重组抗体工程及其在肿瘤靶向及癌症治疗中的应用

在过去的十几年中,重组抗体工程在基础研究、医学和药物生产上已经成为最有希望的领域之一。重组抗体及其片段在正在进行诊断和治疗的临床试验中占所有生物蛋白的30％以上。研究集中在抗体作为理想的癌症靶向试剂方面,最近由于FDA批准使用第一个工程化治疗抗体而使热度达到极点。过去的几年中,在设计、筛选及生产新型工程化抗体方面已经取得了重大进展。改革的筛选方法已经能够分离出高亲和力的癌－靶向及抗病毒的抗体,后能够抑制病毒用于基因治疗。癌症诊断和治疗的另一个策略是将重组抗体片段与放射性同位素、药物、毒素、酶以及生物传感器表面进行融合。双特异性抗体及相关融合蛋白也已经生产出来用于癌症的免疫治疗,在抗癌疫苗以及T细胞补充策略上有效地增强了人免疫应答。     

Modifications of therapeutic proteins: challenges and prospects

The production of therapeutic proteins is one of the fastest growing sectors of the pharmaceutical industry. However, most proteins used in drug therapy require complex post-translational modifications for efficient secretion, drug efficacy and stability. Common protein modifications include variable glycosylation, misfolding and aggregation, oxidation of methionine, deamidation of asparagine and glutamine, and proteolysis. These modifications not only pose challenges for accurate and consistent bioprocessing, but also may have consequences for the patient in that incorrect modifications or aggregation may lead to an immune response to the protein therapeutic. This review provides examples of analytical and preventative advances that have been devised to meet these challenges, and insights into how further advances can improve the efficiency and safety in manufacturing recombinant proteins.     

Macromolecular immunosuppressants

Recombinant immunosuppressants have come of age and represent a significant class of quite diverse drugs. They target extracellular molecules, and either label or inhibit them. Those targets are soluble factors or membrane proteins almost all of which are components of a very complex molecular network of communication and amplification. Notably, many recombinant immunosuppressants have been developed in a rather short period of time. This is due to the fact that developing the actual drug is left to nature, so to speak; either it is a human protein as is the case for Anakinra or it is an antibody or antibody derivative "developed" by the immune system of mice or rats following exposure to the antigen. The challenge for developing recombinant immunosuppressants is to identify relevant targets. It is no longer very difficult to generate proteins to targets, once those are identified. The clinical use of recombinant immunosuppressants has yet to show which targets are truly relevant and which drugs prove effective.     

A murine monoclonal antibody produced in transgenic plants with plant-specific glycans is not immunogenic in mice

Previous studies have shown that the production of recombinant antibodies in plants is highly efficient and presents numerous therapeutic applications. It is, however, known that plant glycoproteins display different glycosylation patterns to those exhibited by mammalian glycoproteins. Thus, it is important to know if these plant recombinant antibodies could induce undesirable immune responses in mammal; and to date no report has documented the potential immunogenicity of parenterally administered plant recombinant antibodies in animals. In order to answer this question, mice were immunised subcutaneously with a recombinant mouse monoclonal antibody produced in tobacco plants, together with alum as adjuvant. Two control groups were immunised in the same way with either the original murine monoclonal antibody or horseradish peroxidase (a plant glycoprotein). Analyses by direct immunoassay, competition immunoassay and real-time surface plasmon resonance, showed undetectable levels of antibody directed against both the protein and the glycan part of the plant recombinant antibody. These results have a direct relevance for the application of plant recombinant proteins as therapeutic agents and vaccines in humans.     

Re-engineering biopharmaceuticals for delivery to brain with molecular Trojan horses

Biopharmaceuticals, including recombinant proteins, monoclonal antibody therapeutics, and antisense or RNA interference drugs, cannot be developed as drugs for the brain, because these large molecules do not cross the blood-brain barrier (BBB). Biopharmaceuticals must be re-engineered to cross the BBB, and this is possible with genetically engineered molecular Trojan horses. A molecular Trojan horse is an endogenous peptide, or peptidomimetic monoclonal antibody (mAb), which enters brain from blood via receptor-mediated transport on endogenous BBB transporters. Recombinant neurotrophins, single chain Fv antibodies, or therapeutic enzymes may be re-engineered as IgG fusion proteins. The engineering of IgG-avidin fusion proteins enables the BBB delivery of biotinylated drugs. The IgG fusion proteins are new chemical entities that are dual or triple function molecules that bind multiple receptors. The fusion proteins are able both to enter the brain, by binding an endogenous BBB receptor, and to induce the desired pharmacologic effect in brain, by binding target receptors in the brain behind the BBB. The development of molecular Trojan horses for BBB drug delivery allows the re-engineering of biopharmaceuticals that, owing to the BBB problem, could not otherwise be developed as new drugs for the human brain.     

Glycoprotein production in moss bioreactors

Complex multimeric recombinant proteins such as therapeutic antibodies require a eukaryotic expression system. Transgenic plants may serve as promising alternatives to the currently favored mammalian cell lines or hybridomas. In contrast to prokaryotic systems, posttranslational modifications of plant and human proteins resemble each other largely, among those, protein N-glycosylation of the complex type. However, a few plant-specific sugar residues may cause immune reactions in humans, representing an obstacle for the broad use of plant-based systems as biopharmaceutical production hosts. The moss Physcomitrella patens represents a flexible tissue-culture system for the contained production and secretion of recombinant biopharmaceuticals in photobioreactors. The recent synthesis of therapeutic proteins as a scFv antibody fragment or the large and heavily modified complement regulator factor H demonstrate the versatility of this expression system. A uniquely efficient gene targeting mechanism can be employed to precisely engineer the glycosylation machinery for recombinant products. In this way, P. patens lines with non-immunogenic optimized glycan structures were created. Therapeutic antibodies produced in these strains exhibited antibody-dependent cellular cytotoxicity superior to the same molecules synthesized in mammalian cell lines.     

Mesenchymal stem cells as carrier of the therapeutic agent in the gene therapy of blood disorders

Nonhematopoietic stem cells as a delivery platform of therapeutic useful genes have attracted widespread attention in recent years, owing to gained a long lifespan, easy separation, high proliferation, and high transfection capacity. Mesenchymal stem/stromal cells (MSCs) are the choice of the cells for gene and cell therapy due to high self-renewal capacity, high migration rate to the site of the tumor, and with immune suppressive and anti-inflammatory properties. Hence, it has a high potential of safety genetic modification of MSCs for antitumor gene expression and has paved the way for the clinical application of these cells to target the therapy of cancers and other diseases. The aim of gene therapy is targeted treatment of cancers and diseases through recovery, change, or enhancement cell performance to the sustained secretion of useful therapeutic proteins and induction expression of the functional gene in intended tissue. Recent developments in the vectors designing leading to the increase and durability of expression and improvement of the safety of the vectors that overcome a lot of problems, such as durability of expression and the host immune response. Nowadays, gene therapy approach is used by MSCs as a delivery vehicle in the preclinical and the clinical trials for the secretion of erythropoietin, recombinant antibodies, coagulation factors, cytokines, as well as angiogenic inhibitors in many blood disorders like anemia, hemophilia, and malignancies. In this study, we critically discuss the status of gene therapy by MSCs as a delivery vehicle for the treatment of blood disorders. Finally, the results of clinical trial studies are assessed, highlighting promising advantages of this emerging technology in the clinical setting.     

Optimisation of prime-boost immunization in mice using novel protein-based and recombinant vaccinia (tiantan)-based HBV vaccine

Background

A therapeutic vaccine for chronic hepatitis B virus (HBV) infection that enhances virus-specific cellular immune responses is urgently needed. The “prime–boost” regimen is a widely used vaccine strategy against many persistence infections. However, few reports have addressed this strategy applying for HBV therapeutic vaccine development.

Methodology/Principal Findings

To develop an effective HBV therapeutic vaccine, we constructed a recombinant vaccinia virus (Tiantan) containing the S+PreS1 fusion antigen (RVJSS1) combined with the HBV particle-like subunit vaccine HBVSS1 to explore the most effective prime–boost regimen against HBV. The immune responses to different prime–boost regimens were assessed in C57BL/C mice by ELISA, ELISpot assay and Intracellular cytokine staining analysis. Among the combinations tested, an HBV protein particle vaccine priming and recombinant vaccinia virus boosting strategy accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody) titres as well as the strongest multi-antigen (PreS1, and S)-specific cellular immune response. HBSS1 protein prime/RVJSS1 boost immunization was also generated more significant level of both CD4+ and CD8+ T cell responses for Th1 cytokines (TNF-α and IFN-γ).

Conclusions

The HBSS1 protein-vaccine prime plus RVJSS1 vector boost elicits specific antibody as well as CD4 and CD8 cells secreting Th1-like cytokines, and these immune responses may be important parameters for the future HBV therapeutic vaccines.     

SLPI and elafin: multifunctional antiproteases of the WFDC family

SLPI (secretory leucoprotease inhibitor) and elafin represent the archetypal members of the WFDC [WAP (whey acidic protein) four disulfide core] family of proteins, and were originally characterized as protease inhibitors but have since been shown to possess a wider repertoire of activities. These functions include antimicrobial and immunomodulatory properties, suggesting that these proteins may play key roles in the innate immune response, and indicate the potential to develop some of these proteins as novel therapeutics. Susceptibility to host and bacterial protease cleavage may, however, limit the efficacy of recombinant protein therapies in diseases with a high protease burden such as CF (cystic fibrosis) lung disease. To overcome this problem, further refinement of the native proteins will be required to provide effective treatment strategies.     

Optimization of protein–protein docking for predicting Fc–protein interactions

The antibody crystallizable fragment (Fc) is recognized by effector proteins as part of the immune system. Pathogens produce proteins that bind Fc in order to subvert or evade the immune response. The structural characterization of the determinants of Fc–protein association is essential to improve our understanding of the immune system at the molecular level and to develop new therapeutic agents. Furthermore, Fc‐binding peptides and proteins are frequently used to purify therapeutic antibodies. Although several structures of Fc–protein complexes are available, numerous others have not yet been determined. Protein–protein docking could be used to investigate Fc–protein complexes; however, improved approaches are necessary to efficiently model such cases. In this study, a docking‐based structural bioinformatics approach is developed for predicting the structures of Fc–protein complexes. Based on the available set of X‐ray structures of Fc–protein complexes, three regions of the Fc, loosely corresponding to three turns within the structure, were defined as containing the essential features for protein recognition and used as restraints to filter the initial docking search. Rescoring the filtered poses with an optimal scoring strategy provided a success rate of approximately 80% of the test cases examined within the top ranked 20 poses, compared to approximately 20% by the initial unrestrained docking. The developed docking protocol provides a significant improvement over the initial unrestrained docking and will be valuable for predicting the structures of currently undetermined Fc–protein complexes, as well as in the design of peptides and proteins that target Fc.     

美国、欧盟和中国生物技术药物的比较

按照相同表达系统表达的相同氨基酸序列的产品视为同种产品,而不同表达系统表达的相同氨基酸序列的产品视为不同产品的原则,归纳总结了美国、欧盟和中国已批准上市的生物技术药物。美国FDA批准的以基因工程产品、抗体工程产品和细胞工程产品为主要代表的生物技术药物共79种(18种为大肠杆菌表达,8种为酵母表达,53种为哺乳动物细胞培养生产),其中基因重组蛋白质药物为64种。欧盟批准了49种基因重组酶、激素或细胞因子,11种基因重组治疗性抗体和5种基因重组疫苗。在欧美60％-70％的产品由哺乳动物细胞表达。中国批准了27种生物技术药物。比较了美国、欧盟和中国生物制药的特点。     

Attenuated Recombinant Influenza A Virus Expressing HPV16 E6 and E7 as a Novel Therapeutic Vaccine Approach

Persistent infection with high-risk human papillomavirus (HPV) types, most often HPV16 and HPV18, causes all cervical and most anal cancers, and a subset of vulvar, vaginal, penile and oropharyngeal carcinomas. Two prophylactic virus-like particle (VLPs)-based vaccines, are available that protect against vaccine type-associated persistent infection and associated disease, yet have no therapeutic effect on existing lesions or infections. We have generated recombinant live-attenuated influenza A viruses expressing the HPV16 oncogenes E6 and E7 as experimental immunotherapeutic vaccine candidates. The influenza A virus life cycle lacks DNA intermediates as important safety feature. Different serotypes were generated to ensure efficient prime and boost immunizations. The immune response to vaccination in C57BL/6 mice was characterized by peptide ELISA and IFN-γ ELISpot, demonstrating induction of cell-mediated immunity to HPV16 E6 and E7 oncoproteins. Prophylactic and therapeutic vaccine efficacy was analyzed in the murine HPV16-positive TC-1 tumor challenge model. Subcutaneous (s.c.) prime and boost vaccinations of mice with recombinant influenza A serotypes H1N1 and H3N2, followed by challenge with TC-1 cells resulted in complete protection or significantly reduced tumor growth as compared to control animals. In a therapeutic setting, s.c. vaccination of mice with established TC-1 tumors decelerated tumor growth and significantly prolonged survival. Importantly, intralesional vaccine administration induced complete tumor regression in 25% of animals, and significantly reduced tumor growth in 50% of mice. These results suggest recombinant E6E7 influenza viruses as a promising new approach for the development of a therapeutic vaccine against HPV-induced disease.     

Recombinant viral vaccines for cancer

Cancer arises from 'self' in a series of steps that are all subject to immunoediting. Therefore, therapeutic cancer vaccines must stimulate an immune response against tumour antigens that have already evaded the body's immune defences. Vaccines presenting a tumour antigen in the context of obvious danger signals seem more likely to stimulate a response. This approach can be facilitated by genetic engineering using recombinant viral vectors expressing tumour antigens, cytokines, or both, from an immunogenic virus particle. We overview clinical attempts to use these agents for systemic immunisation and contrast the results with strategies employing direct intratumoural administration. We focus on the challenge of producing an effective response within the immune-suppressive tumour microenvironment, and discuss how the technology can overcome these obstacles.     

Selective induction of immune responses by cytokines coexpressed in recombinant fowlpox virus.

Avipoxviruses have recently been studied as potential vectors for the delivery of heterologous vaccine antigen. Because these viruses abortively infect mammalian cells yet still effectively present encoded foreign genes to the host immune system, they offer a safer but effective alternative to other live virus vectors. We have examined the effect of coexpressing the cytokine interleukin-6 or gamma interferon on immune responses to a recombinant fowlpox virus expressing influenza virus hemagglutinin. The encoded cytokine was expressed for prolonged periods in infected cell culture with little cytopathic effect due to the abortive nature of the infection. In mice, vector-expressed cytokine dramatically altered immune responses induced by the coexpressed hemagglutinin antigen. Expression of interleukin-6 augmented both primary systemic and mucosal antibody responses and primed for enhanced recall responses. In contrast, expression of gamma interferon markedly inhibited antibody responses without affecting the generation of cell-mediated immunity. The safety of these constructs was demonstrated in mice with severe combined immunodeficiency, and no side effects due to cytokine expression were observed. In summary, fowlpox virus vectors encoding cytokines represent a safe and effective vaccine strategy which may be used to selectively manipulate the immune response.     

Recombinant human alpha-1 proteinase inhibitor: towards therapeutic use

Summary. Human alpha-1-proteinase inhibitor is a well-characterized protease inhibitor with a wide spectrum of anti-protease activity. Its major physiological role is inhibition of neutrophil elastase in the lungs, and its deficiency is associated with progressive ultimately fatal emphysema. Currently in the US, only plasma-derived human alpha-1-proteinase inhibitor is available for augmentation therapy, which appears to be insufficient to meet the anticipated clinical demand. Moreover, despite effective viral clearance steps in the manufacturing process, the potential risk of contamination with new and unknown pathogens still exists. In response, multiple efforts to develop recombinant versions of human alpha-1-proteinase inhibitor, as an alternative to the plasma-derived protein, have been reported. Over the last two decades, various systems have been used to express the human gene for alpha-1-proteinase inhibitor. This paper reviews the recombinant versions of human alpha-1-proteinase inhibitor produced in various hosts, considers current major safety and efficacy issues regarding recombinant glycoproteins as potential therapeutics, and the factors that are impeding progress in this area¹. ¹ The opinions expressed in this paper reflect the authors’ personal views, based on published data and the information available from the public domains, and have no relation to the official statements, if any, held by the US FDA, National Institutes of Health, or the US Department of Health and Human Services. FDA official recommendations for plasma protein therapeutics and recombinant proteins regarding safety, purity, and potency of new drugs and biologics produced by recombinant DNA technology are referred below as the US FDA Guidances.     

An antigenic HIV-1 peptide sequence engineered into the surface structure of transferrin does not elicit an antibody response.

One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein such as human serum transferrin (HST). However, a potential drawback is that the HST may increase the immunoreactivity of the peptide, in the same way that carrier proteins can be used to generate highly immunogenic peptide hapten conjugates. In this study we have generated a recombinant HST carrier protein that contains a peptide substrate of HIV-1 protease (VSQNYPIVL). The protein retained native HST function, and the peptide was surface exposed since it was immunoreactive in native dot blots, and was cleaved by HIV-1 protease. Immunisation of rabbits with the recombinant protein elicited only a very poor anti-peptide immune response. In contrast, strong anti-peptide immune responses were raised against both the peptide alone, and a chemical conjugate of the peptide with HST. These data demonstrate that it is possible to attenuate the immune response normally directed against an immunogenic peptide sequence by engineering into a surface exposed loop of HST. These findings may have an important impact on the future design of peptide delivery systems.     

Identification of novel tumor antigens with patient-derived immune-selected antibodies

The identification of tumor antigens capable of eliciting an immune response in vivo may be an effective method to identify therapeutic cancer targets. We have developed a method to identify such antigens using frozen tumor-draining lymph node samples from breast cancer patients. Immune responses in tumor-draining lymph nodes were identified by immunostaining lymph node sections for B-cell markers (CD20&CD23) and Ki67 which revealed cell proliferation in germinal center zones. Antigen-dependent somatic hypermutation (SH) and clonal expansion (CE) were present in heavy chain variable (VH) domain cDNA clones obtained from these germinal centers, but not from Ki67 negative germinal centers. Recombinant VH single-domain antibodies were used to screen tumor proteins and affinity select potential tumor antigens. Neuroplastin (NPTN) was identified as a candidate breast tumor antigen using proteomic identification of affinity selected tumor proteins with a recombinant VH single chain antibody. NPTN was found to be highly expressed in approximately 20% of invasive breast carcinomas and 50% of breast carcinomas with distal metastasis using a breast cancer tissue array. Additionally, NPTN over-expression in a breast cancer cell line resulted in a significant increase in tumor growth and angiogenesis in vivo which was related to increased VEGF production in the transfected cells. These results validate NPTN as a tumor-associated antigen which could promote breast tumor growth and metastasis if aberrantly expressed. These studies also demonstrate that humoral immune responses in tumor-draining lymph nodes can provide antibody reagents useful in identifying tumor antigens with applications for biomarker screening, diagnostics and therapeutic interventions.     

Transient co-expression for fast and high-yield production of antibodies with human-like N-glycans in plants

Plant-based transient expression is potentially the most rapid and cost-efficient system for the production of recombinant pharmaceutical proteins, but safety concerns associated with plant-specific N -glycosylation have hampered its adoption as a commercial production system. In this article, we describe an approach based on the simultaneous transient co-expression of an antibody, a suppressor of silencing and a chimaeric human β1,4-galactosyltransferase targeted for optimal activity to the early secretory pathway in agroinfiltrated Nicotiana benthamiana leaves. This strategy allows fast and high-yield production of antibodies with human-like N -glycans and, more generally, provides solutions to many critical problems posed by the large-scale production of therapeutic and vaccinal proteins, specifically yield, volume and quality.     

Immunogenicity of Leishmania donovani iron superoxide dismutase B1 and peroxidoxin 4 in BALB/c mice: the contribution of Toll-like receptor agonists as adjuvant

In this study, we assessed the immune response of two Leishmania donovani recombinant proteins: iron superoxide dismutase B1 (SODB1) and peroxidoxin 4 (Pxn4) in BALB/c mice. Assessment of the immunogenicity of these proteins alone or combined with Toll-like receptor 9 (TLR-9) agonist (CpG ODN) or TLR-4 agonist (GLA-SE) showed that they elicit specific antibody as well as cytokine production in response to the respective antigen in vitro. The use of adjuvants augmented immunogenicity of these antigens and more importantly, skewed the immune response to a Th1-type. These results indicate that recombinant SODB1 and Pxn4 proteins are potential vaccine candidates when administered with appropriate adjuvants.