Engineering antibody heavy chain CDR3 to create a phage display Fab library rich in antibodies that bind charged carbohydrates

A number of small charged carbohydrate moieties have been associated with inflammation and cancer. However, the development of therapeutic Abs targeting these moieties has been hampered by their low immunogenicity and their structural relationship to self-Ag. We report the design of an Ab repertoire enriched in Abs binding to small charged carbohydrates and the construction of a human Fab phagemid library, "FAB-CCHO." This library combines L chain Ig sequences from human donors and H chain synthetic diversity constructed in key Ag contact sites in CDRs 1, 2, and 3 of the human framework V(H)3-23. The H chain CDR3 has been engineered to enrich the library in Abs that bind charged carbohydrates by the introduction of basic residues at specific amino acid locations. These residues were selected on the basis of anti-carbohydrate Ab sequence alignment. The success of this design is demonstrated by the isolation of phage Abs against charged carbohydrate therapeutic target Ags such as sulfated sialyl-Lewis X glycan and heparan sulfate.     

Molecular characterization of the anti-idiotypic immune response of a relapse-free neuroblastoma patient following antibody therapy: a possible vaccine against tumors of neuroectodermal origin?

Neuroblastoma treatment with chimeric antidisialoganglioside GD2 Ab ch14.18 showed objective antitumor responses. Production of anti-idiotypic Abs (Ab2) against ch14.18 (Ab1) in some cases was positively correlated with a more favorable prognosis. According to Jerne's network theory, a subset of anti-idiotypic Abs (Ab2beta) carries an "internal image" of the Ag and induces Abs (Ab3) against the original Ag. The molecular origin of an anti-idiotypic Ab response in tumor patients was not investigated previously. To clone anti-idiotypic Abs, B cells of a ch14.18-treated neuroblastoma patient with Ab2 serum reactivity were used to construct Ab phage display libraries. After repeated biopannings on ch14.18 and its murine relative, anti-GD2 mAb 14G2a, we selected 40 highly specific clones. Sequence analysis revealed at least 10 of 40 clones with different Ig genes. Identities to putative germline genes ranged between 94.90 and 100% for V(H) and between 93.90 and 99.60% for V(L). An overall high rate of replacement mutations suggested a strong Ag-driven maturation of the anti-idiotypic Abs. Two clones that were analyzed further, GK2 and GK8, inhibited binding of ch14.18 to GD2 just as the patient's serum did. GK8 alone inhibited >80% of the patient's anti-idiotypic serum Abs in binding to ch14.18. Rabbits vaccinated with GK8 or GK2 (weaker) produced Ab3 against the original target Ag GD2. GK8 may be useful as a tumor vaccine for GD2-positive [corrected] tumors.     

Cloning the arterial IgA antibody response during acute Kawasaki disease

Kawasaki disease (KD) is the most common acquired cardiac disease in children in developed nations. The etiology of KD is unknown but likely to be a ubiquitous microbial agent. Previously, we showed that oligoclonal IgA plasma cells infiltrate coronary arteries and other inflamed tissues in acute KD. We demonstrated that a synthetic Ab made using an alpha H chain sequence prevalent in acute KD arterial tissue detected Ag in acute KD coronary arteries, lung, and other inflamed tissues and that Ag localized to cytoplasmic inclusion bodies in the acute KD ciliated bronchial epithelium. In this study, we synthesized a panel of mAbs from alpha and kappa chain sequences present in the KD arterial wall and tested the Abs for binding to acute KD tissues. We report that all of the synthetic mAbs that bind to acute KD tissues detect Ag in cytoplasmic inclusion bodies in the acute KD ciliated bronchial epithelium. Abs made from alpha sequences that were prevalent in KD arterial tissue show stronger binding to acute KD tissues than Abs made from less prevalent sequences. These findings highlight the likely importance of the inclusion bodies in the etiopathogenesis of acute KD, confirm that the IgA Ab response in acute KD is Ag driven, and demonstrate the usefulness of cloning the Ab response in diseased tissues to identify disease-relevant Ags.     

Plasticity within the antigen-combining site may manifest as molecular mimicry in the humoral immune response

Structural and physiological facets of carbohydrate-peptide mimicry were addressed by analyzing the Ab response to alpha-d-mannopyranoside. mAbs against alpha-d-mannopyranoside were generated and screened with the carbohydrate-mimicking 12 mer (DVFYPYPYASGS) peptide. Three mAbs, 2D10, 1H11, and 1H7, which were subjected to detailed analysis, exhibit diverse V gene usage, indicating their independent germline origins. Although the mAb 1H7 was specific in binding only to the immunizing Ag, the Abs 2D10 and 1H11 recognize the 12 mer peptide as well as the immunogen, alpha-d-mannopyranoside. The Abs that recognize mimicry appear to bind to a common epitope on the peptide and do not share the mode of peptide binding with Con A. Binding kinetics and thermodynamics of Ag recognition suggest that the Ab that does not recognize peptide-carbohydrate mimicry probably has a predesigned mannopyranoside-complementing site. In contrast, the mimicry-recognizing Abs adopt the Ag-combining site only on exposure to the sugar, exploiting the conformational flexibility in the CDRs. Although the mAb 1H7 showed unique specificity toward mannopyranoside, the mimicry-recognizing Abs 2D10 and 1H11 exhibited degenerate specificities with regard to other sugar moieties. It is proposed that the degeneracy of specificity arising from the plasticity at the Ag-combining site in a subset of the Ab clones may be responsible for exhibiting molecular mimicry in the context of Ab response.     

The primary antibody repertoire represents a linked network of degenerate antigen specificities

In this study, germline Abs were used to select clones from a random dodecapeptide phage-display library. This revealed a much greater heterogeneity of binders than could be obtained with mutated daughter Abs that presumably had been selected in vivo by nominal Ag during active immune responses. We demonstrate that the pluripotency of germline Abs can subsequently be optimized by binding interactions that correlate with thermodynamic changes indicative of structural adaptations at the interface. This singular feature confers on each Ab a distinct window of Ag specificities, where the entropic space explored constitutes a thermodynamic signature of that particular Ab. Combining site plasticity may facilitate overlaps in such windows, with independent Abs converging onto common determinants with near identical binding affinities. In addition to providing for an amplified recognition potential, this networking of individual spectra of Ag specificities simultaneously facilitates the rapid recognition of Ag. Importantly, it also ensures that the primary response is composed of Abs with a high degree of "evolvability."     

Molecular analysis of the autoantibody response in peptide-induced autoimmunity

Immunization of nonautoimmune BALB/c mice with multimeric DWEYSVWLSN, a peptide mimotope of DNA, induces anti-DNA and other lupus-associated Abs. To further investigate the pathogenesis of the autoantibody response induced by peptide immunization, we generated hybridomas from peptide-immunized mice that bound peptide, dsDNA, cardiolipin, Sm/ribonucleoprotein (RNP), or some combination of these Ags. Analysis of 24 IgM Abs led to the identification of three groups of Abs: 1) Abs reactive with peptide alone, 2) anti-peptide Abs cross-reactive with one or more autoantigens, and 3) autoantibodies that do not bind to peptide. The gene families and particular VH-VL combinations used in those hybridomas binding DNA were similar to those used in the anti-DNA response in spontaneous murine lupus. Another similarity to the spontaneous anti-DNA response was the generation of arginines in the complementarity-determining region-3 of DNA-binding hybridomas. Interestingly, one Ab had the VH-VL combination present in the original R4A anti-DNA Ab used to select the DWEYSVWLSN peptide from a phage display library. Many of the heavy and light chains displayed evidence of somatic mutation, suggesting that they were made by Ag-activated B cells. Analysis of the Ab repertoire in peptide-induced autoimmunity may provide insights into the generation of anti-DNA Abs following exposure to foreign Ag. Furthermore, the recovery of an Ab with the heavy and light chain combination of the Ab originally used to isolate the immunizing peptide confirms the utility of phage display peptide libraries in generating true molecular mimics.     

Expression vectors for the introduction of highly diverged sequences into the six complementarity-determining regions of an antibody

We prepared three kinds of phagemid vector that permit the simultaneous introduction of highly diverged sequences into six complementarity-determining regions (CDRs) of an antibody (Ab) by the polymerase chain reaction (PCR) with degenerate oligodeoxynucleotide (oligo) primers. The phages expressed either the Fv, single-chain Fv (sc Fv) or Fab form of an Ab fused with a half-molecule of cpIII on the surface of M13 phage. A phage-display library, composed of 2 × 10⁸ independent clones, was constructed; the phages that were specific for hen egg-white lysozyme (HEL) were selected by three rounds of panning; and 20 clones were isolated. The isolated clones consisted of 17 different clones. Among them, 16 clones expressed proteins that were able to bind to HEL. The association constants for binding of the encoded proteins to HEL ranged from 1.48 × 10⁶ to 7.71 × 10⁶/M. These vectors allowed us to prepare many libraries of artificial Ab in which the sequences of six CDRs were very different and reflected the artificial sequences that had been designed for the degenerate oligo that we used as primers for PCR. The libraries should be also useful for the analysis of relationships between the sequences of the CDRs and antigen (Ag) specificity.     

A phage‐displayed single‐chain Fab library optimized for rapid production of single‐chain IgGs

Phage‐displayed synthetic antibody (Ab) repertoires have become a major source of affinity reagents for basic and clinical research. Specific Abs identified from such libraries are often screened as fragments antigen binding (Fabs) produced in bacteria, and those with desired biochemical characteristics are reformatted for production as full‐length immunoglobulin G (IgG) in mammalian cells. The conversion of Fabs to IgGs is a cumbersome and often rate‐limiting step in the development of Abs. Moreover, biochemical properties required for lead IgG development are not always shared by the Fabs, and these issues are not uncovered until a significant effort has been spent on Abs that ultimately will not be useful. Thus, there is a need for simple and rapid techniques to convert phage‐displayed Fabs to IgGs at an early stage of the Ab screening process. We report the generation of a highly diverse phage‐displayed synthetic single‐chain Fab (scFab) library, in which the light and heavy chains were tethered with an optimized linker. Following selection, pools of scFabs were converted to single‐chain IgGs (scIgGs) en masse, enabling facile screening of hundreds of phage‐derived scIgGs. We show that this approach can be used to rapidly screen for and select scIgGs that target cell‐surface receptors, and scIgGs behave the same as conventional IgGs.     

Analysis and modeling of the variable region of camelid single-domain antibodies

Camelids have a special type of Ab, known as heavy chain Abs, which are devoid of classical Ab light chains. Relative to classical Abs, camelid heavy chain Abs (cAbs) have comparable immunogenicity, Ag recognition diversity and binding affinities, higher stability and solubility, and better manufacturability, making them promising candidates for alternate therapeutic scaffolds. Rational engineering of cAbs to improve therapeutic function requires knowledge of the differences of sequence and structural features between cAbs and classical Abs. In this study, amino acid sequences of 27 cAb variable regions (V(H)H) were aligned with the respective regions of 54 classical Abs to detect amino acid differences, enabling automatic identification of cAb V(H)H CDRs. CDR analysis revealed that the H1 often (and sometimes the H2) adopts diverse conformations not classifiable by established canonical rules. Also, although the cAb H3 is much longer than classical H3 loops, it often contains common structural motifs and sometimes a disulfide bond to the H1. Leveraging these observations, we created a Monte Carlo-based cAb V(H)H structural modeling tool, where the CDR H1 and H2 loops exhibited a median root-mean-square deviation to natives of 3.1 and 1.5 ?, respectively. The protocol generated 8-12, 14-16, and 16-24 residue H3 loops with a median root-mean-square deviation to natives of 5.7, 4.5, and 6.8 ?, respectively. The large deviation of the predicted loops underscores the challenge in modeling such long loops. cAb V(H)H homology models can provide structural insights into interaction mechanisms to enable development of novel Abs for therapeutic and biotechnological use.     

Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions

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.     

Grafting of "abbreviated" complementarity-determining regions containing specificity-determining residues essential for ligand contact to engineer a less immunogenic humanized monoclonal antibody

Murine mAb COL-1 reacts with carcinoembryonic Ag (CEA), expressed on a wide range of human carcinomas. In preclinical studies in animals and clinical trials in patients, murine COL-1 showed excellent tumor localization. To circumvent the problem of immunogenicity of the murine Ab in patients, a humanized COL-1 (HuCOL-1) was generated by grafting the complementarity-determining regions (CDRs) of COL-1 onto the frameworks of the variable light and variable heavy regions of human mAbs. To minimize anti-V region responses, a variant of HuCOL-1 was generated by grafting onto the human frameworks only the "abbreviated" CDRs, the stretches of CDR residues that contain the specificity-determining residues that are essential for the surface complementarity of the Ab and its ligand. In competition RIAs, the recombinant variant completely inhibited the binding of radiolabeled murine and humanized COL-1 to CEA. The HuCOL-1 and its variant showed no difference in their binding ability to the CEA expressed on the surface of a CEA-transduced tumor cell line. Compared with HuCOL-1, the HuCOL-1 variant showed lower reactivity to patients' sera carrying anti-V region Abs to COL-1. The final variant of the HuCOL-1, which retains its Ag-binding reactivity and shows significantly lower serum reactivity than that of the parental Ab, can serve as a prototype for the development of a potentially useful clinical reagent.     

Generation of humanized anti-DNA hydrolyzing catalytic antibodies by complementarity determining region grafting

Humanization of nonhuman antibodies (Abs) has been carried out mainly for Abs which bind to antigen without catalytic activity. Here we report humanization of mouse-originated 3D8 (m3D8) mAbs (scFv, VH, and VL) with DNA hydrolyzing catalytic activity by grafting the complementarity determining regions (CDRs) into the corresponding regions of a fixed human framework scaffold, generating humanized 3D8 (h3D8) Abs in the respective format of scFv, VH, and VL. h3D8 Abs retained comparable DNA binding and hydrolyzing activities to those of the corresponding m3D8 Abs. Our results suggest that CDRs of anti-DNA hydrolyzing Abs might possess the intrinsic properties of DNA binding and hydrolyzing activities.     

High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold

Phage-displayed synthetic antibody libraries were built on a single human framework by introducing synthetic diversity at solvent-exposed positions within the heavy chain complementarity-determining regions (CDRs). The design strategy of mimicking natural diversity using tailored codons had been validated previously with scFv libraries, which produced antibodies that bound to antigen, murine vascular endothelial growth factor (mVEGF), with affinities in the 100nM range. To improve library performance, we constructed monovalent and bivalent antigen-binding fragment (Fab) libraries, and explored different CDR-H3 diversities by varying the amino acid composition and CDR length. A Fab with sub-nanomolar affinity for mVEGF was obtained from a library with CDR-H3 diversity designed to contain all 20 naturally occurring amino acids. We then expanded the library by increasing the variability of CDR-H3 length and using tailored codons that mimicked the amino acid composition of natural CDR-H3 sequences. The library was tested against a panel of 13 protein antigens and high-affinity Fabs were obtained for most antigens. Furthermore, the heavy chain of an anti-mVEGF clone was recombined with a library of light chain CDRs, and the affinity was improved from low nanomolar to low picomolar. The results demonstrated that high-affinity human antibodies can be generated from libraries with completely synthetic CDRs displayed on a single scaffold.     

Isotype can affect the fine specificity of an antibody for a polysaccharide antigen

Ab specificity is determined by V region sequence. The murine Mab 18B7 (IgG1) binds to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan and produces annular immunofluorescence (IF) on yeast cells. The heavy and light V regions of 18B7 were expressed with the human C regions micro, gamma 1, gamma 2, gamma 3, gamma 4, and alpha1, and the specificity and binding properties of these mouse-human chimeric (ch) Abs was determined. The chIgG1, chIgG2, chIgG4, and the chIgA produced annular IF, whereas the IgM and IgG3 produced punctate IF, despite identical V region sequences. Competition experiments with murine Abs that competed with mAb 18B7 and binding assays to peptide mimetics of glucuronoxylomannan provided additional evidence for altered specificity in some of the ch Abs. Expression of 18B7 heavy V region with murine micro C region produced IgM with a punctate IF, indicating that a change in fine specificity also accompanied the change from murine IgG1 to IgM. Our results show that Ab fine specificity can be a function of isotype. This phenomenon may be most apparent for Abs that bind to Ag with repeating epitopes, such as polysaccharides, where the quarternary structure of the Ag-Ab complex may be influenced by such constraints as Fab-Fab angles, Fc-Fc interactions, Ab size, and solvent accessibility to exposed surfaces. Alterations in Ab fine specificity following isotype change could have important implications for current concepts on the generation of secondary Ab responses to certain Ags and for the isotype preference observed in Abs to polysaccharides.     

Characterization of the antibody response against NeuGcGM3 ganglioside elicited in non-small cell lung cancer patients immunized with an anti-idiotype antibody

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.     

The potency of erythropoietin-mimic antibodies correlates inversely with affinity

Preclinical animal studies have shown that Ab12.6, an agonistic human Ab targeting the erythropoietin receptor (EPOR), exhibits several potential dosing and safety features that make it an attractive clinical candidate for the treatment of anemia. Ab12.6 was derived by yeast display affinity maturation of parental Ab12, a strategy initially intended to improve off-rate and affinity for EPOR, thereby enhancing erythropoietic activity. Analysis of full-length IgGs derived from yeast clones identified sequences within Ab12 CDRH2 that independently influenced both affinity and potency. The Ab12.6 derivative displayed improved in vitro potency and in vivo efficacy, although its binding affinity to the EPOR was lower than that of the parent Ab12. Additional Ab12 derivatives also exhibited an inverse correlation between affinity and potency. These results suggest that for this class of agonistic Abs, faster off-rates may permit continuous receptor stimulation and more efficient erythropoiesis.     

The autoreactivity of anti-phosphorylcholine antibodies for atherosclerosis-associated neo-antigens and apoptotic cells

Abs specific for phosphorylcholine (PC) are known to contribute to the immune defense against a variety of microbial infections. To assess for other types of binding interactions, we performed surveys of anti-PC Abs of diverse biologic origins and structural diversity and demonstrated a common autoreactivity for oxidatively modified low density lipoprotein and other oxidation-specific structures containing PC-Ags. We also found that cells undergoing apoptosis sequentially express a range of oxidation-specific neo-self PC determinants. Whereas natural Abs to PC recognized cells at early stages of apoptosis, by contrast, an IgG anti-PC Ab, representative of a T cell-dependent response, recognized PC determinants primarily associated with late stages of apoptosis. Cumulatively, these results demonstrate a fundamental paradigm in which Abs from both the innate and the T cell-dependent tiers of the B cell compartment recognize a minimal molecular motif arrayed both on microbes and as neo-self Ags linked to atherosclerosis and autoimmune disease.     

Structural effects of framework mutations on a humanized anti-lysozyme antibody.

A humanized version of the mouse anti-lysozyme Ab D1.3 was previously constructed as an Fv fragment and its structure was crystallographically determined in the free form and in complex with lysozyme. Here we report five new crystal structures of single-amino acid substitution mutants of the humanized Fv fragment, four of which were determined as Fv-lysozyme complexes. The crystals were isomorphous with the parent forms, and were refined to free R values of 28-31% at resolutions of 2.7-2.9 A. Residue 27 in other Abs has been implicated in stabilizing the conformation of the first complementarity-determining region (CDR) of the H chain, residues 31-35. We find that a Phe-to-Ser mutation at 27 alters the conformation of immediately adjacent residues, but this change is only weakly transmitted to Ag binding residues in the nearby CDR. Residue 71 of the H chain has been proposed to control the relative disposition of H chain CDRs 1 and 2, based on the bulk of its side chain. However, in structures we determined with Val, Ala, or Arg substituted in place of Lys at position 71, no significant change in the conformation of CDRs 1 and 2 was observed.     

Crystal structure of a hydrophobic immunodominant antigenic site on hepatitis C virus core protein complexed to monoclonal antibody 19D9D6

The first crystal structure of a complex between a hepatitis C virus (HCV) core protein-derived peptide (residues 13-40) and the Ab fragment of a murine mAb (19D9D6) has been solved, allowing determination of the recognized epitope and elucidation of its conformation. This Ab, raised against the first 120 residues of the core protein, recognizes core particles and strongly competes with anticore human Abs, suggesting that it is highly representative of the human anti-HCV core response. Its epitope lies within the first 45 aa of the protein, the major antigenic segment of core recognized both by murine and human Abs. Surprisingly, the recognized epitope (29-37: QIVGGVYLL) has an unusual preponderance of hydrophobic residues, some of which are buried in a small hydrophobic core in the nuclear magnetic resonance structure of the peptide (2-45) in solution, suggesting that the Ab may induce a structural rearrangement upon recognition. The flexibility may reside entirely within the Ag, since the Fab'-peptide complex structure at 2.34 A shows that the Ab binding site is hardly perturbed by complexation. Given that the recognized residues are unlikely to be solvent exposed, we are left with the interesting possibility that Ab-core interactions may take place in a nonaqueous environment.     

Characterization of the anti-tissue transglutaminase antibody response in nonobese diabetic mice

Type 1 diabetes mellitus is an autoimmune disorder characterized by destruction of insulin-producing pancreatic beta cells by T lymphocytes. In nonobese diabetic (NOD) mice, a role has been hypothesized for dietary gluten proteins in the onset of diabetes, and because gluten dependence is the major feature of celiac disease, together with production of Abs to the autoantigen tissue transglutaminase (tTG), we looked for the presence of anti-tTG Abs in the serum of NOD mice and, to establish their origin, analyzed the Ab repertoire of NOD mice using phage display Ab libraries. We found significant levels of serum anti-tTG Abs and were able to isolate single-chain Ab fragments to mouse tTG mainly from the Ab libraries made from intestinal lymphocytes and to a lesser extent from splenocytes. Data from NOD mice on a gluten-free diet suggest that the anti-tTG response is not gluten-dependent. The intestinal Ab response to tTG is a feature of NOD mice, but the underlying mechanisms remain obscure.