Serum amyloid P (PTX2) attenuates hepatic fibrosis in mice by inhibiting the activation of fibrocytes and HSCs.

Background: Liver fibrosis is caused by chronic toxic or cholestatic liver injury. Fibrosis results from the recruitment of myeloid cells into the injured liver, the release of inflammatory and fibrogenic cytokines, and the activation of myofibroblasts, which secrete extracellular matrix, mostly collagen type I. Hepatic myofibroblasts originate from liver-resident mesenchymal cells, including HSCs and bone marrow-derived CD45+ collagen type I+ expressing fibrocytes.

Targeting CD74 in B-cell non-Hodgkin lymphoma with the antibody-drug conjugate STRO-001.

Overexpression of CD74, a type II transmembrane glycoprotein involved in MHC class II antigen presentation, has been reported in many B-cell non-Hodgkin lymphomas (NHLs) and in multiple myeloma (MM). STRO-001 is a site-specific, predominantly single-species antibody-drug conjugate (ADC) that targets CD74 and has demonstrated efficacy in xenograft models of MM and tolerability in non-human primates. Here we report results of preclinical studies designed to elucidate the potential role of STRO-001 in B-cell NHL.

Discovery of STRO-002, a Novel Homogeneous ADC Targeting Folate Receptor Alpha, for the Treatment of Ovarian and Endometrial Cancers.

STRO-002 is a novel homogeneous folate receptor alpha (FolRα) targeting antibody-drug conjugate (ADC) currently being investigated in the clinic as a treatment for ovarian and endometrial cancers. Here, we describe the discovery, optimization, and antitumor properties of STRO-002. STRO-002 was generated by conjugation of a novel cleavable 3-aminophenyl hemiasterlin linker-warhead (SC239) to the nonnatural amino acid para-azidomethyl-L-phenylalanine incorporated at specific positions within a high affinity anti-FolRα antibody using Sutro's XpressCF+, which resulted in a homogeneous ADC with a drug-antibody ratio (DAR) of 4.

Site-Specific Zr- and In-Radiolabeling and In Vivo Evaluation of Glycan-free Antibodies by Azide-Alkyne Cycloaddition with a Non-natural Amino Acid.

Antibody-drug conjugates (ADCs) are a class of targeted therapeutics consisting of a monoclonal antibody coupled to a cytotoxic payload. Various bioconjugation methods for producing site-specific ADCs have been reported recently, in efforts to improve immunoreactivity and pharmacokinetics and minimize batch variance-potential issues associated with first-generation ADCs prepared via stochastic peptide coupling of lysines or reduced cysteines. Recently, cell-free protein synthesis of antibodies incorporating -azidomethyl phenylalanine (pAMF) at specific locations within the protein sequence has emerged as a means to generate antibody-drug conjugates with strictly defined drug-antibody-ratio, leading to ADCs with markedly improved stability, activity, and specificity.

Targeting CD74 in multiple myeloma with the novel, site-specific antibody-drug conjugate STRO-001.

STRO-001 is a site-specific, predominantly single-species, fully human, aglycosylated anti-CD74 antibody-drug conjugate incorporating a non-cleavable linker-maytansinoid warhead with a drug-antibody ratio of 2 which was produced by a novel cell-free antibody synthesis platform. We examined the potential pharmacodynamics and anti-tumor effects of STRO-001 in multiple myeloma (MM). CD74 expression was assessed in MM cell lines and primary bone marrow (BM) MM biopsies.

Pentraxin-2 suppresses c-Jun/AP-1 signaling to inhibit progressive fibrotic disease.

Pentraxin-2 (PTX-2), also known as serum amyloid P component (SAP/APCS), is a constitutive, antiinflammatory, innate immune plasma protein whose circulating level is decreased in chronic human fibrotic diseases. Here we show that recombinant human PTX-2 (rhPTX-2) retards progression of chronic kidney disease in mutant mice with Alport syndrome, reducing blood markers of kidney failure, enhancing lifespan by 20%, and improving histological signs of disease. Exogenously delivered rhPTX-2 was detected in macrophages but also in tubular epithelial cells, where it counteracted macrophage activation and was cytoprotective for the epithelium.

Recombinant human serum amyloid P in healthy volunteers and patients with pulmonary fibrosis.

PRM-151, recombinant human Pentraxin-2 (PTX-2) also referred to as serum amyloid P (SAP), is under development for treatment of fibrosis. A First-in-Human (FIH) trial was performed to assess the safety, tolerability, and pharmacokinetics of single ascending intravenous doses of PRM-151 administered to healthy subjects, using a randomized, blinded, placebo controlled study design. Each cohort included three healthy subjects (PRM-151:placebo; 2:1).

Host responses in tissue repair and fibrosis.

Myofibroblasts accumulate in the spaces between organ structures and produce extracellular matrix (ECM) proteins, including collagen I. They are the primary "effector" cells in tissue remodeling and fibrosis. Previously, leukocyte progenitors termed fibrocytes and myofibroblasts generated from epithelial cells through epithelial-to-mesenchymal transition (EMT) were considered the primary sources of ECM-producing myofibroblasts in injured tissues.

PRM-151 (recombinant human serum amyloid P/pentraxin 2) for the treatment of fibrosis.

Serum amyloid P or pentraxin 2 (PTX2) is a highly phylogenetically conserved, naturally circulating plasma protein and a soluble pattern recognition receptor of the innate immune system. The unique binding activities of PTX2 suggest that it may localize specifically to sites of injury and function to aid in the removal of damaged tissue. The recent discovery of its ability to regulate certain monocyte differentiation states has identified PTX2 as a novel and potentially powerful antifibrotic agent.

Investigational approaches to therapies for idiopathic pulmonary fibrosis.

Importance Of The Field: In fibrosing diseases, scar tissue begins to replace normal tissue, causing tissue dysfunction. For instance, in lung fibrosis, foci of what resembles scar tissue form in the lungs, impeding the ability of patients to breathe. These conditions represent a significant source of morbidity and mortality.

Serum amyloid P inhibits fibrosis through Fc gamma R-dependent monocyte-macrophage regulation in vivo.

New therapies that target chronic inflammation with fibrosis are urgently required. Increasing evidence points to innate activation of inflammatory cells in driving chronic organ fibrosis. Serum amyloid P is a naturally circulating soluble pattern recognition receptor, a member of the family of pentraxin proteins.

Bone marrow Ly6Chigh monocytes are selectively recruited to injured kidney and differentiate into functionally distinct populations.

Roles for monocyte/macrophages (Mphi) in directing the development of tissue fibrosis are increasingly recognized. Macrophages form a heterogeneous group of inflammatory leukocytes, and the mechanisms by which they acquire heterogeneity and its functional significance are unclear. We used the unilateral ureteral obstruction model of progressive kidney fibrosis to explore macrophage heterogeneity and function further.

Targeting integrin structure and function in disease.

Initially linked to the pathogenesis of inflammatory and hematologic diseases, integrins have become validated drug targets with the approval of five drugs. Moreover, there are several promising drug candidates in preclinical and clinical stages of development for multiple clinical indications. Integrins are attractive drug targets as their antagonism can block several steps in disease progression or maintenance.

Regulation of fibrosis by the immune system.

Inflammation and fibrosis are two inter-related conditions with many overlapping mechanisms. Three specific cell types, macrophages, T helper cells, and myofibroblasts, each play important roles in regulating both processes. Following tissue injury, an inflammatory stimulus is often necessary to initiate tissue repair, where cytokines released from resident and infiltrating leukocytes stimulate proliferation and activation of myofibroblasts.

Crystal structure of the A domain from complement factor B reveals an integrin-like open conformation.

Complement factor B is a 90 kDa protein consisting of three domains: a three-module complement control protein, a von Willebrand factor A domain, and a C-terminal serine protease (SP) domain that adopts a default inactive (zymogen) conformation. The interaction between factor B and pathogen-bound C3b is mediated by its A domain, triggering a conformational change in factor B that ultimately creates the "C3 convertase" of the alternative complement pathway. We report the crystal structure of the A domain from factor B and show that it contains an integrin-like MIDAS motif that adopts the "open" conformation typical of integrin-ligand complexes, with an acidic residue (provided by a fortuitous crystal contact) completing the coordination of the metal ion.

Cellular activation of leukocyte function-associated antigen-1 and its affinity are regulated at the I domain allosteric site.

The I domain of the integrin LFA-1 possesses a ligand binding interface that includes the metal ion-dependent adhesion site. Binding of the LFA-1 ligand, ICAM-1 to the metal ion-dependent adhesion site is regulated by the I domain allosteric site (IDAS). We demonstrate here that intracellular signaling leading to activation of LFA-1 binding to ICAM-1 is regulated at the IDAS.

Cbl-transforming variants trigger a cascade of molecular alterations that lead to epithelial mesenchymal conversion.

Dispersal of epithelial cells is an important aspect of tumorigenesis, and invasion. Factors such as hepatocyte growth factor induce the breakdown of cell junctions and promote cell spreading and the dispersal of colonies of epithelial cells, providing a model system to investigate the biochemical signals that regulate these events. Multiple signaling proteins are phosphorylated in epithelial cells during hepatocyte growth factor-induced cell dispersal, including c-Cbl, a protooncogene docking protein with ubiquitin ligase activity.

NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding.

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal alpha-helix.

The linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells.

The protooncogene product Cbl has emerged as a negative regulator of tyrosine kinases. We have shown previously that Cbl binds to ZAP-70 through its N-terminal tyrosine kinase binding (TKB) domain. In this study, we demonstrate that overexpression of Cbl in Jurkat T cells decreases the TCR-induced phosphorylation of ZAP-70 and other cellular phosphoproteins.

The Cbl proto-oncogene product negatively regulates the Src-family tyrosine kinase Fyn by enhancing its degradation.

Fyn is a prototype Src-family tyrosine kinase that plays specific roles in neural development, keratinocyte differentiation, and lymphocyte activation, as well as roles redundant with other Src-family kinases. Similar to other Src-family kinases, efficient regulation of Fyn is achieved through intramolecular binding of its SH3 and SH2 domains to conserved regulatory regions. We have investigated the possibility that the tyrosine kinase regulatory protein Cbl provides a complementary mechanism of Fyn regulation.

Ox40-ligand has a critical costimulatory role in dendritic cell:T cell interactions.

The tumor necrosis factor family molecule Ox40-ligand (Ox40L) has been identified as a potential costimulatory molecule and also has been implicated in T cell homing and B cell activation. To ascertain the essential functions of Ox40L, we generated and characterized Ox40L-deficient mice. Mice lacking Ox40L exhibit an impaired contact hypersensitivity response, a dendritic cell-dependent T cell-mediated response, due to defects in T cell priming and cytokine production.

The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase.

The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear.

The evolutionarily conserved N-terminal region of Cbl is sufficient to enhance down-regulation of the epidermal growth factor receptor.

The mammalian proto-oncoprotein Cbl and its homologues in Caenorhabditis elegans and Drosophila are evolutionarily conserved negative regulators of the epidermal growth factor receptor (EGF-R). Overexpression of wild-type Cbl enhances down-regulation of activated EGF-R from the cell surface. We report that the Cbl tyrosine kinase-binding (TKB) domain is essential for this activity.

The Cbl protooncoprotein: a negative regulator of immune receptor signal transduction.

The Cbl protooncoprotein has recently emerged as a component of tyrosine kinase-mediated signal transduction in a variety of cell types. Here, we discuss evidence that supports a role for Cbl as a novel negative regulator of immune receptor signaling, and present models for its mode of function.

Cbl-mediated negative regulation of the Syk tyrosine kinase. A critical role for Cbl phosphotyrosine-binding domain binding to Syk phosphotyrosine 323.

The proto-oncogene product Cbl has emerged as a potential negative regulator of the Syk tyrosine kinase; however, the nature of physical interactions between Cbl and Syk that are critical for this negative regulation remains unclear. Here we show that the phosphotyrosine-binding (PTB) domain within the N-terminal transforming region of Cbl (Cbl-N) binds to phosphorylated Tyr323 in the linker region between the Src homology 2 and kinase domains of Syk, confirming recent results by another laboratory using the yeast two-hybrid approach (Deckert, M., Elly, C.