High-affinity Human Antibodies from Phage-displayed Synthetic Fab Libraries with a Single Framework Scaffold (original) (raw)
Related papers
Nature Biotechnology, 2005
Combinatorial libraries of rearranged hypervariable V H and V L sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of V H s and V L s. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found. Lengthy in vitro affinity maturation is often needed to improve antibodies from such libraries 1,2. We report the construction of human Fab libraries having a unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2. The success of this strategy is demonstrated by identifying many monovalent Fabs against multiple therapeutic targets that show higher affinities than approved therapeutic antibodies 3-6. This very often circumvents the need for affinity maturation, accelerating discovery of antibody drug candidates. The human germline V H 3-23 segment is very frequently found in rearranged antibodies, is well expressed and pairs with almost all light chains 7. We synthesized heavy-chain complementarity-determining region (CDR)1 and CDR2 DNA sequences in the V H 3-23 framework and used codon-based mutagenesis 8 to introduce diversity into selected CDR residues. From a 3D model 9 , we determined that the side chains of amino acids (amino acids 31, 33 and 35 (Kabat numbering (http:// www.hgmp.mrc.ac.uk)) of CDR1 point toward the antigen-combining site. All amino acid types except cysteine were allowed at each of these positions, exceeding the diversity of germline V H and of immunoglobulin sequences in mature B-cell populations. In CDR2, amino acids 50, 52, and 52a vary between germline V H s, and amino acids 56 and 58 are known to be surface exposed and highly varied by somatic mutation in antibodies derived from in vivo responses 10. At 50 and 52, amino acid types most frequently found in germline V H s (tyrosine, arginine, tryptophan, valine, glycine, serine) were allowed. At 52a, proline or serine (the most common types in germ lines) were allowed. At 56 and 58, all types except cysteine were allowed. This synthetic diversity mimics the pattern of mutations seen in immunoglobulins after immunization and clonal selection. It has been reported that the structural differences found in four monoclonal antibodies bind hen egg white lysozyme with various affinities 11. The researchers concluded that all CDRs are involved in binding protein antigens, higher affinity antibodies have more hydrophobic surface buried and higher affinity antibodies match the shape of the antigen to a higher degree. Germ line 3-23 has S 31 , A 33 , S 35 , A 50 , S 52 , G 52a , S 56 and Y 58 , all except Y 58 being small and hydrophilic. Our variegation of heavy chain CDR1 and CDR2 allows these positions to change shape and to become selectively hydrophobic, increasing the likelihood of recovering high-affinity Fabs. A library of phage-displayed Fab fragments having one V L and synthetic diversity in one V H has been produced and antibodies that bind fluorescein with K d s between 1,000 and 100 nM have been selected 12. In naturally occurring human antibodies, heavy chain CDR3 varies from 4 to over 35 residues 13 and has nonrandom 14 sequence diversity. Long heavy chain CDR3s often contain diversity (D) segments. Germline D segments have been selected to foster proper folding and binding, and inclusion of D segments in a library is desirable. Although libraries containing heavy chain CDR3 length diversity have been reported 15,16 , it is impossible to synthesize DNA encoding both the sequence and the length diversity found in natural heavy chain CDR3 repertoires 17,18. In devising the library with heavy chain CDR3 length diversity, one group 16 biased the amino acids to those found in natural antibodies, included heavy chain CDR3s with lengths ranging from 7 to 19, but the library lacks the sequences found in D segments. Hence,
Phage-displayed Antibody Libraries of Synthetic Heavy Chain Complementarity Determining Regions
Journal of Molecular Biology, 2004
A structure-based approach was used to design libraries of synthetic heavy chain complementarity determining regions (CDRs). The CDR libraries were displayed as either monovalent or bivalent single-chain variable fragments (scFvs) with a single heavy chain variable domain scaffold and a fixed light chain variable domain. Using the structure of a parent antibody as a guide, we restricted library diversity to CDR positions with significant exposure to solvent. We introduced diversity with tailored degenerate codons that ideally only encoded for amino acids commonly observed in natural antibody CDRs. With these design principles, we reasoned that we would produce libraries of diverse solvent-exposed surfaces displayed on stable scaffolds with minimal structural perturbations. The libraries were sorted against a panel of proteins and yielded multiple unique binding clones against all six antigens tested. The bivalent library yielded numerous unique sequences, while the monovalent library yielded fewer unique clones. Selected scFvs were converted to the Fab format, and the purified Fab proteins retained high affinity for antigen. The results support the view that synthetic heavy chain diversity alone may be sufficient for the generation of high-affinity antibodies from phage-displayed libraries; thus, it may be possible to dispense with the light chain altogether, as is the case in natural camelid immunoglobulins.
Journal of Molecular Biology, 2000
By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V H and seven V L (four Vk and three Vl) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversi®cation, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites¯anking the CDRs. Molecular modeling veri®ed that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A ®rst set of antibody phage display libraries totalling 2 Â 10 9 members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V H and V L CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high af®nities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes.
Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries
Nature Biotechnology, 2000
Results and discussion Library design using proofread gene segments. We have constructed an scFv antibody fragment gene library of 2 × 10 9 members, designated n-CoDeR. The genes were built using the VH DP47 and the VL DPL3 immunoglobulin genes as single master frameworks 12 , since they are (1) well expressed/displayed in bacterial/phage systems 6 and (2) present in vivo in most human individuals 13-16. Sequences encoding in vivo-formed CDRs from rearranged immunoglobulin genes of different germline origin were combined into the master framework. This was achieved by amplification of CDRs with primers specific for the DP47 and DPL3 frameworks 11 and overlap extension of single-stranded DNA (ssDNA) to create antibody genes encoding variable light and heavy chains 9,11,12. The principle is outlined (Fig. 1). An extremely large genetic variation can be created by random combination of six in vivo-formed CDR sequences, although an exact calculation of the variation cannot be performed, since human germline genes acquire additional genetic variation in vivo from somatic mutation processes and V(D)J recombination. The assembled scFv were cloned into the pFAB5c-His display vector 17 and
Journal of Molecular Biology, 2013
We have generated large libraries of single-chain Fv antibody fragments (N 10 10 transformants) containing unbiased amino acid diversity that is restricted to the central combining site of the stable, well-expressed DP47 and DPK22 germline V-genes. Library WySH2A was constructed to examine the potential for synthetic complementarity-determining region (CDR)-H3 diversity to act as the lone source of binding specificity. Library WySH2B was constructed to assess the necessity for diversification in both the H3 and L3. Both libraries provided diverse, specific antibodies, yielding a total of 243 unique hits against 7 different targets, but WySH2B produced fewer hits than WySH2A when selected in parallel. WySH2A also consistently produced hits of similar quality to WySH2B, demonstrating that the diversification of the CDR-L3 reduces library fitness. Despite the absence of deliberate bias in the library design, CDR length was strongly associated with the number of hits produced, leading to a functional loop length distribution profile that mimics the biases observed in the natural repertoire. A similar trend was also observed for the CDR-L3. After target selections, several key amino acids were enriched in the CDR-H3 (e.g., small and aromatic residues) while others were reduced (e.g., strongly charged residues) in a manner that was specific to position, preferentially occurred in CDR-H3 stem positions, and tended towards residues associated with loop stabilization. As proof of principle for the WySH2 libraries to produce viable lead candidate antibodies, 114 unique hits were produced against Delta-like ligand 4 (DLL4). Leads exhibited nanomolar binding affinities, highly specific staining of DLL4 + cells, and biochemical neutralization of DLL4-NOTCH1 interaction.
Optimal Design Features of Camelized Human Single-domain Antibody Libraries
Journal of Biological Chemistry, 2001
We have constructed a human V H library based on a camelized V H sequence. The library was constructed with complete randomization of 19 of the 23 CDR3 residues and was panned against two monoclonal antibody targets to generate V H sequences for determination of the antigen contact residue positions. Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the library showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disulfide formation was universal. However, surface plasmon resonance and NMR data showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the 1 H-15 N HSQC spectra, indicated that the V H sequence for library construction was a good choice. These results should be useful in the design of V H libraries with optimal features.
Journal of Molecular Biology, 1995
We have constructed a large (3.6×10 8 clones) phage display library of human 1 Department of Immunology and 2 Eykman-Winkler single chain Fv (scFv) antibody fragments by combining 49 germline V H genes with synthetic heavy chain CDR3 (HCDR3) regions and seven light Institute for Clinical and chains. The HCDR3 regions varied in length between 6 and 15 residues and Medical Microbiology University of Utrecht, PO were designed to contain fully randomized stretches of amino acid residues flanked by regions of limited residue variability that were composed of Box 85500, 3508 GA Utrecht The Netherlands amino acid residues that frequently occur in natural antibodies. We reasoned that this approach would increase the frequency of functional molecules in our library and, in addition, permit us to efficiently utilize available cloning space. By direct selection on solid phase-bound antigens we obtained phage antibodies with binding activities to 13 different antigens, including Von Willebrand factor, the DNA-binding HMG box of transcription factor TCF-1 and the tumor antigen EGP-2. In addition, we applied a competitive selection procedure to target phage antibodies to the desired portion of a recombinant fusion protein and to select phage antibodies capable of discriminating between the two highly homologous homeobox proteins PBX1a and PBX2. The functional capacity of monoclonal phage antibodies was assessed in immuno-histochemical staining of tissue specimens, Western blotting assays and immunofluorescent analysis of cells by flow cytometry. The results demonstrate that this large human phage antibody library contains a broad assortment of binding specificities that can be applied in a variety of biochemical assays.
Generation and analysis of the improved human HAL9/10 antibody phage display libraries
BMC Biotechnology, 2015
Background: Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries-in theory-antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library. Results: The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×10 10 independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations. Conclusion: The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.
Journal of Molecular Biology, 2007
We have previously established a minimalist approach to antibody engineering by using a phage-displayed framework to support complementarity determining region (CDR) diversity restricted to a binary code of tyrosine and serine. Here, we systematically augmented the original binary library with additional levels of diversity and examined the effects. The diversity of the simplest library, in which only heavy chain CDR positions were randomized by the binary code, was expanded in a stepwise manner by adding diversity to the light chain, by diversifying non-paratope residues that may influence CDR conformations, and by adding additional chemical diversity to CDR-H3. The additional diversity incrementally improved the affinities of antibodies raised against human vascular endoethelial growth factor and the structure of an antibody-antigen complex showed that tyrosine side-chains are sufficient to mediate most of the interactions with antigen, but a glycine residue in CDR-H3 was critical for providing a conformation suitable for high-affinity binding. Using new high-throughput procedures and the most complex library, we produced multiple high-affinity antibodies with dissociation constants in the single-digit nanomolar range against a wide variety of protein antigens. Thus, this fully synthetic, minimalist library has essentially recapitulated the capacity of the natural immune system to generate high-affinity antibodies. Libraries of this type should be highly useful for proteomic applications, as they minimize inherent complexities of natural antibodies that have hindered the establishment of high-throughput procedures. Furthermore, analysis of a large number of antibodies derived from these well-defined and simplistic libraries allowed us to uncover statistically significant trends in CDR sequences, which provide valuable insights into antibody library design and into factors governing protein-protein interactions.
Construction of a human antibody domain (VH) library
Highly diverse antibody (Fab or scFv) libraries have become vital sources to select antibodies with high affinity and novel properties. Combinatorial strategies provide efficient ways of creating antibody libraries containing a large number of individual clones. These strategies include the reassembly of naturally occurring genes encoding the heavy and light chains from either immune or nonimmune B-cell sources, or introduction of synthetic diversity to either the framework regions (FRs) or the complementarity-determining regions (CDRs) of the variable domains of antibodies. In the late 1980s, the smallest known antigen-binding fragment was identified when a murine VH repertoire was screened for binding to lysozyme. This fragment (~15 kDa), called a " domain antibody " , or " dAb " , is approximately four times smaller than an Fab and half the size of an scFv. Here, we describe the construction of a phage-displayed VH library and an approach to introduce genetic diversity in this library, where both diverse human CDRs and synthetic CDRs are combined into a single domain (VH) framework.