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.

Nitric oxide and geriatrics:Implications in diagnostics and treatment of the elderly

The nation’s aging population is growing rapidly.By 2030,the number of adults age 65 and older will nearly double to 70 million.Americans are living longer and older adults can now live for many years with multiple chronic illnesses but with a substantial cost to health care.Twenty percent of the Medicare population has at least five chronic conditions i.e.,hypertension,diabetes,arthritis,etc.Studies in experimental models and even humans reveal that constitutive production of nitric oxide(NO)is reduced with aging and this circumstance may be relevant to a number of diseases that plague the aging population.NO is a multifunctional signaling molecule,intricately involved with maintaining a host of physiological processes including,but not limited to,host defense,neuronal communication and the regulation of vascular tone.NO is one of the most important signaling molecules in our body,and loss of NO function is one of the earliest indicators or markers of disease.Clinical studies provide evidence that insufficient NO production is associated with all major cardiovascular risk factors,such as hyperlipidemia,diabetes,hypertension,smoking and severity of atherosclerosis,and also has a profound predictive value for disease progression including cardiovascular and Alzheimers disease.Thirty plus years after its discovery and over 13 years since a Nobel Prize was awarded for its discovery,there have been no hallmark therapeutic breakthroughs or even NO based diagnostics.We will review the current state of the science surrounding NO in the etiology of a number of different diseases in the geriatric patient.From these observations,it can be concluded that enzymatic production of NO declines steadily with increasing age in healthy human subjects.Implementing strategies to diagnose and treat NO insufficiency may provide enormous benefit to the geriatric patient.

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.

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.

Curcumin induces differentiation of embryonic stem cells through possible modulation of nitric oxide-cyclic GMP pathway

Curcumin,an active ingredient of dietary spice used in curry,has been shown to exhibit anti-oxidant,anti-inflammatory and anti-proliferative properties.Using EB directed differentiation protocol of H-9 human em-bryonic stem(ES)cells;we evaluated the effect of cur-cumin(0-20μmol/L)in enhancing such differentiation.Our results using real time PCR,western blotting and immunostaining demonstrated that curcumin signifi-cantly increased the gene expression and protein levels of cardiac specific transcription factor NKx2.5,cardiac troponin I,myosin heavy chain,and endothelial nitric oxide synthase during ES cell differentiation.Further-more,an NO donor enhanced the curcumin-mediated induction of NKx2.5 and other cardiac specific proteins.Incubation of cells with curcumin led to a dose depend-ent increase in intracellular nitrite to the same extent as giving an authentic NO donor.Functional assay for second messenger(s)cyclic AMP(cAMP)and cyclic GMP(cGMP)revealed that continuous presence of cur-cumin in differentiated cells induced a decrease in the baseline levels of cAMP but it significantly elevated baseline contents of cGMP.Curcumin addition to a cell free assay significantly suppressed cAMP and cGMP degradation in the extracts while long term treatment of intact cells with curcumin increased the rates of cAMP and cGMP degradation suggesting that this might be due to direct suppression of some cyclic nucleo-tide-degrading enzyme(phosphodiesterase)by curcu-min.These studies demonstrate that polyphenol curcu-min may be involved in differentiation of ES cells partly due to manipulation of nitric oxide signaling.

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.

Beta-adrenergic signaling on neuroendocrine differentiation, angiogenesis, and metastasis in prostate cancer progression

Prostate cancer is a complex, heterogeneous disease that mainly affects the older male population with a high-mortality rate. The mechanisms underlying prostate cancer progression are still incompletely understood. Beta-adrenergic signaling has been shown to regulate multiple cellular processes as a mediator of chronic stress. Recently, beta-adrenergic signaling has been reported to affect the development of aggressive prostate cancer by regulating neuroendocrine differentiation, angiogenesis, and metastasis. Here, we briefly summarize and discuss recent advances in these areas and their implications in prostate cancer therapeutics. We aim to provide a better understanding of the contribution of beta-adrenergic signaling to the progression of aggressive prostate cancer.

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.

A novel therapeutic anti-HBV antibody with increased binding to human FcRn improves in vivo PK in mice and monkeys

Antibody immunotherapy is a well-established therapeutic modality for cancer, acute viral infections （Marasco and Sui, 2007） and persistent viral infection such as HIV （Barouch et al., 2013） and HCMV （Freed et al., 2013）. To reduce immunogenicity of rodent antibodies （Abs）, approved anti-body drugs entering clinical trials are of human origin or are humanized versions of rodent antibodies （Reichert, 2008）.

Are endophytes an important link between airborne spores and allergen exposure?

Grasses represent one of the Earth’s most common plant groups,and natural and cultivated habitats dominated by grasses cover about 40%of the land surface.In turn,each grass species hosts multiple fungal species which can behave as endophytes.An analysis of the endophytic taxa identified in surveys conducted in 14 grass species showed that some of the most frequent taxa on each grass were also present across several host grasses.These taxa were Alternaria,Epicoccum,Cladosporium,Fusarium,and a few others.A similar analysis of airborne fungi surveyed at 41 different locations throughout the world showed that some of the most geographically widespread,and most locally frequent airborne fungi belonged to the same genera that are dominant endophytes in grasses(i.e.Cladosporium,Alternaria,Fusarium,etc.).Therefore,airborne spores of genera that are ubiquitous in grasses are common worldwide and attain high atmospheric concentrations.In addition,spores of the above mentioned fungi are also important respiratory allergens.Direct observation indicates that saprobic colonization and sporulation of non-systemic grass endophytes could become unrestrained when their host plant tissue dies.Subsequently,when appropriate environmental conditions favour sporulation on grass host surfaces,the natural cycle for airborne conidia initiates,and large numbers of these conidia disperse as inoculum for new endophytic infections.Therefore,the cycle of endophytism may be an important link between climate,plant biology and public health.

＂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.