HCC treatment. SIGNIFICANCE: These findings highlight CAF-expressed endosialin as a primary regulator of macrophage recruitment and polarization and demonstrate endosialin inhibition as a potential treatment strategy for HCC. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/18/3892/F1.large.jpg.

Simple strategy for single-chain fragment antibody-conjugated probe construction.

AIMS: A combination of biomarker and instrument technology diagnosis methods, especially antigen-targeted imaging methods, is required to increase the accuracy of the diagnosis of cancer. Currently, the targeting efficiency is limited by the conjugation methods used for the conjugation of antibodies and imaging materials. Here, a simple strategy for the conjugation of a probe and a single-chain fragment antibody (scFv) that does not change the characteristics of the antibody was shown. MAIN METHODS: An ScFv was conjugated with superparamagnetic iron oxide (SPIO) or indocyanine green (ICG) via a linker by utilizing the reaction between cysteine and maleimide. The characterization of the probe was performed by flow cytometry, confocal imaging, optical imaging and magnetic resonance imaging (MRI). KEY FINDINGS: After conjugation, the scFv retained high affinity, antigen specificity, and strong internalization ability. The application of the conjugated probe was also confirmed by optical imaging and MRI. SIGNIFICANCE: The proposed strategy provides a simple method for the production of high efficiency antigen-targeted imaging probes for tumor diagnosis.