Antibody Therapies Targeting Complex Membrane Proteins

In analyses of protein families that may serve as drug targets,membrane-associated G-protein-coupled receptors(GPCRs)dominate,followed by ion channels,transporters,and—to a lesser extent—membrane-bound enzymes.However,various challenges put such membrane proteins among key groups of underutilized opportunities for the application of therapeutic antibodies.Antibodies hold the promise of exquisite specificity,as they are able to target even specific conformations of a particular membrane protein,as well as adaptability through engineering into various antibody formats.However,the ease of raising and isolating specific,effective antibodies targeting membrane proteins depends on many factors.In particular,the generation of specific antibodies is easier when targeting larger,simpler,extracellular domains with greater uniqueness of amino acid sequence.The rareness of such ideal conditions is illustrated by the limited number of approved biologics for targeting GPCRs and other complex membrane proteins.Challenges in developing antibodies to complex membrane proteins such as GPCRs,ion channels,transporters,and membrane-bound enzymes can be addressed by the design of the antigen,antibody-generation strategies,lead optimization technologies,and antibody modalities.A better understanding of the membrane proteins being targeted would facilitate mechanism-based drug discovery.This review describes the advantages and challenges of targeting complex membrane proteins with antibodies and discusses the preparation of membrane protein antigens and antibody generation,illustrated by select examples of success.

Trastuzumab-Doxorubicin Conjugate Provides Enhanced Anti-Cancer Potency and Reduced Cardiotoxicity

Since trastuzumab monotherapy for treatment of breast cancer with HER2/ErbB2 over-expression has been shown to have limited efficacy, combined treatment of trastuzumab with chemotherapy is widely practiced in clinic. However, certain combination treatments of trastuzumab and chemotherapy (i.e. doxorubicin) are not recommended due to high risk of cardiotoxicity. Antibody-drug conjugates (ADCs) offer selective delivery of cytotoxic agents into targeted cancer cells, thereby allowing for reduced general cellular cytotoxicity caused by chemotherapeutic agents through antibody mediated specific recognition of tumor antigens. In this study, we constructed a trastuzumab-doxorubicin conjugate (T-Dox) using a thioether linkage and characterized both biophysical stability and anti-cancer potency of the T-Dox using a panel of HER2 expressing cancer cell lines. The T-Dox conjugate showed significantly improved anti-cancer potency in comparison with trastuzumab. The results demonstrated for the first time that there were significant differences in the uptake of T-Dox among high HER2 expression cancer cells and higher T-Dox uptake also showed stronger anti-cancer potency. Similar to trastuzumab, T-Dox selectively bound to HER2 overexpressing cancer cells and low HER2 expression cells had no detectable uptake of T-Dox. Consistent to the uptake data, human cardiomyocyte cells had no detectable HER2 expression and T-Dox showed minimal cytotoxic effects. On the contrary, a treatment with combination of trastuzumab and doxorubicin showed severe cytotoxicity to human cardiomyocytes (>90% cell death after 3 day exposure). This study demonstrated that trastuzumab conjugated with doxorubicin (T-Dox) can provide valuable alternative to the combination treatment with doxorubicin and trastuzumab for high HER2 expressing cancer patients.

Novel Approach for Quantitative Measurement of Matrix Metalloprotease-1 (MMP1) in Human Breast Cancer Cells Using Mass Spectrometry

Identification and quantification of low abundance growth factors and regulators in complex biological samples still present a challenging task in analytical biochemistry. Immunoassays are often used for such purpose but immunoassays face limitation of both availability and qualities of antibody reagents that are necessary for development of immune assays. With genomics data base available, mass spectrometry (MS) can analyze protein tryptic peptides directly for quantitative determination of proteins. In this study, we report a method for detection of matrix metalloproteinase 1 (MMP1), an important extracellular matrix modulator, in human breast cancer cells by quadrupole time-of-flight (Q-TOF) MS. Absolute quantification of MMP1 was conducted using the selected reaction monitoring (SRM) on a triple quadrupole (Triple-Quad) MS via transitions selected from MMP1 tryptic peptides using non isotope labeled MMP1 protein as a titration standard. In comparison with immune based assay, this MS method showed picogram level sensitivity for quantitative determination of MMP1 intotal cell lysates. Our results demonstrated the feasibility of absolute quantification of low abundance proteins using label-free protein standard by mass spectrometry. Therefore, this method provides not only advantages of high sensitivity but also cost saving in comparison with the commonly used mass spectrometry that currently employs isotype labeled proteins for quantitative analysis.

Multi-functional stimuli-responsive biomimetic flower assembled from CLCE and MOF-based pedals

Simulating the structures and behaviors of living organisms are of great significance to develop novel multi-functional intelligent devices. However, the development of biomimetic devices with complex deformable structures and synergistic properties is still on the way. Herein, we propose a simple and effective approach to create the multi-functional stimuli-responsive biomimetic devices with independently pre-programmable colorful visual patterns, complex geometries and morphable modes. The metal organic framework(MOF)-based composite film acts as a rigidity actuation substrate to support and mechanically guide the spatial configuration of the soft chiral nematic liquid crystal elastomer(CLCE) sheet. We can directly program the structural color of the CLCE sheet by adjusting the thickness distribution without tedious chemical modification. By using this coordination strategy, we fabricate an artificial flower, which exhibits a synergistic effect of both shape transformation and color change like paeonia ‘Coral Sunset’at different flowering stages, and can even perform different flowering behaviors by bending, twisting and curling petals. The assembled bionic flower is innovatively demonstrated to respond to local stimuli of humidity, heat or ultraviolet irradiation. Therefore, the spatial assembly of CLCE combined with functional MOF materials has a wide range of potential application in multi-functional integrated artificial systems.

Antibody-drug conjugates： recent advances in conjugation and linker chemistries

The antibody-drug conjugate （ADC）, a humanized or human monoclonal antibody conjugated with highly cytotoxic small molecules （payloads） through chemical linkers, is a novel therapeutic format and has great potential to make a paradigm shift in cancer chemother- apy. This new antibody-based molecular platform enables selective delivery of a potent cytotoxic payload to target cancer cells, resulting in improved efficacy, reduced systemic toxicity, and preferable pharmacokinetics （PK）I pharmacodynamics （PD） and biodistribution compared to traditional chemotherapy. Boosted by the successes of FDA-approved Adcetris~ and Kadcyla~, this drug class has been rapidly growing along with about 60 ADCs cur- rently in clinical trials. In this article, we briefly review molecular aspects of each component （the antibody, payload, and linker） of ADCs, and then mainly discuss traditional and new technologies of the conjugation and linker chemistries for successful construction of clini- cally effective ADCs. Current efforts in the conjugation and linker chemistries will provide greater insights into molecular design and strategies for clinically effective ADCs from medicinal chemistry and pharmacology standpoints. The development of site-specific conjuga- tion methodologies for constructing homogeneous ADCs is an especially promising path to improving ADC design, which will open the way for novel cancer therapeutics.

Monoclonal antibodies-a proven and rapidly expanding therapeutic modality for human diseases

The study of antibodies has been a focal point in modern biology and medicine since the early 1900s.However,progress in therapeutic antibody development was slow and intermittent until recently.The first antibody therapy,murine-derived murononab OKT3 for acute organ rejection,was approved by the US Food and Drug Administration(FDA)in 1986,more than a decade after César Milstein and Georges Köhler developed methods for the isolation of mouse monoclonal antibodies from hybridoma cells in 1975.As a result of the scientific,technological,and clinical breakthroughs in the 1980s and 1990s,the pace of therapeutic antibody discovery and development accelerated.Antibodies are becoming a major drug modality with more than two dozen therapeutic antibodies in the clinic and hundreds more in development.Despite the progress,need for improvement exists at every level.Antibody therapeutics provides fertile ground for protein scientists to fulfill the dream of personalized medicine through basic scientific discovery and technological innovation.

HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners.Unlike other members in the family,the kinase domain of HER3 lacks key amino acid residues for catalytic activity.As a result,HER3 is suggested to serve as an allosteric activator of other EGFR family members which include EGFR,HER2 and HER4.To study the role of intracellular domains in HER3 dimerization and activation of downstream signaling pathways,we constructed HER3/HER2 chimeric receptors by replacing the HER3 kinase domain(HER3-2-3)or both the kinase domain and the C-terminal tail(HER3-2-2)with the HER2 counterparts and expressed the chimeric receptors in Chinese hamster ovary(CHO)cells.While over expression of the intact human HER3 transformed CHO cells with oncogenic properties such as AKT/ERK activation and increased proliferation and migration,CHO cells expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2 dimerization and decreased phosphorylation of both AKT and ERK1/2 in the presence of neuregulin-1(NRG-1).In contrast,CHO cells expressing the HER3-2-2 chimeric receptor resulted in a total loss of downstream AKT activation in response to NRG-1,but maintained partial activation of ERK1/2.The results demonstrate that the intracellular domains play a crucial role in HER3’s function as an allosteric activator and its role in downstream signaling.

Molecular and functional analysis of monoclonal antibodies in support of biologics development

Monoclonal antibody （mAb）-based therapeutics are playing an increasingly important role in the treatment or pre- vention of many important diseases such as cancers, autoimmune disorders, and infectious diseases. Multi- domain mAbs are far more complex than small molecule drugs with intrinsic heterogeneities. The critical quality attributes of a given mAb, including structure, post-trans- lational modifications, and functions at biomolecular and cellular levels, need to be defined and profiled in details during the developmental phases of a biologics. These critical quality attributes, outlined in this review, serve an important database for defining the drug properties during commercial production phase as well as post licensure life cycle managemenL Specially, the molecular characteriza- tion, functional assessment, and effector function analysis of mAbs, are reviewed with respect to the critical parame- ters and the methods used for obtaining them. The three groups of analytical methods are three essential and inte. gral facets making up the whole analytical package for a mAIPbased drug. Such a package is critically important for the licensure and the post-licensurs life cycle management of a therapeutic or prophylactic biologics. In addition, the basic principles on the evaluation of biosimilar mAbs were discussed briefly based on the recommendations by the World Health Organization.

Pharmacological applications of a novel neoepitope antibody to a modified amyloid precursor protein-derived beta-secretase product

We have previously described a novel artificial NFEVβ-secretase(BACE1)cleavage site,which when introduced into the amyloid-βprecursor protein(APP),significantly enhances APP cleavage by BACE1 in in vitro and cellular assays.In this study,we describe the identification and characterization of a single chain fragment of variable region(scFv),specific to the EV neo-epitope derived from BACE1 cleavage of the NFEV-containing peptide,and its conversion to IgG1.Both the scFv displayed on phage and EV-IgG1 show exquisite specificity for binding to the EV neoepitope without cross-reactivity to other NFEV containing peptides or WT-APP KMDA cleavage products.EV-IgG1 can detect as little as 0.3 nmol/L of the EV peptide.EV-IgG1 antibody was purified,conjugated with alkaline phosphatase and utilized in various biological assays.In the BACE1 enzymatic assay using NFEV substrate,a BACE1 inhibitor MRK-3 inhibited cleavage with an IC50 of 2.4 nmol/L with excellent reproducibility.In an APP_NFEV stable SH-SY5Y cellular assay,the EC_(50) for inhibition of EV-Aβ peptide secretion with MRK-3 was 236 nmol/L,consistent with values derived using an EV polyclonal antibody.In an APP_NFEV knock-in mouse model,both Aβ_EV40 and Aβ_EV42 peptides in brain homogenate showed excellent gene dosage dependence.In conclusion,the EV neoepitope specific monoclonal antibody is a novel reagent for BACE1 inhibitor discovery for both in vitro,cellular screening assays and in vivo biochemical studies.The methods described herein are generally applicable to novel synthetic substrates and enzyme targets to enable robust screening platforms for enzyme inhibitors.

＂Magic Bullets＂ at the center stage of immune therapy： a special issue on therapeutic antibodies

Therapeutic antibodies represent one of the most significant advances in the history of medicine. Progress in the antibody therapy field was initially slow and intermittent. The first therapeutic antibody, murine-derived Murononab OKT3 for acute organ rejection, was approved by the FDA in 1986, more than a decade after the discovery of the hybridoma technology by Milstein and Kohler in 1975 （Kohler and Mil- stein, 1975.

Investigation of a special neutralizing epitope of HEV E2s

Dear Editor After 14-years of development, the first prophylactic vaccine against the Hepatitis E virus （HEY） has been marketed since 2012 （Wu et al., 2012）o However, the neutralizing epitopes of HEY are not completely defined. E2s, a protruding homodi- mer domain of HEV capsid protein, is responsible for inter- acting with host cells to initiate infection （Li et al., 2009; Li et al., 2005）. It was shown that two monoclonal antibodies （mAb）, 8Cll and 8H3, could neutralize the infectivity of HEV in rhesus, and the two mAbs bind to different neutralizing conformational epitopes on E2s （Zhang et al., 2005）.