Engineering of a novel anti-CD40L domain antibody for treatment of autoimmune diseases.

CD40-CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE. Our results show that the interaction of the 5c8 wild-type IgG1 Fc domain with FcγRs is responsible for platelet activation, as measured by induction of PAC-1 and CD62P. A version of 5c8 with a mutated IgG1 tail was identified that showed minimal FcγR binding and platelet activation while maintaining full binding to CD40L. To address whether Fc effector function is required for immunosuppression, a potent Ab fragment, termed a "domain Ab" (dAb), against murine CD40L was identified and fused to a murine IgG1 Fc domain containing a D265A mutation that lacks Fc effector function. In vitro, this dAb-Fc demonstrated comparable potency to the benchmark mAb MR-1 in inhibiting B cell and dendritic cell activation. Furthermore, the anti-CD40L dAb-Fc exhibited a notable efficacy comparable to MR-1 in various preclinical models, such as keyhole limpet hemocyanin-induced Ab responses, alloantigen-induced T cell proliferation, "heart-to-ear" transplantation, and NZB × NZW F1 spontaneous lupus. Thus, our data show that immunosuppression and TE can be uncoupled and that a CD40L dAb with an inert Fc tail is expected to be efficacious for treating autoimmune diseases, with reduced risk for TE.

A monovalent anti-human CD28 domain antibody antagonist: preclinical efficacy and safety.

Targeting the CD28-CD80/86 pathway with an anti-CD28 antagonist is a promising alternative to current therapies for autoimmunity. However, attempts at generating conventional anti-CD28 mAbs lacking stimulatory activity has been challenging. In this study, we describe anti-human CD28 receptor antagonist domain Abs (dAbs) that are specific for human CD28. These dAbs are potent inhibitors of T cell activation, with an EC50 of 35 ± 14 ng/ml for inhibition of proliferation. The EC50 of 53 ± 11 ng/ml in an ex vivo CD28 receptor occupancy assay corresponds with in vitro functional activity, suggesting a direct correlation. The anti-CD28 dAb is equipotent in the inhibition of CD80- and CD86-mediated T cell proliferation and does not interfere with CTLA-4-mediated downmodulation of CD86 expression on APCs. The anti-CD28 dAbs are monomeric and do not demonstrate any evidence of agonism or costimulatory activity. In cynomolgus monkeys, the anti-CD28 dAb demonstrated pharmacodynamic activity, as measured by the inhibition of a T cell-dependent Ab response, without evidence of T cell depletion or cytokine release. Furthermore, there was a strong correlation between systemic exposure, duration, and extent of CD28 receptor occupancy, and pharmacodynamic activity. Taken together, these data support clinical evaluation of this novel anti-CD28 dAb for the treatment of autoimmune diseases.

A universal surrogate peptide to enable LC-MS/MS bioanalysis of a diversity of human monoclonal antibody and human Fc-fusion protein drug candidates in pre-clinical animal studies.

For the development of human antibody Fc (fraction crystallizable) region-containing therapeutic protein candidates, which can be either monoclonal antibodies (mAbs) or pharmacologically active proteins/peptides fused to the Fc region of human Immunoglobulin G (IgG), reliable quantification of these proteins in animal pharmacokinetic study plasma samples is critical. LC-MS/MS has emerged as a promising assay platform for this purpose. LC-MS/MS assays used for bioanalysis of human antibody Fc region-containing therapeutic protein candidates frequently rely upon quantification of a 'signature' surrogate peptide whose sequence is unique to the protein analyte of interest. One drawback of the signature peptide approach is that a new LC-MS/MS assay must be developed for each new human Fc region-containing therapeutic protein. To address this issue, we propose an alternative 'universal surrogate peptide' approach for the quantification of human antibody Fc region-containing therapeutic protein candidates in plasma samples from all nonclinical species. A single surrogate tryptic peptide was identified in the Fc region of most human antibody Fc-containing therapeutic protein candidates. An LC-MS-MS method based upon this peptide was shown to be capable of supporting bioanalysis of a diversity of human Fc region-containing therapeutic protein candidates in plasma samples of all commonly used animal species.

Preclinical Evaluation of IMGC936, a Next-Generation Maytansinoid-based Antibody-drug Conjugate Targeting ADAM9-expressing Tumors.

ADAM metallopeptidase domain 9 (ADAM9) is a member of the ADAM family of multifunctional, multidomain type 1 transmembrane proteins. ADAM9 is overexpressed in many cancers, including non-small cell lung, pancreatic, gastric, breast, ovarian, and colorectal cancer, but exhibits limited expression in normal tissues. A target-unbiased discovery platform based on intact tumor and progenitor cell immunizations, followed by an IHC screen, led to the identification of anti-ADAM9 antibodies with selective tumor-versus-normal tissue binding. Subsequent analysis revealed anti-ADAM9 antibodies were efficiently internalized and processed by tumor cells making ADAM9 an attractive target for antibody-drug conjugate (ADC) development. Here, we describe the preclinical evaluation of IMGC936, a novel ADC targeted against ADAM9. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21-C, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable tripeptide linker, at a drug antibody ratio of approximately 2.0. In addition, the YTE mutation (M252Y/S254T/T256E) was introduced into the CH2 domain of the antibody Fc to maximize in vivo plasma half-life and exposure. IMGC936 exhibited cytotoxicity toward ADAM9-positive human tumor cell lines, as well as bystander killing, potent antitumor activity in human cell line-derived xenograft and patient-derived xenograft tumor models, and an acceptable safety profile in cynomolgus monkeys with favorable pharmacokinetic properties. Our preclinical data provide a strong scientific rationale for the further development of IMGC936 as a therapeutic candidate for the treatment of ADAM9-positive cancers. A first-in-human study of IMGC936 in patients with advanced solid tumors has been initiated (NCT04622774).

Multispecific, Multivalent Antibody-Based Molecules Engineered on the DART® and TRIDENTTM Platforms.

Multispecific antibodies bind two or more different antigens and enable new therapeutic applications that cannot be replicated with conventional monoclonal antibodies, such as bridging different cells or bringing soluble proteins in close proximity. The DART and TRIDENT platforms enable the engineering of such antibodies. A DART molecule combines two independent antigen-binding sites in a stabilized, diabody-like structure. A DART molecule can be expressed with or without an Fc domain and thus can be tailored to have a long or short half-life in vivo and to induce or ablate effector function. Linking two DART units or a DART unit and a Fab domain (the latter structure is called TRIDENT format) via an Fc domain creates a monospecific, bispecific, trispecific, or tetraspecific molecule with up to tetravalent targeting of antigens. This article focuses on the design of DART and TRIDENT molecules that target two or three different antigens. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Design and generation of expression plasmids encoding DART and TRIDENT molecules Basic Protocol 2: Expression of DART and TRIDENT molecules by transient transfection of CHO cells Basic Protocol 3: Purification of DART and TRIDENT molecules from CHO cell supernatants.

Preclinical Development of MGC018, a Duocarmycin-based Antibody-drug Conjugate Targeting B7-H3 for Solid Cancer.

B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non-small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival. In this article, we report the preclinical development of MGC018, an antibody-drug conjugate targeted against B7-H3. MGC018 is comprised of the cleavable linker-duocarmycin payload, valine-citrulline-seco duocarmycin hydroxybenzamide azaindole (vc-seco-DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa mAb through reduced interchain disulfides, with an average drug-to-antibody ratio of approximately 2.7. MGC018 exhibited cytotoxicity toward B7-H3-positive human tumor cell lines, and exhibited bystander killing of target-negative tumor cells when cocultured with B7-H3-positive tumor cells. MGC018 displayed potent antitumor activity in preclinical tumor models of breast, ovarian, and lung cancer, as well as melanoma. In addition, antitumor activity was observed toward patient-derived xenograft models of breast, prostate, and head and neck cancer displaying heterogeneous expression of B7-H3. Importantly, MGC018 exhibited a favorable pharmacokinetic and safety profile in cynomolgus monkeys following repeat-dose administration. The antitumor activity observed preclinically with MGC018, together with the positive safety profile, provides evidence of a potentially favorable therapeutic index and supports the continued development of MGC018 for the treatment of solid cancers. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/11/2235/F1.large.jpg.

Characterization of S-thiolation on secreted proteins from E. coli by mass spectrometry.

S-thiolation is a reversible post-translational modification in which thiol metabolites of low molecular masses are linked to protein sulfhydryl groups through disulfide bonds. This modification is commonly observed in recombinant proteins secreted from E. coli cells. Since it can alter protein functions and introduce molecular heterogeneity, S-thiolation is undesirable for recombinant protein production. To date, few published studies have characterized thiol modifiers or investigated the mechanism of S-thiolation in recombinant proteins. In this work, reversed-phase liquid chromatography and mass spectrometry were used to characterize four of the most abundant thiol modifiers on recombinant proteins secreted from E. coli BL21 (DE3) strain. These thiol modifiers have been identified as glutathione, 4-phosphopantetheine, gluconoylated glutathione, and dephosphorylated coenzyme A. S-thiolation by these thiol modifiers increases protein mass by 305, 356, 483, and 685 Da, respectively. These specific mass increases can be used as markers for identifying S-thiolation in recombinant proteins.

Involvement of DPP-IV catalytic residues in enzyme-saxagliptin complex formation.

The inhibition of DPP-IV by saxagliptin has been proposed to occur through formation of a covalent but reversible complex. To evaluate further the mechanism of inhibition, we determined the X-ray crystal structure of the DPP-IV:saxagliptin complex. This structure reveals covalent attachment between S630 and the inhibitor nitrile carbon (C-O distance <1.3 A). To investigate whether this serine addition is assisted by the catalytic His-Asp dyad, we generated two mutants of DPP-IV, S630A and H740Q, and assayed them for ability to bind inhibitor. DPP-IV H740Q bound saxagliptin with an approximately 1000-fold reduction in affinity relative to DPP-IV WT, while DPP-IV S630A showed no evidence for binding inhibitor. An analog of saxagliptin lacking the nitrile group showed unchanged binding properties to the both mutant proteins, highlighting the essential role S630 and H740 play in covalent bond formation between S630 and saxagliptin. Further supporting mechanism-based inhibition by saxagliptin, NMR spectra of enzyme-saxagliptin complexes revealed the presence of three downfield resonances with low fractionation factors characteristic of short and strong hydrogen bonds (SSHB). Comparison of the NMR spectra of various wild-type and mutant DPP-IV:ligand complexes enabled assignment of a resonance at approximately 14 ppm to H740. Two additional DPP-IV mutants, Y547F and Y547Q, generated to probe potential stabilization of the enzyme-inhibitor complex by this residue, did not show any differences in inhibitor binding either by ITC or NMR. Together with the previously published enzymatic data, the structural and binding data presented here strongly support a histidine-assisted covalent bond formation between S630 hydroxyl oxygen and the nitrile group of saxagliptin.

Differential activation of recombinant human acetyl-CoA carboxylases 1 and 2 by citrate.

The role of citrate as a physiological modulator of mammalian acetyl-CoA carboxylases (ACCs) has been well studied; however, the mechanism has not been clearly defined. In the current study, we found that citrate activated recombinant human ACC2 by more than approximately 1000-fold, but activated recombinant human ACC1 only by approximately 4-fold. The data fit best to a model which accounts for cooperative binding of two citrate molecules. Citrate activates ACCs at lower concentrations and inhibits at higher concentrations with apparent K(d) values of 0.8+/-0.3 and 3.4+/-0.6 mM, and apparent K(i) values of 20+/-8 and 38 +/-8 mM for ACC1 and ACC2, respectively. In the absence of added citrate, both ACC1 and ACC2 were inactivated by avidin rapidly and completely. Addition of 10 mM citrate protected ACC2 from avidin inactivation; however, protection by citrate was less pronounced for ACC1. In response to citrate treatment, different aggregation patterns for the two isoforms were also observed by dynamic light scattering. Although formation of aggregates by both isoforms was sensitive to citrate, with Mg2+ and Mg-citrate addition only formation of the ACC2 aggregates showed a dependence on citrate concentration. Mass spectrometry data indicated phosphorylation of Ser79 of ACC1 (a serine known to regulate activity), and the corresponding Ser221 of ACC2. Taken together, these data suggest that recombinant human ACC1 and ACC2 are differentially activated by citrate, most likely through conformational changes leading to aggregation, with ACC2 being more sensitive to this activator.

Is exogenous tissue plasminogen activator necessary for antithrombotic efficacy of an inhibitor of thrombin activatable fibrinolysis inhibitor (TAFI) in rats?

INTRODUCTION: TAFI indirectly reduces the action of tPA on plasminogen. Whether exogenous tPA is necessary for TAFI inhibitor efficacy is unclear. Potato carboxypeptidase inhibitor (PCI), a TAFI inhibitor, has shown variable tPA dependence in rat models of arteriovenous shunt thrombosis (required) and microthrombosis (not required). This study was designed to further explore the importance of exogenous tPA in revealing PCI activity in rat models of venous and arterial thrombosis and provoked bleeding. METHODS: PCI was given as a bolus (5, 10 mg/kg) +/- infusion (5, 10 mg/kg/h) and with or without low dose tPA (5, 10, 25 microg/kg/min). In each instance tPA was adjusted to produce subthreshold thrombus reduction. Arterial thrombosis was induced by FeCl2; venous thrombosis by tissue factor or FeCl2. Bleeding was induced by kidney incision with PCI given (5 mg + 5 mg/kg/h) in the presence or absence of tPA (10, 150, 200 microg/kg/min). RESULTS: PCI was ineffective without exogenous tPA in all tested thrombosis models. With exogenous tPA, PCI decreased thrombus weight 85% in tissue factor thrombosis, 59% in FeCl2 thrombosis, and 46% in arterial thrombosis. PCI prolonged bleeding only when combined with a relatively high tPA dose (200 microg/kg/min) that increased bleeding alone. CONCLUSIONS: If the current results predict clinical efficacy, the need for exogenous tPA in combination with TAFI inhibition is a potential problem. However, in acute settings where intravenous fibrinolytics are administered, or indications in which tPA production increases, TAFI inhibitors may prove to be safe and moderately effective profibrinolytic agents.

Functional Antagonism of Human CD40 Achieved by Targeting a Unique Species-Specific Epitope.

Current clinical anti-CD40 biologic agents include both antagonist molecules for the treatment of autoimmune diseases and agonist molecules for immuno-oncology, yet the relationship between CD40 epitope and these opposing biological outcomes is not well defined. This report describes the identification of potent antagonist domain antibodies (dAbs) that bind to a novel human CD40-specific epitope that is divergent in the CD40 of nonhuman primates. A similarly selected anti-cynomolgus CD40 dAb recognizing the homologous epitope is also a potent antagonist. Mutagenesis, biochemical, and X-ray crystallography studies demonstrate that the epitope is distinct from that of CD40 agonists. Both the human-specific and cynomolgus-specific molecules remain pure antagonists even when formatted as bivalent Fc-fusion proteins, making this an attractive therapeutic format for targeting hCD40 in autoimmune indications.

Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody.

Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy). The large PEG dominates the properties of the dAb-PEG conjugate such as a hydrodynamic radius that corresponds to a globular protein over four times its size and a much reduced association rate. We have used AFM single-molecule studies to determine the mechanism of PEG-dependent reductions in the effectiveness of the dAb observed by SPR kinetic studies. Recognition imaging showed that all of the PEGylated dAb molecules are active, suggesting that some may transiently become inactive if PEG sterically blocks binding. This helps explain the disconnect between the SPR, determined kinetically, and the force microscopy and ITC results that demonstrated that PEG does not change the binding energy.

Discovery of novel P1 groups for coagulation factor VIIa inhibition using fragment-based screening.

A multidisciplinary, fragment-based screening approach involving protein ensemble docking and biochemical and NMR assays is described. This approach led to the discovery of several structurally diverse, neutral surrogates for cationic factor VIIa P1 groups, which are generally associated with poor pharmacokinetic (PK) properties. Among the novel factor VIIa inhibitory fragments identified were aryl halides, lactams, and heterocycles. Crystallographic structures for several bound fragments were obtained, leading to the successful design of a potent factor VIIa inhibitor with a neutral lactam P1 and improved permeability.

Dual universal peptide approach to bioanalysis of human monoclonal antibody protein drug candidates in animal studies.

BACKGROUND: There is a need for general and reliable LC-MS assays capable of supporting the bioanalysis of a variety of human monoclonal antibody-based therapeutic drug candidates in animal PK/TK studies. RESULTS: We present herein improvements in our previously reported universal peptide approach to the bioanalysis of human monoclonal antibody protein drug candidates in animal studies. These improvements include incorporation of a second, light chain-based universal peptide into the assay, thus introducing the concept of a dual universal peptide assay; and incorporation of a universal stable isotope-labeled monoclonal antibody into the assay. CONCLUSION: Improvements reported herein to the universal peptide assay will enable more reliable quantification of human monoclonal antibody protein drug candidates in animal studies.

Pellet digestion: a simple and efficient sample preparation technique for LC-MS/MS quantification of large therapeutic proteins in plasma.

BACKGROUND: There is a need for a simple and efficient sample preparation technique for LC-MS/MS quantification of large therapeutic proteins in plasma. RESULTS: The sample preparation technique presented here is based upon trypsin digestion of the pellet obtained following precipitation of the protein analyte from plasma. The pellet digestion technique was shown to facilitate efficient digestion of large therapeutic proteins, with concomitant removal of a substantial amount of potentially problematic plasma phospholipids. The technique was successfully applied to a pharmacokinetic study of a large therapeutic protein. CONCLUSION: This simple sample preparation approach will be beneficial to bioanalytical laboratories engaged in the LC-MS/MS quantification of large therapeutic proteins in biological matrices.

Discovery of 6-(aminomethyl)-5-(2,4-dichlorophenyl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamides as potent, selective dipeptidyl peptidase-4 (DPP4) inhibitors.

Continued structure-activity relationship (SAR) exploration within our previously disclosed azolopyrimidine containing dipeptidyl peptidase-4 (DPP4) inhibitors led us to focus on an imidazolopyrimidine series in particular. Further study revealed that by replacing the aryl substitution on the imidazole ring with a more polar carboxylic ester or amide, these compounds displayed not only increased DPP4 binding activity but also significantly reduced human ether-a-go-go related gene (hERG) and sodium channel inhibitory activities. Additional incremental adjustment of polarity led to permeable molecules which exhibited favorable pharmacokinetic (PK) profiles in preclinical animal species. The active site binding mode of these compounds was determined by X-ray crystallography as exemplified by amide 24c. A subsequent lead molecule from this series, (+)-6-(aminomethyl)-5-(2,4-dichlorophenyl)-N-(1-ethyl-1H-pyrazol-5-yl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamide (24s), emerged as a potent, selective DPP4 inhibitor that displayed excellent PK profiles and in vivo efficacy in ob/ob mice.

Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes.

Acetyl coenzyme A (acetyl-CoA) carboxylase isozyme 1 (ACC1) and acetyl-CoA carboxylase isozyme 2 (ACC2) are critical for de novo fatty acid synthesis and for the regulation of beta-oxidation. Emerging evidence indicates that one or both isozymes might be therapeutic targets for the treatment of obesity, type 2 diabetes, and dyslipidemia. One of the major obstacles in the field is the lack of readily-available source of recombinant human ACC enzymes to support systematic drug discovery efforts. Here, we describe an efficient and optimal protocol for expressing and isolating recombinant mammalian ACCs with high yield and purity. The resultant human ACC2, human ACC1, and rat ACC2 possess high specific activities, are properly biotinylated, and exhibit kinetic parameters very similar to the native ACC enzymes. We believe that the current study paves a road to a systematic approach for drug design revolving around the ACC inhibition mechanism.