PDGFRα-lineage origin directs monocytes to trafficking proficiency to support peripheral immunity.

Multiple embryonic precursors give rise to leukocytes in adults while the lineage-based functional impacts are underappreciated. Mesodermal precursors expressing PDGFRα appear transiently during E7.5-8.5 descend to a subset of Lin- Sca1+ Kit+ hematopoietic progenitors found in adult BM. By analyzing a PDGFRα-lineage tracing mouse line, we here report that PDGFRα-lineage BM F4/80+ SSClo monocytes/macrophages are solely Ly6C+ LFA-1hi Mac-1hi monocytes enriched on the abluminal sinusoidal endothelium while Ly6C- LFA-1lo Mac-1lo macrophages are mostly from non-PDGFRα-lineage in vivo. Monocytes with stronger integrin profiles outcompete macrophages for adhesion on an endothelial monolayer or surfaces coated with ICAM-1-Fc or VCAM-1-Fc. Egress of PDGFRα-lineage-rich monocytes and subsequent differentiation to peripheral macrophages spatially segregates them from non-PDGFRα-lineage BM-resident macrophages and allows functional specialization since macrophages derived from these egressing monocytes differ in morphology, phenotype, and functionality from BM-resident macrophages in culture. Extravasation preference for blood PDGFRα-lineage monocytes varies by tissues and governs the local lineage composition of macrophages. More PDGFRα-lineage classical monocytes infiltrated into skin and colon but not into peritoneum. Accordingly, transcriptomic analytics indicated augmented inflammatory cascades in dermatitis skin of BM-chimeric mice harbouring only PDGFRα-lineage leukocytes. Thus, the PDGFRα-lineage origin biasedly generates monocytes predestined for BM exit to support peripheral immunity following extravasation and macrophage differentiation.

Assessment of the platelet-derived growth factor receptor alpha antibody olaratumab in a panel of patient-derived soft tissue sarcoma xenografts.

BACKGROUND: Soft tissue sarcoma (STS) comprises a family of rare, heterogeneous tumors of mesenchymal origin. Single-agent doxorubicin remains the first-line standard-of-care treatment for advanced and inoperable STS, but response rates are only around 15%. In 2016, phase Ib/II clinical trial results reported an overall survival benefit of 11.8 months when combining doxorubicin and the platelet-derived growth factor receptor alpha (PDGFRA)-directed antibody olaratumab compared to doxorubicin alone, without providing a scientific rationale for such unprecedented therapeutic effect. We decided to evaluate the efficacy of olaratumab in a panel of STS patient-derived xenografts (PDX). METHODS: NMRI nu/nu mice were bilaterally transplanted with tumor tissue of patient-derived xenograft models expressing PDGFRA, including models of leiomyosarcoma (UZLX-STS22), malignant peripheral nerve sheath tumor (UZLX-STS39), myxofibrosarcoma (UZLX-STS59) and undifferentiated pleomorphic sarcoma (UZLX-STS84). Mice were randomly divided into four different treatment groups: (1) control, (2) doxorubicin (3 mg/kg once weekly), (3) anti-PDGFRA [olaratumab (60 mg/kg twice weekly) + mouse anti-PDGFRA antibody 1E10 (20 mg/kg twice weekly)] and (4) the combination of doxorubicin and anti-PDGFRA (same dose/schedule as in the single treatment arms). Tumor volume, histopathology and Western blotting were used to assess treatment efficacy. RESULTS: Anti-PDGFRA treatment as a single agent did not reduce tumor growth and did not result in significant anti-proliferative or pro-apoptotic activity. Combining doxorubicin and anti-PDGFRA did not reduce tumor burden, though a mild inhibition of proliferation was observed in UZLX-STS39 and -STS59. A pro-apoptotic effect was observed in all models except UZLX-STS22. Antitumor effects on histology were not significantly different comparing doxorubicin and the combination treatment. Moreover, anti-PDGFRA treatment, both as a single agent as well as combined with doxorubicin, did not result in inhibition of the downstream MAPK and PI3K/AKT signaling pathways. CONCLUSIONS: We were not able to demonstrate significant antitumor effects of anti-PDGFRA treatment in selected STS PDX models, neither alone nor in combination with doxorubicin. This is in line with the very recent results of the phase III clinical trial NCT02451943 ANNOUNCE, which did not confirm the clinical benefit of olaratumab in combination with doxorubicin over single agent doxorubicin.

Recently Discovered Interstitial Cell Population of Telocytes: Distinguishing Facts from Fiction Regarding Their Role in the Pathogenesis of Diverse Diseases Called "Telocytopathies".

In recent years, the interstitial cells telocytes, formerly known as interstitial Cajal-like cells, have been described in almost all organs of the human body. Although telocytes were previously thought to be localized predominantly in the organs of the digestive system, as of 2018 they have also been described in the lymphoid tissue, skin, respiratory system, urinary system, meninges and the organs of the male and female genital tracts. Since the time of eminent German pathologist Rudolf Virchow, we have known that many pathological processes originate directly from cellular changes. Even though telocytes are not widely accepted by all scientists as an individual and morphologically and functionally distinct cell population, several articles regarding telocytes have already been published in such prestigious journals as Nature and Annals of the New York Academy of Sciences. The telocyte diversity extends beyond their morphology and functions, as they have a potential role in the etiopathogenesis of different diseases. The most commonly described telocyte-associated diseases (which may be best termed "telocytopathies" in the future) are summarized in this critical review. It is difficult to imagine that a single cell population could be involved in the pathogenesis of such a wide spectrum of pathological conditions as extragastrointestinal stromal tumors ("telocytomas"), liver fibrosis, preeclampsia during pregnancy, tubal infertility, heart failure and psoriasis. In any case, future functional studies of telocytes in vivo will help to understand the mechanism by which telocytes contribute to tissue homeostasis in health and disease.

PDGFRα monoclonal antibody: Assessment of embryo-fetal toxicity and time-dependent placental transfer of a murine surrogate antibody of olaratumab in mice.

BACKGROUND: Olaratumab (Lartruvo™) is a recombinant human IgG1 monoclonal antibody that specifically binds PDGFRα. The maternal and in utero embryo-fetal toxicity and toxicokinetics of a human anti-mouse PDGFRα antibody (LSN3338786) were investigated in pregnant mice. METHODS: A pilot study was used to set doses for the definitive study. In the definitive study, mice were administered vehicle, 5, 50, or 150 mg/kg LSN3338786 by intravenous injection on gestation days (GD) 6, 9, 12, and 15. Fetal tissues and/or serum samples were collected on GD 10, 12, 15, and 18 to evaluate exposure of antibody. RESULTS: There were no adverse maternal effects at 50 and 150 mg/kg although maternal deaths and adverse clinical signs were observed at 5 mg/kg. LSN3338786 crossed the placenta as early as GD 10 during organogenesis. Elimination half-life of LSN3338786 in dams decreased between GD 6 and 15. On GD 18, fetal serum concentrations of antibody were substantially higher than maternal serum concentrations at all doses. Increased incidences of malformations consisting of open and partially open eye and increased incidences of skeletal variation frontal/parietal additional ossification site occurred in fetuses from mid- and high-dose groups. CONCLUSIONS: The majority of transplacental migration of antibody occurred in concert with rapid maternal serum clearance before parturition. The no-observed effect level for teratogenicity of 5 mg/kg was associated with GD 15 maternal serum concentrations 3-11 times lower than clinical exposure of olaratumab, suggesting that olaratumab may cause fetal harm when administered to pregnant women.

Anti-Platelet-Derived Growth Factor Receptor Alpha Chain Antibodies Predict for Response to Nilotinib in Steroid-Refractory or -Dependent Chronic Graft-Versus-Host Disease.

Imatinib has clinical activity in chronic graft-versus-host disease (cGVHD), a significant complication of allogeneic hematopoietic cell transplant. Nilotinib is a tyrosine kinase inhibitor that targets the same receptors as imatinib but with different affinities. We tested the hypothesis that nilotinib is safe and has clinical activity in cGVHD. Thirty-three participants were enrolled in a phase I/II dose escalation and dose extension clinical trial of nilotinib for the treatment of steroid-refractory or- dependent cGVHD (ClinicalTrials.gov, NCT01155817). We assessed safety, clinical response, and pretreatment anti-platelet-derived growth factor receptor alpha chain (anti-PDGFRA) antibody levels. The 200-mg dose was identified as the maximum tolerated dose and used for the phase II dose extension study. At 6 months the incidence of failure-free survival (FFS), cGVHD progression, and nilotinib intolerance resulting in its discontinuation was 50%, 23%, and 23%, respectively. cGVHD responses in skin, joints, and mouth were observed at 3 and 6 months based on improvement in respective National Institutes of Health organ severity scores. Pretreatment anti-PDGFRA antibody levels ≥ .150 optical density as measured by ELISA correlated with longer FFS time (P < .0005) and trended with time until cGVHD progression (P < .06) but not drug intolerance. Nilotinib may be effective for corticosteroid-resistant or -refractory cGVHD in some patients, but its use is limited by intolerable side effects. Selection of patients with high pretreatment anti-PDGFRA antibody levels might improve the risk-to-benefit ratio of nilotinib and better justify its side effects.

Olaratumab for soft tissue sarcoma.

INTRODUCTION: Soft tissue sarcomas (STS) are rare malignant tumors. Unfortunately, the first-line doxorubicin-based treatment has not been improved since the 1970s. Platelet-derived growth factor (PDGF) receptor alpha (PDGFR-α) and its ligands are co-expressed in many types of cancer, including sarcomas. They are involved in stimulating growth and regulating stromal-derived fibroblasts and angiogenesis. PDGFR-α and its ligand may play an important role in tumorigenesis and be a potential target in the treatment of sarcomas. Olaratumab is a fully human IgG1-type anti-PDGFR-α monoclonal antibody with a high affinity and a low 50% inhibitory concentration (IC50). Areas covered: The authors review the role of olaratumab in the treatment of STS by focusing on the recent, randomized Phase II JDGD trial that challenged patients with unresectable or metastatic STS with doxorubicin in the presence or absence of olaratumab. This trial showed a great improvement in overall survival (OS), with an increase in survival from 14.7 months to 26.5 months for patients in the experimental arm and showed acceptable toxicity. Expert opinion: Results seem promising. However, it must be qualified, as the study includes several uncertainties. These uncertainties should be addressed by the ongoing Phase 3 JGDJ confirmatory trial, for which the final efficacy analysis is expected by 2019.

Olaratumab for advanced soft tissue sarcoma.

INTRODUCTION: Olaratumab is a humanized IgG1 monoclonal antibody that blocks the platelet-derived growth factor receptor alpha (PDGFRα). Its antagonistic behavior inhibits the receptor's tyrosine kinase activity, thereby, turning off the downstream signaling cascades responsible for soft tissue sarcoma tumorigenesis. In October 2016, olaratumab received Food and Drug Administration (FDA) approval for its use in combination with doxorubicin for treatment of advanced soft tissue sarcoma. Areas covered: This drug profile takes a comprehensive look at the clinical studies leading to FDA approval of olaratumab as well as its safety and efficacy as a front-line treatment option for sarcoma patients. The literature search was primarily conducted using PubMed. Expert commentary: The combination of olaratumab plus doxorubicin has provided a new front-line therapeutic option for soft tissue sarcoma patients. An open-label phase Ib and randomized phase II trial in patients with advanced soft tissue sarcoma demonstrated that the addition of olaratumab to doxorubicin prolonged progression-free survival by 2.5 months and overall survival by 11.8 months when compared to doxorubicin alone. Of importance, this clinically meaningful increase in overall survival did not come at the expense of a significantly greater number of toxicities. A phase III confirmatory trial (ClinicalTrials.gov Identifier NCT02451943) will be completed in 2020.

Characterization of binding and quantification of human autoantibodies to PDGFRα using a biosensor-based approach.

Systemic sclerosis (SSc) is a chronic autoimmune disease of the connective tissue. The variety and clinical relevance of autoantibodies in SSc patients have been extensively studied, eventually identifying agonistic autoantibodies targeting the platelet-derived growth factor receptor alpha (PDGFRα), and representing potential biomarkers for SSc. We used a resonant mirror biosensor to characterize the binding between surface-blocked PDGFRα and PDGFRα-specific recombinant human monoclonal autoantibodies (mAbs) produced by SSc B cells, and detect/quantify serum autoimmune IgG with binding characteristics similar to the mAbs. Kinetic data showed a conformation-specific, high-affinity interaction between PDGFRα and mAbs, with equilibrium dissociation constants in the low-to-high nanomolar range. When applied to total serum IgG, the assay discriminated between SSc patients and healthy controls, and allowed the rapid quantification of autoimmune IgG in the sera of SSc patients, with anti-PDGFRα IgG falling in the range 3.20-4.67 neq/L of SSc autoantibodies. The test was validated by comparison to direct and competitive anti-PDGFRα antibody ELISA. This biosensor assay showed higher sensibility with respect to ELISA, and other major advantages such as the specificity, rapidity, and reusability of the capturing surface, thus representing a feasible approach for the detection and quantification of high affinity, likely agonistic, SSc-specific anti-PDGFRα autoantibodies.

Olaratumab: First Global Approval.

Olaratumab (Lartruvo™) is a fully human IgG1 monoclonal antibody targeted against the human platelet-derived growth factor (PDGF) receptor α (PDGFRα). It was developed by Eli Lilly and Co. (previously ImClone Systems) after PDGFRα was identified as a potential therapeutic target in a variety of cancers. Olaratumab acts by selectively binding PDGFRα, thereby blocking PDGF ligand binding and inhibiting PDGFRα activation and downstream signalling. In October 2016, olaratumab received its first global approval, in the USA, for use in combination with doxorubicin for the treatment of adult patients with soft tissue sarcoma. The approval was granted by the US FDA under its Accelerated Approval Program based on the results of the JGDG phase II trial (NCT01185964). In addition, the EMA granted conditional approval for olaratumab in this indication in November 2016 following a review under the EMA's Accelerated Assessment Program. An international, confirmatory phase III trial in patients with soft tissue sarcoma is ongoing (ANNOUNCE; NCT02451943). Olaratumab has also been investigated in phase II trials in several other cancers. This article summarizes the milestones in the development of olaratumab leading to this first approval, for use in combination with doxorubicin for the treatment of soft tissue sarcoma in adults.

Variability in Platelet-Derived Growth Factor Receptor Alpha Antibody Specificity May Impact Clinical Utility of Immunohistochemistry Assays.

Aberrant regulation of the receptor tyrosine kinase platelet-derived growth factor alpha (PDGFRα) is implicated in several types of cancer. Inhibition of the PDGFRα pathway may be a beneficial therapy, and detection of PDGFRα in tumor biopsies may lead to insights about which patients respond to therapy. Exploratory or clinical biomarker use of PDGFRα IHC has been frequently reported, often with polyclonal antibody sc-338. An sc-338-based assay was systematically compared with anti-PDGFRα rabbit monoclonal antibody D13C6 using immunoblot profiling and IHC in formalin-fixed and paraffin-embedded human tumor cell lines. Application of sc-338 to blots of whole cell lysates showed multiple bands including some of unknown origin, whereas application of D13C6 resulted in a prominent band at the expected molecular mass of PDGFRα. The IHC assay using D13C6 showed appropriate staining in cell lines, whereas the assay using sc-338 suggested nonspecific detection of proteins. An optimized IHC assay using D13C6 showed a range of staining in the tumor stromal compartment in lung and ovarian carcinomas. These observations suggest that use of clone sc-338 produced unreliable results and should not be used for an IHC research grade assay. In addition, this precludes its use as a potential antibody for a clinical diagnostic tool.

A Phase I study of MEDI-575, a PDGFRα monoclonal antibody, in Japanese patients with advanced solid tumors.

PURPOSE: MEDI-575 is a fully human monoclonal antibody that selectively binds to platelet-derived growth factor receptor alpha (PDGFRα). This open-label Phase I study assessed the safety and tolerability of MEDI-575 in Japanese patients with advanced solid tumors. METHODS: The study comprised two parts: Part A, dose escalation; Part B, dose expansion in patients with hepatocellular cancer. In Part A, patients were enrolled into three cohorts: MEDI-575 was administered intravenously over a 21-day treatment cycle at doses of 9 and 15 mg/kg/week (cohorts 1, 2) and 35 mg/kg/3-weekly (cohort 3). In Part B, MEDI-575 25 mg/kg/3-weekly was administered. Secondary measures included assessment of the maximum tolerated dose, pharmacokinetics, immunogenicity and anti-tumor activity. RESULTS: Ten and 12 patients were treated in Parts A and B, respectively. There were no dose-limiting toxicities; the maximum tolerated dose was not determined. Common treatment-related adverse events were fatigue (30%) and decreased appetite (20%) in Part A and decreased appetite (33.3%) in Part B. All treatment-related adverse events were grade 1 or 2 in severity. No patients discontinued MEDI-575 because of an adverse event and there were no patient deaths due to adverse events. MEDI-575 binding with PDGFRα resulted in a dose-dependent increase in PDGF-AA ligand, with plateau levels observed within 2 days and sustained during the dosing interval. None of the patients in Part A or B experienced complete or partial responses to treatment. CONCLUSIONS: MEDI-575 once weekly and 3-weekly was well tolerated with a favorable pharmacokinetic profile in Japanese patients with advanced solid tumors. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT01102400.

Epitope Specificity Determines Pathogenicity and Detectability of Anti-Platelet-Derived Growth Factor Receptor α Autoantibodies in Systemic Sclerosis.

OBJECTIVE: To identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor α (PDGFRα) in systemic sclerosis (SSc) and develop novel assays for detection of serum anti-PDGFRα autoantibodies. METHODS: Epstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFRα and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFRα IgG were cloned into an IgG expression vector to generate distinct recombinant human monoclonal autoantibodies (mAb), which were characterized by binding and functional assays. The epitopes of anti-PDGFRα recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFRα extracellular domains, and expression analyses of alanine-scanned PDGFRα mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFRα autoantibodies or, selectively, the agonistic ones. RESULTS: Three types of anti-PDGFRα recombinant human mAb, with the same VH but distinct VL chains, were generated. Nonagonistic VH PAM-Vκ 13B8 recognized 1 linear epitope, whereas agonistic VH PAM-Vλ 16F4 and VH PAM-Vκ 16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFRα antibodies were detected in 66 of 70 patients with SSc, 63 of 130 healthy controls, 11 of 26 patients with primary Raynaud's phenomenon (RP), and 13 of 29 patients with systemic lupus erythematosus (SLE). Serum VH PAM-Vκ 16F4-like antibodies were found in 24 of 34 patients with SSc, but not in healthy controls, patients with primary RP, or patients with SLE. Peptides composing the VH PAM-Vκ 16F4 epitope inhibited PDGFRα signaling triggered by serum IgG from SSc patients. CONCLUSION: Agonistic anti-PDGFRα autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFRα signaling in patients with SSc.

A phase I dose-escalation study of MEDI-575, a PDGFRα monoclonal antibody, in adults with advanced solid tumors.

PURPOSE: The purpose of the study was to evaluate safety and determine the maximum tolerated dose (MTD) of MEDI-575, a fully human monoclonal antibody that selectively binds to platelet-derived growth factor receptor-α (PDGFRα), in patients with advanced solid tumors. METHODS: This phase I multicenter, open-label, single-arm study enrolled adults in a 3 + 3 dose escalation design to receive MEDI-575 (3, 6, 9, 12, or 15 mg/kg) once weekly (QW) until toxicity or disease progression occurred. One 0.5-mg/kg dose was given before the first dose in the 3-mg/kg cohort to determine pharmacokinetics (PK) and pharmacodynamics under unsaturated conditions. After completion of dose escalation in the QW cohorts, patients were enrolled in two additional cohorts and received MEDI-575 25 or 35 mg/kg every 3 weeks (Q3W). Secondary measures included assessments of PK, immunogenicity, and antitumor activity. RESULTS: A total of 35 patients received MEDI-575 QW (n = 23) or Q3W (n = 12). Most treatment-related adverse events were grade 1 or 2 in severity across all dose levels, with fatigue (n = 12) and nausea (n = 8) being reported most frequently. With no reports of dose-limiting toxicities (DLTs), the MTD was not reached. MEDI-575 exhibited a nonlinear PK profile and increased plasma platelet-derived growth factor-AA levels in a dose-dependent manner with limited immunogenicity. Stable disease was reported as the best tumor response in 9 of 29 evaluable patients; however, no objective responses were reported. CONCLUSION: Administration of MEDI-575 QW or Q3W resulted in a favorable safety profile, including a lack of DLTs, but without evidence of antitumor activity in patients with refractory solid tumors.

Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation.

Pancreatic cancer is characterized by aggressive local invasion and early metastasis formation. Active migration of the pancreatic cancer cells is essential for these processes. We have shown previously that the pancreatic cancer cells lines CFPAC1 and IMIM-PC2 show high migratory activity, and we have investigated herein the reason for this observation. Cell migration was assessed using a three-dimensional, collagen-based assay and computer-assisted cell tracking. The expression of receptor tyrosine kinases was determined by flow-cytometry and cytokine release was measured by an enzyme-linked immunoassay. Receptor function was blocked by antibodies or pharmacological enzyme inhibitors. Both cells lines express the epidermal growth factor receptor (EGFR) as well as its family-member ErbB2 and the platelet-derived growth factor receptor (PDGFR)α, whereas only weak expression was detected for ErbB3 and no expression of PDGFRß. Pharmacological inhibition of the EGFR or ErbB2 significantly reduced the migratory activity in both cell lines, as did an anti-EGFR antibody. Interestingly, combination of the latter with an anti-PDGFR antibody led to an even more pronounced reduction. Both cell lines release detectable amounts of EGF. Thus, the high migratory activity of the investigated pancreatic cancer cell lines is due to autocrine EGFR activation and possibly of other receptor tyrosine kinases.

Biologic effects of platelet-derived growth factor receptor α blockade in uterine cancer.

PURPOSE: Platelet-derived growth factor receptor α (PDGFRα) expression is frequently observed in many kinds of cancer and is a candidate for therapeutic targeting. This preclinical study evaluated the biologic significance of PDGFRα and PDGFRα blockade (using a fully humanized monoclonal antibody, 3G3) in uterine cancer. EXPERIMENTAL DESIGN: Expression of PDGFRα was examined in uterine cancer clinical samples and cell lines, and biologic effects of PDGFRα inhibition were evaluated using in vitro (cell viability, apoptosis, and invasion) and in vivo (orthotopic) models of uterine cancer. RESULTS: PDGFRα was highly expressed and activated in uterine cancer samples and cell lines. Treatment with 3G3 resulted in substantial inhibition of PDGFRα phosphorylation and of downstream signaling molecules AKT and mitogen-activated protein kinase (MAPK). Cell viability and invasive potential of uterine cancer cells were also inhibited by 3G3 treatment. In orthotopic mouse models of uterine cancer, 3G3 monotherapy had significant antitumor effects in the PDGFRα-positive models (Hec-1A, Ishikawa, Spec-2) but not in the PDGFRα-negative model (OVCA432). Greater therapeutic effects were observed for 3G3 in combination with chemotherapy than for either drug alone in the PDGFRα-positive models. The antitumor effects of therapy were related to increased apoptosis and decreased proliferation and angiogenesis. CONCLUSIONS: These findings identify PDGFRα as an attractive target for therapeutic development in uterine cancer.

A phase I study of olaratumab, an anti-platelet-derived growth factor receptor alpha (PDGFRα) monoclonal antibody, in patients with advanced solid tumors.

PURPOSE: The platelet-derived growth factor receptor (PDGFR) has an important role in tumorigenesis and tumor progression. Olaratumab (IMC-3G3) is a fully human monoclonal antibody that selectively binds human PDGFRα and blocks ligand binding. This phase I study assessed the safety, maximum tolerated dose (MTD), recommended phase II dose (RP2D), pharmacokinetics, and preliminary antitumor activity of olaratumab in patients with advanced solid tumors. METHODS: Patients were enrolled into five dose-escalating cohorts of 3-6 patients each. Olaratumab was administered intravenously weekly at 4, 8, or 16 mg/kg (cohorts 1-3) or once every other week at 15 or 20 mg/kg (cohorts 4-5), with 4 weeks/cycle. RESULTS: Nineteen patients were treated in five cohorts. There were no dose-limiting toxicities; the MTD was not identified with the doses studied. The most common olaratumab-related adverse events (AE) were fatigue and infusion reactions (10.5 % each). With the exception of 1 patient (20 mg/kg) experiencing two grade 3 drug-related AEs after the dose-limiting toxicity assessment period, all drug-related AEs were grade 1 or 2. The trough concentrations (C min) for 16 mg/kg weekly and 20 mg/kg biweekly were higher than 155 µg/mL, and the concentration found to be efficacious in preclinical xenograft models. Twelve patients (63.2 %) had a best response of stable disease [median duration of 3.9 months (95 % CI 2.3-8.7)]. CONCLUSIONS: Olaratumab was well tolerated and showed preliminary antitumor activity. RP2Ds are 16 mg/kg weekly and 20 mg/kg biweekly. Phase II studies of olaratumab as monotherapy and in combination are ongoing in several tumor types.

Identification and elimination of target-related matrix interference in a neutralizing anti-drug antibody assay.

Biopharmaceuticals administered to the human body have the potential to trigger the production of anti-drug (also called anti-therapeutic) antibodies (ADA) that can neutralize the therapeutic activity. For antibody therapeutics, cell-based neutralizing ADA assays are frequently used to evaluate ADA in clinical studies. We developed a method to detect neutralizing antibodies against MEDI-575, a fully human IgG2κ antagonistic antibody against PDGFR-α. We evaluated three assay formats, two of which measured late responses, cell proliferation and apoptosis, whereas the third assay detected an early signaling event, phosphorylation of PDGFR-α. Measuring phosphorylation provided a superior assay window and therefore was developed as a neutralizing ADA (NAb) assay. Matrix interference, however, was significant, and could be identified to be caused by PDGF-AA and PDGF-AB, apparently the two most abundant ligands of PDGFR-α present in human serum samples. A simple pre-treatment step, addition of an inhibitory antibody to PDGF-A, a subunit present in PDGF-AA and PDGF-AB, was found to eliminate matrix interference, increasing assay reliability and sensitivity. We integrated the pre-treatment step into assay development and qualified a robust NAb assay.

Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia.

Fibroblast activation protein (FAP) is a candidate universal target antigen because it has been reported to be selectively expressed in nearly all solid tumors by a subset of immunosuppressive tumor stromal fibroblasts. We verified that 18/18 human tumors of various histologies contained pronounced stromal elements staining strongly for FAP, and hypothesized that targeting tumor stroma with FAP-reactive T cells would inhibit tumor growth in cancer-bearing hosts. T cells genetically engineered with FAP-reactive chimeric antigen receptors (CARs) specifically degranulated and produced effector cytokines upon stimulation with FAP or FAP-expressing cell lines. However, adoptive transfer of FAP-reactive T cells into mice bearing a variety of subcutaneous tumors mediated limited antitumor effects and induced significant cachexia and lethal bone toxicities in two mouse strains. We found that FAP was robustly expressed on PDGFR-α(+), Sca-1(+) multipotent bone marrow stromal cells (BMSCs) in mice, as well as on well-characterized, clinical-grade multipotent human BMSCs. Accordingly, both mouse and human multipotent BMSCs were recognized by FAP-reactive T cells. The lethal bone toxicity and cachexia observed after cell-based immunotherapy targeting FAP cautions against its use as a universal target. Moreover, the expression of FAP by multipotent BMSCs may point toward the cellular origins of tumor stromal fibroblasts.

Human monoclonal antibodies in cancer therapy: a review of recent developments.

In the last decade, phage-display technology for the generation of monoclonal antibodies (mAbs) has improved significantly. Several novel human mAbs directed to a wide range of targets have been generated for the treatment of common malignancies. These targets include antigens associated with apoptosis, angiogenesis and solid tumors, as well as tumor growth-related antigens, insulin-like growth factor I receptor and hepatocyte growth factor. The safety, pharmacokinetics, and pharmacodynamics of several human mAbs have been evaluated in patients with advanced solid tumors. In conclusion, significant advances in the generation and application of human mAbs in cancer therapy have been made in the last decade.

PDGF receptor-α does not promote HCMV entry into epithelial and endothelial cells but increased quantities stimulate entry by an abnormal pathway.

Epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFRα) were reported to mediate entry of HCMV, including HCMV lab strain AD169. AD169 cannot assemble gH/gL/UL128-131, a glycoprotein complex that is essential for HCMV entry into biologically important epithelial cells, endothelial cells, and monocyte-macrophages. Given this, it appeared incongruous that EGFR and PDGFRα play widespread roles in HCMV entry. Thus, we investigated whether PDGFRα and EGFR could promote entry of wild type HCMV strain TR. EGFR did not promote HCMV entry into any cell type. PDGFRα-transduction of epithelial and endothelial cells and several non-permissive cells markedly enhanced HCMV TR entry and surprisingly, promoted entry of HCMV mutants lacking gH/gL/UL128-131 into epithelial and endothelial cells. Entry of HCMV was not blocked by a panel of PDGFRα antibodies or the PDGFR ligand in fibroblasts, epithelial, or endothelial cells or by shRNA silencing of PDGFRα in epithelial cells. Moreover, HCMV glycoprotein induced cell-cell fusion was not increased when PDGFRα was expressed in cells. Together these results suggested that HCMV does not interact directly with PDGFRα. Instead, the enhanced entry produced by PDGFRα resulted from a novel entry pathway involving clathrin-independent, dynamin-dependent endocytosis of HCMV followed by low pH-independent fusion. When PDGFRα was expressed in cells, an HCMV lab strain escaped endosomes and tegument proteins reached the nucleus, but without PDGFRα virions were degraded. By contrast, wild type HCMV uses another pathway to enter epithelial cells involving macropinocytosis and low pH-dependent fusion, a pathway that lab strains (lacking gH/gL/UL128-131) cannot follow. Thus, PDGFRα does not act as a receptor for HCMV but increased PDGFRα alters cells, facilitating virus entry by an abnormal pathway. Given that PDGFRα increased infection of some cells to 90%, PDGFRα may be very useful in overcoming inefficient HCMV entry (even of lab strains) into the many difficult-to-infect cell types.