Objective. Lubricin, also referred to as superficial zone protein and PRG4, is a synovial glycoprotein that supplies a friction-resistant, antiadhesive coating to the surfaces of articular cartilage, thereby protecting against arthritis-associated tissue wear and degradation. This study was undertaken to generate and characterize a novel recombinant lubricin protein construct, LUB:1, and to evaluate its therapeutic efficacy following intraarticular delivery in a rat model of osteoarthritis (OA).Methods. Binding and localization of LUB:1 to cartilage surfaces was assessed by immunohistochemistry. The cartilage-lubricating properties of LUB:1 were determined using a custom friction testing apparatus. A cell-binding assay was performed to quantify the ability of LUB:1 to prevent cell adhesion. Efficacy studies were conducted in a rat meniscal tear model of OA. One week after the surgical induction of OA, LUB:1 or phosphate buffered saline vehicle was administered by intraarticular injection for 4 weeks, with dosing intervals of either once per week or 3 times per week. OA pathology scores were determined by histologic analysis.Results. LUB:1 was shown to bind effectively to cartilage surfaces, and facilitated both cartilage boundary lubrication and inhibition of synovial cell adhesion. Treatment of rat knee joints with LUB:1 resulted in significant disease-modifying, chondroprotective effects during the progression of OA, by markedly reducing cartilage degeneration and structural damage.Conclusion. Our findings demonstrate the potential use of recombinant lubricin molecules in novel biotherapeutic approaches to the treatment of OA and associated cartilage abnormalities.Osteoarthritis (OA) severely restricts the daily activities, mobility, and overall quality of life of millions of patients worldwide, imposing a high societal burden that reflects the current lack of effective medical therapies. OA is characterized by escalated degeneration and loss of articular cartilage, the specialized connective tissue covering the ends of interfacing bones within joints. To help withstand formidable biomechanical forces and loads, articular cartilage surfaces possess an inherently low coefficient of friction, which is facilitated in part by localization of the boundary lubricant lubricin (1). Lubricin was originally identified as a lubricating glycoprotein present in synovial fluid (2), and it is now recognized to have a major protective role in preventing cartilage wear and synovial cell adhesion and proliferation (3). Lubricin is encoded by the PRG4 gene, and PRG4-nullifying mutations can cause OA-like symptoms in mice and humans (3,4). Lubricin synthesis/localization (and therefore function) is also down-regulated in sheep (5), guinea pig (6), and rat (7)

Myostatin is a member of the bone morphogenetic protein/transforming growth factor-β (BMP/TGFβ) super-family of secreted differentiation factors. Myostatin is a negative regulator of muscle mass as shown by increased muscle mass in myostatin deficient mice. Interestingly, these mice also exhibit increased bone mass suggesting that myostatin may also play a role in regulating bone mass. To investigate the role of myostatin in bone, young adult mice were administered with either a myostatin neutralizing antibody (Mstn-mAb), a soluble myostatin decoy receptor (ActRIIB-Fc) or vehicle. While both myostatin inhibitors increased muscle mass, only ActRIIB-Fc increased bone mass. Bone volume fraction (BV/TV), as determined by microCT, was increased by 132% and 27% in the distal femur and lumbar vertebrae, respectively. Histological evaluation demonstrated that increased BV/TV in both locations was attributed to increased trabecular thickness, trabecular number and bone formation rate. Increased BV/TV resulted in enhanced vertebral maximum compressive force compared to untreated animals. The fact that ActRIIB-Fc, but not Mstn-mAb, increased bone volume suggested that this soluble decoy receptor may be binding a ligand other than myostatin, that plays a role in regulating bone mass. This was confirmed by the significant increase in BV/TV in myostatin deficient mice treated with ActRIIB-Fc. Of the other known ActRIIB-Fc ligands, BMP3 has been identified as a negative regulator of bone mass. However, BMP3 deficient mice treated with ActRIIB-Fc showed similar increases in BV/TV as wild type (WT) littermates treated with ActRIIB-Fc. This result suggests that BMP3 neutralization is not the mechanism responsible for increased bone mass. The results of this study demonstrate that ActRIIB-Fc increases both muscle and bone mass in mice. Therefore, a therapeutic that has this dual activity represents a potential approach for the treatment of frailty.

Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART ® ) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90˝apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (~4.4 days in human FcRn knock-in mice), high stability (T m 1 > 68˝C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.

CD3 bispecific antibody constructs recruit cytolytic T cells to kill tumor cells, offering a potent approach to treat cancer. T cell activation is driven by the formation of a trimolecular complex (trimer) between drugs, T cells, and tumor cells, mimicking an immune synapse. A translational quantitative systems pharmacology (QSP) model is proposed for CD3 bispecific molecules capable of predicting trimer concentration and linking it to tumor cell killing. The model was used to quantify the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of a CD3 bispecific targeting P-cadherin (PF-06671008). It describes the disposition of PF-06671008 in the central compartment and tumor in mouse xenograft models, including binding to target and T cells in the tumor to form the trimer. The model incorporates T cell distribution to the tumor, proliferation, and contraction. PK/PD parameters were estimated for PF-06671008 and a tumor stasis concentration (TSC) was calculated as an estimate of minimum efficacious trimer concentration. TSC values ranged from 0.0092 to 0.064 pM across mouse tumor models. The model was translated to the clinic and used to predict the disposition of PF-06671008 in patients, including the impact of binding to soluble P-cadherin. The predicted terminal half-life of PF-06671008 in the clinic was approximately 1 day, and P-cadherin expression and number of T cells in the tumor were shown to be sensitive parameters impacting clinical efficacy. A translational QSP model is presented for CD3 bispecific molecules, which integrates in silico, in vitro and in vivo data in a mechanistic framework, to quantify and predict efficacy across species.

A full-length transcript encoding a functional type II GnRH receptor was cloned from the pituitary of the sea lamprey, Petromyzon marinus. The current study is the first to identify a pituitary GnRH receptor transcript in an agnathan, which is the oldest vertebrate lineage. The cloned receptor retains the conserved structural features and amino acid motifs of other known GnRH receptors and notably includes a C-terminal intracellular tail of approximately 120 amino acids, the longest C-terminal tail of any vertebrate GnRH receptor identified to date. The lamprey GnRH receptor was shown to activate the inositol phosphate (IP) signaling system; stimulation with either lamprey GnRH-I or lamprey GnRH-III led to dose-dependent responses in transiently transfected COS7 cells. Furthermore, analyses of serially truncated lamprey GnRH receptor mutants indicate perturbations of the C-terminal tail disrupts IP accumulation, however, the tailless lamprey GnRH receptor was not only functional but was also capable of stimulating IP levels equal to wild type. Expression of the receptor transcript was demonstrated in the pituitary and testes using RT-PCR, whereas in situ hybridization showed expression and localization of the transcript in the proximal pars distalis of the pituitary. The phylogenetic placement and structural and functional features of this GnRH receptor suggest that it is representative of an ancestral GnRH receptor. In addition to having an important role in lamprey reproductive processes, the extensive C-terminal tail of this lamprey GnRH receptor may have great significance for understanding the evolutionary change of this vital structural feature within the GnRH receptor family.

Strong evidence exists supporting the important role T cells play in the immune response against tumors. Still, the ability to initiate tumor-specific immune responses remains a challenge. Recent clinical trials suggest that bispecific antibody-mediated retargeted T cells are a promising therapeutic approach to eliminate hematopoietic tumors. However, this approach has not been validated in solid tumors. PF-06671008 is a dual-affinity retargeting (DART)-bispecific protein engineered with enhanced pharmacokinetic properties to extend in vivo half-life, and designed to engage and activate endogenous polyclonal T cell populations via the CD3 complex in the presence of solid tumors expressing P-cadherin. This bispecific molecule elicited potent P-cadherin expression-dependent cytotoxic T cell activity across a range of tumor indications in vitro, and in vivo in tumor-bearing mice. Regression of established tumors in vivo was observed in both cell line and patient-derived xenograft models engrafted with circulating human T lymphocytes. Measurement of in vivo pharmacodynamic markers demonstrates PF-06671008-mediated T cell activation, infiltration and killing as the mechanism of tumor inhibition.

Purpose: Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy. Experimental Design: PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, and in vivo in established subcutaneous and orthotopic human colorectal cancer xenograft tumors with adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using human CD3e transgenic mice. IHC and mass cytometry were performed to demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of immune evasion. Combination studies were performed with anti-PD-1/PD-L1 and anti-VEGF antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque. Results: We demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3e bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T-cell-mediated in vitro activity and in vivo efficacy in multiple colorectal cancer human xenograft tumor models, including KRASand BRAF-mutant tumors, as well as in the immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced when combined with anti-PD-1/ PD-L1 treatment or in combination with antiangiogenic therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile. Conclusions: These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in colorectal cancer and other gastrointestinal malignancies.

LOX‐1 is a scavenger receptor that functions as the primary receptor for oxidized low‐density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX‐1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX‐1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand‐binding function of cell surface LOX‐1, we generated a series of recombinant human LOX‐1–crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand‐binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX‐1 is required for high‐avidity binding of ligands, we found that LOX‐1–Fc fusion protein containing four ligand‐binding domains per Fc dimer, but not the one containing two ligand‐binding domains, exhibited ligand‐binding activity. Optimal ligand‐binding activity could be achieved via crosslinking of LOX‐1–Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX‐1–Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX‐1. These findings demonstrate that functional oligomerization is required for recombinant LOX‐1–Fc to function as an effective antagonist. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7213199: LOX1 (uniprotkb:http://www.ebi.uniprot.org/entry/P78380) and LOX1 (uniprotkb:http://www.ebi.uniprot.org/entry/P78380) physically interact (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0914) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)