Generation of native bovine mAbs by phage display

Modeling of disease pathogenesis and immunity often is carried out in large animals that are natural targets for pathogens of human or economic relevance. Although murine mAbs are a valuable tool in identifying certain host/pathogen interactions, progress in comparative immunology would be enhanced by the use of mAbs isolated from the host species. Such antibodies would reflect an authentic host immune response to infection or vaccination, and as they are host derived, would allow the application of in vivo experiments that previously have been unrealizable in large animals because of induction of an antispecies immune response. The advent of antibody phage display technology provides a way of producing host-derived mAbs in animals where the molecular genetics of Ig formation are known. Exploiting recent advances in the molecular immunology of cattle, we report here the design of an optimized phage display vector, pComBov, for the construction of combinatorial libraries of bovine Ig antigen-binding fragments (Fab) of native sequence. By using this system, we initially have generated and characterized a panel of bovine mAbs against a model antigen glutathione S-transferase. The isolated mAbs showed features typical of bovine Igs and recognized glutathione S-transferase with high specificity in ELISA and by Western blotting. The pComBov expression system can be readily adapted for the preparation of libraries from related ruminant species and advances the use of monoclonal reagents derived in this way for comparative studies in animals of economic importance.     

Phage Display Technology as a Powerful Platform for Antibody Drug Discovery

Antibody drugs with a high affinity and specificity are effective and safe for intractable diseases, such as cancers and autoimmune diseases. Furthermore, they have played a central role in drug discovery, currently accounting for eight of the top 20 pharmaceutical products worldwide by sales. Forty years ago, clinical trials on antibody drugs that were thought to be a magic bullet failed, partly due to the immunogenicity of monoclonal antibodies produced in mice. The recent breakthrough in antibody drugs is largely because of the contribution of phage display technology. Here, we reviewed the importance of phage display technology as a powerful platform for antibody drug discovery from various perspectives, such as the development of human monoclonal antibodies, affinity enhancement of monoclonal antibodies, and the identification of therapeutic targets for antibody drugs.     

Circumventing tolerance to generate autologous monoclonal antibodies to the prion protein.

Prion diseases are disorders of protein conformation and do not provoke an immune response. Raising antibodies to the prion protein (PrP) has been difficult due to conservation of the PrP sequence and to inhibitory activity of alpha-PrP antibodies toward lymphocytes. To circumvent these problems, we immunized mice in which the PrP gene was ablated (Prnp 0/0) and retrieved specific monoclonal antibodies (mAbs) through phage display libraries. This approach yielded alpha-PrP mAbs that recognize mouse PrP. Studies with these mAbs suggest that cellular PrP adopts an unusually open structure consistent with the conformational plasticity of this protein.     

Phage display of a catalytic antibody to optimize affinity for transition-state analog binding

Catalytic antibodies have shown great promise for catalyzing a tremendously diverse set of natural and unnatural chemical transformations. However, few catalytic antibodies have efficiencies that approach those of natural enzymes. In principle, random mutagenesis procedures such as phage display could be used to improve the catalytic activities of existing antibodies; however, these studies have been hampered by difficulties in the recombinant expression of antibodies. Here, we have grafted the antigen binding loops from a murine-derived catalytic antibody, 17E8, onto a human antibody framework in an effort to overcome difficulties associated with recombinant expression and phage display of this antibody. “Humanized” 17E8 retained similar catalytic and hapten binding properties as the murine antibody while levels of functional Fab displayed on phage were 200-fold higher than for a murine variable region/human constant region chimeric Fab. This construct was used to prepare combinatorial libraries. Affinity panning of these resulted in the selection of variants with 2- to 8-fold improvements in binding affinity for a phosphonate transition-state analog. Surprisingly, none of the affinity-matured variants was more catalytically active than the parent antibody and some were significantly less active. By contrast, a weaker binding variant was identified with 2-fold greater catalytic activity and incorporation of a single substitution (Tyr-100a_H → Asn) from this variant into the parent antibody led to a 5-fold increase in catalytic efficiency. Thus, phage display methods can be readily used to optimize binding of catalytic antibodies to transition-state analogs, and when used in conjunction with limited screening for catalysis can identify variants with higher catalytic efficiencies.     

Humanized antibodies for antiviral therapy.

Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.     

Isolation of human monoclonal antibodies by mammalian cell display

Due to their low immunogenicity in patients, humanized or fully human mAbs are becoming increasingly important for the treatment of a growing number of diseases, including cancer, infections, and immune disorders. Here, we describe a technology allowing for the rapid isolation of fully human mAbs. In contrast to previously described methods, B cells specific for an antigen of interest are directly isolated from peripheral blood mononuclear cells (PBMC) of human donors. Recombinant, antigen-specific single-chain Fv (scFv) libraries are generated from this pool of B cells and screened by mammalian cell surface display by using a Sindbis virus expression system. This method allows isolating antigen-specific antibodies by a single round of FACS. The variable regions (VRs) of the heavy chains (HCs) and light chains (LCs) are isolated from positive clones and recombinant fully human antibodies produced as whole IgG or Fab fragments. In this manner, several hypermutated high-affinity antibodies binding the Qβ virus like particle (VLP), a model viral antigen, as well as antibodies specific for nicotine were isolated. All antibodies showed high expression levels in cell culture. The human nicotine-specific mAbs were validated preclinically in a mouse model. Thus, the technology presented here allows for rapid isolation of high-affinity, fully human antibodies with therapeutic potential from human volunteers.     

High affinity mouse-human chimeric Fab against hepatitis B surface antigen

AIM: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. METHODS: We cloned the VH and VL genes of this mouse antibody, and fused them with CH1 domain of human IgG1 and CL domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. coli. RESULTS: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. This chimeric antibody fragment was further expressed in different strains of E. coli to increase the yield. CONCLUSION: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a full-length chimeric antibody for therapeutic uses.     

Bispecific antibody based therapeutics: Strengths and challenges

Monoclonal antibody-based targeted therapy has greatly improved treatment options for patients. However, long-term efficacy of such antibodies is limited by resistance mechanisms. New insights into the mechanisms by which tumors evade immune control have driven innovative therapeutic strategies to eliminate cancer by re-directing immune cells to tumors. Advances in protein engineering technology have generated multiple bispecific antibody (BsAb) formats capable of targeting multiple antigens as a single agent. Approval of two BsAb and three check point blocking mAbs represent a paradigm shift in the use of antibody constructs. Since BsAbs can directly target immune cells to tumors, drug resistance and severe adverse effects are much reduced. The wave of next generation “bispecific or multispecific antibodies” has advanced multiple candidates into ongoing clinical trials. In this review, we focus on preclinical and clinical studies in hematological malignancies as well as discuss reasons for the limited success of BsAbs against solid tumors.     

Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

AIM:To construct the natural immune Fab antibody phagedisplay libraries of colorectal cancer and to select antibodiesrelated with colorectal cancer.METHODS:Extract total RNA from tissue of local cancermetastasis lymph nodes of petients with colorectal cancer.RT-PCR was used to amplify the heavy chain Fd and lightchain k and the amplification products were insertedsuccessively into the vector pComb3 to construct the humanlibraries of Fab antibodies.They were then panned by phagedisplay technology.By means of Dot immunoblotting andEUSA,the libradse were identified and the Fab phageantibodies binding with antigens of colorectal cancer wereselected.RWSULTS:The amplified fragments of Fd and k gained byRT-PCR were about 650bp.Fd and k PCR products weresubsequently inserted into the vector pComb3,resulting in arecombination rate of 40% and the volume of Fab phagedisplay library reached 1.48×10~6.The libraries wereenriched about 120-fold by 3 cycles of adsorption-elution-multiplication(penning).Dot immunoblotting showed Fabexpressions on the phage libraries and ELISA showed 5clones of Fab phage antibodies which had binding activitieswith antigens of colomctal cancer.CONCLUSION:The nstuml immune Fab antibody phagedisplay libraries of colorectal cancer were constructed.Theycould he used to select the relstive antibodies of colorectalcancer.     

人源性抗-HBc单链噬菌体抗体库的构建

目的 构建人源性单链噬菌体抗体库，为筛选人源性抗—HBc单链抗体奠定基础。方法 利用逆转录—聚合酶链反应(RT—PCR)和噬菌体表面展示技术，直接从乙肝病毒核心抗体(抗—HBc)阳性患者淋巴细胞中提取总RNA，逆转录成cDNA；合成全套人抗体可变区引物扩增抗体可变区基因，并将重、轻链可变区基因进行拼接装配成单链抗体(ScFv)基因，重组于噬菌粒载体叶pHEN1，转化抑制型大肠埃希菌E.coliTG1，以辅助噬菌体援救后，构建成人源性单链噬菌体库。结果 成功地构建了人源性抗—HBc单链噬菌体库，库容量达10^6。结论 利用RT—PCR和噬菌体表面展示技术可以成功构建人源性单链抗体库，并达到建库标准，可进一步从中筛选人源性单链抗体。     

Therapeutic applications of monoclonal antibodies

Researchers have sought therapeutic applications for monoclonal antibodies since their development in 1975. However, murine-derived monoclonal antibodies may cause an immunogenic response in human patients, reducing their therapeutic efficacy. Chimeric and humanized antibodies have been developed that are less likely to provoke an immune reaction in human patients than are murine-derived antibodies. Antibody fragments, bispecific antibodies, and antibodies produced through the use of phage display systems and genetically modified plants and animals may aid researchers in developing new uses for monoclonal antibodies in the treatment of disease. Monoclonal antibodies may have a number of promising potential therapeutic applications in the treatment of asthma, autoimmune diseases, cancer, poisoning, septicemia, substance abuse, viral infections, and other diseases.     

Phage-display library selection of high-affinity human single-chain antibodies to tumor-associated carbohydrate antigens sialyl Lewisx and Lewisx

mAbs against tumor-associated carbohydrate antigens have the potential to play a prominent role in cancer immunotherapy. However, it has not been possible to fully exploit the clinical utility of such antibodies primarily, because those of adequate affinity could be derived only from murine sources. To address this problem, we prepared a single-chain Fv (scFv) antibody library from the peripheral blood lymphocytes of 20 patients with various cancer diseases. Completely human high-affinity scFv antibodies were then selected by using synthetic sialyl Lewisx and Lewisx BSA conjugates. These human scFv antibodies were specific for sialyl Lewisx and Lewisx, as demonstrated by ELISA, BIAcore, and flow cytometry binding to the cell surface of pancreatic adenocarcinoma cells. Nucleotide sequencing revealed that at least four unique scFv genes were obtained. The Kd values ranged from 1.1 to 6.2 x 10(-7) M that were comparable to the affinities of mAbs derived from the secondary immune response. These antibodies could be valuable reagents for probing the structure and function of carbohydrate antigens and in the treatment of human tumor diseases.     

ORFeome Phage Display Reveals a Major Immunogenic Epitope on the S2 Subdomain of SARS-CoV-2 Spike Protein

The development of antibody therapies against SARS-CoV-2 remains a challenging task during the ongoing COVID-19 pandemic. All approved therapeutic antibodies are directed against the receptor binding domain (RBD) of the spike, and therefore lose neutralization efficacy against emerging SARS-CoV-2 variants, which frequently mutate in the RBD region. Previously, phage display has been used to identify epitopes of antibody responses against several diseases. Such epitopes have been applied to design vaccines or neutralize antibodies. Here, we constructed an ORFeome phage display library for the SARS-CoV-2 genome. Open reading frames (ORFs) representing the SARS-CoV-2 genome were displayed on the surface of phage particles in order to identify enriched immunogenic epitopes from COVID-19 patients. Library quality was assessed by both NGS and epitope mapping of a monoclonal antibody with a known binding site. The most prominent epitope captured represented parts of the fusion peptide (FP) of the spike. It is associated with the cell entry mechanism of SARS-CoV-2 into the host cell; the serine protease TMPRSS2 cleaves the spike within this sequence. Blocking this mechanism could be a potential target for non-RBD binding therapeutic anti-SARS-CoV-2 antibodies. As mutations within the FP amino acid sequence have been rather rare among SARS-CoV-2 variants so far, this may provide an advantage in the fight against future virus variants.     

Monoclonal Antibodies as Therapeutic Agents: Advances and Challenges

Despite the major advances in conventional forms of treatment (i.e. surgical techniques, radiotherapy and chemotherapy) and improved survival rates, cancer is still the second leading cause of death in developing countries. One major limitation of cytotoxic drugs and radiation in the treatment of cancer patients is their inability to discriminate between malignant and normal tissues. This in turn prevents the delivery of the optimal (therapeutic) dose of such agents to malignant tissues for their eradication. With the advent of hybridoma technology in 1975, it has been possible for the first time to produce large amounts of an antibody (i.e. monoclonal antibody) against any antigens of interest. Since each antibody is highly specific for a particular antigen, this typical feature of the antibodies has resulted in their widespread use in diagnostic kits, medical research (e.g. to unravel the function of the antigen in physiological and pathological conditions), and more recently, for the management of a wide range of human diseases such as autoimmune disease and human cancers. Thanks to recent advances in genetic engineering, the immunogenicity of rodent antibodies was reduced by producing the chimeric or humanized version of such antibodies or by developing the fully human antibodies. In other instances, as intact antibodies are too large for rapid penetration into solid tumours, it has been possible to develop a smaller fragment of such antibodies (e.g. Fab, scFv, VHH) with greater potential for use in cancer imaging and therapy. Depending on the target antigens and the antibody format, monoclonal antibodies can induce their anti-tumour activities by several mechanisms including activation of the host effector cells. To date, several mAbs have been approved for management of human cancers including: anti-EGFR antibody cetuximab and anti-VEGF antibody bevacizumab for treatment of metastatic colorectal cancer, anti-HER-2 antibody trastuzumab for metastatic breast cancer, anti-CD20 antibodies rituximab and ibritumomab tituxetan for non-Hodgkin lymphoma, anti- CD52 antibody alemeutumab for chronic lymphocytic leukaemia, and anti-CD33 antibody gemutuzumab ozogamicin for the treatment of acute myeloid leukaemia patients. Monoclonal antibodies currently account for about 30% of all new drugs in development, with more than 500 antibodies at different stages of clinical trials worldwide. In this review, the characteristic features of some of the therapeutic antibodies and the antigens recognised by such antibodies will be discussed as well as several challenges that need to be addressed in order to facilitate their widespread use as “magic bullets” in the management of human diseases and in particular human cancers.     

Chimeric antibodies with extended half‐life in ferrets

Background

Ferrets have long been used as a disease model for the study of influenza vaccines, but a more recent use has been for the study of human monoclonal antibodies directed against influenza viruses. Published data suggest that human antibodies are cleared unusually quickly from the ferret and that immune responses may be partially responsible. This immunogenicity increases variability within groups and may present an obstacle to long-term studies.

Objective

Our aim was to identify an antibody design with reduced immunogenicity and longer circulating half-life in ferrets.

Methods

The constant region coding sequences for ferret immunoglobulin G were cloned, and chimeric human/ferret antibodies were expressed and purified. Some of the chimeric antibodies included substitutions that have been shown to extend the half-life of human IgG antibodies. These chimeric antibodies were tested for binding to recombinant ferret FcRn receptor and then evaluated in pharmacokinetic studies in ferrets.

Results

A one-residue substitution in the ferret Fc domain, S252Y, was identified that increased binding affinity to the ferret neonatal receptor by 24-fold and extended half-life from 65 ± 27 to 206 ± 28 hours or ∼9 days. Ferrets dosed twice with this surrogate antibody showed no indications of an immune response.

Conclusion

Expressing the variable region of a candidate human therapeutic antibody with ferret constant regions containing the S252Y substitution can offer long half-life and limit immunogenicity.     

Oligopeptide M13 Phage Display in Pathogen Research

Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline.     

Utilization of monoclonal antibodies in the treatment of leukemia and lymphoma

The generation of murine monoclonal antibodies reactive with human leukemia and lymphoma cells has recently led to clinical trials that have begun to evaluate the use of these reagents in the treatment of various leukemias and lymphomas. Several of these studies have demonstrated that infusion of monoclonal antibody can cause the rapid and specific clearance of leukemic cells from the peripheral blood. Intravenously administered antibody also rapidly binds to bone marrow lymphoblasts, and in one instance, has resulted in the partial regression of tumor cell infiltrates in lymph nodes and skin. Unfortunately, clinically significant responses have not in general been achieved, but these clinical studies have identified specific factors that result in the development of resistance to antibody- mediated lysis in vivo. These factors include the presence of circulating antigen, antigenic modulation, reactivity of monoclonal antibody with normal cells, immune response to murine antibody, and the inefficiency of natural immune effector mechanisms. Current research is now being directed towards developing methods to circumvent each of these obstacles. Future clinical studies utilizing antibodies in vitro or with different specificity may demonstrate greater therapeutic efficacy. In addition, monoclonal antibodies can be used as carriers of other cytotoxic agents and in conjunction with other agents that will reduce the total load. Monoclonal antibodies represent new and powerful reagents that may in the near future become an additional therapeutic modality for patients with malignant disease.     

Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody

The botulinum neurotoxins (BoNTs) cause the paralytic human disease botulism and are one of the highest-risk threat agents for bioterrorism. To generate a pharmaceutical to prevent or treat botulism, monoclonal antibodies (mAbs) were generated by phage display and evaluated for neutralization of BoNT serotype A (BoNT/A) in vivo. Although no single mAb significantly neutralized toxin, a combination of three mAbs (oligoclonal Ab) neutralized 450,000 50% lethal doses of BoNT/A, a potency 90 times greater than human hyperimmune globulin. The potency of oligoclonal Ab was primarily due to a large increase in functional Ab binding affinity. The results indicate that the potency of the polyclonal humoral immune response can be deconvoluted to a few mAbs binding nonoverlapping epitopes, providing a route to drugs for preventing and treating botulism and diseases caused by other pathogens and biologic threat agents.     

人源性抗结核分枝杆菌噬菌体展示单链抗体文库的构建

目的 构建人源抗结核分枝杆菌噬菌体展示单链抗体文库,为结核特异性单链抗体的筛选奠定基础. 方法 从抗结核分枝杆菌抗体阳性患者淋巴细胞中提取总RNA,逆转录成cDNA,PCR扩增获得抗体的重、轻链可变区基因,然后拼接装配成单链抗体基因,重组于噬菌粒载体pCANTAB5S,转化大肠埃希菌E.coli TG1,以辅助噬菌体拯救后,构建人源性抗结核分枝杆菌噬菌体展示单链抗体库. 结果 成功构建人源性抗结核分枝杆菌噬菌体展示单链抗体文库,库容量达107. 结论 以结核患者淋巴细胞免疫球蛋白可变区基因片段和噬菌粒展示载体pCANTAB5S为基础,成功构建人源性抗结核分枝杆菌噬菌体展示单链抗体库,并达到建库标准,可进一步从中筛选获得结核特异性单链抗体.     

Production and characterization of human monoclonal anti-idiotype antibodies to anti-dsDNA antibodies

Anti-dsDNA autoantibodies are the hallmark of systemic lupus erythematosus (SLE) and frequently correlate with disease activity. In this study we report the isolation and characterization of human anti-Id monoclonal antibody fragments as single-chain Fv fragments (scFv) against anti-dsDNA antibody. The anti-Id monoclonal antibodies, specific for anti-dsDNA antibodies, have been cloned from phage display antibody scFv libraries derived from a patient with SLE. The V gene repertoires were derived from the RNA obtained from the B cells of an SLE patient with anti-Ro/SSA and anti-La/SSB antibodies. Affinity-purified anti-dsDNA antibodies were used for selection of bacterial clones producing specific scFv antibody fragments against anti-dsDNA antibodies and little reactivity with normal IgG and other IgG antibodies by ELISA. The anti-Id antibody recognizes a public idiotope that is broadly cross-reactive with polyclonal and monoclonal anti-dsDNA antibodies. This binding was largely inhibited by dsDNA antigen. The anti-Id antibody inhibited anti-dsDNA binding to dsDNA antigen in immunoassays and in the Crithidia luciliae assay. The anti-Id scFv antibody fragments derived from human genes could modulate the pathogenicity of anti-dsDNA autoantibodies and may have therapeutic implications in SLE. They may also be used as probes in studies of the structure of the idiotype.