LTßR Agonism Promotes Anti-Tumor Immune Responses via Modulation of the Tumor Microenvironment.

The presence of high endothelial venules (HEV) and tertiary lymphoid structures (TLS) in solid tumors is correlated with favorable prognosis and better responses to immune-checkpoint blockade (ICB) in many cancer types. Elucidation of the molecular mechanisms underlying intratumoral HEV and TLS formation and their contribution to anti-tumor responses may facilitate development of improved treatment strategies. Lymphotoxin beta receptor (LTßR) signaling is a critical regulator of lymph node organogenesis and can cooperate with antiangiogenic and ICB treatment to augment tumor-associated HEV formation. Here, we demonstrated that LTßR signaling modulates the tumor microenvironment via multiple mechanisms to promote anti-tumor T cell responses. Systemic activation of the LTßR pathway via agonistic antibody treatment induced tumor-specific HEV formation, upregulated the expression of TLS-related chemokines, and enhanced dendritic cell (DC) and T cell infiltration and activation in syngeneic tumor models. In vitro studies confirmed direct effects of LTßR agonism on DC activation and maturation and associated DC-mediated T cell activation. Single agent LTßR agonist treatment inhibited syngeneic tumor growth in a CD8+ T cell- and HEV-dependent manner, and the LTßR agonist enhanced anti-tumor effects of anti-PD-1 and CAR T cell therapies. An in vivo tumor screen for TLS-inducing cytokines revealed that the combination of LTßR agonism and lymphotoxin alpha (LT⍺) expression promoted robust intratumoral TLS induction and enhanced tumor responses to anti-CTLA-4 treatment. Collectively, this study highlights crucial functions of LTßR signaling in modulating the tumor microenvironment and could inform future HEV/TLS-based strategies for cancer treatments.

A Tetravalent Bispecific Antibody Selectively Inhibits Diverse FGFR3 Oncogenic Variants.

The receptor tyrosine kinase FGFR3 is frequently mutated in bladder cancer and is a validated therapeutic target. Although pan-FGFR tyrosine kinase inhibitors (TKI) have shown clinical efficacy, toxicity and acquired resistance limit the benefit of these agents. While antibody-based therapeutics can offer superior selectivity than TKIs, conventional ligand-blocking antibodies are usually ineffective inhibitors of constitutively active receptor tyrosine kinases. Furthermore, the existence of multiple oncogenic variants of FGFR3 presents an additional challenge for antibody-mediated blockade. Here, we developed a tetravalent FGFR3×FGFR3 bispecific antibody that inhibited FGFR3 point mutants and fusion proteins more effectively than any of the conventional FGFR3 antibodies that we produced. Each arm of the bispecific antibody contacted two distinct epitopes of FGFR3 through a cis mode of binding. The antibody blocked dimerization of the most common FGFR3 oncogenic variant (S249C extracellular domain mutation) and inhibited the function of FGFR3 variants that are resistant to pan-FGFR TKIs. The antibody was highly effective in suppressing growth of FGFR3-driven tumor models, providing efficacy comparable to that of the FDA-approved TKI erdafitinib. Thus, this bispecific antibody may provide an effective approach for broad and highly selective inhibition of oncogenic FGFR3 variants. Significance: Development of a bispecific antibody that broadly inhibits gain-of-function FGFR3 variants provides a therapeutic strategy to target tumors with oncogenic FGFR3 point mutations and fusions, a particularly difficult case for antibody blockade.

Generic versus disease-specific adverse event reporting: a comparison of the NSQIP and SAVES databases for the identification of acute care adverse events in adult spine surgery.

OBJECTIVE: The accurate identification and reporting of adverse events (AEs) is crucial for quality improvement. A myriad of AE systems are utilized. There is a lack of understanding of the differences between prospective versus retrospective, disease-specific versus generic, and point-of-care versus chart-abstracted systems. The objective of this study was to compare the benefits and limitations between the prospective, disease-specific, point-of-care Spine Adverse Events Severity System (SAVES) and the retrospective, generic, and chart-abstracted National Surgical Quality Improvement Program (NSQIP) for the identification and reporting of AEs in adult patients undergoing spinal surgery. METHODS: The authors conducted an observational ambidirectional cohort study of adult patients undergoing spine surgery other than for trauma between 2011 and 2019 in a quaternary spine center. Patients were identified using Current Procedural Terminology codes in the NSQIP database and matched using unique medical record numbers to their corresponding record in SAVES. The incidence of AEs and per-patient AEs as recorded in NSQIP and SAVES was the primary outcome of interest. Comparable AEs were identified by matching NSQIP AEs to equivalent ones in SAVES. Chi-square tests were used to test for significant differences in the incidence of overall and comparable AEs between the databases. RESULTS: There were 2198 patients identified in NSQIP, of whom 2033 also had complete records in SAVES. SAVES identified 5342 individual AEs in 1484 patients (73%) compared with 1291 individual AEs in 807 patients (39.7%) with the NSQIP database (p < 0.001). SAVES identified 250 intraoperative and 422 postoperative spine-specific AEs that NSQIP did not record. NSQIP captured a greater number of AEs beyond 30 days, including prolonged length of stay > 30 days, unplanned readmission, unplanned reoperation, and death later than 30 days after surgery compared with SAVES. CONCLUSIONS: SAVES captures a greater incidence of peri- and intraoperative spine-specific AEs than NSQIP, while NSQIP identifies a greater number of AEs beyond 30 days. While a prospective, disease-specific, point-of-care AE system such as SAVES is specific for guiding quality improvement in spine surgery, it incurs greater time and financial costs. Conversely, a retrospective, generic, and chart-abstracted system such as NSQIP provides equivocal cross-institutional comparability with reduced time and financial costs. Specific contextual and aim-specific needs should guide the choice and implementation of an AE system.

IL-6-GP130 signaling protects human hepatocytes against lipid droplet accumulation in humanized liver models.

Liver steatosis is an increasing health issue with few therapeutic options, partly because of a paucity of experimental models. In humanized liver rodent models, abnormal lipid accumulation in transplanted human hepatocytes occurs spontaneously. Here, we demonstrate that this abnormality is associated with compromised interleukin-6 (IL-6)-glycoprotein 130 (GP130) signaling in human hepatocytes because of incompatibility between host rodent IL-6 and human IL-6 receptor (IL-6R) on donor hepatocytes. Restoration of hepatic IL-6-GP130 signaling, through ectopic expression of rodent IL-6R, constitutive activation of GP130 in human hepatocytes, or humanization of an Il6 allele in recipient mice, substantially reduced hepatosteatosis. Notably, providing human Kupffer cells via hematopoietic stem cell engraftment in humanized liver mice also corrected the abnormality. Our observations suggest an important role of IL-6-GP130 pathway in regulating lipid accumulation in hepatocytes and not only provide a method to improve humanized liver models but also suggest therapeutic potential for manipulating GP130 signaling in human liver steatosis.

A Bispecific METxMET Antibody-Drug Conjugate with Cleavable Linker Is Processed in Recycling and Late Endosomes.

Most antibody-drug conjugates (ADC) approved for the treatment of cancer contain protease-cleavable linkers. ADCs that traffic to lysosomes traverse highly acidic late endosomes, while ADCs that recycle to the plasma membrane traffic through mildly acidic sorting and recycling endosomes. Although endosomes have been proposed to process cleavable ADCs, the precise identity of the relevant compartments and their relative contributions to ADC processing remain undefined. Here we show that a METxMET biparatopic antibody internalizes into sorting endosomes, rapidly traffics to recycling endosomes, and slowly reaches late endosomes. In agreement with the current model of ADC trafficking, late endosomes are the primary processing site of MET, EGFR, and prolactin receptor ADCs. Interestingly, recycling endosomes contribute up to 35% processing of the MET and EGFR ADCs in different cancer cells, mediated by cathepsin-L, which localizes to this compartment. Taken together, our findings provide insight into the relationship between transendosomal trafficking and ADC processing and suggest that receptors that traffic through recycling endosomes might be suitable targets for cleavable ADCs.

Preclinical Study of a Biparatopic METxMET Antibody-Drug Conjugate, REGN5093-M114, Overcomes MET-driven Acquired Resistance to EGFR TKIs in EGFR-mutant NSCLC.

PURPOSE: MET amplification is a frequent mechanism of resistance to EGFR tyrosine kinase inhibitors (TKI) in patients with EGFR-mutated non-small cell lung cancer (NSCLC), and combined treatment with EGFR TKIs and MET TKIs has been explored as a strategy to overcome resistance. However, durable response is invariably limited by the emergence of acquired resistance. Here, we investigated the preclinical activity of REGN5093-M114, a novel antibody-drug conjugate targeting MET in MET-driven patient-derived models. EXPERIMENTAL DESIGN: Patient-derived organoids, patient-derived cells, or ATCC cell lines were used to investigate the in vitro/in vivo activity of REGN5093-M114. RESULTS: REGN5093-M114 exhibited significant antitumor efficacy compared with MET TKI or unconjugated METxMET biparatopic antibody (REGN5093). Regardless of MET gene copy number, MET-overexpressed TKI-naïve EGFR-mutant NSCLC cells responded to REGN5093-M114 treatment. Cell surface MET expression had the most predictive power in determining the efficacy of REGN5093-M114. REGN5093-M114 potently reduced tumor growth of EGFR-mutant NSCLC with PTEN loss or MET Y1230C mutation after progression on prior osimertinib and savolitinib treatment. CONCLUSIONS: Altogether, REGN5093-M114 is a promising candidate to overcome the challenges facing functional MET pathway blockade.

A Biparatopic Antibody-Drug Conjugate to Treat MET-Expressing Cancers, Including Those that Are Unresponsive to MET Pathway Blockade.

Lung cancers harboring mesenchymal-to-epithelial transition factor (MET) genetic alterations, such as exon 14 skipping mutations or high-level gene amplification, respond well to MET-selective tyrosine kinase inhibitors (TKI). However, these agents benefit a relatively small group of patients (4%-5% of lung cancers), and acquired resistance limits response durability. An antibody-drug conjugate (ADC) targeting MET might enable effective treatment of MET-overexpressing tumors (approximately 25% of lung cancers) that do not respond to MET targeted therapies. Using a protease-cleavable linker, we conjugated a biparatopic METxMET antibody to a maytansinoid payload to generate a MET ADC (METxMET-M114). METxMET-M114 promotes substantial and durable tumor regression in xenografts with moderate to high MET expression, including models that exhibit innate or acquired resistance to MET blockers. Positron emission tomography (PET) studies show that tumor uptake of radiolabeled METxMET antibody correlates with MET expression levels and METxMET-M114 efficacy. In a cynomolgus monkey toxicology study, METxMET-M114 was well tolerated at a dose that provides circulating drug concentrations that are sufficient for maximal antitumor activity in mouse models. Our findings suggest that METxMET-M114, which takes advantage of the unique trafficking properties of our METxMET antibody, is a promising candidate for the treatment of MET-overexpressing tumors, with the potential to address some of the limitations faced by the MET function blockers currently in clinical use.

Monomeric/dimeric forms of Fgf15/FGF19 show differential activity in hepatocyte proliferation and metabolic function.

Human Fibroblast Growth Factor 19 (FGF19) and mouse ortholog Fgf15 play similar roles in liver regeneration and metabolism via the activation of Fgfr4/b-klotho (Klb). Monomeric FGF19 and dimeric Fgf15 are both necessary for liver regeneration and proper bile acid (BA) metabolism. FGF19 elicits stronger effects than Fgf15 on glucose and fatty acid metabolism and only FGF19 induces hepatocellular carcinoma (HCC). However, inhibiting FGF19/FGFR4 signaling in HCC patients is associated with toxicity due to elevated BA levels. Here, we examine the structure/function relationship in Fgf15/FGF19 to better understand the molecular basis for their distinct functions. We demonstrate that FGF19 is a more effective activator of Fgfr4 and of downstream signaling (Erk, Plcg1) than Fgf15. Furthermore, we use site-directed mutagenesis to show that the presence or absence of an unpaired cysteine in Fgf15/19 modulates ligand structure and determines the ability of these molecules to induce hepatocyte proliferation, with monomers being more potent activators. Consistent with these findings, an engineered dimeric variant of FGF19 is less effective than wild-type FGF19 at inducing liver growth in cooperation with the Wnt-enhancer RSPO3. In contrast to effects on proliferation, monomeric and dimeric ligands equally inhibited the expression of Cyp7a1, the enzyme catalyzing the rate limiting step in BA production. Thus, structure and function of Fgf15/FGF19 are intricately linked, explaining why FGF19, but not Fgf15, induces liver tumorigenesis. Our data provide insight into FGF19/FGFR4 signaling and may inform strategies to target this pathway while limiting on-target toxicity due to dysregulation of BA production or induction of hepatocyte proliferation.

Tumor-targeted CD28 bispecific antibodies enhance the antitumor efficacy of PD-1 immunotherapy.

Monoclonal antibodies that block the programmed cell death 1 (PD-1) checkpoint have revolutionized cancer immunotherapy. However, many major tumor types remain unresponsive to anti-PD-1 therapy, and even among responsive tumor types, most of the patients do not develop durable antitumor immunity. It has been shown that bispecific antibodies activate T cells by cross-linking the TCR/CD3 complex with a tumor-specific antigen (TSA). The class of TSAxCD3 bispecific antibodies have generated exciting results in early clinical trials. We have recently described another class of "costimulatory bispecifics" that cross-link a TSA to CD28 (TSAxCD28) and cooperate with TSAxCD3 bispecifics. Here, we demonstrate that these TSAxCD28 bispecifics (one specific for prostate cancer and the other for epithelial tumors) can also synergize with the broader anti-PD-1 approach and endow responsiveness-as well as long-term immune memory-against tumors that otherwise do not respond to anti-PD-1 alone. Unlike CD28 superagonists, which broadly activate T cells and induce cytokine storm, TSAxCD28 bispecifics display little or no toxicity when used alone or in combination with a PD-1 blocker in genetically humanized immunocompetent mouse models or in primates and thus may provide a well-tolerated and "off the shelf" combination approach with PD-1 immunotherapy that can markedly enhance antitumor efficacy.

Spinal Pleomorphic Xanthoastrocytoma: Case Report and Literature Review.

Pleomorphic xanthoastrocytoma (PXA) is an uncommon central nervous system neoplasm with an overall favorable survival prognosis. Metastatic spread of PXA to the spinal cord and the cauda equina is rare and may have a different clinicopathologic course. Treatment and prognostic outcomes, in this context, are not well defined. We discuss a case of a 30-year-old patient with known cerebral PXA presenting with metastatic spinal anaplastic PXA and present a literature analysis of treatment outcomes.

A Biparatopic Antibody That Modulates MET Trafficking Exhibits Enhanced Efficacy Compared with Parental Antibodies in MET-Driven Tumor Models.

PURPOSE: Recent clinical data demonstrate that tumors harboring MET genetic alterations (exon 14 skip mutations and/or gene amplification) respond to small-molecule tyrosine kinase inhibitors, validating MET as a therapeutic target. Although antibody-mediated blockade of the MET pathway has not been successful in the clinic, the failures are likely the result of inadequate patient selection strategies as well as suboptimal antibody design. Thus, our goal was to generate a novel MET blocking antibody with enhanced efficacy. EXPERIMENTAL DESIGN: Here, we describe the activity of a biparatopic MET×MET antibody that recognizes two distinct epitopes in the MET Sema domain. We use a combination of in vitro assays and tumor models to characterize the effect of our antibody on MET signaling, MET intracellular trafficking, and the growth of MET-dependent cells/tumors. RESULTS: In MET-driven tumor models, our biparatopic antibody exhibits significantly better activity than either of the parental antibodies or the mixture of the two parental antibodies and outperforms several clinical-stage MET antibodies. Mechanistically, the biparatopic antibody inhibits MET recycling, thereby promoting lysosomal trafficking and degradation of MET. In contrast to the parental antibodies, the biparatopic antibody fails to activate MET-dependent biological responses, consistent with the observation that it recycles inefficiently and induces very transient downstream signaling. CONCLUSIONS: Our results provide strong support for the notion that biparatopic antibodies are a promising therapeutic modality, potentially having greater efficacy than that predicted from the properties of the parental antibodies.

Autophagy protects tumors from T cell-mediated cytotoxicity via inhibition of TNFα-induced apoptosis.

Although T cell checkpoint inhibitors have transformed the treatment of cancer, the molecular determinants of tumor cell sensitivity to T cell-mediated killing need further elucidation. Here, we describe a mouse genome-scale CRISPR knockout screen that identifies tumor cell TNFα signaling as an important component of T cell-induced apoptosis, with NF-κB signaling and autophagy as major protective mechanisms. Knockout of individual autophagy genes sensitized tumor cells to killing by T cells that were activated via specific TCR or by a CD3 bispecific antibody. Conversely, inhibition of mTOR signaling, which results in increased autophagic activity, protected tumor cells from T cell killing. Autophagy functions at a relatively early step in the TNFα signaling pathway, limiting FADD-dependent caspase-8 activation. Genetic inactivation of tumor cell autophagy enhanced the efficacy of immune checkpoint blockade in mouse tumor models. Thus, targeting the protective autophagy pathway might sensitize tumors to T cell-engaging immunotherapies in the clinic.

Genome-scale CRISPR activation screen uncovers tumor-intrinsic modulators of CD3 bispecific antibody efficacy.

Bispecific antibodies (bsAb) that bridge tumor cells and CD3-positive effector T cells are being developed against many tumor cell targets. While tumor cell factors other than target expression level appear to play a role in determining the efficacy of CD3 bsAb, the identity of such factors remains largely unknown. Using a co-culture system of primary human T cells and B lymphoma cell lines, we demonstrate a range of sensitivities to CD20xCD3 bsAb that is independent of CD20 surface expression. To identify genes that modulate tumor cell sensitivity to CD3 bsAb, we employed a genome-scale CRISPR activation screen in a CD20xCD3-sensitive human B lymphoma cell line. Among the most highly enriched sgRNAs were those targeting genes with predicted effects on cell-cell adhesion, including sialophorin (SPN). Increased expression of SPN impeded tumor cell clustering with T cells, thereby limiting CD3 bsAb-mediated tumor cell lysis. This inhibitory effect of SPN appeared to be dependent on sialylated core 2 O-glycosylation of the protein. While SPN is not endogenously expressed in the majority of B cell lymphomas, it is highly expressed in acute myeloid leukemia. CRISPR-mediated SPN knockout in AML cell lines facilitated T cell-tumor cell clustering and enhanced CD3 bsAb-mediated AML cell lysis. In sum, our data establish that the cell cross-linking mechanism of CD3 bsAb is susceptible to subversion by anti-adhesive molecules expressed on the tumor cell surface. Further evaluation of anti-adhesive pathways may provide novel biomarkers of clinical response and enable the development of effective combination regimens for this promising therapeutic class.

Glioblastoma Presenting as Spontaneous Subarachnoid Hemorrhage: Technical Case Note of Combined Endovascular and Microsurgical Vision-Sparing Treatment.

BACKGROUND: Clinical utility of endovascular adjunct for tumor resection is well established, but its role in acute subarachnoid hemorrhage secondary to neoplastic pseudoaneurysm rupture has not been reported. CASE DESCRIPTION: We discuss a 46-year-old patient presenting with a World Federation of Neurological Surgeons grade 1 subarachnoid hemorrhage from a ruptured posterior cerebral artery pseudoaneurysm due to glioblastoma tumor invasion. CONCLUSIONS: A combined targeted endovascular embolization with microsurgical resection to spare the calcarine artery was used to avoid disruption to the optic radiation fiber pathway.

Perioperative care for lumbar microdiscectomy: a survey of Australasian neurosurgeons.

BACKGROUND: Lumbar microdiscectomy is the most commonly performed spine surgery procedure. Over time it has evolved to a minimally invasive procedure. Traditionally patients were advised to restrict activity following lumbar spine surgery. However, post-operative instructions are heterogeneous. The purpose of this report is to assess, by survey, the perioperative care practices of Australasian neurosurgeons in the minimally invasive era. METHODS: A survey was conducted by email invitation sent to all full members of the Neurosurgical Society of Australasia (NSA). This consisted of 11 multi-choice questions relating to operative indications, technique, and post-operative instructions for lumbar microdiscectomy answered by an electronically distributed anonymized online survey. RESULTS: The survey was sent to all Australasian Neurosurgeons. In total, 68 complete responses were received (28.9%). Most surgeons reported they would consider a period of either 4 to 8 weeks (42.7%) or 8 to 12 weeks (32.4%) as the minimum duration of radicular pain adequate to offer surgery. Unilateral muscle dissection with unilateral discectomy was practiced by 76.5%. Operative microscopy was the most commonly employed method of magnification (76.5%). The majority (55.9%) always refer patients to undergo inpatient physiotherapy. Sitting restrictions were advised by 38.3%. Lifting restrictions were advised by 83.8%. CONCLUSIONS: Australasian neurosurgical lumbar microdiscectomy perioperative care practices are generally consistent with international practices and demonstrate a similar degree of heterogeneity. Recommendation of post-operative activity restrictions by Australasian neurosurgeons is still common. This suggests a role for the investigation of the necessity of such restrictions in the era of minimally invasive spine surgery.

Angiopoietins bind thrombomodulin and inhibit its function as a thrombin cofactor.

Angiopoietin-1 (Ang1) and Angiopoietin-2 (Ang2) are ligands for Tie2, an endothelial-specific receptor tyrosine kinase that is an essential regulator of angiogenesis. Here we report the identification, via expression cloning, of thrombomodulin (TM) as another receptor for Ang1 and Ang2. Thrombomodulin is an endothelial cell surface molecule that plays an essential role as a coagulation inhibitor via its function as a cofactor in the thrombin-mediated activation of protein C, an anticoagulant protein, as well as thrombin-activatable fibrinolysis inhibitor (TAFI). Ang1 and Ang2 inhibited the thrombin/TM-mediated generation of activated protein C and TAFI in cultured endothelial cells, and inhibited the binding of thrombin to TM in vitro. Ang2 appears to bind TM with higher affinity than Ang1 and is a more potent inhibitor of TM function. Consistent with a potential role for angiopoietins in coagulation, administration of thrombin to mice rapidly increased plasma Ang1 levels, presumably reflecting release from activated platelets (previously shown to contain high levels of Ang1). In addition, Ang1 levels were significantly elevated in plasma prepared from wound blood, suggesting that Ang1 is released from activated platelets at sites of vessel injury. Our results imply a previously undescribed role for angiopoietins in the regulation of hemostasis.

Prevalence of Human Papillomavirus Genotypes and Abnormal Pap Smears Among Women in the Military Health System.

Human papillomavirus infection (HPV) is the most common sexually transmitted infection among United States Military Servicemembers, and present in the majority of cervical cancers. Many of these infections are preventable, but HPV immunization is not mandatory during military service. The objective of this study was to examine the prevalence of vaccine-preventable cervical disease among women enrolled in the San Antonio Military Health System. This is a retrospective cross-sectional study of Pap smear results and HPV genotyping data among Military Servicewomen and beneficiaries. Simple descriptive statistics and logistic regression were used to assess the association between demographics, cervical pathology and high-risk HPV (hrHPV) infection. Pap smears were obtained by 16.9% of women and cervical pathology was present in 28.8% of samples. Compared to the 25-34 year group, 35-44 year-olds were more likely to have an abnormal Pap smear (OR 1.25, CI 1.05-1.50). Of the samples tested, 10.5% were positive for hrHPV. Adjusted multivariable analysis revealed that hrHPV infection was more likely among the 23-34 year group when compared to 35-44 (OR 0.50, CI 0.38-0.67), 45-54 (0.40. CI 0.28-0.59) and 55-65 year groups (0.46, CI 0.30-0.71). Active Duty Servicewomen were more likely to test positive for hrHPV when compared to Active Duty Family Members (OR 0.59, CI 0.45-0.79) and Retiree Family Members (OR 0.59, CI 0.41-0.83). Younger women and Active Duty Servicewomen are significantly more likely to have cervical infection with hrHPV. Future studies should assess the cost-effectiveness of mandatory HPV immunization for military members.

Pentosan polysulfate binds to STRO-1+ mesenchymal progenitor cells, is internalized, and modifies gene expression: a novel approach of pre-programing stem cells for therapeutic application requiring their chondrogenesis.

BACKGROUND: The pharmaceutical agent pentosan polysulfate (PPS) is known to induce proliferation and chondrogenesis of mesenchymal progenitor cells (MPCs) in vitro and in vivo. However, the mechanism(s) of action of PPS in mediating these effects remains unresolved. In the present report we address this issue by investigating the binding and uptake of PPS by MPCs and monitoring gene expression and proteoglycan biosynthesis before and after the cells had been exposed to limited concentrations of PPS and then re-established in culture in the absence of the drug (MPC priming). METHODS: Immuno-selected STRO-1+ mesenchymal progenitor stem cells (MPCs) were prepared from human bone marrow aspirates and established in culture. The kinetics of uptake, shedding, and internalization of PPS by MPCs was determined by monitoring the concentration-dependent loss of PPS media concentrations using an enzyme-linked immunosorbent assay (ELISA) and the uptake of fluorescein isothiocyanate (FITC)-labelled PPS by MPCs. The proliferation of MPCs, following pre-incubation and removal of PPS (priming), was assessed using the Wst-8 assay method, and proteoglycan synthesis was determined by the incorporation of 35SO4 into their sulphated glycosaminoglycans. The changes in expression of MPC-related cell surface antigens of non-primed and PPS-primed MPCs from three donors was determined using flow cytometry. RNA sequencing of RNA isolated from non-primed and PPS-primed MPCs from the same donors was undertaken to identify the genes altered by the PPS priming protocol. RESULTS: The kinetic studies indicated that, in culture, PPS rapidly binds to MPC surface receptors, followed by internalisation and localization within the nucleus of the cells. Following PPS-priming of MPCs and a further 48 h of culture, both cell proliferation and proteoglycan synthesis were enhanced. Reduced expression of MPC-related cell surface antigen expression was promoted by the PPS priming, and RNA sequencing analysis revealed changes in the expression of 42 genes. CONCLUSION: This study has shown that priming of MPCs with low concentrations of PPS enhanced chondrogenesis and MPC proliferation by modifying their characteristic basal gene and protein expression. These findings offer a novel approach to re-programming mesenchymal stem cells for clinical indications which require the repair or regeneration of cartilaginous tissues such as in osteoarthritis and degenerative disc disease.

Bispecific Antibodies and Antibody-Drug Conjugates (ADCs) Bridging HER2 and Prolactin Receptor Improve Efficacy of HER2 ADCs.

The properties of cell surface proteins targeted by antibody-drug conjugates (ADCs) have not been fully exploited; of particular importance are the rate of internalization and the route of intracellular trafficking. In this study, we compared the trafficking of HER2, which is the target of the clinically approved ADC ado-trastuzumab emtansine (T-DM1), with that of prolactin receptor (PRLR), another potential target in breast cancer. In contrast to HER2, we found that PRLR is rapidly and constitutively internalized, and traffics efficiently to lysosomes, where it is degraded. The PRLR cytoplasmic domain is necessary to promote rapid internalization and degradation, and when transferred to HER2, enhances HER2 degradation. In accordance with these findings, low levels of cell surface PRLR (∼30,000 surface receptors per cell) are sufficient to mediate effective killing by PRLR ADC, whereas cell killing by HER2 ADC requires higher levels of cell surface HER2 (∼106 surface receptors per cell). Noncovalently cross-linking HER2 to PRLR at the cell surface, using a bispecific antibody that binds to both receptors, dramatically enhances the degradation of HER2 as well as the cell killing activity of a noncompeting HER2 ADC. Furthermore, in breast cancer cells that coexpress HER2 and PRLR, a HER2xPRLR bispecific ADC kills more effectively than HER2 ADC. These results emphasize that intracellular trafficking of ADC targets is a key property for their activity and, further, that coupling an ADC target to a rapidly internalizing protein may be a useful approach to enhance internalization and cell killing activity of ADCs. Mol Cancer Ther; 16(4); 681-93. ©2017 AACR.

Opposing actions of angiopoietin-2 on Tie2 signaling and FOXO1 activation.

Angiopoietin-2 (ANG2) regulates blood vessel remodeling in many pathological conditions through differential effects on Tie2 signaling. While ANG2 competes with ANG1 to inhibit Tie2, it can paradoxically also promote Tie2 phosphorylation (p-Tie2). A related paradox is that both inactivation and overactivation of Tie2 can result in vascular remodeling. Here, we reconciled these opposing actions of ANG2 by manipulating conditions that govern its actions in the vasculature. ANG2 drove vascular remodeling during Mycoplasma pulmonis infection by acting as a Tie2 antagonist, which led to p-Tie2 suppression, forkhead box O1 (FOXO1) activation, increased ANG2 expression, and vessel leakiness. These changes were exaggerated by anti-Tie2 antibody, inhibition of PI3K signaling, or ANG2 overexpression and were reduced by anti-ANG2 antibody or exogenous ANG1. In contrast, under pathogen-free conditions, ANG2 drove vascular remodeling by acting as an agonist, promoting high p-Tie2, low FOXO1 activation, and no leakage. Tie1 activation was strong under pathogen-free conditions, but infection or TNF-α led to Tie1 inactivation by ectodomain cleavage and promoted the Tie2 antagonist action of ANG2. Together, these data indicate that ANG2 activation of Tie2 supports stable enlargement of normal nonleaky vessels, but reduction of Tie1 in inflammation leads to ANG2 antagonism of Tie2 and initiates a positive feedback loop wherein FOXO1-driven ANG2 expression promotes vascular remodeling and leakage.