BackgroundCD47 is a broadly expressed cell surface glycoprotein associated with immune evasion. Interaction with the inhibitory receptor signal regulatory protein alpha (SIRPα), primarily expressed ...on myeloid cells, normally serves to restrict effector function (eg, phagocytosis and immune cell homeostasis). CD47/SIRPα antagonists, commonly referred to as ‘macrophage checkpoint’ inhibitors, are being developed as cancer interventions. SRF231 is an investigational fully human IgG4 anti-CD47 antibody that is currently under evaluation in a phase 1 clinical trial. The development and preclinical characterization of SRF231 are reported here.MethodsSRF231 was characterized in assays designed to probe CD47/SIRPα blocking potential and effects on red blood cell (RBC) phagocytosis and agglutination. Additionally, SRF231-mediated phagocytosis and cell death were assessed in macrophage:tumor cell in vitro coculture systems. Further mechanistic studies were conducted within these coculture systems to ascertain the dependency of SRF231-mediated antitumor activity on Fc receptor engagement vs CD47/SIRPα blockade. In vivo, SRF231 was evaluated in a variety of hematologic xenograft models, and the mechanism of antitumor activity was assessed using cytokine and macrophage infiltration analyses following SRF231 treatment.ResultsSRF231 binds CD47 and disrupts the CD47/SIRPα interaction without causing hemagglutination or RBC phagocytosis. SRF231 exerts antitumor activity in vitro through both phagocytosis and cell death in a manner dependent on the activating Fc-gamma receptor (FcγR), CD32a. Through its Fc domain, SRF231 engagement with macrophage-derived CD32a serves dual purposes by eliciting FcγR-mediated phagocytosis of cancer cells and acting as a scaffold to drive CD47-mediated death signaling into tumor cells. Robust antitumor activity occurs across multiple hematologic xenograft models either as a single agent or in combination with rituximab. In tumor-bearing mice, SRF231 increases tumor macrophage infiltration and induction of the macrophage cytokines, mouse chemoattractant protein 1 and macrophage inflammatory protein 1 alpha. Macrophage depletion results in diminished SRF231 antitumor activity, underscoring a mechanistic role for macrophage engagement by SRF231.ConclusionSRF231 elicits antitumor activity via apoptosis and phagocytosis involving macrophage engagement in a manner dependent on the FcγR, CD32a.
Titanium alloys provide excellent corrosion resistance and favorable mechanical properties well suited for a variety of biomaterial applications. The native oxide surface on titanium alloys has been ...shown to be less than ideal and surface modification is often needed. Previously, an optimized anodization process was shown to form a porous phosphorus‐enhanced anatase oxide layer on commercially pure Ti grade 4. The anodized layer was shown to improve osseointegration and to reduce bacteria attachment when photocatalytically activated with UVA preillumination. The primary objective of the present study was to create a similar phosphorus‐enhanced anatase oxide layer on series of titanium alloys including commercially pure Ti grade 4, Ti‐6Al‐7Nb, Ti‐6Al‐4V ELI, alpha + beta Ti‐15Mo, beta Ti‐15Mo, and Ti‐35Nb‐7Zr‐5Ta. Phosphorus‐enhanced anatase oxide layers were formed on each titanium substrate. Anatase formation was shown to generally increase with oxide thickness, except on substrate alloys containing niobium. Phosphorus uptake was shown to be dependent on the titanium alloy chemistry or microstructure. Anodized layers formed on beta‐structured titanium alloys revealed the lowest phosphorus uptake and the most nanosized surface porosity. A methylene blue degradation assay showed anodized layers on commercially pure Ti and both Ti‐15Mo alloys to exhibit the highest levels of photocatalytic activity. Given the range of mechanical properties available with the commercially pure Ti and Ti‐15Mo alloys, the results of this study indicate the benefits of phosphorus‐enhanced anatase oxide coatings may be applicable to a wide variety of biomaterial applications.
Aim: To evaluate the efficacy and safety of alogliptin, a potent and highly selective dipeptidyl peptidase‐4 (DPP‐4) inhibitor, in combination with glyburide in patients with type 2 diabetes ...inadequately controlled by sulphonylurea monotherapy.
Methods: After a 2‐week screening period, adult patients 18–80 years of age entered a 4‐week run‐in/stabilization period in which they were switched from their own sulphonylurea medication to an equivalent dose of glyburide (open label) plus placebo (single blind). After the run‐in period, patients were randomly assigned to double‐blind treatment with alogliptin 12.5 mg (n = 203), alogliptin 25 mg (n = 198), or placebo (n = 99) for 26 weeks. The primary end‐point was change from baseline to week 26 in glycosylated haemoglobin (HbA1c). Secondary end‐points included clinical response rates and changes in fasting plasma glucose, β‐cell function (fasting proinsulin, insulin, proinsulin/insulin ratio, and C‐peptide, and homeostasis model assessment β‐cell function), body weight, and safety end‐points adverse events (AEs), clinical laboratory tests, vital signs and electrocardiographic readings.
Results: The study population had a mean age of 57 years and a mean disease duration of 8 years; it was well balanced for gender (52% women) and was mainly white (71%). The mean baseline HbA1c was approximately 8.1% in each group. Significantly greater least squares (LS) mean reductions in HbA1c were seen at week 26 with alogliptin 12.5 mg (−0.38%) and 25 mg (−0.52%) vs. placebo (+0.01%; p < 0.001), and more patients in the alogliptin 25‐mg group had HbA1c levels ≤7.0% at week 26 (34.8%, p = 0.002) vs. placebo (18.2%). Proportionately more patients in the alogliptin 12.5 mg (47.3%) and 25 mg (50.5%) groups had an HbA1c reduction ≥0.5% from baseline compared with patients in the placebo group (26.3%; p < 0.001). Minor improvements in individual markers of β‐cell function were seen with alogliptin, but no significant treatment group differences were noted relative to placebo. Minor LS mean changes in body weight were noted across groups (placebo, −0.20 kg; alogliptin 12.5 mg, +0.60 kg; alogliptin 25 mg, +0.68 kg). AEs were reported for 63–64% of patients receiving alogliptin and 54% of patients receiving placebo. Few AEs were treatment limiting (2.0–2.5% across groups), and serious AEs (2.0–5.6%) were infrequent, similar across groups, and generally considered not related to treatment. The incidences of hypoglycaemia for placebo, alogliptin 12.5 mg and alogliptin 25 mg groups were 11.1, 15.8 and 9.6% respectively.
Conclusions: In patients with type 2 diabetes inadequately controlled by glyburide monotherapy, the addition of alogliptin resulted in clinically significant reductions in HbA1c without increased incidence of hypoglycaemia.
The updated affiliation list is shown below and has been updated in the article. 1Istituto Nazionale Tumori IRCCS Fondazione 'G. Pascale', Naples, Italy 2Earle A. Chiles Research Institute, ...Providence Cancer Institute, Portland, Oregon, USA 3Harvard Medical School, Boston, Massachusetts, USA 4Georgetown Lombardi Comprehensive Cancer Center, Washington DC, USA 5University of Wisconsin Clinical Cancer Center, Madison, Wisconsin, USA 6Johns Hopkins University School of Medicine, Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USA 7Research, Parker Institute for Cancer Immunotherapy, San Francisco, California, USA 8ESSA Pharma Inc, Redwood City, California, USA 9IGM Biosciences Inc, Mountain View, California, USA 10Medical Oncology - Amsterdam University Medical Centers, Vrije Universiteit-Cancer Center Amsterdam, Amsterdam, The Netherlands 11AIVITA Biomedical, Inc, Irvine, California, USA 12Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York, USA 13UPMC Hillman Cancer Center, Pittsburgh, Pennsylvania, USA 14Pathology and Medicine, Immunology and Cancer Program, University of Chicago, Chicago, Illinois, USA 15National Cancer Institute, Bethesda, Maryland, USA 16Dana Farber Cancer Institute, Boston, Massachusetts, USA 17University of Texas MD Anderson Cancer Center, Houston, Texas, USA 18Bill & Melinda Gates Medical Research Institute, Cambridge, Massachusetts, USA 19Immuneering Corp New York, New York, New York, USA 20University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA 21Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, USA 22Medical Oncology, City of Hope National Medical Center, Duarte, California, USA 23Refuge Biotechnologies, Menlo Park, California, USA 24Thomas Jefferson Medical College, Philadelphia, Pennsylvania, USA 25Massachusetts General Hospital Cancer Center, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, USA 26ESSA Pharma Inc, Palo Alto, California, USA 27Oncology, University of Lausanne, Lausanne, VD, Switzerland 28Pediatrics, University of Wisconsin Madison, Madison, Wisconsin, USA 29Massachusetts Institute of Technology Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts, USA 30Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut, USA 31Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, Iowa, USA 32MacroGenics Inc, Rockville, Maryland, USA 33Laura and Isaac Perlmutter Comprehensive Cancer Center, NYU Langone Medical Center, New York, New York, USA 3) In the main text, The sentence ‘The hypoxia and profound inflammatory response associated with the pneumonitis observed with the severe acute respiratory virus coronavirus-2 SARS-COV-2 virus…’ now reads ‘The hypoxia and profound inflammatory response associated with the pneumonitis observed with the SARS-CoV-2 virus…’ The sentence ‘…including tocilizumab and sarilumab for use on a compassionate basis to critically ill hospitalized COVID-19-infected patients during this extraordinary situation’ now reads ‘…including tocilizumab and sarilumab for use on a compassionate basis to critically ill hospitalized SARS-CoV-2-infected patients during this extraordinary situation’ 4) To acknowledge medical writing support, the acknowledgment section has been updated to read: ‘The authors thank the clinicians working tirelessly on the frontlines of the COVID-19 pandemic. The authors also acknowledge SITC staff for their contributions including Sam Million Weaver, PhD for medical writing and editorial support and Angela Kilbert for project management and assistance.
Abstract
MERTK, a member of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases, is a pleiotropic immune modulator that controls efferocytosis. Engagement of MERTK with its ligand GAS6, ...found anchored to phosphatidylserine exposed on the outer membrane of apoptotic cells, triggers MERTK phosphorylation and signaling events that culminate in the removal of apoptotic debris. Recent studies have highlighted the expression of MERTK on tumor-associated macrophages, and Mertk-deficient mice show reduced tumor cell growth accompanied by inflammatory cytokine production and alterations in macrophage activation. Thus, MERTK has emerged as a promising therapeutic target for augmenting innate antitumor immune responses. MERTK is also expressed in retinal pigmented epithelium (RPE) cells of the eye where it mediates phagocytosis of photoreceptor outer segment fragments. Mutations in MERTK that disrupt its expression or kinase activity lead to marked retinal degeneration and blindness in mice, rats, and humans. Due to known differences in blood-retinal permeability, we explored whether therapeutic antibodies targeting MERTK could inhibit macrophage-mediated efferocytosis and promote antitumor activity while sparing RPE toxicity. A diverse panel of high-affinity antibodies was developed to explore MERTK blockade in vitro and in vivo. Multiple antibodies disrupted MERTK-GAS6 binding and blocked human and murine macrophage-mediated efferocytosis. Two antibodies targeting distinct GAS6 binding epitopes were selected for further characterization. Both antibodies demonstrated antitumor activity in murine CT26 and MC38 syngeneic colorectal cancer models and led to alterations in immune cell-related gene expression. To investigate potential effects on RPE biology with MERTK antibodies, a multi-dose, 4-week cynomolgus monkey study with several in-life and post-mortem ophthalmologic endpoints was designed. While no abnormal ophthalmic or electroretinography (ERG) findings were detected, all animals treated with either MERTK antibody at all doses showed histological abnormalities of the retina, including vacuolation of the outer segments of photoreceptors, displacement of RPE cells, and single cell necrosis of the outer nuclear layer. These data suggest that inhibition of efferocytosis by antibody-mediated blockade of MERTK can promote immune activation and inhibit tumor growth in vivo; however, retinal toxicity consistent with histological observations made in Mertk mutant animals is an on-target effect. As several therapeutics that block MERTK function are currently in preclinical development, a thorough evaluation of retinal toxicity is warranted.
Citation Format: Kerry F. White, Matthew Rausch, Jing Hua, Katherine H. Walsh, Christine E. Miller, Christopher C. Wells, Devapregasan Moodley, Benjamin H. Lee, Scott C. Chappel, Pamela M. Holland, Jonathan A. Hill. MERTK-specific antibodies that have therapeutic antitumor activity in mice disrupt the integrity of the retinal pigmented epithelium in cynomolgus monkeys abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 558.
Follicle stimulating hormone (FSH) receptor clones were isolated from a human testis cDNA library. Characterization of the cDNA clones showed that the DNA and predicted amino acid sequences of the ...long open reading frame differed from a previously published human ovarian FSH receptor sequence (Minegish et al. (1991) Biochem. Biophys. Res. Commun. 175, 1125-1130) by seven nucleotides and five amino acids. A human FSH receptor splice variant was also identified and characterized. A full-length human FSH receptor cDNA was engineered for expression in COS-7, CHO, and Y-1 cells. In transient transfections of COS-7 cells and stable transfections of Y-1 cells, efficient FSH receptor mRNA accumulation and isolation of FSH-responsive cell lines occurred only when an intron was included in the 5' untranslated region of the FSH receptor transcription unit. Y-1 cells stably transfected with the FSH receptor responded to FSH treatment by rounding up and by synthesizing increased amounts of progesterone. Stably transfected CHO cell lines, which responded to FSH by synthesizing increased amounts of cAMP, were isolated irrespective of the presence of the heterologous intron. The FSH-responsive CHO and Y-1 cell lines may be suitable for the development of better in vitro FSH bioassays. These cells also constitute a convenient source of human FSH receptor protein for use in radioreceptor assays and in studies of receptor-ligand interactions.
The transmembrane protein CD47 is an immunoglobulin superfamily member involved in multiple cellular processes, including cell migration, adhesion and T cell function. The interaction between CD47 ...and signal regulatory protein alpha (SIRPα), an inhibitory protein expressed on macrophages, prevents phagocytosis of CD47-expressing cells. CD47 was originally identified as a tumor antigen on human ovarian cancer and was subsequently shown to be expressed on multiple human tumor types, including both hematologic and solid tumors. In many hematologic cancers, high CD47 expression is associated with poor clinical outcomes. CD47 is also expressed at low levels on virtually all non-malignant cells, and loss of expression or changes in membrane distribution may serve as markers of aged or damaged cells, particularly on red blood cells (RBC). High expression of CD47 on tumors blocks phagocytic uptake, subsequent antigen cross-presentation and T cell activation, which collectively contribute to tumor immune evasion. Agents that block the CD47-SIRPα interaction can restore phagocytic uptake of CD47+ target cells and lower the threshold for macrophage activation, which can enhance the efficacy of therapeutic antibodies with ADCC-enabling activity. We developed and characterized a CD47 blocking antibody and evaluated its activity in multiple hematologic models.
SRF231 is a fully human monoclonal antibody that binds with high affinity to human CD47 and blocks the CD47-SIRP□ interaction. SRF231 promotes macrophage-mediated phagocytic clearance of several hematologic primary tumor samples and cell lines in vitro. For example, SRF231 increases phagocytosis of Raji tumor cell line targets with an EC50 of ~300 ng/ml. Enhanced phagocytosis is preferential for tumor cells over normal leukocytes and RBC. Tumor cell phagocytosis can be enhanced in the presence of opsonizing antibodies (e.g., anti-CD20 Ab) when co-administered with SRF231.
In vivo efficacy of SRF231 was assessed in several preclinical murine xenograft models of hematologic malignancies. Notably, SRF231 administration led to profound tumor growth inhibition in models of multiple myeloma, diffuse large B cell lymphoma and Burkitt's lymphoma as a single agent and in combination with opsonizing antibodies. In the Raji xenograft model, single agent therapy leads to 112% tumor growth inhibition. This anti-tumor activity is at least partially dependent on macrophages, as depletion of macrophages via clodronate administration leads to reduced tumor growth inhibition. Tumor-associated macrophage (TAM) numbers and polarization status are also modulated by SRF231 treatment.
In summary, the CD47 mAb SRF231 induces robust tumor cell phagocytosis and tumor clearance both alone and in combination with opsonizing antibodies in pre-clinical models of myeloma and lymphoma. These properties warrant further development of SRF231 in hematologic malignancies. SRF231 is currently in IND-enabling studies and is expected to enter clinical trials in 2017.
No relevant conflicts of interest to declare.
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