Engineering Fully Human Monoclonal Antibodies from Murine Variable Regions

Fully human monoclonal antibodies (mAbs) derived from transgenic mice or human antibody libraries are the current state of the art for reducing the immunogenicity risk of antibody drugs. Here, we describe a novel method for generating fully human mAbs from nonhuman variable regions using information from the human germline repertoire. Central to our strategy is the rational engineering of residues within and proximal to CDRs and the V_H/V_L interface by iteratively exploring substitutions to the closest human germline sequences using semi-automated computational methods. Starting from the parent murine variable regions of three currently marketed mAbs targeting CD25, vascular endothelial growth factor, and tumor necrosis factor alpha, we have generated fully human antibodies with 59, 46, and 45 substitutions, respectively, compared to the parent murine sequences. A large number of these substitutions were in the CDRs, which are typically avoided in humanization methods. Antigen affinities of the fully human variants were comparable to the chimeric mAbs in each case. Furthermore, in vitro functional characterization indicated that all retain potency of the chimeric mAbs and have comparable activity to their respective marketed drugs daclizumab, bevacizumab, and infliximab. Based on local and global sequence identity, the sequences of our engineered mAbs are indistinguishable from those of fully human mAbs isolated from transgenic mice or human antibody libraries. This work establishes a simple rational engineering methodology for generating fully human antibody therapeutics from murine mAbs produced from standard hybridoma technology.     

Diminution of antibodies directed against tumor cell surface epitopes: a single chain Fv fusion molecule specifically recognizes the extracellular domain of the c-erbB-2 receptor.

We are evaluating strategies for the inhibition of growth or the selective killing of tumor cells. Cell surface antigens which are exclusively expressed or which are enhanced in their expression in tumor cells might provide the means to target cytotoxic or cytostatic agents to these cells. Few tumor specific cell surface antigens have been found, but the enhanced expression of growth factor receptors has been described for several types of tumors. A prominent example is the overexpression of the c-erbB-2 receptor in a high percentage of primary breast and ovarian carcinomas. We have derived monoclonal antibodies against the extracellular domain of the c-erbB-2 receptor. The antibody molecules were genetically engineered to minimize their size and to allow for their functional modification. For this purpose the cDNA sequences corresponding to the variable domains of one monoclonal antibody (FRP5) were molecularly cloned and joined by a short linker. The resulting single chain antibody molecule (scFv) was expressed in bacteria and purified. We show in an immunoprecipitation experiment that this molecule retains its ability to recognize the c-erbB-2 extracellular domain. This molecule could become a valuable vehicle to specifically transport anti-tumor agents to breast cancer cells.     

Recombinant IgE antibody engineering to target EGFR

Monoclonal antibodies have become a mainstay for the targeted treatment of cancer today. Some of the most successful targets of monoclonal antibodies are constituted by the epidermal growth factor receptor family spearheaded by the epidermal growth factor receptor (EGFR). Prompted by studies indicating that IgE compared to IgG may harness alternate effector functions to eradicate malignant cells, we addressed the establishment, engineering, and the potential tumoricidal effects of recombinant anti-EGFR IgE. Therefore, two different therapeutic EGFR-specific antibodies, 225 and 425, were chosen for re-cloning into different chimeric IgE and IgG formats and produced in human cells. Simultaneous antibody binding to the sEGFR demonstrated accessibility of both epitopes for recombinant IgE. Proliferation and cytotoxicity assays demonstrated signal blocking and effector mediating capability of IgE isotypes. Pronounced degranulation in the presence of sEGFR upon activation exclusively with two IgE antibodies verified the epitope proximity and provides evidence that tumor-targeting by anti-EGFR IgE is safe with regard to soluble target structures. Degranulation mediated by tumor cells expressing EGFR could be demonstrated for singular and combined IgE antibodies; however, use of two IgE specificities was not superior to use of one IgE alone. The data suggest that the surface distribution of EGFR is optimally suited to mount a robust effector cell trigger and corroborate the potential and specificity of the IgE/IgE receptor network to react to xenobiotic or pathogenic patterns for targeting malignancies.     

Protein Engineering of Antibodies

Abstract

This article reviews the technical advances in antibody engineering and the clinical applications of these molecules. Recombinant DNA technology facilitates the construction and expression of engineered antibodies. These novel molecules are designed to meet specific applications. Although genomic and cDNA cloning have been used widely in the past to isolate the relevant antibody V domains, at present, the PCR-based cloning is the preferred system. Bacterial and mammalian expression systems are used commonly for the production of antibodies, antibody fragments, and antibody fusion proteins. A range of chimeric antibodies with murine V domains joined to C regions from human and other species have been produced and found to exhibit the expected binding characteristics and effector functions. Humanized antibodies have been developed to minimize the HAMA response, and bifunctional immunoglobulins are being used in tumor therapy and diagnosis. Single chain antibodies and fusion proteins with antibody specificities joined to nonimmunoglobulin sequences provide a source of antibody-like molecules with novel properties. The potential applications of minimal recognition units and antigenized antibodies are described. Combinatorial libraries produced in bacteriophage present an alternative to hybridomas for the production of antibodies with the desired antigen binding specificities. Future developments in this field are discussed also.     

抗体药物的发展与应用

早期抗体药物是鼠源单克隆抗体,存在免疫原性强、半衰期短等问题。历经数十年的发展,抗体药物从最初的鼠源单抗,逐步发展为人鼠嵌合抗体、人源化抗体及全人源化抗体。通过片段重组、位点修饰、药物偶联等方法,科研人员研发了包括抗体融合蛋白、抗体偶联药物、双特异性抗体、小分子抗体片段等形式多样的抗体药物。抗体药物在恶性肿瘤、自身免疫病、感染性疾病的治疗上发挥重要作用。通过对抗体药物人源化历程,不同类型的抗体结构和特点,以及抗体药物在新型冠状病毒肺炎治疗中的应用进行综述,并对抗体药物的发展前景进行展望,以期为我国抗体药物的研发提供参考。     

Utilization of monoclonal antibody-targeted nanomaterials in the treatment of cancer

Due to their excellent specificity for a single epitope, monoclonal antibodies (mAbs) present a means of influencing the function of cells at the molecular level. In particular they show great promise in the treatment of cancer because they can inhibit cancer cell proliferation, tumor angiogenesis, invasiveness and malignant spread of cancerous cells. Many mAbs are in various stages of testing and 11 are currently marketed in the US or Europe for the treatment of cancers that express particular antigens such as human epidermal growth factor receptor-2, CD20, epidermal growth factor receptor and vascular endothelial growth factor. Strategies to conjugate mAbs to toxins, radioactive isotopes and chemotherapeutic drugs to improve efficacy are under intense investigation and numerous immunoconjugates have been studied in the clinical setting. However, the molecules have limitations, and so nanomaterials (NMs), which potentially offer more flexibility of design and functionality in providing platforms for binding of multiple therapeutic agents in a single structure, are being examined as an alternative. Studies utilizing mAb-targeted NMs have shown that they exhibit focused targeting, improved pharmacokinetics and improved “passive” drug delivery via leaky vasculature. Nevertheless, before they can be utilized to treat cancer, potential NM toxicity must be thoroughly investigated. Thus, rigorous testing of NM-mAb conjugates in both in vitro and in vivo systems is underway to determine how NM-mAb conjugates will interact with cells and tissues of the body. In this review, we discuss the broad range of nanomaterials that are under investigation as potential platforms for the presentation of mAbs either as single therapeutics or in combination with other drugs and their advantages and limitations in specifically targeting cancer.     

Protein engineering of antibodies.

This article reviews the technical advances in antibody engineering and the clinical applications of these molecules. Recombinant DNA technology facilitates the construction and expression of engineered antibodies. These novel molecules are designed to meet specific applications. Although genomic and cDNA cloning have been used widely in the past to isolate the relevant antibody V domains, at present, the PCR-based cloning is the preferred system. Bacterial and mammalian expression systems are used commonly for the production of antibodies, antibody fragments, and antibody fusion proteins. A range of chimeric antibodies with murine V domains joined to C regions from human and other species have been produced and found to exhibit the expected binding characteristics and effector functions. Humanized antibodies have been developed to minimize the HAMA response, and bifunctional immunoglobulins are being used in tumor therapy and diagnosis. Single chain antibodies and fusion proteins with antibody specificities jointed to nonimmunoglobulin sequences provide a source of antibody-like molecules with novel properties. The potential applications of minimal recognition units and antigenized antibodies are described. Combinatorial libraries produced in bacteriophage present an alternative to hybridomas for the production of antibodies with the desired antigen binding specificities. Future developments in this field are discussed also.     

High level expression of a functional human/mouse chimeric anti-CD20 monoclonal antibody in milk of transgenic mice

Rituximab, a chimeric anti-CD20 monoclonal antibody, is one of the most successful biomedicines and has been used to treat at least 370,000 patients with indolent, aggressive non-Hodgkin's lymphoma and other malignant diseases. However, the global demand for rituximab and other therapeutic monoclonal antibodies is exponentially increasing and barely able to be met by current manufacturing capacities of mammalian cell culture. The mammary gland bioreactor has been regarded as an ideal substitute for mammalian cell culture to mass-produce recombinant monoclonal antibodies at the lowest possible cost. Here, we show a feasible model to produce recombinant anti-CD20 antibodies in the mammary glands of transgenic animals. Six lines of transgenic mice were generated by co-microinjection of the two expression cassettes that can specially express the chimeric light and heavy chain of anti-CD20 mAbs in the milk of transgenic animals. The recombinant antibodies were detected in the milk of transgenic mice with the highest expression level up to 17 microg/mul and could specifically bind the CD20 surface antigens on human B-lymphoma cells.     

In vivo cervical cancer growth inhibition by genetically engineered cytotoxic T cells

Purpose: The CD44 v7/8 splice variant that is frequently expressed in cervical carcinoma and rarely expressed in normal tissues displays promising properties as a target antigen for cancer immune therapy. In this study, cytotoxic T lymphocytes (CTLs) were genetically engineered to gain CD44v7/8 target specificity. Methods: Clone 96 (CI96), an established murine cytotoxic T-cell line, and naïve murine T cells were retrovirally transduced with a fusion gene construct encoding for the single chain fragment scFv of the monoclonal antibody VFF17 and for the chain of the T-cell receptor (TCR). The therapeutic potential of genetically engineered T cells was tested in vitro and in vivo. Results: Surface expression of the chimeric TCR on infected Cl96 and naïve T cells was shown by FACS analysis. CD44v7/8-positive target cells were efficiently lysed by transduced Cl96 and naïve T cells, demonstrating the functionality and specificity of the chimeric TCR. In a xenograft BALB/c mouse model, efficient growth retardation of CD44v7/8-positive tumours was mediated by genetically engineered Cl96(VFF17)cyYZ cells. Conclusions: We were able to reprogramme the target specificity of recombinant Cl96 and naïve CTLs resulting in efficient cytolysis of CD44v7/8-positive cervical cancer cells. High transduction rates and the specific cytolysis of CD44v7/8-redirected CTLs are promising tools for an immune gene therapy approach for advanced cervical cancer.Abbreviations Ab Antibody - CTL Cytolytic T lymphocyte - mAb Monoclonal antibody - TCR T-cell receptor     

Effective Therapeutic Approach for Head and Neck Cancer by an Engineered Minibody Targeting the EGFR Receptor

Cetuximab, a chimeric monoclonal antibody developed for targeting the Epidermal Growth Factor Receptor (EGFR), has been intensively used to treat cancer patients with metastatic colorectal cancer and head and neck cancer. Intact immunoglobulin G (IgG) antibody like cetuximab, however, has some limitations such as high production cost and low penetration rate from vasculature into solid tumor mass due to its large size. In attempt to overcome these limitations, we engineered cetuximab to create single chain variable fragments (scFv-CH3; Minibody) that were expressed in bacterial system. Among three engineered minibodies, we found that MI061 minibody, which is composed of the variable heavy (V_H) and light (V_L) region joined by an 18-residue peptide linker, displays higher solubility and better extraction properties from bacterial lysate. In addition, we validated that purified MI061 significantly interferes ligand binding to EGFR and blocks EGFR''s phosphorylation. By using a protein microarray composed of 16,368 unique human proteins covering around 2,400 plasma membrane associated proteins such as receptors and channels, we also demonstrated that MI061 only recognizes the EGFR but not other proteins as compared with cetuximab. These results indicated that engineered MI061 retains both binding specificity and affinity of cetuximab for EGFR. Although it had relatively short half-life in serum, it was shown to be highly significant anti-tumor effect by inhibiting ERK pathway in A431 xenograft model. Taken together, our present study provides compelling evidence that engineered minibody is more effective and promising agent for in vivo targeting of solid tumors.     

Recombinant immunotoxins and retargeted killer cells: employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors

Over the past years, monoclonal antibodies have attracted enormous interest as targeted therapeutics, and a number of such reagents are in clinical use. However, responses could not be achieved in all patients with tumors expressing high levels of the respective target antigens, suggesting that other factors such as limited recruitment of endogenous immune effector mechanisms can also influence treatment outcome. This justifies the search for alternative, potentially more effective reagents. Antibody-toxins and cytolytic effector cells genetically modified to carry antibody-based receptors on the surface, represent such tailor-made targeting vehicles with the potential of improved tumor localization and enhanced efficacy. In this way, advances in recombinant antibody technology have made it possible to circumvent problems inherent in chemical coupling of antibodies and toxins, and have allowed construction via gene fusion of recombinant molecules which combine antibody-mediated recognition of tumor cells with specific delivery of potent protein toxins of bacterial or plant origin. Likewise, recombinant antibody fragments provide the basis for the construction of chimeric antigen receptors that, upon expression in cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells, link antibody-mediated recognition of tumor antigens with these effector cells potent cytolytic activities, thereby making them promising cellular therapeutics for adoptive cancer therapy. Here, general principles for the derivation of cytotoxic proteins and effector cells with antibody-dependent tumor specificity are summarized, and current strategies to employ these molecules and cells for directed cancer therapy are discussed, focusing mainly on the tumor-associated antigens epidermal growth factor receptor (EGFR) and the closely related ErbB2 (HER2) as targets.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Construction, expression, and biologic activity of murine/human chimeric antibodies with specificity for the human alpha/beta T cell receptor.

Murine/human chimeric antibodies with specificity for the human TCR-alpha/beta have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 were isolated and inserted into mammalian expression vectors containing the human kappa and gamma 1 or gamma 4 C region exons. The chimeric genes were transfected into murine Sp2/O hybridoma cells by electroporation and transfectomas secreting chimeric antibody were isolated. Secretion levels ranged from 1 to 7 pg/cell/24 h. The chimeric antibodies bound specifically to T cells and competed effectively with the parental murine mAb for binding to these sites. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced in the chimeric antibodies as compared with murine BMA 031. C-dependent cytolysis, however, was not detectable with any of the antibodies. Chimeric BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft-vs-host disease, autoimmune diseases and other T cell-related disorders.     

重组抗体药物研究进展及应用

重组抗体药物的发展经历了鼠源单克隆抗体（McAb）、人 鼠嵌合抗体、人源化抗体和全人抗体等阶段,目前初步应用于抗肿瘤、抗自身免疫病、抗感染等领域。保持和提高抗体的亲和力、降低抗体的免疫原性是抗体药物基因工程改造的两大原则。在嵌合抗体成功的基础上,通过CDR移植、表面修饰、抗体库以及转基因鼠技术,逐步提高人源化程度至100%。然而,实验室水平的研究结果与实际应用仍然存在一定差距。就重组抗体药物的基本概况、现存的问题与可能的解决办法以及在肿瘤、病毒性疾病和阿尔茨海默病治疗上的应用情况等进行了综述。     

Chimeric antigen receptor–engineered regulatory T lymphocytes: promise for immunotherapy of autoimmune disease

Regulatory T lymphocytes (Tregs) exist as natural ideal immunosuppressors in the immune system. Autologous or allogeneic Treg transfusion therapy has been carried out in animal models and humans as a new strategy for treating autoimmune disease. Recent studies have shown that Tregs can be engineered with chimeric antigen receptors to be antigen-specific, which are more effective than polyclonal Tregs with fewer target limitations and a lack of major histocompatibility complex restriction. This review describes the potential for applying chimeric antigen receptor–engineered regulatory T cells in autoimmune diseases.     

Preparation of a monoclonal antibody to a melanoma growth-stimulatory activity released into serum-free culture medium by Hs0294 malignant melanoma cells

Autostimulatory growth factors may contribute to the ability of malignant cells to escape normal growth controls. We have previously shown that Hs0294 human malignant melanoma cells release into culture medium an acid-soluble, heat-stable, trypsin-sensitive, autostimulatory monolayer mitogen which can be purified from acetic acid extracts of conditioned medium by gel filtration, reverse-phase high-performance liquid chromatography, and preparative electrophoresis. The majority of this melanoma growth-stimulatory activity (MGSA) resides in a 16-Kd moiety, though bioactivity is also associated with 24-26 and less than 14-Kd forms of MGSA (Richmond and Thomas: J Cell Physiol 129:375, 1986). In order to further characterize this growth factor, monoclonal antibodies were prepared against a partially purified preparation of the autostimulatory melanoma mitogen. Monoclonal antibody clones were selected based on supernate inhibition of 3H-thymidine incorporation in serum-free Hs0294 melanoma cultures. One of these, termed FB2AH7, slows, but does not completely block, the growth of Hs0294 cells in serum-free medium in a dose-dependent manner. This antibody does not slow the growth of normal rat kidney fibroblasts, which neither produce nor require this mitogen, in either serum-free medium or medium containing 0.8% calf serum. This monoclonal antibody also blocks the mitogenic effects of partially purified preparations of this melanoma growth stimulatory activity (MGSA) on both Hs0294 cells and normal rat kidney fibroblasts. The FB2AH7 antibody has been demonstrated to bind MGSA by Western blot and by immunoprecipitation procedures. Western blot analysis of reverse-phase high-performance liquid chromatography purified growth factor demonstrated that FB2AH7 antibody binds to the 16-Kd and approximately 13-14-Kd forms of MGSA. FB2AH7 antibody can be used in immunoprecipitation experiments to bind the approximately 13-16-Kd forms of MGSA. The specificity of the binding of FB2AH7 antibody for MGSA but not other growth factors has been demonstrated in a modified dot blot assay. These data thus support the hypothesis that MGSA is an autostimulatory melanoma mitogen distinct from other growth factors.     

Directed molecular evolution of antibodies

Antibodies play a key role in the immune system, are characterized by a homogeneous overall structure and by their ability to interact with an almost unlimited number of compounds. Encoded by a fixed number of genes, they acquire their specificity and affinity of recognition after a succession of genetic recombination and molecular mutation processes. Since the pioneer works of Kohler and Milstein in 1975 describing the possibility of producing monoclonal antibodies with pre-determined specificity, the use of antibodies in the fields of research, diagnosis and therapy has never stopped increasing. Thus, about twenty monoclonal antibodies have yet been authorized to be used in human immunotherapy. However, a majority of these molecules have been engineered to bring them into line with their clinical use: chimerization, humanization, recombinant expression of single or fused fragments. Furthermore, the recent development of in vitro molecular evolution approaches now make it possible to engineer the affinity, the specificity as well as the stability of monoclonal antibodies. The potential of in vitro molecular evolution of antibodies will be illustrated through the example of the specificity improvement of an anti-progesterone antibody.     

Construction,expression and characterization of the engineered antibody against tumor surface antigen,p185^c—erbB—2

The c-erbB-2 proto-oncogene encodes a 185kD protein p185,which belongs to epidermal growth factor receptor family.Amplification of this gene has been shown to correlate with poor clinical prognosis for certain cancer patients.The monoclonal antibody A21 which directed against p185 specifically inhibits proliferation of tumor cells overexpressing p185,hence allows it to be a candidate for targeted therapy.In order to overcome several drawbacks of murine MAb,we cloned its VH and VL genes and constructed the single-chain FV(scFv)through a peptide linker.The recombinant scFv A21 was expressed in Escherichia coli and purified by the affinity column.Subsequently it was characterized by ELISA,Western blot,cell immunohistochemistry and FACS.All these assays showed the binding activity to extracellular domain(ECD)of p185.Based on those properties of scFvA21,we further constructed the scFv-Fc fusion molecule with a homodimer form and the recombinant product was expressed in mammalian cells.In a series of subsequent analysis this fusion protein showed identical antigen binding site and activity with the parent antibody.These anti-p185 engineered antibodies have promised to be further modified as a tumor targeting drugs,with a view of application in the diagnosis and treatment of human breast cancer.     

上皮生长因子受体和癌胚抗原分子内血型Y配基表达的免疫生化分析

以抗CEA、EGF-γ和血型前体Y配基抗原的单抗进行分析,结果表明:结肠癌组织中表达CEA和血型前体Y配基较强,而EGF-γ表达水平极低;进一步鉴定分析,识别血型前体Y配基抗原决定簇的单抗C_(14)与肿瘤具有较好的反应性。并发现这种决定簇存在于各种生物大分子中,包括以糖蛋白和糖脂形式存在,并表达于EGF-γ与CEA分子中。这一结果提示血型前体抗原过量表达于肿瘤细胞中,不但是血型组织相容抗原,可能也参与到一些特殊生物分子中,应深入研究和鉴定其功能。     

Interactions between growth factor receptors and corresponding monoclonal antibodies in human tumors

Monoclonal antibodies (MAbs) to the human epidermal growth factor (EGF) receptor, the type I insulin-like growth factor (IGF) receptor, and the nerve growth factor (NGF) receptor were used to study the growth regulation of malignant cells. Anti-EGF receptor MAb 425 inhibited the growth of A 431 squamous carcinoma cells which express high numbers of EGF receptors on their surfaces. Growth inhibition induced by MAb 425 was accompanied by alterations of the cell-cycle distribution of these cells, indicating the ability of a monoclonal antibody to act as a biologically active ligand. Growth stimulation of melanoma cells by EGF was unrelated to EGF receptor expression on the cell surface. Insulin- and IGF-I-induced growth stimulation of melanoma cells was inhibited by MAb alpha IR-3 which reacts with the type I IGF receptor. This result indicates that the type I IGF receptor mediated growth stimulation not only by IGF-I but also by insulin. Normal melanocytes and cells of all stages of tumor progression expressed in tissue culture the receptor for NGF, but no effect on the growth of these cells has been observed.