从抗TNF抗体的CDR肽库中获得拮抗TNF的短肽

为设计来自抗体的短肽 ,以抗肿瘤坏死因子 (TNF)嵌合抗体 (cA2 )CDRs为模板 ,在其两侧各加 3个随机氨基酸残基 ( X3 CDR X3 ) ,构建了 6个以CDR为基础的肽库 .经过 3轮亲和选择 ,挑取单克隆 ,进一步经ELISA检测TNF阳性噬菌体克隆 ,分离得到 7个ELISA阳性较好的噬菌体肽克隆 ,分别命名为CDR2L1、CDR2L2、CDR2L3、CDR1L1、CDR2H1、CDR3H1、CDR3H 2 .应用MTT方法 ,检测 7个克隆对TNF生物学活性的拮抗作用 .结果显示 :来自CDR2L ,CDR3H肽库中的CDR2L2、CDR2L3,CDR3H2噬菌体肽具有明显的拮抗TNF诱导L92 9细胞的细胞毒作用 ,其中以CDR2L2噬菌体肽的拮抗活性最强 .而来源于CDR1L ,CDR2H肽库的CDR1L1和CDR2H1噬菌体肽和来自CDR2L ,CDR3H肽库中的CDR2L1和CDR3H1噬菌体肽没有明显的拮抗TNF作用 .研究结果初步表明 :从cA2抗体CDR肽库中筛选得到的噬菌体CDR模拟肽具有亲本抗体相似的结合活性和生物学效应 ,从而为开发已知抗体 (特别是治疗用抗体 )CDR为基础的肽药物创建一个技术平台奠定基础     

一种构建改形单域抗体的方法

为验证一种构建改形单域抗体的实用新方法,与以往方法不同的是,该方法不需要对抗体进行空间结构模拟,以确定人源抗体的FRs接受序列及在人源FRs接受序列中哪些氨基酸残基需要突变,并且该方法将抗体的改形与亲和力成熟于同一过程完成,利用该方法构建了改形抗CD28重链单域抗体,根据一种鼠源抗CD28重链单域抗体的氨基酸序列,于GenBank中查得两条与之最同源的人源抗体序列,利用其中一条的FRs作为改形抗体的主框架进行改形构建,将鼠源抗体的CDR区插入到人源FR区后,对人源FR区的一些氨基酸残基进行替换突变,替换的氨基酸残基数及替换原则主要是根据对查到的人源抗体序列,鼠源抗体序列,以及这些序列与Kabat分类中的种属序列进行的比较,为了增加改形抗体基因的多样性,对要被替换的氨基酸残基在基因合成中采用简并的方式,使要被替换的氨基酸残基和替换的氨基酸残基都有机会出现,二者出现的几率各为50%,同时,在将大小不同的合成核苷酸片段采用重叠PCR扩增以获得完整改形抗体基因时,采用高Mg^2 浓度下和使用TaqDNA聚合酶,以进一步随机引入突变,利用重叠PCR产物构建了一个噬菌体抗体库,经过3轮淘选后,获得了几个具有较高免疫学活性的改形抗体,对其中的两个抗体进行了进一步研究,将两个抗体的基因在大肠杆菌BL21（DE3）中表达,复性后的表达蛋白仍具有较高的免疫学活性,结果表明该方法是有效可行的。     

Construction and expression of a reshaped VH domain against human CD28 molecules

Compared with the amino acid sequence of a mouse anti-human CD28 VH domain antibody, the two most homologous sequences of human antibodies were pulled out from Genbank. One of them was used as the main template for the framework regions of the reshaped VH domain. While the original mouse antibody CDRs were inserted into the human acceptor FRs, some residues in human acceptor FRs, which were different from those of the original mouse FRs in corresponding positions, were then determined or, alternatively, mutagenized to their conservative properties in kappa classification. Based on the amino acid sequences of the designed VH domain, the nucleotide sequence was deduced by using E. coli bias codons. The sequence was split into ten 30 to 60 nucleotide fragments for synthesizing, then annealed and amplified by overlap PCR. Taq DNA polymerase was used in a buffer with high Mg2+ concentration to induce more random mutations, both in FRs and CDRs. A phage display library was constructed by cloning these PCR products. After three rounds of panning, several reshaped VH with high antigen binding activity were obtained. One of them had the same CDR amino acid sequences as that of the original mouse VH domain. Further study showed that it retained a high antigen binding affinity after being expressed in E. coli BL21 (DE3).     

Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation.

A mouse monoclonal antibody (mAb 425) with therapeutic potential was 'humanized' in two ways. Firstly the mouse variable regions from mAb 425 were spliced onto human constant regions to create a chimeric 425 antibody. Secondly, the mouse complementarity-determining regions (CDRs) from mAb 425 were grafted into human variable regions, which were then joined to human constant regions, to create a reshaped human 425 antibody. Using a molecular model of the mouse mAb 425 variable regions, framework residues (FRs) that might be critical for antigen-binding were identified. To test the importance of these residues, nine versions of the reshaped human 425 heavy chain variable (VH) regions and two versions of the reshaped human 425 light chain variable (VL) regions were designed and constructed. The recombinant DNAs coding for the chimeric and reshaped human light and heavy chains were co-expressed transiently in COS cells. In antigen-binding assays and competition-binding assays, the reshaped human antibodies were compared with mouse 425 antibody and to chimeric 425 antibody. The different versions of 425-reshaped human antibody showed a wide range of avidities for antigen, indicating that substitutions at certain positions in the human FRs significantly influenced binding to antigen. Why certain individual FR residues influence antigen-binding is discussed. One version of reshaped human 425 antibody bound to antigen with an avidity approaching that of the mouse 425 antibody.     

A universal combinatorial design of antibody framework to graft distinct CDR sequences: a bioinformatics approach

Antibody (Ab) humanization is crucial to generate clinically relevant biologics from hybridoma-derived monoclonal antibodies (mAbs). In this study, we integrated antibody structural information from the Protein Data Bank with known back-to-mouse mutational data to build a universal consensus of framework positions (10 heavy and 7 light) critical for the preservation of the functional conformation of the Complimentarity Determining Region of antibodies. On the basis of FR consensus, we describe here a universal combinatorial library suitable for humanizing exogenous antibodies by CDR-grafting. The six CDRs of the murine anti-human EGFR Fab M225 were grafted onto a distinct (low FR sequence similarity to M225) human FR sequence that incorporates at the 17 FR consensus positions the permutations of the naturally observed amino acid diversities. Ten clones were selected from the combinatorial library expressing phage-displayed humanized M225 Fabs. Surprisingly, 2 of the 10 clones were found to bind EGFR with stronger affinity than M225. Cell-based assays demonstrated that the 10 selected clones retained epitope specificity by blocking EGFR phosphorylation and thus hindering cellular proliferation. Our results suggest that there is a universal and structurally rigid near-CDR set of FR positions that cooperatively support the binding conformation of CDRs.     

Antibody humanization by framework shuffling

We report here the humanization of a mouse monoclonal antibody (mAb B233) using a new technique which we call framework shuffling. mAb B233 was raised against the human receptor tyrosine kinase EphA2 which is selectively up-regulated in many cancer cell lines and as such constitutes an attractive target for cancer therapy. The six CDRs of B233 were fused in-frame to pools of corresponding individual human frameworks. These human frameworks encompassed all known heavy and light (kappa) chain human germline genes. The resulting Fab combinatorial libraries were then screened for binding to the antigen. A two-step selection process, in which the light and heavy chains of the parental mAb were successively humanized, resulted in the identification of several humanized variants that retained binding to EphA2. More precisely, after conversion to human IgG1, the dissociation constants of three select fully humanized variants ranged from 3 to 48 nM. This brings the best framework-shuffled, humanized binder within 5-fold of the avidity of parental mAb B233. Importantly, these humanized IgGs also possessed biochemical activities similar to those of parental mAb B233 as judged by induction of EphA2 phosphorylation. Thus, without requiring any rational design or structural information, this new humanization approach allows to rapidly identify various human framework combinations able to support the structural feature(s) of the CDRs which are essential for binding and functional activity.     

Structural consensus among antibodies defines the antigen binding site

The Complementarity Determining Regions (CDRs) of antibodies are assumed to account for the antigen recognition and binding and thus to contain also the antigen binding site. CDRs are typically discerned by searching for regions that are most different, in sequence or in structure, between different antibodies. Here, we show that ~20% of the antibody residues that actually bind the antigen fall outside the CDRs. However, virtually all antigen binding residues lie in regions of structural consensus across antibodies. Furthermore, we show that these regions of structural consensus which cover the antigen binding site are identifiable from the sequence of the antibody. Analyzing the predicted contribution of antigen binding residues to the stability of the antibody-antigen complex, we show that residues that fall outside of the traditionally defined CDRs are at least as important to antigen binding as residues within the CDRs, and in some cases, they are even more important energetically. Furthermore, antigen binding residues that fall outside of the structural consensus regions but within traditionally defined CDRs show a marginal energetic contribution to antigen binding. These findings allow for systematic and comprehensive identification of antigen binding sites, which can improve the understanding of antigenic interactions and may be useful in antibody engineering and B-cell epitope identification.     

Positive darwinian selection observed at the variable-region genes of immunoglobulins

To examine Gojobori and Nei's hypothesis that the immunoglobulin heavy- chain variable-region (VH) genes in mammals are subject to diversity- enhancing selection, we studied the rates of synonymous and nonsynonymous nucleotide substitution in the complementarity- determining regions (CDRs) and in the framework regions (FRs) of mouse and human VH genes. The results obtained indicate that the non- synonymous rate is higher than the synonymous rate in CDRs, whereas the reverse is true in FRs. This observation supports Gojobori and Nei's hypothesis and suggests that diversity-enhancing selection (similar to overdominant selection) operates mainly in CDRs and that this is one of the evolutionary factors that increase antibody diversity.      

Display of somatostatin-related peptides in the complementarity determining regions of an antibody light chain

Peptide display in antibody complementarity determining regions (CDRs) offers several advantages over other peptide display systems including the potential to graft heterologous peptide sequences into multiple positions in the same backbone molecule. Despite the presence of six CDRs in an antibody variable domain, the majority of insertions reported have been made in heavy chain CDR3 (h-CDR3) which may be explained in part by the highly variable length and sequence diversity found in h-CDR3 in native antibodies. The ability to graft peptide sequences into CDRs is restricted by amino acids in these loops that make structural contacts to framework regions or are oriented towards the hydrophobic interior and are important for the proper folding of the antibody. To identify such positions in human kappa-light chain CDR1 (kappa-CDR1) and CDR2 (kappa-CDR2), we performed alignments of 1330 kappa-light chain variable region amino acid sequences and 19 variable region X-ray crystal structures. From analyses of these alignments, we predict insertion points where sequences can be grafted into kappa-CDR1 and kappa-CDR2 to prepare synthetic antibody molecules. We then tested these predictions by inserting somatostatin and somatostatin-related sequences into kappa-CDR1 and kappa-CDR2, and analyzing the expression and ability of the modified antibodies to bind to membranes containing somatostatin receptor 5. These results expand the repertoire of CDRs that can be used for the display of heterologous peptides in the CDRs of antibodies.     

A Two-in-One antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions

A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.     

A Two-in-One antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions

A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.     

A novel TNFalpha antagonizing peptide-Fc fusion protein designed based on CDRs of TNFalpha neutralizing monoclonal antibody

The variable regions of antibody molecules bind antigens with high affinity and specificity. The binding sites are imparted largely to the hypervariable portions (i.e., CDRs) of the variable region. Peptides derived from CDRs can bind antigen with similar specificity acting as mimic of antibody and become drug-designing core, although with markedly lower affinity. In order to increase the affinity and bioactivity, in this study, a novel peptide (PT) designed on CDRs of a TNFalpha neutralizing monoclonal antibody Z12 was linked with Fc fragment of human IgG1. The interaction mode of PT-linker-Fc (PLF) with TNFalpha was analyzed with computer-guided molecular modeling method. After expression in Escherichia coli and purification, recombinant PT-linker-Fc could bind directly with the TNFalpha coated on the ELISA plates. Furthermore, PLF could competitively inhibit the binding of Z12 to TNFalpha and also inhibit the TNFalpha-induced cytotoxicity on L929 cells. The TNFalpha antagonizing activity of PLF was significantly higher than that of the free peptide. This study highlights the potential of human Fc to enhance the potency of peptides designed on the CDRs of antibodies and could be useful in developing new TNFalpha antagonists.     

Design and synthesis of germline-based hemi-humanized single-chain Fv against the CD18 surface antigen

The 6.7 murine monoclonal antibody (mAb) recognizes the human CD18 antigen and is therefore of interest as an anti-inflammatory agent. The 6.7 heavy variable chain (VH) was humanized using the closest human germline sequence as the template on to which to graft the murine complementary determining regions (CDRs). Two versions were proposed, one in which the residue proline 45 of the murine form was maintained and another in which this framework residue was changed to the leucine found in the human sequence. These VH humanized versions were expressed in the yeast Pichia pastoris as hemi-humanized single-chain Fv (scFvs), with the VL from the murine antibody. The scFv from the murine antibody was also expressed. The binding activities of the murine and both hemi-humanized scFvs were determined by flow cytometry analysis. All the constructions were able to recognize human lymphocytes harboring CD18, indicating successful humanization with transfer of the original binding capability. Some differences between the two hemi-humanized versions were observed. The method used was simple and straightforward, with no need for refined structural analyses and could be used for the humanization of other antibodies.     

Small antibody mimetics comprising two complementarity-determining regions and a framework region for tumor targeting

Here we show that fusion of two complementarity-determining regions (CDRs), VHCDR1 and VLCDR3, through a cognate framework region (VHFR2) yields mimetics that retain the antigen recognition of their parent molecules, but have a superior capacity to penetrate tumors. The antigen-recognition abilities of these approximately 3 kDa mimetics surpass those of comparable fragments lacking the framework region. In vivo activities of the mimetics suggests that the structural orientation of their CDRs approximates the conformation of the CDRs in the complex of the parent antibody with antigen. We linked the antibody mimetics to the bacterial toxin colicin Ia to create fusion proteins called "pheromonicins," which enable targeted inhibition of tumor growth. In mice bearing human malignant tumors, pheromonicins directed against tumor-specific surface markers show greater capacity to target and penetrate tumors than their parent antibodies. Rational recombination of selected VH/VL binding sites and their framework regions might provide useful targeting moieties for cytotoxic cancer therapies.     

Construction and characterization of a high-affinity humanized SM5-1 monoclonal antibody

SM5-1 is a mouse monoclonal antibody which has a high specificity for melanoma, hepatocellular carcinoma, and breast cancer, making it a promising candidate for cancer targeting therapy. We have therefore attempted to construct a humanized antibody of SM5-1 to minimize its immunogenicity for potential clinical use. Using a molecular model of SM5-1 built by computer-assisted homology modeling, framework region (FR) residues of potential importance to the antigen binding were identified. Then, a humanized version of SM5-1 was generated by transferring these mouse key FR residues onto a human framework that was selected based on homology to the mouse framework, together with the mouse complementarity-determining region (CDR) residues. This humanized antibody retained only six murine residues outside of the CDRs but was shown to possess affinity and specificity comparable to that of the parental antibody, suggesting that it might have the potential to be developed for future clinical use.     

Construction, affinity maturation, and biological characterization of an anti-tumor-associated glycoprotein-72 humanized antibody

The tumor-associated glycoprotein (TAG)-72 is expressed in the majority of human adenocarcinomas but is rarely expressed in most normal tissues, which makes it a potential target for the diagnosis and therapy of a variety of human cancers. Here we describe the construction, affinity maturation, and biological characterization of an anti-TAG-72 humanized antibody with minimum potential immunogenicity. The humanized antibody was constructed by grafting only the specificity-determining residues (SDRs) within the complementarity-determining regions (CDRs) onto homologous human immunoglobulin germ line segments while retaining two mouse heavy chain framework residues that support the conformation of the CDRs. The resulting humanized antibody (AKA) showed only about 2-fold lower affinity compared with the original murine monoclonal antibody CC49 and 27-fold lower reactivity to patient serum compared with the humanized antibody HuCC49 that was constructed by CDR grafting. The affinity of AKA was improved by random mutagenesis of the heavy chain CDR3 (HCDR3). The highest affinity variant (3E8) showed 22-fold higher affinity compared with AKA and retained the original epitope specificity. Mutational analysis of the HCDR3 residues revealed that the replacement of Asn(97) by isoleucine or valine was critical for the affinity maturation. The 3E8 labeled with (125)I or (131)I showed efficient tumor targeting or therapeutic effects, respectively, in athymic mice with human colon carcinoma xenografts, suggesting that 3E8 may be beneficial for the diagnosis and therapy of tumors expressing TAG-72.     

Deep mutational scanning of an antibody against epidermal growth factor receptor using mammalian cell display and massively parallel pyrosequencing

We developed a method for deep mutational scanning of antibody complementarity-determining regions (CDRs) that can determine in parallel the effect of every possible single amino acid CDR substitution on antigen binding. The method uses libraries of full length IgGs containing more than 1000 CDR point mutations displayed on mammalian cells, sorted by flow cytometry into subpopulations based on antigen affinity and analyzed by massively parallel pyrosequencing. Higher, lower and neutral affinity mutations are identified by their enrichment or depletion in the FACS subpopulations. We applied this method to a humanized version of the anti-epidermal growth factor receptor antibody cetuximab, generated a near comprehensive data set for 1060 point mutations that recapitulates previously determined structural and mutational data for these CDRs and identified 67 point mutations that increase affinity. The large-scale, comprehensive sequence-function data sets generated by this method should have broad utility for engineering properties such as antibody affinity and specificity and may advance theoretical understanding of antibody-antigen recognition.     

Length-independent structural similarities enrich the antibody CDR canonical class model

Complementarity-determining regions (CDRs) are antibody loops that make up the antigen binding site. Here, we show that all CDR types have structurally similar loops of different lengths. Based on these findings, we created length-independent canonical classes for the non-H3 CDRs. Our length variable structural clusters show strong sequence patterns suggesting either that they evolved from the same original structure or result from some form of convergence. We find that our length-independent method not only clusters a larger number of CDRs, but also predicts canonical class from sequence better than the standard length-dependent approach.

To demonstrate the usefulness of our findings, we predicted cluster membership of CDR-L3 sequences from 3 next-generation sequencing datasets of the antibody repertoire (over 1,000,000 sequences). Using the length-independent clusters, we can structurally classify an additional 135,000 sequences, which represents a ～20% improvement over the standard approach. This suggests that our length-independent canonical classes might be a highly prevalent feature of antibody space, and could substantially improve our ability to accurately predict the structure of novel CDRs identified by next-generation sequencing.     

Concerted evolution of the immunoglobulin VH gene family

With the aim of understanding the concerted evolution of the immunoglobulin VH multigene family, a phylogenetic tree for the DNA sequences of 16 mouse and five human germ line genes was constructed. This tree indicates that all genes in this family have undergone substantial evolutionary divergence. The most closely related genes so far identified in the mouse genome seem to have diverged about 6 million years (MY) ago, whereas the most distantly related genes diverged about 300 MY ago. This suggests that gene duplication caused by unequal crossing-over or gene conversion occurs very slowly in this gene family. The rate of occurrence of gene duplication in the VH gene family has been estimated to be 5 x 10(-7) per gene per year, which seems to be at least about 100 times lower than that for the rRNA gene family. This low rate of concerted evolution in the VH gene family helps retain intergenic genetic variability that in turn contributes to antibody diversity. Because of accumulation of destructive mutations, however, about one-third of the mouse and human VH genes seem to have become nonfunctional. Many of these pseudogenes have apparently originated recently, but some of them seem to have existed in the genome for more than 10 MY. The rate of nucleotide substitution for the complementarity-determining regions (CDRs) is as high as that of pseudogenes. This suggests that there is virtually no purifying selection operating in the CDRs and that germ line mutations are effectively used for generating antibody diversity.      

Characterization of the expressed immunoglobulin IGHV repertoire in the New World marmoset Callithrix jacchus

The common marmoset (Callithrix jacchus jacchus) is a member of the Callithrichinae, a family of outbred New World primates with limited MHC polymorphisms and a propensity to develop spontaneous or experimentally induced autoimmunity. C. jacchus marmosets are susceptible to experimental allergic encephalomyelitis (EAE), and spontaneously develop autoimmune colitis and thyroiditis. Such disease models approximate the complexity of human autoimmune disorders, and allow an investigation of the respective roles of T-cell and antibody responses to self-antigens in outbred species. A key issue for further definition of the pathogenic antibody responses in human autoimmunity is to understand the diversity of the immunoglobulin repertoire in primate models. Here, we characterized the expressed immunoglobulin IGHV repertoire of the C. jacchus marmoset. Six IGHV subgroups were identified which show a high degree of sequence similarity to their human IGHV counterparts (IGHV1, IGHV3, IGHV4, IGHV5, IGHV6, and IGHV7). As in the expressed human IGHV repertoire, the framework regions are more conserved when compared to the complementarity-determining regions (CDRs), with the greatest degree of variability located in CDR3. Predicted structural features are highly conserved between C. jacchus and human IGHV. This information now provides a framework for studies of the antigen-specific repertoire of pathogenic antibodies in EAE and other immune-mediated diseases.