CB307: A Dual Targeting Costimulatory Humabody VH Therapeutic for Treating PSMA-Positive Tumors.

PURPOSE: CD137 is a T- and NK-cell costimulatory receptor involved in consolidating immunologic responses. The potent CD137 agonist urelumab has shown clinical promise as a cancer immunotherapeutic but development has been hampered by on-target off-tumor toxicities. A CD137 agonist targeted to the prostate-specific membrane antigen (PSMA), frequently and highly expressed on castration-resistant metastatic prostate cancer (mCRPC) tumor cells, could bring effective immunotherapy to this immunologically challenging to address disease. EXPERIMENTAL DESIGN: We designed and manufactured CB307, a novel half-life extended bispecific costimulatory Humabody VH therapeutic to elicit CD137 agonism exclusively in a PSMA-high tumor microenvironment (TME). The functional activity of CB307 was assessed in cell-based assays and in syngeneic mouse antitumor pharmacology studies. Nonclinical toxicology and toxicokinetic properties of CB307 were assessed in a good laboratory practice (GLP) compliant study in cynomolgus macaques. RESULTS: CB307 provides effective CD137 agonism in a PSMA-dependent manner, with antitumor activity both in vitro and in vivo, and additional activity when combined with checkpoint inhibitors. A validated novel PSMA/CD137 IHC assay demonstrated a higher prevalence of CD137-positive cells in the PSMA-expressing human mCRPC TME with respect to primary lesions. CB307 did not show substantial toxicity in nonhuman primates and exhibited a plasma half-life supporting weekly clinical administration. CONCLUSIONS: CB307 is a first-in-class immunotherapeutic that triggers potent PSMA-dependent T-cell activation, thereby alleviating toxicologic concerns against unrestricted CD137 agonism.

Isolation of Potent CGRP Neutralizing Antibodies Using Four Simple Assays.

Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets.

Versatility of homogeneous time-resolved fluorescence resonance energy transfer assays for biologics drug discovery.

Identification of potential lead antibodies in the drug discovery process requires the use of assays that not only measure binding of the antibody to the target molecule but assess a wide range of other characteristics. These include affinity ranking, measurement of their ability to inhibit relevant protein-protein interactions, assessment of their selectivity for the target protein, and determination of their species cross-reactivity profiles to support in vivo studies. Time-resolved fluorescence resonance energy transfer is a technology that offers the flexibility for development of such assays, through the availability of donor and acceptor fluorophore-conjugated reagents for detection of multiple tags or fusion proteins. The time-resolved component of the technology reduces potential assay interference, allowing screening of a range of different crude sample types derived from the bacterial or mammalian cell expression systems often used for antibody discovery projects. Here we describe the successful application of this technology across multiple projects targeting soluble proteins and demonstrate how it has provided key information for the isolation of potential therapeutic antibodies with the desired activity profile.

Application of the mirrorball high-sensitivity cytometer to multiplexed assays for antibody drug discovery.

Highly sensitive, high-throughput assay technologies are required for the identification of antibody therapeutics. Multiplexed assay systems are particularly advantageous because they allow evaluation of several parameters within 1 well, increasing throughput and reducing hands-on laboratory time. The mirrorball (TTP Labtech), using high-throughput fluorometric microvolume assay technology, offers simultaneous scanning with up to 3 lasers as well as laser scatter detection. This makes the mirrorball especially suitable for the development of highly sensitive and multiplexed assays. We have developed bead- and cell-based binding assays that demonstrate how the multilaser capability of the mirrorball can be exploited to enhance assay sensitivity. In addition, using the multilaser simultaneous scanning capability, we have established multiplexed cytokine quantitation assays and antibody-cell binding assays. Our results demonstrate the potential utility of this technology to improve the sensitivity and efficiency of biologics screening, resulting in streamlining of the lead antibody selection process.

A combination of in vitro techniques for efficient discovery of functional monoclonal antibodies against human CXC chemokine receptor-2 (CXCR2).

BACKGROUND: Development of functional monoclonal antibodies against intractable GPCR targets. RESULTS: Identification of structured peptides mimicking the ligand binding site, their use in panning to enrich for a population of binders, and the subsequent challenge of this population with receptor overexpressing cells leads to functional monoclonal antibodies. CONCLUSION: The combination of techniques provides a successful strategic approach for the development of functional monoclonal antibodies against CXCR2 in a relatively small campaign. SIGNIFICANCE: The presented combination of techniques might be applicable for other, notoriously difficult, GPCR targets. SUMMARY: The CXC chemokine receptor-2 (CXCR2) is a member of the large 'family A' of G-protein-coupled-receptors and is overexpressed in various types of cancer cells. CXCR2 is activated by binding of a number of ligands, including interleukin 8 (IL-8) and growth-related protein α (Gro-α). Monoclonal antibodies capable of blocking the ligand-receptor interaction are therefore of therapeutic interest; however, the development of biological active antibodies against highly structured GPCR proteins is challenging. Here we present a combination of techniques that improve the discovery of functional monoclonal antibodies against the native CXCR2 receptor. The IL-8 binding site of CXCR2 was identified by screening peptide libraries with the IL-8 ligand, and then reconstructed as soluble synthetic peptides. These peptides were used as antigens to probe an antibody fragment phage display library to obtain subpopulations binding to the IL-8 binding site of CXCR2. Further enrichment of the phage population was achieved by an additional selection round with CXCR2 overexpressing cells as a different antigen source. The scFvs from the CXCR2 specific phage clones were sequenced and converted into monoclonal antibodies. The obtained antibodies bound specifically to CXCR2 expressing cells and inhibited the IL-8 and Gro-α induced ß-arrestin recruitment with IC50 values of 0.3 and 0.2 nM, respectively, and were significantly more potent than the murine monoclonal antibodies (18 and 19 nM, respectively) obtained by the classical hybridoma technique, elicited with the same peptide antigen. According to epitope mapping studies, the antibody efficacy is largely defined by N-terminal epitopes comprising the IL-8 and Gro-α binding sites. The presented strategic combination of in vitro techniques, including the use of different antigen sources, is a powerful alternative for the development of functional monoclonal antibodies by the classical hybridoma technique, and might be applicable to other GPCR targets.

Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes.

Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and ß-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.

Tailored amino acid diversity for the evolution of antibody affinity.

Antibodies are a unique class of proteins with the ability to adapt their binding sites for high affinity and high specificity to a multitude of antigens. Many analyses have been performed on antibody sequences and structures to elucidate which amino acids have a predominant role in antibody interactions with antigens. These studies have generally not distinguished between amino acids selected for broad antigen specificity in the primary immune response and those selected for high affinity in the secondary immune response. By studying a large data set of affinity matured antibodies derived from in vitro directed evolution experiments, we were able to specifically highlight a subset of amino acids associated with affinity improvements. In a comparison of affinity maturations using either tailored or full amino acid diversification, the tailored approach was found to be at least as effective at improving affinity while requiring fewer mutagenesis libraries than the traditional method. The resulting sequence data also highlight the potential for further reducing amino acid diversity for high affinity binding interactions.

Inhibition of the human chemokine receptor CCR5 by variecolin and variecolol and isolation of four new variecolin analogues, emericolins A-D, from Emericella aurantiobrunnea.

An extract from the fungus Emericella aurantiobrunnea was found to compete with macrophage inflammatory protein (MIP)-1alpha for binding to human CCR5 in a scintillation proximity assay (SPA). Bioassay-guided fractionation led to the isolation of variecolin (1) and variecolol (2), which had IC50 values of 9 and 32 microM, respectively. An X-ray crystal structure of variecolin (1) was obtained for the first time. Also isolated were four new inactive analogues, emericolin A (3), B (4), C (5), and D (6), and the relative stereochemistry of these compounds was determined by NMR methods using ROESY spectra and 1H/1H coupling constants.

Phenolic derivatives from Wigandia urens with weak activity against the chemokine receptor CCR5.

Three compounds, 2,3-dihydroxy-4-methoxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (1), 8-methoxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-6-ol (2) and 4-methoxy-3-(3-methyl-2-butenyl)-benzoic acid (3), have been isolated from Wigandia urens. The structures of compounds 1, 2 and 3 were determined from spectroscopic data and showed activity in a CCR5 assay with IC(50) values of 33, 46 and 26 muM respectively.

10-Methoxydihydrofuscin, fuscinarin, and fuscin, novel antagonists of the human CCR5 receptor from Oidiodendron griseum.

Two new compounds, 10-methoxydihydrofuscin (1) and fuscinarin (2), and one known compound, fuscin (3), have been isolated from the soil fungus Oidiodendron griseum. These compounds were found to compete effectively with macrophage inflammatory protein (MIP)-1 alpha for binding to human CCR5, an important anti HIV-1 target that interferes with HIV entry into cells. The structures of these compounds were elucidated by spectroscopic methods.