Combination of two novel blocking antibodies, anti-PD-1 antibody ezabenlimab (BI 754091) and anti-LAG-3 antibody BI 754111, leads to increased immune cell responses.

Upregulation of inhibitory receptors, such as lymphocyte activation gene-3 (LAG-3), may limit the antitumor activity of therapeutic antibodies targeting the programmed cell death protein-1 (PD-1) pathway. We describe the binding properties of ezabenlimab, an anti-human PD-1 antibody, and BI 754111, an anti-human LAG-3 antibody, and assess their activity alone and in combination. Ezabenlimab bound with high affinity to human PD-1 (KD = 6 nM) and blocked the interaction of PD-1 with PD-L1 and PD-L2. Ezabenlimab dose-dependently increased interferon-γ secretion in human T cells expressing PD-1 in co-culture with PD-L1-expressing dendritic cells. Administration of ezabenlimab to human PD-1 knock-in mice dose-dependently inhibited growth of MC38 tumors. To reduce immunogenicity, ezabenlimab was reformatted from a human IgG4 to a chimeric variant with a mouse IgG1 backbone (BI 905725) for further in vivo studies. Combining BI 905725 with anti-mouse LAG-3 antibodies improved antitumor activity versus BI 905725 monotherapy in the MC38 tumor model. We generated BI 754111, which bound with high affinity to human LAG-3 and prevented LAG-3 interaction with its ligand, major histocompatibility complex class II. In an in vitro model of antigen-experienced memory T cells expressing PD-1 and LAG-3, interferon-γ secretion increased by an average 1.8-fold versus isotype control (p = 0.027) with BI 754111 monotherapy, 6.9-fold (p < 0.0001) with ezabenlimab monotherapy and 13.2-fold (p < 0.0001) with BI 754111 plus ezabenlimab. Overall, ezabenlimab and BI 754111 bound to their respective targets with high affinity and prevented ligand binding. Combining ezabenlimab with BI 754111 enhanced in vitro activity versus monotherapy, supporting clinical investigation of this combination (NCT03156114; NCT03433898).

Effect of the ADCC-Modulating Mutations and the Selection of Human IgG Isotypes on Physicochemical Properties of Fc.

Monoclonal antibodies, particularly IgGs and Ig-based molecules, are a well-established and growing class of biotherapeutic drugs. In order to improve efficacy, potency and pharmacokinetics of these therapeutic drugs, pharmaceutical industries have investigated significantly in engineering fragment crystallizable (Fc) domain of these drugs to optimize the interactions of these drugs and Fc gamma receptors (FcγRs) in recent ten years. The biological function of the therapeutics with the antibody-dependent cellular cytotoxicity (ADCC) enhanced double mutation (S239D/I332E) of isotype IgG1, the ADCC reduced double mutation (L234A/L235A) of isotype IgG1, and ADCC reduced isotype IgG4 has been well understood. However, limited information regarding the effect of these mutations or isotype difference on physicochemical properties (PCP), developability, and manufacturability of therapeutics bearing these different Fc regions is available. In this report, we systematically characterize the effects of the mutations and IgG4 isotype on conformation stability, colloidal stability, solubility, and storage stability at accelerated conditions in two buffer systems using six Fc variants. Our results provide a basis for selecting appropriate Fc region during development of IgG or Ig-based therapeutics and predicting effect of the mutations on CMC development process.

An optimally designed anti-human CD40 antibody with potent B cell suppression for the treatment of autoimmune diseases.

Antibodies targeting the CD40-CD40L pathway have great potential for treating autoimmune diseases like rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), and inflammatory bowel diseases (IBD). However, in addition to the known difficulty in generating a purely antagonistic CD40 antibody, the presence of CD40 and CD40L on platelets creates additional unique challenges for the safety, target coverage, and clearance of antibodies targeting this pathway. Previously described therapeutic antibodies targeting this pathway have various shortcomings, and the full therapeutic potential of this axis has yet to be realized. Herein, we describe the generation and characterization of BI 655064, a novel, purely antagonistic anti-CD40 antibody that potently neutralizes CD40-CD40L-dependent B-cell stimulation without evidence of impacting platelet functions. This uniquely optimized antibody targeting a highly challenging pathway was obtained by applying stringent functional and biophysical criteria during the lead selection process. BI 655064 has favorable target-mediated drug disposition (TMDD)-saturation pharmacokinetics, consistent with that of a high-quality therapeutic monoclonal antibody.

Crystal structure and receptor-interacting residues of MYDGF - a protein mediating ischemic tissue repair.

Myeloid-derived growth factor (MYDGF) is a paracrine-acting protein that is produced by bone marrow-derived monocytes and macrophages to protect and repair the heart after myocardial infarction (MI). This effect can be used for the development of protein-based therapies for ischemic tissue repair, also beyond the sole application in heart tissue. Here, we report the X-ray structure of MYDGF and identify its functionally relevant receptor binding epitope. MYDGF consists of a 10-stranded ß-sandwich with a folding topology showing no similarities to other cytokines or growth factors. By characterizing the epitope of a neutralizing antibody and utilizing functional assays to study the activity of surface patch-mutations, we were able to localize the receptor interaction interface to a region around two surface tyrosine residues 71 and 73 and an adjacent prominent loop structure of residues 97-101. These findings enable structure-guided protein engineering to develop modified MYDGF variants with potentially improved properties for clinical use.

Design and characterization of Zweimab and Doppelmab, high affinity dual antagonistic anti-TSLP/IL13 bispecific antibodies.

Patients with severe Th2 type asthma often have a steroid resistant phenotype and are prone to acute exacerbations. Current novel therapies have only marginal therapeutic effects. One of the hypotheses for lack of major efficacy in most patients is targeting only one redundant pathway leaving others active. Hence, we have designed and developed novel highly potent bispecific anti-TSLP/IL13 antibodies called Zweimabs (monovalent bispecific) and Doppelmabs (bivalent bispecific) that concurrently inhibits the signaling by these two cytokines.

Generation and functional characterization of anti-human and anti-mouse IL-36R antagonist monoclonal antibodies.

Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, ß and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation.

Efficient Qualitative and Quantitative Determination of Antigen-induced Immune Responses.

To determine the effectiveness of immunization strategies used in therapeutic antibody or vaccine development, it is critical to assess the quality of immunization-induced polyclonal antibody responses. Here, we developed a workflow that uses sensitive methods to quantitatively and qualitatively assess immune responses against foreign antigens with regard to antibody binding affinity and epitope diversity. The application of such detailed assessments throughout an immunization campaign can significantly reduce the resources required to generate highly specific antibodies. Our workflow consists of the following two steps: 1) the use of surface plasmon resonance to quantify antigen-specific antibodies and evaluate their apparent binding affinities, and 2) the recovery of serum IgGs using an automated small scale purification system, followed by the determination of their epitope diversity using hydrogen deuterium exchange coupled with mass spectrometry. We showed that these methods were sensitive enough to detect antigen-specific IgGs in the nanogram/µl range and that they provided information for differentiating the antibody responses of the various immunized animals that could not be obtained by conventional methods. We also showed that this workflow can guide the selection of an animal that produces high affinity antibodies with a desired epitope coverage profile, resulting in the generation of potential therapeutic monoclonal antibody clones with desirable functional profiles. We postulate that this workflow will be an important tool in the development of effective vaccines to combat the highly sophisticated evasion mechanisms of pathogens.

Selective targeting of the IL23 pathway: Generation and characterization of a novel high-affinity humanized anti-IL23A antibody.

Herein, we describe the generation and characterization of BI 655066, a novel, highly potent neutralizing anti-interleukin-23 (IL23) monoclonal antibody in clinical development for autoimmune conditions, including psoriasis and Crohn's disease. IL23 is a key driver of the differentiation, maintenance, and activity of a number of immune cell subsets, including T helper 17 (Th17) cells, which are believed to mediate the pathogenesis of several immune-mediated disorders. Thus, IL23 neutralization is an attractive therapeutic approach. Designing an antibody for clinical activity and convenience for the patient requires certain properties, such as high affinity, specificity, and solubility. These properties were achieved by directed design of the immunization, lead identification, and humanization procedures. Favorable substance and pharmacokinetic properties were established by biophysical assessments and studies in cynomolgus monkeys.

A steric antagonism of actin polymerization by a salmonella virulence protein.

Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.

The determination of high-affinity protein/inhibitor binding constants by electrospray ionization hydrogen/deuterium exchange mass spectrometry.

Recently, a hydrogen/deuterium exchange method termed SUPREX (Stability of Unpurified Proteins from Rates of hydrogen/deuterium EXchange), capable of measuring protein/ligand binding constants, which utilizes matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), has been reported. Unlike more conventional approaches, SUPREX is inherently capable of measuring Kd values of tight binding ligands. Here we present a SUPREX-based method, incorporating automation and electrospray ionization (ESI)-MS, to measure Kd values for very potent inhibitors of the kinase PKCtheta. The use of ESI offers an alternative to MALDI, with the advantages of improved mass measurement precision for larger proteins, and amenability to automation. Kd values generated by this method are in good agreement with those generated by a molecular protein kinase assay.

Conformational changes of the glucocorticoid receptor ligand binding domain induced by ligand and cofactor binding, and the location of cofactor binding sites determined by hydrogen/deuterium exchange mass spectrometry.

HXMS (hydrogen/deuterium exchange mass spectrometry) of the glucocorticoid receptor ligand-binding domain (GR LBD) complexed with the agonist dexamethasone and the antagonist RU-486 is described. Variations in the rates of exchange were observed in regions consistent with the published crystal structures of GR LBD complexed with RU-486 when compared with the GR dexamethasone complex. We also report the HXMS results for agonist-bound GR LBD with the coactivator transcriptional intermediary factor 2 (TIF2) and anatagonist-bound GR LBD with nuclear receptor corepressor (NCoR). Alterations in exchange rates observed for agonist-bound GR LBD with TIF2 present were consistent with the published crystal structural contacts for the complex. Alterations in exchange rates observed for antagonist-bound GR LBD with NCoR were a subset of those observed with TIF2 binding, suggesting a common or overlapping binding site for coactivator and corepressor.

Discovery and characterization of a substrate selective p38alpha inhibitor.

A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-dependent phosphorylation (K(i)(app) = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K(i)(app) > 20 microM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D(2)O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues (E160 and D161), and a Mg(2+) ion cofactor binding residue (D168). Although the exact mechanism of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.

Catalysis and function of the p38 alpha.MK2a signaling complex.

The p38 mitogen-activated protein kinase (p38) pathway is required for the production of proinflammatory cytokines (TNFalpha and IL-1) that mediate the chronic inflammatory phases of several autoimmune diseases. Potent p38 inhibitors, such as the slow tight-binding inhibitor BIRB 796, have recently been reported to block the production of TNFalpha and IL-1beta. Here we analyze downstream signaling complexes and molecular mechanisms, to provide new insight into the function of p38 signaling complexes and the development of novel inhibitors of the p38 pathway. Catalysis, signaling functions, and molecular interactions involving p38alpha and one of its downstream signaling partners, mitogen-activated protein kinase-activated protein kinase 2 (MK2), have been explored by steady-state kinetics, surface plasmon resonance, isothermal calorimetry, and stopped-flow fluorescence. Functional 1/1 signaling complexes (Kd = 1-100 nM) composed of activated and nonactivated forms of p38alpha and a splice variant of MK2 (MK2a) were characterized. Catalysis of MK2a phosphorylation and activation by p38alpha was observed to be efficient under conditions where substrate is saturating (kcat(app) = 0.05-0.3 s(-1)) and nonsaturating (kcat(app)/KM(app) = 1-3 x 10(6) M(-1) s(-1)). Specific interactions between the carboxy-terminal residues of MK2a (370-400) and p38alpha precipitate formation of a high-affinity complex (Kd = 20 nM); the p38alpha-dependent MK2a phosphorylation reaction was inhibited by the 30-amino acid docking domain peptide of MK2a (IC50 = 60 nM). The results indicate that the 30-amino acid docking domain peptide of MK2a is required for the formation of a tight, functional p38alpha.MK2a complex, and that perturbation of the tight-docking interaction between these signaling partners prevents the phosphorylation of MK2a. The thermodynamic and steady-state kinetic characterization of the p38alpha.MK2a signaling complex has led to a clear understanding of complex formation, catalysis, and function on the molecular level.

Characterization of the binding site for inhibitors of the HPV11 E1-E2 protein interaction on the E2 transactivation domain by photoaffinity labeling and mass spectrometry.

An indandione-containing class of inhibitors abrogates DNA replication of human papillomavirus (HPV) types 6 and 11 by binding reversibly to the transactivation domain (TAD) of the viral E2 protein and inhibiting its interaction with the viral E1 helicase. To locate the binding site of this class of protein-protein interaction inhibitors, a benzophenone derivative was used to generate an irreversibly labeled E2-TAD polypeptide. The single site of covalent modification of the E2-TAD was identified by proteolytic digestions using trypsin, LysC, and V8 proteases and characterization of the resulting peptides by LC-MS procedures. Through this methodology, the benzophenone attachment point was located at the terminal methyl of residue Met101. Evidence further pinpointed the site of photoaffinity attachment to the terminal carbon atom, which is significant in providing a definitive example of the ability to locate photoinduced cross-linking to a polypeptide with atomic resolution using solely mass spectrometric detection. The location of the inhibitor binding site vis-à-vis the Glu39 and Glu100 residues sensitive to mutation for HPV 11 E2-TAD is discussed in relation to the crystal structure of the E2-TAD from the related HPV type 16.

Micro-high-performance liquid chromatography/Fourier transform mass spectrometry with electron-capture dissociation for the analysis of protein enzymatic digests.

Electron-capture dissociation (ECD) Fourier transform mass spectrometry (FTMS) employed to generate comprehensive sequence information for the chromatographic analysis of enzymatic protein digests is described. A pepsin digest of cytochrome c was separated by reversed-phase micro-high-performance liquid chromatography (microHPLC) and ionized 'on-line' by electrospray ionization (ESI). The ions thus formed were transferred to and trapped in the FTMS analyzer cell. Typically, no precursor ion isolation was performed. The trapped ions were subjected to a pulse of electrons to induce fragmentation. Mass spectra were acquired continuously to produce a three-dimensional LC/MS data set. The spectra were dominated by c and, to a lesser degree, z ions, which provided near complete sequence coverage. External calibration provided good mass accuracy and resolution, typical of FTMS. Thus microHPLC/ECD - FTMS is shown to be a highly informative method for the analysis of enzymatic protein digests.