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1.
《MABS-AUSTIN》2013,5(3):256-265
Monoclonal antibodies represent an attractive therapeutic tool as they are highly specific for their targets, convey effector functions and enjoy robust manufacturing procedures. Humanization of murine monoclonal antibodies has vastly improved their in vivo tolerability. Humanization, the replacement of mouse constant regions and V framework regions for human sequences, results in a significantly less immunogenic product. However, some humanized and even fully human sequence-derived antibody molecules still carry immunological risk. To more fully understand the immunologic potential of humanized and human antibodies, we analyzed CD4+ helper T cell epitopes in a set of eight humanized antibodies. The antibodies studied represented a number of different VH and VL family members carrying unique CDR regions. In spite of these differences, CD4+ T cell epitopes were found only in CDR-sequence containing regions. We were able to incorporate up to two amino acid modifications in a single epitope that reduced the immunogenic potential while retaining full biologic function. We propose that immunogenicity will always be present in some antibody molecules due to the nature of the antigen-specific combining sites. A consequence of this result is modifications to reduce immunogenicity will be centered on the affinity-determining regions. Modifications to CDR regions can be designed that reduce the immunogenic potential while maintaining the bioactivity of the antibody molecule.  相似文献   

2.
We previously constructed a humanized antibody, HuS10, by grafting the complementarity-determining regions (CDRs) of a parental murine monoclonal antibody into the homologous human antibody sequences. This process is termed CDR grafting. Some residues that were thought to affect the CDR loops and stabilize the structure of the variable regions were retained in the framework region. HuS10 exhibited in vivo virus-neutralizing activity, but its murine content had the potential to elicit immune responses in patients. In this study, to minimize the immunogenic potential of HuS10, we replaced 17 mouse residues in HuS10 with the comparable human residues using specificity-determining residue (SDR)-grafting and de-immunization methods. The resultant humanized antibody, HzS-III, had the same affinity and epitope specificity as HuS10 and had reduced immunogenic potential, as assessed by T-cell epitope analysis. Thus, SDR grafting in combination with de-immunization may be a useful strategy for minimizing the immunogenicity of humanized antibodies. In addition, HzS-III may be a good candidate for immunoprophylaxis of HBV infection.  相似文献   

3.
More than twenty recombinant monoclonal antibodies are approved as therapeutics. Almost all of these are based on the whole IgG isotype format, but vary in the origin of the variable regions between mouse (chimeric), humanized mouse and fully human sequences; all of those with whole IgG format employ human constant region sequences. Currently, the opposing merits of the four IgG subclasses are considered with respect to the in vivo biological activities considered to be appropriate to the disease indication being treated. Human heavy chain genes also exhibit extensive structural polymorphism(s) and, being closely linked, are inherited as a haplotype. Polymorphisms (allotypes) within the IgG isotype were originally discovered and described using serological reagents derived from humans; demonstrating that allotypic variants can be immunogenic and provoke antibody responses as a result of allo-immunization. The serologically defined allotypes differ widely within and between population groups; therefore, a mAb of a given allotype will, inevitably, be delivered to a cohort of patients homozygous for the alternative allotype. This publication reviews the serologically defined human IgG allotypes and considers the potential for allotype differences to contribute to or potentiate immunogenicity.Key words: human IgG, polymorphisms, IgG allotypes, antibody therapeutics, immunogenicity, anti-therapeutic antibody, IgG glycosylation  相似文献   

4.
Immunogenicity of engineered antibodies   总被引:12,自引:0,他引:12  
Administration of a therapeutic antibody can lead to an anti-antibody response (AAR). Much effort has been applied to engineering antibodies with as little as possible non-human structure to minimize such responses. Here, we review reported AAR to murine, mouse-human chimeric, and humanized antibodies. Replacement of mouse immunoglobulin constant regions with human ones effects the largest immunogenicity reduction. Humanization of variable domains effects a further decrease.  相似文献   

5.
Shahab U  Ahmad S  Moinuddin  Dixit K  Habib S  Alam K  Ali A 《PloS one》2012,7(2):e31199

Background

The oxidation of proteins by endogenously generated free radicals causes structural modifications in the molecules that lead to generation of neo-antigenic epitopes that have implications in various autoimmune disorders, including rheumatoid arthritis (RA). Collagen induced arthritis (CIA) in rodents (rats and mice) is an accepted experimental model for RA.

Methodology/Principal Findings

Hydroxyl radicals were generated by the Fenton reaction. Collagen type II (CII) was modified by OH radical (CII-OH) and analysed by ultraviolet-visible (UV-VIS), fluorescence and circular dichroism (CD) spectroscopy. The immunogenicity of native and modified CII was checked in female Lewis rats and specificity of the induced antibodies was ascertained by enzyme linked immunosorbent assay (ELISA). The extent of CIA was evaluated by visual inspection. We also estimated the oxidative and inflammatory markers in the sera of immunized rats. A slight change in the triple helical structure of CII as well as fragmentation was observed after hydroxyl radical modification. The modified CII was found to be highly arthritogenic and immunogenic as compared to the native form. The CII-OH immunized rats exhibited increased oxidative stress and inflammation as compared to the CII immunized rats in the control group.

Conclusions/Significance

Neo-antigenic epitopes were generated on OH modified CII which rendered it highly immunogenic and arthritogenic as compared to the unmodified form. Since the rodent CIA model shares many features with human RA, these results illuminate the role of free radicals in human RA.  相似文献   

6.
Secukinumab is a human monoclonal antibody that selectively targets interleukin-17A and has been demonstrated to be highly efficacious in the treatment of moderate to severe plaque psoriasis, starting at early time points, with a sustained effect and a favorable safety profile. Biotherapeutics—including monoclonal antibodies (mAbs)—can be immunogenic, leading to formation of anti-drug antibodies (ADAs) that can result in unwanted effects, including hypersensitivity reactions or compromised therapeutic efficacy. To gain insight into possible explanations for the clinically observed low immunogenicity of secukinumab, we evaluated its immunogenicity potential by applying 2 different in vitro assays: T-cell activation and major histocompatibility complex–associated peptide proteomics (MAPPs). For both assays, monocyte-derived dendritic cells (DCs) from healthy donors were exposed in vitro to biotherapeutic proteins. DCs naturally process proteins and present the derived peptides in the context of human leukocyte antigen (HLA)-class II. HLA-DR–associated biotherapeutic-derived peptides, representing potential T–cell epitopes, were identified in the MAPPs assay. In the T-cell assay, autologous CD4+ T cells were co-cultured with secukinumab-exposed DCs and T-cell activation was measured by proliferation and interleukin-2 secretion. In the MAPPs analysis and T-cell activation assays, secukinumab consistently showed relatively low numbers of potential T-cell epitopes and low T-cell response rates, respectively, comparable to other biotherapeutics with known low clinical immunogenicity. In contrast, biotherapeutics with elevated clinical immunogenicity rates showed increased numbers of potential T-cell epitopes and increased T-cell response rates in T-cell activation assays, indicating an approximate correlation between in vitro assay results and clinical immunogenicity incidence.  相似文献   

7.
Hou S  Li B  Wang L  Qian W  Zhang D  Hong X  Wang H  Guo Y 《Journal of biochemistry》2008,144(1):115-120
4C8 is a new mouse anti-human CD34 monoclonal antibody (mAb), which recognizes class II CD34 epitopes and can be used for clinical hematopoietic stem/progenitor cell selection. In an attempt to improve its safety profiles, we have developed a humanized antibody of 4C8 by complementarity-determining region (CDR) grafting method in this study. Using a molecular model of 4C8 built by computer-assisted homology modelling, framework region (FR) residues of potential importance to the antigen binding were identified. A humanized version of 4C8, denoted as h4C8, was generated by transferring these key murine FR residues onto a human antibody framework that was selected based on homology to the mouse antibody framework, together with the mouse CDR residues. The resultant humanized antibody was shown to possess antigen-binding affinity and specificity similar to that of the original murine antibody, suggesting that it might be an alternative to mouse anti-CD34 antibodies routinely used clinically.  相似文献   

8.
《Cytotherapy》2022,24(4):393-404
Background aimsGiven their low immunogenicity, immunoregulatory effects and multiple differentiation capacity, mesenchymal stromal cells (MSCs) have the potential to be used for “off-the-shelf” cell therapy to treat various diseases. However, the allorejection of MSCs indicates that they are not fully immune-privileged. In this study, the authors investigated the immunogenicity of human adipose-derived MSCs (Ad-MSCs) and identified potential immunogenic molecules.MethodsTo evaluate the immunogenicity of human Ad-MSCs in vivo, cells were transplanted into humanized mice (hu-mice), then T-cell infiltration and clearance of human Ad-MSCs were observed by immunofluorescence and bioluminescence imaging. One-way mixed lymphocyte reaction and flow cytometry were performed to evaluate the immunogenicity of human Ad-MSCs in vitro. High-throughput T-cell receptor (TCR) repertoire sequencing and mass spectrometry were applied to identified potential immunogenic molecules.ResultsThe authors observed that allogeneic Ad-MSCs recruited human T cells and caused faster clearance in hu-mice than non-humanized NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice. The proliferation and activation of T cells were significantly enhanced during in vitro co-culture with human Ad-MSCs. In addition, the level of HLA-II expression on human Ad-MSCs was dramatically increased after co-culture with human peripheral blood mononuclear cells (PBMCs). High-throughput sequencing was applied to analyze the TCR repertoire of the Ad-MSC-recruited T cells to identify dominant TCR CDR3 sequences. Using synthesized TCR CDR3 peptides, the authors identified several potential immunogenic candidates, including alpha-enolase (ENO1). The ENO1 expression level of Ad-MSCs significantly increased after co-culture with PBMCs, whereas ENO1 inhibitor (ENOblock) treatment decreased the expression level of ENO1 and Ad-MSC-induced proliferation of T cells.ConclusionsThe authors’ findings improve the understanding of the immunogenicity of human Ad-MSCs and provide a theoretical basis for the safe clinical application of allogeneic MSC therapy.  相似文献   

9.
人源化抗体研究历程及发展趋势   总被引:7,自引:0,他引:7  
单克隆抗体从问世到目前广泛应用于临床,经历了一段曲折的发展历程。其中人源化抗体是一个重要的里程碑,并伴随着一系列重大的技术革新,如PCR技术、抗体库技术、转基因动物等。人源化抗体的形式也从最初的嵌合抗体、改型抗体等逐步发展为今天的人抗体。抗体人源化已经成为治疗性抗体的发展趋势,同时各种抗体衍生物也不断涌现,它们从不同角度克服抗体本身的应用局限,也为治疗人类疾病提供了更多利器。对单克隆抗体进行改造使之应用于临床治疗,不仅需要对抗体效应机制进行更细致深入的研究,同时还有赖于对人类免疫系统调控机制的全面精确认识。  相似文献   

10.
Anti-CD52 therapy has been shown to be effective in the treatment of a number of B cell malignancies, hematopoietic disorders and autoimmune diseases (including rheumatoid arthritis and multiple sclerosis); however the current standard of treatment, the humanized monoclonal antibody alemtuzumab, is associated with the development of anti-drug antibodies in a high proportion of patients. In order to address this problem, we have identified a novel murine anti-CD52 antibody which has been humanized using a process that avoids the inclusion within the variable domains of non-human germline MHC class II binding peptides and known CD4+ T cell epitopes, thus reducing its potential for immunogenicity in the clinic. The resultant humanized antibody, ANT1034, was shown to have superior binding to CD52 expressing cells than alemtuzumab and was more effective at directing both antibody dependent and complement dependent cell cytotoxicity. Furthermore, when in the presence of a cross-linking antibody, ANT1034 was more effective at directly inducing apoptosis than alemtuzumab. ANT1034 also showed superior activity in a SCID mouse/human CD52 tumour xenograft model where a single 1 mg/Kg dose of ANT1034 led to increased mouse survival compared to a 10 mg/Kg dose of alemtuzumab. Finally, ANT1034 was compared to alemtuzumab in in vitro T cell assays in order to evaluate its potential to stimulate proliferation of T cells in peripheral blood mononuclear cells derived from a panel of human donors: whereas alemtuzumab stimulated proliferation in a high proportion of the donor cohort, ANT1034 did not stimulate proliferation in any of the donors. Therefore we have developed a candidate therapeutic humanized antibody, ANT1034, that may have the potential to be more efficacious and less immunogenic than the current standard anti-CD52 therapy.  相似文献   

11.
The immunogenicity of clinically administered antibodies has clinical implications for the patients receiving them, ranging from mild consequences, such as increased clearance of the drug from the circulation, to life-threatening effects. The emergence of methods to engineer variable regions resulting in the generation of humanised and fully human antibodies as therapeutics has reduced the potential for adverse immunogenicity. However, due to differences in sequence referred to as allotypic variation, antibody constant regions are not homogeneous within the human population, even within sub-classes of the same immunoglobulin isotype. For therapeutically administered antibodies, the potential exists for an immune response from the patient to the antibody if the allotype of patient and antibody do not match. Allotypic distribution in the human population varies within and across ethnic groups making the choice of allotype for a therapeutic antibody difficult. This study investigated the potential of human IgG1 allotypes to stimulate responses in human CD4+ T cells from donors matched for homologous and heterologous IgG1 allotypes. Allotypic variants of the therapeutic monoclonal antibody trastuzumab were administered to genetically defined allotypic matched and mismatched donor T cells. No significant responses were observed in the mismatched T cells. To investigate the lack of T-cell responses in relation to mismatched allotypes, HLA-DR agretopes were identified via MHC associated peptide proteomics (MAPPs). As expected, many HLA-DR restricted peptides were presented. However, there were no peptides presented from the sequence regions containing the allotypic variations. Taken together, the results from the T-cell assay and MAPPs assay indicate that the allotypic differences in human IgG1 do not represent a significant risk for induction of immunogenicity.  相似文献   

12.
Design of humanized antibodies: from anti-Tac to Zenapax   总被引:3,自引:0,他引:3  
Since the introduction of hybridoma technology, monoclonal antibodies have become one of the most important tools in the biosciences, finding diverse applications including their use in the therapy of human disease. Initial attempts to use monoclonal antibodies as therapeutics were hampered, however, by the potent immunogenicity of mouse (and other rodent) antibodies in humans. Humanization technology has made it possible to remove the immunogenicity associated with the use of rodent antibodies, or at least to reduce it to an acceptable level for clinical use in humans, thus facilitating the application of monoclonal antibodies to the treatment of human disease. To date, nine humanized monoclonal antibodies have been approved for use as human therapeutics in the United States. In this paper, we describe procedures for antibody humanization with an emphasis on strategies for designing humanized antibodies with the aid of computer-guided modeling of antibody variable domains, using as an example the humanized anti-CD25 monoclonal antibody, Zenapax.  相似文献   

13.
The concept of peptide‐based vaccines against cancer has made noteworthy progress. Metadherin (MTDH) overexpression and its role in the development of diverse cancers make it an attractive target for cancer immunotherapy. In the current study, six different T cell epitope prediction tools were run to identify MTDH peptides with multiple immunogenic regions. Further, molecular docking was performed to assess HLA‐peptide binding interactions. Nine and eleven peptides fragments containing multiple CD8 + and CD4 + T‐cell epitopes, ranging from 9 to 20 amino acids, respectively, were obtained using a consensus immunoinformatics approach. The three peptides that were finally identified as having overlapping CD4 + and CD8 + T‐ cell epitopes are ARLREMLSVGLGFLRTELG, FLLGYGWAAACAGAR, YIDDEWSGLNGLSSADP. These peptides were found to not only have multiple T cell epitopes but also to have binding affinity with wide HLA molecules. A molecular docking study revealed that the predicted immunogenic peptides (with single or multiple T cell epitopes) of MTDH have comparable binding energies with naturally bound peptides for both HLA classes I and II. Thus, these peptides have the potential to induce immune responses that could be considered for developing synthetic peptide vaccines against multiple cancers.  相似文献   

14.
Since the first monoclonal antibody, muromonab-CD3, was approved for therapeutic use in 1986, numerous molecules have been targeted using therapeutic antibody technology, resulting in 26 therapeutic antibodies being approved by the US FDA as of November, 2009. Initial concerns regarding antibody drugs focused on immunogenicity, short serum half-life, and weak efficacy. As the types of antibodies progressed from murine to chimeric, humanized, and fully human antibodies, great progress has been made in immunogenicity and in vivo instability issues. For example, humanized antibodies, such as bevacizumab, exhibit less than 0.2% immunogenicity and a 20 day serum half-life, which is comparable to native immunoglobulin. Some recently developed antibodies are exceedingly efficacious and have become first-line therapy for their target diseases. Here, we address and analyze all clinically approved therapeutic antibodies to date by discussing immunogenicity, half-life, and efficacy.  相似文献   

15.
HLA-A*11:01 is one of the most prevalent human leukocyte antigens (HLAs), especially in East Asian and Oceanian populations. It is also highly expressed in Indigenous people who are at high risk of severe influenza disease. As CD8+ T cells can provide broadly cross-reactive immunity to distinct influenza strains and subtypes, including influenza A, B and C viruses, understanding CD8+ T cell immunity to influenza viruses across prominent HLA types is needed to rationally design a universal influenza vaccine and generate protective immunity especially for high-risk populations. As only a handful of HLA-A*11:01-restricted CD8+ T cell epitopes have been described for influenza A viruses (IAVs) and epitopes for influenza B viruses (IBVs) were still unknown, we embarked on an epitope discovery study to define a CD8+ T cell landscape for HLA-A*11:01-expressing Indigenous and non-Indigenous Australian people. Using mass-spectrometry, we identified IAV- and IBV-derived peptides presented by HLA-A*11:01 during infection. 79 IAV and 57 IBV peptides were subsequently screened for immunogenicity in vitro with peripheral blood mononuclear cells from HLA-A*11:01-expressing Indigenous and non-Indigenous Australian donors. CD8+ T cell immunogenicity screening revealed two immunogenic IAV epitopes (A11/PB2320-331 and A11/PB2323-331) and the first HLA-A*11:01-restricted IBV epitopes (A11/M41-49, A11/NS1186-195 and A11/NP511-520). The immunogenic IAV- and IBV-derived peptides were >90% conserved among their respective influenza viruses. Identification of novel immunogenic HLA-A*11:01-restricted CD8+ T cell epitopes has implications for understanding how CD8+ T cell immunity is generated towards IAVs and IBVs. These findings can inform the development of rationally designed, broadly cross-reactive influenza vaccines to ensure protection from severe influenza disease in HLA-A*11:01-expressing individuals.  相似文献   

16.
Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly‐immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor‐targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralizing antibodies and evidence of cell‐mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen‐specific CD4+ and CD8+ T‐cell proliferation, IFN‐γ and antibody production. The purified polymeric fraction of dengue PIGS (D‐PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low‐affinity Fcγ receptors on antigen‐presenting cells. These results show that the plant‐expressed D‐PIGS have the potential for translation towards a safe and easily scalable single antigen‐based tetravalent dengue vaccine.  相似文献   

17.
Posttranslational modifications regulate the function and stability of proteins, and the immune system is able to recognize some of these modifications. Therefore, the presence of posttranslational modifications increases the diversity of potential immune responses to a determinant antigen. The stimulation of tumor-specific CD4+ helper T lymphocytes (HTLs) is considered important for the production of anti-tumor antibodies by B cells and for the generation and persistence of CD8+ cytotoxic T lymphocytes, and in some instances, HTLs can directly reduce tumor cell growth. Identification of MHC class II-restricted peptide epitopes from tumor-associated antigens including those generated from posttranslational protein modifications should enable the improvement of peptide-based cancer immunotherapy. We describe here an MHC class II binding peptide from the tumor protein p53, which possesses an acetylated lysine at position 120 (p53110-124/AcK120) that is effective in eliciting CD4+ T cell responses specific for the acetylated peptide. Most importantly, the acetylated peptide-reactive CD4 HTLs recognized the corresponding naturally processed posttranslational modified epitope presented by either dendritic cells loaded with tumor cell lysates or directly on tumors expressing p53 and the restricting MHC class II molecules. Treatment of tumor cells with a histone deacetylase inhibitor augmented their recognition by the p53110-124/AcK120-reactive CD4+ T cells. These findings prove that the epitope p53110-124/AcK120 is immunogenic for anti-tumor responses and is likely to be useful for cancer immunotherapy.  相似文献   

18.
Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application.  相似文献   

19.
Yi-Fan Zhang 《MABS-AUSTIN》2017,9(3):419-429
Rabbit monoclonal antibodies (RabMAbs) can recognize diverse epitopes, including those poorly immunogenic in mice and humans. However, there have been only a few reports on RabMAb humanization, an important antibody engineering step usually done before clinical applications are investigated. To pursue a general method for humanization of RabMAbs, we analyzed the complex structures of 5 RabMAbs with their antigens currently available in the Protein Data Bank, and identified antigen-contacting residues on the rabbit Fv within the 6 Angstrom distance to its antigen. We also analyzed the supporting residues for antigen-contacting residues on the same heavy or light chain. We identified “HV4” and “LV4” in rabbit Fvs, non-complementarity-determining region (CDR) loops that are structurally close to the antigen and located in framework 3 of the heavy chain and light chain, respectively. Based on our structural and sequence analysis, we designed a humanization strategy by grafting the combined Kabat/IMGT/Paratome CDRs, which cover most antigen-contacting residues, into a human germline framework sequence. Using this strategy, we humanized 4 RabMAbs that recognize poorly immunogenic epitopes in the cancer target mesothelin. Three of the 4 humanized rabbit Fvs have similar or improved functional binding affinity for mesothelin-expressing cells. Interestingly, 4 immunotoxins composed of the humanized scFvs fused to a clinically used fragment of Pseudomonas exotoxin (PE38) showed stronger cytotoxicity against tumor cells than the immunotoxins derived from their original rabbit scFvs. Our data suggest that grafting the combined Kabat/IMGT/Paratome CDRs to a stable human germline framework can be a general approach to humanize RabMAbs.  相似文献   

20.
Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted anti-myostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall “humanness” was increased for both the light and heavy chain variable regions.  相似文献   

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