Primate cerebrospinal fluid CHI3L1 reflects brain TREM2 agonism.

Introduction: Triggering receptor expressed on myeloid cells 2 (TREM2) agonists are being clinically evaluated as disease-modifying therapeutics for Alzheimer's disease. Clinically translatable pharmacodynamic (PD) biomarkers are needed to confirm drug activity and select the appropriate therapeutic dose in clinical trials.

Methods: We conducted multi-omic analyses on paired non-human primate brain and cerebrospinal fluid (CSF), and stimulation of human induced pluripotent stem cell-derived microglia cultures after TREM2 agonist treatment, followed by validation of candidate fluid PD biomarkers using immunoassays.

Neutral or Detrimental Effects of TREM2 Agonist Antibodies in Preclinical Models of Alzheimer's Disease and Multiple Sclerosis.

Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia.

High-Throughput Analyses of Therapeutic Antibodies Using High-Field Asymmetric Waveform Ion Mobility Spectrometry Combined with SampleStream and Intact Protein Mass Spectrometry.

Intact protein mass spectrometry (MS) coupled with liquid chromatography was applied to characterize the pharmacokinetics and stability profiles of therapeutic proteins. However, limitations from chromatography, including throughput and carryover, result in challenges with handling large sample numbers. Here, we combined intact protein MS with multiple front-end separations, including affinity capture, SampleStream, and high-field asymmetric waveform ion mobility spectrometry (FAIMS), to perform high-throughput and specific mass measurements of a multivalent antibody with one antigen-binding fragment (Fab) fused to an immunoglobulin G1 (IgG1) antibody.

Systematic discovery of neoepitope-HLA pairs for neoantigens shared among patients and tumor types.

The broad application of precision cancer immunotherapies is limited by the number of validated neoepitopes that are common among patients or tumor types. To expand the known repertoire of shared neoantigen-human leukocyte antigen (HLA) complexes, we developed a high-throughput platform that coupled an in vitro peptide-HLA binding assay with engineered cellular models expressing individual HLA alleles in combination with a concatenated transgene harboring 47 common cancer neoantigens. From more than 24,000 possible neoepitope-HLA combinations, biochemical and computational assessment yielded 844 unique candidates, of which 86 were verified after immunoprecipitation mass spectrometry analyses of engineered, monoallelic cell lines.

Mini-PBPK-Based Population Model and Covariate Analysis to Assess the Complex Pharmacokinetics and Pharmacodynamics of RO7449135, an Anti-KLK5/KLK7 Bispecific Antibody in Cynomolgus Monkeys.

RO7449135, an anti-kallikrein (KLK)5/KLK7 bispecific antibody, is in development as a potential therapy against Netherton's syndrome (NS). In cynomolgus monkey studies, RO7449135 bound to KLK5 and KLK7, causing considerable accumulation of total KLKs, but with non-dose-proportional increase. To understand the complex PKPD, a population model with covariate analysis was developed accounting for target binding in skin and migration of bound targets from skin to blood.

Adapting an Electronic STI Risk Assessment Program for Use in Pediatric Primary Care.

Objectives: Sexually transmitted infections (STIs) are an ongoing public health issue in adolescents. the Centers for Disease Control and Prevention and American Academy of Pediatrics have and continue to recommend STI screening in at-risk adolescents, however screening and testing continues to lag behind the need. We previously designed and implemented an electronic risk assessment tool to support STI testing in our pediatric emergency department.

Direct Tie2 Agonists Stabilize Vasculature for the Treatment of Diabetic Macular Edema.

Purpose: Diabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients.

Nonclinical Pharmacokinetics, Pharmacodynamics, and Translational Model of RO7297089, A Novel Anti-BCMA/CD16A Bispecific Tetravalent Antibody for the Treatment of Multiple Myeloma.

RO7297089, an anti-B-cell maturation antigen (BCMA)/CD16A bispecific tetravalent antibody, is being developed as a multiple myeloma (MM) therapeutic. This study characterized nonclinical pharmacokinetics (PK), pharmacodynamics (PD), soluble BCMA (sBCMA), and soluble CD16 (sCD16) changes following administration of RO7297089 to support clinical trials. Unbound and total RO7297089 concentrations were measured in cynomolgus monkeys.

Translational Approaches for Brain Delivery of Biologics via Cerebrospinal Fluid.

Delivery of biologics via cerebrospinal fluid (CSF) has demonstrated potential to access the tissues of the central nervous system (CNS) by circumventing the blood-brain barrier and blood-CSF barrier. Developing an effective CSF drug delivery strategy requires optimization of multiple parameters, including choice of CSF access point, delivery device technology, and delivery kinetics to achieve effective therapeutic concentrations in the target brain region, whereas also considering the biologic modality, mechanism of action, disease indication, and patient population. This review discusses key preclinical and clinical examples of CSF delivery for different biologic modalities (antibodies, nucleic acid-based therapeutics, and gene therapy) to the brain via CSF or CNS access routes (intracerebroventricular, intrathecal-cisterna magna, intrathecal-lumbar, intraparenchymal, and intranasal), including the use of novel device technologies.

High-throughput identification of conditional MHCI ligands and scaled-up production of conditional MHCI complexes.

Despite the need to monitor the impact of Cancer Immunotherapy (CI)/Immuno-Oncology (IO) therapeutics on neoantigen-specific T-cell responses, very few clinical programs incorporate this aspect of immune monitoring due to the challenges in high-throughput (HTP) generation of Major Histocompatibility Complex Class I (MHCI) tetramers across a wide range of HLA alleles. This limitation was recently addressed through the development of MHCI complexes with peptides containing a nonnatural UV cleavable amino acid (conditional MHCI ligands) that enabled HTP peptide exchange upon UV exposure. Despite this advancement, the number of alleles with known conditional MHCI ligands is limited.

Preclinical optimization of Ly6E-targeted ADCs for increased durability and efficacy of anti-tumor response.

Early success with brentuximab vedotin in treating classical Hodgkin lymphoma spurred an influx of at least 20 monomethyl auristatin E (MMAE) antibody-drug conjugates (ADCs) into clinical trials. While three MMAE-ADCs have been approved, most of these conjugates are no longer being investigated in clinical trials. Some auristatin conjugates show limited or no efficacy at tolerated doses, but even for drugs driving initial remissions, tumor regrowth and metastasis often rapidly occur.

The Immunoglobulin Superfamily Receptome Defines Cancer-Relevant Networks Associated with Clinical Outcome.

Cell surface receptors and their interactions play a central role in physiological and pathological signaling. Despite its clinical relevance, the immunoglobulin superfamily (IgSF) remains uncharacterized and underrepresented in databases. Here, we present a systematic extracellular protein map, the IgSF interactome.

Trem2 Deletion Reduces Late-Stage Amyloid Plaque Accumulation, Elevates the Aβ42:Aβ40 Ratio, and Exacerbates Axonal Dystrophy and Dendritic Spine Loss in the PS2APP Alzheimer's Mouse Model.

is an Alzheimer's disease (AD) risk gene expressed in microglia. To study the role of in a mouse model of β-amyloidosis, we compared PS2APP transgenic mice versus PS2APP mice lacking (PS2APP;Trem2) at ages ranging from 4 to 22 months. Microgliosis was impaired in PS2APP;Trem2 mice, with -deficient microglia showing compromised expression of proliferation/Wnt-related genes and marked accumulation of ApoE.

A Platform for Extracellular Interactome Discovery Identifies Novel Functional Binding Partners for the Immune Receptors B7-H3/CD276 and PVR/CD155.

Receptors expressed on the plasma membrane and their interacting partners critically regulate cellular communication during homeostasis and disease, and as such represent main therapeutic targets. Despite its importance for drug development, receptor-ligand proteomics has remained a daunting field, in part because of the challenges associated to the study of membrane-expressed proteins. Here, to enable sensitive detection of receptor-ligand interactions in high throughput, we implement a new platform, the Conditioned Media AlphaScreen, for interrogation of a library consisting of most single transmembrane human proteins.

Widespread brain distribution and activity following i.c.v. infusion of anti-β-secretase (BACE1) in nonhuman primates.

Background And Purpose: The potential for therapeutic antibody treatment of neurological diseases is limited by poor penetration across the blood-brain barrier. I.c.

High throughput screening identifies novel, cell cycle-arresting small molecule enhancers of transient protein expression.

Transient gene expression in mammalian cells is an efficient process for producing recombinant proteins for various research applications to support large molecule therapeutics development. For the first time, we report a high throughput small molecule (SM) screen to identify novel compounds that increase antibody titers after polyethylenimine (PEI) transient transfection of a HEK293 cell line. After screening 31,413 SMs in a 50 μL scaled-down process, we validated 164 SMs to improve yields by up to twofold.

Taming hyperactive hDNase I: Stable inducible expression of a hyperactive salt- and actin-resistant variant of human deoxyribonuclease I in CHO cells.

While the most common causes of clonal instability are DNA copy number loss and silencing, toxicity of the expressed protein(s) may also induce clonal instability. Human DNase I (hDNase I) is used therapeutically for the treatment of cystic fibrosis (CF) and may have potential benefit for use in systemic lupus erythematosus (SLE). hDNase I is an endonuclease that catalyzes degradation of extracellular DNA and is inhibited by both salt and G-actin.

Provider self-disclosure during contraceptive counseling.

Objectives: Provider self-disclosure (PSD) - defined as providers making statements regarding personal information to patients - has not been well characterized in the context of contraceptive counseling. In this study, we describe the incidence, content and context of contraceptive PSD.

Study Design: This mixed methods analysis used data from the Provider-Patient Contraceptive Counseling study, for which 349 family planning patients were recruited from 2009 to 2012 from six clinics in the San Francisco Bay Area.

The TAM family receptor tyrosine kinase TYRO3 is a negative regulator of type 2 immunity.

Host responses against metazoan parasites or an array of environmental substances elicit type 2 immunity. Despite its protective function, type 2 immunity also drives allergic diseases. The mechanisms that regulate the magnitude of the type 2 response remain largely unknown.

The Wnt Antagonist Dickkopf-1 Promotes Pathological Type 2 Cell-Mediated Inflammation.

Exposure to a plethora of environmental challenges commonly triggers pathological type 2 cell-mediated inflammation. Here we report the pathological role of the Wnt antagonist Dickkopf-1 (Dkk-1) upon allergen challenge or non-healing parasitic infection. The increased circulating amounts of Dkk-1 polarized T cells to T helper 2 (Th2) cells, stimulating a marked simultaneous induction of the transcription factors c-Maf and Gata-3, mediated by the kinases p38 MAPK and SGK-1, resulting in Th2 cell cytokine production.

A neutralizing anti-gH/gL monoclonal antibody is protective in the guinea pig model of congenital CMV infection.

Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2-1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay.

T cell-derived protein S engages TAM receptor signaling in dendritic cells to control the magnitude of the immune response.

Dendritic cell (DC) activation is essential for the induction of immune defense against pathogens, yet needs to be tightly controlled to avoid chronic inflammation and exaggerated immune responses. Here, we identify a mechanism of immune homeostasis by which adaptive immunity, once triggered, tempers DC activation and prevents overreactive immune responses. T cells, once activated, produced Protein S (Pros1) that signaled through TAM receptor tyrosine kinases in DCs to limit the magnitude of DC activation.

A strategy for risk mitigation of antibodies with fast clearance.

A majority of human therapeutic antibody candidates show pharmacokinetic properties suitable for clinical use, but an unexpectedly fast antibody clearance is sometimes observed that may limit the clinical utility. Pharmacokinetic data in cynomolgus monkeys collected for a panel of 52 antibodies showed broad distribution of target-independent clearance values (2.4-61.

DCDT2980S, an anti-CD22-monomethyl auristatin E antibody-drug conjugate, is a potential treatment for non-Hodgkin lymphoma.

Antibody-drug conjugates (ADC), potent cytotoxic drugs linked to antibodies via chemical linkers, allow specific targeting of drugs to neoplastic cells. We have used this technology to develop the ADC DCDT2980S that targets CD22, an antigen with expression limited to B cells and the vast majority of non-Hodgkin lymphomas (NHL). DCDT2980S consists of a humanized anti-CD22 monoclonal IgG1 antibody with a potent microtubule-disrupting agent, monomethyl auristatin E (MMAE), linked to the reduced cysteines of the antibody via a protease cleavable linker, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB).

Total antibody quantification for MMAE-conjugated antibody-drug conjugates: impact of assay format and reagents.

Antibody-drug conjugates (ADCs) are target-specific anticancer agents consisting of cytotoxic drugs covalently linked to a monoclonal antibody. The number of ADCs in the clinic is growing, and therefore thorough characterization of the quantitative assays used to measure ADC concentrations in support of pharmacokinetic, efficacy, and safety studies is of increasing importance. Cytotoxic drugs such as the tubulin polymerization inhibiting auristatin, monomethyl auristatin E, have been conjugated to antibodies via cleavable linkers (MC-vc-PAB) through internal cysteines.