MHC-associated peptide proteomics enabling highly sensitive detection of immunogenic sequences for the development of therapeutic antibodies with low immunogenicity.

Immunogenicity is a key factor capable of influencing the efficacy and safety of therapeutic antibodies. A recently developed method called MHC-associated peptide proteomics (MAPPs) uses liquid chromatography/mass spectrometry to identify the peptide sequences derived from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells, and therefore may induce immunogenicity. In this study, we developed a MAPPs technique (called Ab-MAPPs) that has high throughput and can efficiently identify the MHC II-presented peptides derived from therapeutic antibodies using magnetic nanoparticle beads coated with a hydrophilic polymer in the immunoprecipitation process. The magnetic beads could identify more peptides and sequence regions originating from infliximab and adalimumab in a shorter measurement time than Sepharose beads, which are commonly used for MAPPs. Several sequence regions identified by Ab-MAPPs from infliximab corresponded to immunogenic sequences reported by other methods, which suggests the method's high potential for identifying significant sequences involved in immunogenicity. Furthermore, our study suggests that the Ab-MAPPs method can recognize the difference of a single amino acid residue between similar antibody sequences with different levels of T-cell proliferation activity and can identify potentially immunogenic peptides with high binding affinity to MHC II. In conclusion, Ab-MAPPs is useful for identifying the immunogenic sequences of therapeutic antibodies and will contribute to the design of therapeutic antibodies with low immunogenicity during the drug discovery stage.

In vitro metabolism of alectinib, a novel potent ALK inhibitor, in human: contribution of CYP3A enzymes.

1. The in vitro metabolism of alectinib, a potent and highly selective oral anaplastic lymphoma kinase inhibitor, was investigated. 2. The main metabolite (M4) in primary human hepatocytes was identified, which is produced by deethylation at the morpholine ring. Three minor metabolites (M6, M1a, and M1b) were also identified, and a minor peak of hydroxylated alectinib (M5) was detected as a possible precursor of M4, M1a, and M1b. 3. M4, an important active major metabolite, was produced and further metabolized to M6 by CYP3A, indicating that CYP3A enzymes were the principal contributors to this route. M5 is possibly produced by CYP3A and other isoforms as the primary step in metabolism, followed by oxidation to M4 mainly by CYP3A. Alternatively, M5 could be oxidized to M1a and M1b via an NAD-dependent process. None of the non-CYP3A-mediated metabolism appeared to be major. 4. In conclusion, this study suggests that involvement of multiple enzymes in the metabolism of alectinib reduces its potential for drug-drug interactions.

An anti-glypican 3/CD3 bispecific T cell-redirecting antibody for treatment of solid tumors.

Cancer care is being revolutionized by immunotherapies such as immune checkpoint inhibitors, engineered T cell transfer, and cell vaccines. The bispecific T cell-redirecting antibody (TRAB) is one such promising immunotherapy, which can redirect T cells to tumor cells by engaging CD3 on a T cell and an antigen on a tumor cell. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, TRAB is considered efficacious for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies. However, the success of TRAB in solid tumors has been hampered by the lack of a target molecule with sufficient tumor selectivity to avoid "on-target off-tumor" toxicity. Glypican 3 (GPC3) is a highly tumor-specific antigen that is expressed during fetal development but is strictly suppressed in normal adult tissues. We developed ERY974, a whole humanized immunoglobulin G-structured TRAB harboring a common light chain, which bispecifically binds to GPC3 and CD3. Using a mouse model with reconstituted human immune cells, we revealed that ERY974 is highly effective in killing various types of tumors that have GPC3 expression comparable to that in clinical tumors. ERY974 also induced a robust antitumor efficacy even against tumors with nonimmunogenic features, which are difficult to treat by inhibiting immune checkpoints such as PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T lymphocyte-associated protein-4). Immune monitoring revealed that ERY974 converted the poorly inflamed tumor microenvironment to a highly inflamed microenvironment. Toxicology studies in cynomolgus monkeys showed transient cytokine elevation, but this was manageable and reversible. No organ toxicity was evident. These data provide a rationale for clinical testing of ERY974 for the treatment of patients with GPC3-positive solid tumors.

Preclinical evaluation of the potential for cytochrome P450 inhibition and induction of the selective ALK inhibitor, alectinib.

1. A novel selective anaplastic lymphoma kinase (ALK) inhibitor, alectinib, has shown remarkable efficacy and safety in patients with ALK-positive non-small-cell lung cancer (NSCLC). The purpose of this study was to evaluate in vitro the potential to inhibit and induce cytochrome P450 (CYP) isoforms for alectinib and its major metabolite M4. 2. Alectinib and M4 did not show the meaningful direct inhibition of six major CYP isoforms (CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4) in human liver microsomes (HLM). Alectinib, but not M4, competitively inhibited CYP2C8, by which few marketed drugs are exclusively metabolized, with an inhibition constant of 1.98 µM. 3. Out of the seven CYP isoforms in HLM, alectinib and M4 showed time-dependent inhibition (TDI) of only CYP3A4, which suggests low TDI potential due to low inactivation efficiency. 4. Alectinib exhibited quite smaller induction of mRNA expression of CYP1A2, 2B6 and 3A4 genes in human hepatocytes compared to the respective positive controls, suggesting a low potential of enzyme induction. 5. In summary, the risk of alectinib causing drug-drug interactions with coadministered drugs is expected to be low due to the weak potential of CYP inhibition and induction estimated in the preclinical studies.

A trial proteomics fingerprint analysis of HepaRG cells by FD-LC-MS/MS.

A proteomics profile analysis was performed on a human hepatocyte carcinoma cell line (HepaRG) by using the FD-LC-MS/MS method. One hundred and fifty-eight proteins were newly identified for the first time of which 10 were found to be specific to human hepatocytes. These proteins are a "proteomics fingerprint" that can be used to characterize HepaRG cells.

Comprehensive and temporal analysis of secreted proteins in the medium from IL-6 exposed human hepatocyte.

We have previously identified intracellular secretory acute phase response (sAPR) proteins in human hepatocytes following interleukin-6 (IL-6) induction by fluorogenic derivatization (FD)-liquid chromatography (LC)-tandem mass spectrometry (MS/MS). In this report, we utilized this method, which uses 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) as the FD reagent, to comprehensively and time-dependently analyze secreted proteins in the medium, including sAPR proteins. Since DAABD-Cl selectively reacts with thiol moieties of cysteinyl residues, direct derivatization, high-resolution LC separation and identification of the secreted proteins in the culture medium were successfully achieved without a pretreatment step. As a result, 14 sAPR proteins were identified simultaneously during a 72 h induction by IL-6. The secretion levels of 11 proteins increased, whereas the secretion levels of three important transport proteins decreased (albumin, retinol-binding protein 4 and transthyretin). In addition, the secretion level of a haptoglobin was found to increase significantly between 0 and 6 h by 1.88-fold compared with the control sample. The secretion levels of four cytoplasmic proteins increased: LDH, a known marker for cell damage, and GSTA1, FABP1 and ADH1B, which are marker proteins for hepatocellular damage. The secretion levels of the other two newly identified cytoplasmic proteins, profilin-1 and SOD2, were also found to increase, suggesting that these two proteins represent novel markers for cell damage. These results suggest that the FD-LC-MS/MS proteomics method can be used to analyze comprehensively and time-dependently the secreted proteins and thereby can offer information that aids our understanding of the dynamics of protein secretion affected by the exposure of cytokines such as IL-6.

A versatile method for protein-based antigen bioanalysis in non-clinical pharmacokinetics studies of a human monoclonal antibody drug by an immunoaffinity liquid chromatography-tandem mass spectrometry.

A versatile immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify the total concentration of a protein-based antigen in non-clinical pharmacokinetics (PK) studies of a human monoclonal antibody drug. The method combines using magnetic beads that have been coated with a commercial anti-human Fc region antibody to capture an immune complex of the antigen and antibody drug, with subsequent digestion and quantification of the antigen-derived tryptic peptide via LC-MS/MS. Although a typical immunoassay or an immunoaffinity LC-MS/MS assay requires an antigen-specific antibody that uses a different epitope from the antibody drug, this method requires only a commercial anti-human Fc region antibody. The method was applied to quantify total receptor activator of nuclear factor-κB ligand (RANKL) in the presence of denosumab, a humanized monoclonal antibody (mAb) specific to RANKL. The assay was validated as fit-for-purpose and found to be accurate (<115% interbatch accuracies) and precise (<15%, interbatch coefficient of variation) across a range of 3.13-200ng/mL RANKL. Commercial enzyme-linked immunosorbent assay (ELISA) kit was not able to determine the total RANKL because interference by denosumab decreased recovery. In contrast, the antibody drug had less effect on the LC-MS/MS method. The method now provides a bioanalytical platform for developing other protein-based antigen assays in the early drug stage.

Assessing the impact of HER2 status on the antitumor activity of an HSP90 inhibitor in human tumor xenograft mice using pharmacokinetic-pharmacodynamic modeling.

The purpose of this study is to assess the impact of human epidermal growth factor receptor 2 (HER2) status on the antitumor activity of CH5164840, an orally available heat shock protein 90 (HSP90) inhibitor, using pharmacokinetic-pharmacodynamic modeling. Athymic mice, each implanted with one of eight human tumor xenografts, were treated with CH5164840 once daily at doses of 3.13 to 50 mg/kg. Plasma concentrations of CH5164840 were described by a one-compartment model with first-order absorption rate. Time profiles of tumor growth inhibition in the eight xenograft models were well captured by an indirect response model with a maximum tumor-killing rate constant (Emax model). Threshold plasma concentrations for tumor stasis, which are determined by multiple pharmacodynamic parameters, Emax, EC50 and tumor growth rate constant, were significantly lower in HER2-positive tumors (1.96-3.85 µM) than in HER2-negative tumors (4.48-23.4 µM). The results suggest that CH5164840 was more efficacious in HER2-positive tumors than in HER2-negative tumors in terms of the lower effective concentration of the drug in preclinical animal models.

Injectable hydrogel for sustained protein release by salt-induced association of hyaluronic acid nanogel.

A hyaluronic acid-based anionic nanogel formed by self-assembly of cholesteryl-group-bearing HA is designed for protein delivery. The HA nanogel spontaneously binds various types of proteins without denaturation, such as recombinant human growth hormone, erythropoietin, exendin-4, and lysozyme. The HA nanogel shows unique colloidal properties, in particular that an injectable hydrogel is formed by salt-induced association of the HA nanogel. A pharmacokinetic study in rats shows that an in situ gel formulation, prepared by simply mixing rhGH and HA nanogel in phosphate buffer, maintains plasma rhGH levels within a narrow range over one week. Therefore, HA nanogels offer a simple method for easy formulation of therapeutic proteins and are effective for sustained protein release systems.

Alteration of intracellular secretory acute phase response proteins expressed in human hepatocyte induced by exposure with interleukin-6.

Interleukin-6 (IL-6) is a principal proinflammatory cytokine inducing the acute phase response in various tissues, including liver. Here, we adopt the FD-LC-MS/MS method, consisting of fluorogenic derivatization (FD), separation by liquid chromatography (LC), and identification of proteins by LC-tandem mass spectrometry (MS/MS), to reveal how exposure to IL-6 alters temporally the intracellular secretory acute phase response (sAPR) proteins expressed in human hepatocytes as compared to non-exposure. Nine altered sAPR proteins were identified in cultures in response to IL-6. Seven of them (serum amyloid A protein, haptoglobin, fibrinogen α chain, fibrinogen ß chain, fibrinogen γ chain, α(1)-acid glycoprotein and α(1)-antitrypsin) were significantly increased and two (ß(2)-glycoprotein 1 and transferrin) were significantly decreased in response to IL-6. In addition, the transmission speed of transferrin might be much faster than the other sAPR proteins. These results suggest a different molecular mechanism for protein synthesis and the secretory pathway among the sAPR proteins. In this study, we observed the simultaneously and temporally altered expression of sAPR proteins which had been induced by exposure to IL-6 in human hepatocytes, in contrast to previous reports, in all of which the proteins were tested from the time they were secreted into the medium from the cells.

Bridging from preclinical to clinical studies for tyrosine kinase inhibitors based on pharmacokinetics/pharmacodynamics and toxicokinetics/toxicodynamics.

The purpose of this study was to provide a pharmacokinetics/pharmacodynamics and toxicokinetics/toxicodynamics bridging of kinase inhibitors by identifying the relationship between their clinical and preclinical (rat, dog, and monkey) data on exposure and efficacy/toxicity. For the eight kinase inhibitors approved in Japan (imatinib, gefitinib, erlotinib, sorafenib, sunitinib, nilotinib, dasatinib, and lapatinib), the human unbound area under the concentration-time curve at steady state (AUC(ss,u)) at the clinical dose correlated well with animal AUC(ss,u) at the no-observed-adverse-effect level (NOAEL) or maximum tolerated dose (MTD). The best correlation was observed for rat AUC(ss,u) at the MTD (p < 0.001). E(max) model analysis was performed using the efficacy of each drug in xenograft mice, and the efficacy at the human AUC of the clinical dose was evaluated. The predicted efficacy at the human AUC of the clinical dose varied from far below E(max) to around E(max) even in the tumor for which use of the drugs had been accepted. These results suggest that rat AUC(ss,u) at the MTD, but not the efficacy in xenograft mice, may be a useful parameter to estimate the human clinical dose of kinase inhibitors, which seems to be currently determined by toxicity rather than efficacy.

Comparison of in vitro metabolic conversion of capecitabine to 5-FU in rats, mice, monkeys and humans--toxicological implications.

Capecitabine is an oral anticancer prodrug which is converted to 5-fluorouracil (5-FU) via 3 enzymatic steps, these being 5'-deoxy-5-fluorocytidine (5'-DFCR), 5'-deoxy-5-fluorouridine (5'-DFUR), and finally 5-FU by carboxylesterase (CES), cytidine deaminase (CDA), and thymidine phosphorylase (TP), respectively. Because rats, mice and monkeys are used for preclinical safety studies, we investigated the in vitro conversion from capecitabine to 5-FU by hepatic and intestinal mucosal microsomes and cytosols, to compare their metabolic activity to that of humans. Capecitabine was hydrolyzed to 5'-DFCR in hepatic and intestinal mucosal microsomes in these animal species. In humans and monkeys, CL(int) (V(max)/K(m)) for the hydrolysis of capecitabine in intestine (expressed as µl/min/g tissue) was much lower than that in hepatic microsomes but, in rats and mice, CL(int) was higher in intestine than in liver. In humans and monkeys, similar K(m) values and inhibition patterns by tetrahydrouridine (THU) a CDA inhibitor, were observed in CDA activity of hepatic and intestinal cytosols. However, rats showed very low CDA activity and mice showed non-Michaelis-Menten kinetics and a different inhibition pattern by THU. K(m) values for TP activity were almost similar in rats, mice, monkeys and humans. In conclusion, it was confirmed that monkeys are a suitable animal model for the safety assessment of capecitabine in terms of metabolic enzymes and it was suggested that higher toxic incidences in mouse small intestine were related to high hydrolytic activity of capecitabine in the small intestine.

Antigen-dependent internalization is related to rapid elimination from plasma of humanized anti-HM1.24 monoclonal antibody.

Anti-HM1.24 monoclonal antibody (AHM) is a humanized anti-HM1.24 monoclonal antibody that binds to the HM1.24 antigen, a protein that is highly expressed in multiple myeloma cells. The pharmacokinetics of AHM was determined in experiments in which AHM was administered intravenously to cynomolgus monkeys. The area under the plasma concentration-time curve increased by more than the dose ratio between 2 and 20 mg/kg, and nonlinear pharmacokinetics was observed. The elimination half-life of AHM from the plasma was 7.56 h at 2 mg/kg and 28.6 h at 20 mg/kg, which was shorter than that observed for other therapeutic humanized monoclonal antibodies, such as trastuzumab and bevacizumab. Although antibodies to AHM were detected in all monkeys on or after 10 days of administration, there was a temporal disassociation between the rapid elimination of AHM and the appearance of anti-AHM antibodies. HM1.24 antigen-dependent internalization and intracellular metabolism of AHM were investigated in peripheral blood mononuclear, KPMM2, and U937 cells. In all cases, AHM was rapidly internalized from the cell surface; this internalization was significantly prevented by phenylarsine oxide in KPMM2 cells, an inhibitor of receptor-mediated endocytosis, and the internalized AHM was subsequently degraded within the cells. Furthermore, immunofluorescence microscopy revealed that the internalized AHM is delivered to and degraded in late endosomes/lysosomes. Taken together, our results suggest that the rapid elimination of AHM from plasma in monkey is due to HM1.24 antigen-dependent internalization followed by delivery to the lysosomes.