Repertoire Cloning Research Articles

The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA.

Background: Botulinum neurotoxins (BoNTs) comprise seven agreed-on serotypes, A through G. In 2014, a novel chimeric neurotoxin produced by clostridial strain IBCA10-7060 was reported as BoNT/H, with subsequent names of BoNT/FA or BoNT/HA based on sequence homology of the N-terminus to BoNT/F, the C-terminus to BoNT/A and neutralization studies. The purpose of this study was to define the immunologic identity of the novel BoNT. Methods: monoclonal antibodies (mAbs) to the novel BoNT/H N-terminus were generated by antibody repertoire cloning and yeast display after immunization with BoNT/H LC-HN or BoNT/F LC-HN. Results: 21 unique BoNT/H LC-HN mAbs were obtained; 15 from the BoNT/H LC-HN immunized library (KD 0.78 nM to 182 nM) and six from the BoNT/F-immunized libraries (KD 20.5 nM to 1490 nM). A total of 15 of 21 mAbs also bound catalytically inactive BoNT/H holotoxin. The mAbs bound nine non-overlapping epitopes on the BoNT/H LC-HN. None of the mAbs showed binding to BoNT serotypes A-G, nor any of the seven subtypes of BoNT/F, except for one mAb that weakly bound BoNT/F5. Conclusions: The results, combined with the chimeric structure and neutralization by anti-A, but not anti-F antitoxin indicate that immunologically the novel BoNT is BoNT/HA. This determination has significant implications for existing countermeasures and potential vulnerabilities.

Evidence of Long-Lasting Anti-CD19 Activity of Engrafted CD19 Chimeric Antigen Receptor Modified T Cells in a Phase I Study Targeting Pediatrics with Acute Lymphoblastic Leukemia

Introduction: 90% of relapse/refractory B cell acute lymphatic leukemia (R/R B-ALL) patients can achieve complete remission (CR) after CD19-targeting chimeric antigen receptor T (CAR-T) cell therapy. However, around 50% of them relapse in one year. Persistent CAR-T cell engraftment is considered as the key to remain durable remission. Here we initiated a Phase I study to treat pediatric B-ALL patients using CD19 CAR-T cell. After treatment, CD19+ minimal residual disease (MRD) and CAR-T engraftment were detected monthly aiming to evaluate the anti-CD19 activity of long-term engrafted CAR-T cell clones.Patients and Methods: From July 2016 to December 2017, a total of 10 pediatric patients with CD19+ R/R B-ALL were treated. The median age was 6 (range 3-13 years), none of them has prior hematopoietic stem cell transplant (HSCT). All patients have experienced at least three courses of chemotherapy prior CAR-T infusion (median 4, range 3-10 times). All patients received a standard fludarabine and cyclophosphamide preconditioning regiment, followed by a CAR-T infusion with a median number of 0.5 (0.3-1.58) x 106 cells/kg. We used a 2nd generation CAR including a 4-1BB intracellular domain and a truncated EGFR sequence which can be used to identify and select CAR+ cells. For the first month after infusion, CAR-T engraftment, cytokines profiles, MRD, complete blood counting and other clinical lab evaluation were performed frequently. Afterwards, levels of CAR-T cells and residual leukemia cells in bone marrow (BM) were analyzed once per month. CAR-T engraftment was measured by both flow cytometry and quantitative PCR (qPCR); percentages of MRD were determined by flow cytometry; cytokine profiles were performed by LEGENDplexTM Human inflammation panel from Biolegend company.Result: The median percentage of pre-treatment BM blasts was 59.2% (7.31%-86.2%), excluding one patient only with brain infiltration of leukemia cells (0% BM blasts). Among the ten pts, eight of them reached MRD negative complete remission (CR) on day 28 post-infusion, except one patient who had no obvious response and another patient who withdrawn from the trial at day 5 due to rapid disease progression. All pts experienced cytokine release syndrome (CRS), six pts with grade 1 or 2 CRS, three with grade 3, and one with grade 4; grade 3-4 neurotoxicity also occurred in three patients. Toxicities were managed by supportive care ± tocilizumab and/ or dexamethasone depend on physicians' instruction. The dynamics of cytokine release (TNFα, IFNγ, and IL6) reached to the peak around day +4 to day +7. The peak of CD19 CAR T cell expansion was around day +7 to day +10, and rapidly decreased within 5 to 10 days. The median observation period for 8 CR patients was 261 days (149-733). 4 patients (50%) relapsed, all of them (100%) were associated with loss of cell-surface expression of CD19, including one lineage switch from ALL to myeloid leukemia. Pre-treatment tumor burdens were highly correlated with relapse events. The patient with only brain infiltrating blasts achieved CR and remains for 279 days. The other three patients remaining in remission state have been following up for 243, 619 and 733 days, respectively. Every month after infusion, peripheral and marrow blood were collected to detect the reemergence of residual leukemia cells and persistence of CAR-T engraftment. As Figure 1 shown, in the first treated patient who had the longest remission time (733 days), emergence of CD19+ residual leukemic cells were detected several times during the observation period which were corresponded with reduction of CAR-T cell engraftment. Notably, without any further treatment, anti-CD19 activity was demonstrated obviously by the clearance of CD19+ cells and increase level of CAR-T cell engraftment which reached to a new peak on 20 month after infusion.Conclusion: Our study validates the safety and efficacy of CAR-T cell therapy targeting CD19 in ten pediatric patients, which encourage us to explore more patients with relapse/refractory B-ALL in the future. Importantly, anti-leukemia activity lasting as long as at least two years demonstrates maintenance of durable remission by only CAR-T therapy even without further allo-HSCT. Deep TCR sequence which attempts to identify the TCR repertoire and CAR-T cell cloning which attempts to get immortalized CAR+ T cell line are currently performed in the lab. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Association of the characteristics of B- and T-cell repertoires with papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Complementarity-determining region 3 (CDR3) of B-cell receptors (BCRs) and T-cell receptors are the major site of antigen recognition, which determines a unique clone type, and are considered to be the representative of the disease. In the present study, high-throughput sequencing was used to analyze the association of characteristics of the BCR immunoglobulin heavy chain (IGH) and the T-cell receptor β chain (TRB) CDR3 genes in PTC and corresponding pericarcinous tissues from patients. A difference of CDR3 length distributions of total IGH CDR3 sequences between the two groups was revealed. IGHV3-11/IGHJ6, TRBV2/TRBJ1-2 and TRBV2/TRBJ1-1 may be biomarkers for the development of PTC. Furthermore, it was revealed that the extent of the common clonotype expressions at the amino acid level was slightly higher compared with the nucleotide level. The Shannon entropy demonstrated a diversity reduction in PTC compared with the pericarcinous group, and the highly expended clone (HEC) expression of PTC was higher compared with that of the corresponding pericarcinous group. Additionally, the highest clone frequency percentage of IGH and TRB was at 0.1–1.0% degree of expansion, as HEC expression was higher in PTC compared with the matched group. There was no shared clone of HECs in the two groups either at the amino acid level or at the nucleotide expression level. The differential expression of CDR3 sequences of PTC have been identified in the present study. Further research is required for assessing the immune repertoire size, diversity, cloning tracking and finding public clones of T-cell and B-cell populations in the development of PTC.

A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes.

Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (KD) of 4.06 × 10−9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10−8 M to 1.47×10−12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism.

ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 1. Structural and functional characterization in vitro.

Acquired thrombotic thrombocytopenia purpura (TTP) is a life-threatening illness caused by autoantibodies that decrease the activity of ADAMTS13, the von Willebrand factor-cleaving protease. Despite efficacy of plasma exchange, mortality remains high and relapse is common. Improved therapies may come from understanding the diversity of pathogenic autoantibodies on a molecular or genetic level. Cloning comprehensive repertoires of patient autoantibodies can provide the necessary tools for studying immunobiology of disease and developing animal models. Anti-ADAMTS13 antibodies were cloned from four patients with acquired TTP using phage display and characterized with respect to genetic origin, inhibition of ADAMTS13 proteolytic activity, and epitope specificity. Anti-idiotypic antisera raised to a subset of autoantibodies enabled comparison of their relatedness to each other and to polyclonal immunoglobulin (Ig)G in patient plasma. Fifty-one unique antibodies were isolated comprising epitope specificities resembling the diversity found in circulating patient IgG. Antibodies directed both to the amino terminal domains and to those requiring the ADAMTS13 cysteine-rich/spacer region for binding inhibited proteolytic activity, while those solely targeting carboxy-terminal domains were noninhibitory. Anti-idiotypic antisera raised to a subset of antibody clones crossreacted with and reduced the inhibitory activity of polyclonal IgG from a set of unrelated patients. Anti-ADAMTS13 autoantibodies isolated by repertoire cloning display the diversity of epitope specificities found in patient plasma and provide tools for developing animal models of acquired TTP. Shared idiotypes of inhibitory clones with circulating IgG from multiple patients suggest common features of pathogenic autoantibodies that could be exploited for developing more targeted therapies.

A System for Repertoire Cloning and Phage Display of Murine and Leporid Antibody Fragments

Even though rabbit antibodies (Abs) are known to exceed murine Abs with respect to specificity, affinity, and stability, cloned leporid immune repertoires have been rarely considered in recombinant Ab preparation for environmental analysis. We have developed a set of four tet(p/o)-based phasmid vectors that allow the efficient cloning of both murine and leporid Ab repertoires. These vectors differ in the design of the cloning sites, choice of signal peptides, and antibiotic selection markers. A set of 39 primer oligodeoxynucleotides has been developed for the PCR amplification of rabbit Ab genes, representing the most exhaustive coverage of the leporid immune repertoire described so far. The atrazine-specific murine Fab fragment K411B and a cloned V-gene repertoire from sulfonamide-immunized rabbits were used to compare these phasmids with respect to expression of Fab fragments, phagemid titers, and number of Fab displaying phagemid particles. Our results show that the ratio of recombinant phagemids could be increased up to 65% of total phage titer by utilizing the appropriate phasmid. Based on this system, the selection of two sulfonamide-specific rabbit Abs, SA2 23 and SA2 90, was accomplished after a single phagemid panning round.

Exceptional Amyloid β Peptide Hydrolyzing Activity of Nonphysiological Immunoglobulin Variable Domain Scaffolds

Nucleophilic sites in the paired variable domains of the light and heavy chains (VL and VH domains) of Ig can catalyze peptide bond hydrolysis. Amyloid beta (Abeta)-binding Igs are under consideration for immunotherapy of Alzheimer disease. We searched for Abeta-hydrolyzing human IgV domains (IgVs) in a library containing a majority of single chain Fv clones mimicking physiological VL-VH-combining sites and minority IgV populations with nonphysiological structures generated by cloning errors. Random screening and covalent selection of phage-displayed IgVs with an electrophilic Abeta analog identified rare IgVs that hydrolyzed Abeta mainly at His14-Gln15. Inhibition of IgV catalysis and irreversible binding by an electrophilic hapten suggested a nucleophilic catalytic mechanism. Structural analysis indicated that the catalytic IgVs are nonphysiological structures, a two domain heterodimeric VL (IgVL2-t) and single domain VL clones with aberrant polypeptide tags (IgVL-t'). The IgVs hydrolyzed Abeta at rates superior to naturally occurring Igs by 3-4 orders of magnitude. Forced pairing of the single domain VL with VH or VL domains resulted in reduced Abeta hydrolysis, suggesting catalysis by the unpaired VL domain.Angstrom level amino acid displacements evident in molecular models of the two domain and unpaired VL domain clones explain alterations of catalytic activity. In view of their superior catalytic activity, the VL domain IgVs may help attain clearance of medically important antigens more efficiently than natural Igs.

Recombinant Human IgG antibodies against Human Cytomegalovirus

To study the passive immunization with human monoclonal antibodies as for prophylaxis of human cytomegalovirus (HCMV) infection. Fab monoclonal antibodies to HCMV were recovered by repertoire cloning of mRNA from a HCMV infected individual. Antigen binding specificity, CDR sequence of V(H) and V(L) and neutralizing activity on HCMV AD169 stain were analyzed in vitro. The light and heavy chain Fd fragment genes of Fab antibodies were further cloned into a recombinant baculovirus expression vector pAC-kappa-Fc to express intact IgG. Secreted products were purified with affinity chromatography using protein G. SDS-PAGE and Western blot confirmed the expression of the intact IgG. Immuno-blotting and -precipitation were used to identify HCMV proteins. One Fab monoclonal antibody recognized a conformational HCMV protein. IgG antibodies can neutralize the HCMV AD169 strain efficiently at a titer of 2.5 microg/mL and may prove valuable for passive immunoprophylaxis against HCMV infection in humans.

Humanized Monoclonal Antibodies Derived from Chimpanzee Fabs Protect against Japanese Encephalitis Virus In Vitro and In Vivo

Japanese encephalitis virus (JEV)-specific Fab antibodies were recovered by repertoire cloning from chimpanzees initially immunized with inactivated JE-VAX and then boosted with attenuated JEV SA14-14-2. From a panel of 11 Fabs recovered by different panning strategies, three highly potent neutralizing antibodies, termed Fabs A3, B2, and E3, which recognized spatially separated regions on the virion, were identified. These antibodies reacted with epitopes in different domains: the major determinant for Fab A3 was Lys(179) (domain I), that for Fab B2 was Ile(126) (domain II), and that for Fab E3 was Gly(302) (domain III) in the envelope protein, suggesting that these antibodies neutralize the virus by different mechanisms. Potent neutralizing antibodies reacted with a low number of binding sites available on the virion. These three Fabs and derived humanized monoclonal antibodies (MAbs) exhibited high neutralizing activities against a broad spectrum of JEV genotype strains. Demonstration of antibody-mediated protection of JEV infection in vivo is provided using the mouse encephalitis model. MAb B2 was most potent, with a 50% protective dose (ED(50)) of 0.84 microg, followed by MAb A3 (ED(50) of 5.8 microg) and then MAb E3 (ED(50) of 24.7 microg) for a 4-week-old mouse. Administration of 200 microg/mouse of MAb B2 1 day after otherwise lethal JEV infection protected 50% of mice and significantly prolonged the average survival time compared to that of mice in the unprotected group, suggesting a therapeutic potential for use of MAb B2 in humans.

The Importance of Human FcγRI in Mediating Protection to Malaria

The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcγRI. This important finding documents the capacity of FcγRI to mediate potent antimalaria immunity and supports the development of FcγRI-directed therapy for human malaria.

Codon insertion and deletion functions as a somatic diversification mechanism in human antibody repertoires

It has been suggested that codon insertion and/or deletion may represent a mechanism that, along with hypermutation, contributes to the affinity maturation of antibodies. We used repertoire cloning to examine human antibodies directed against 3 carbohydrate antigens and 1 protein antigen for the presence of such modifications. We find that both the insertion and deletion of codons occur frequently in antigen-specific responses following vaccination. Codon insertions and deletions were observed most often in the complementarity determining regions, and less frequently in the framework regions, of VH, Vκ, and Vλ gene segments, and involved motifs known to be preferred targets of somatic hypermutation. Clonal lineage analysis shows that these events occur through out the course of the somatic maturation of individual antibody clones. We also determined that these alterations of paratope structure have varying effects on the relative affinity of the binding site for its cognate antigen.ReviewersThis article was reviewed by Mark Shlomchik, Deborah Dunn-Walters (nominated by Dr. Andrew Macpherson), and Rachel M. Gerstein.Open peer reviewReviewed by Mark Shlomchik, Deborah Dunn-Walters (nominated by Dr. Andrew Macpherson), and Rachel M. Gerstein. For the full reviews, please go to the Reviewers' comments section.

Antibody responses against wild-type yellow fever virus and the 17D vaccine strain: characterization with human monoclonal antibody fragments and neutralization escape variants

Human monoclonal antibody fragments neutralizing wild-type and vaccine strains of yellow fever (YF) virus (genotypes West Africa I + II, East/Central Africa, 17D-204-WHO) were generated by repertoire cloning from YF patients. Analysis of virus escape variants identified amino acid (aa) 71 in domain II of the envelope glycoprotein (E) as the most critical residue for neutralization, with aa 153–155 in domain I contributing to the epitope. These data confirm the previous mapping of YFV neutralizing epitopes using mouse monoclonal antibodies but suggest that a conformational epitope could be formed by amino acids from domains I and II opposing each other in the dimeric form of the E protein. While the sera of the YF patients showed up to 10-fold reduced neutralizing activity against the 17D escape variants, sera from 17D vaccinees retained their neutralizing titers. Mutations in this major neutralizing epitope of YFV thus do not seem to carry the risk of immune escape in persons immunized with the YFV-17D vaccine.

Platelet integrin α2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display

Antibodies are a powerful tool for structure/function studies of platelet proteins. However, classic immunisation frequently elicits antibody responses against domains of minor functional interest. Robust strategies to generate antibodies against defined domains would be of significant interest in post-genome research. In this study, we report a new strategy using a combination of DNA vaccination and V gene phage display that allows the rapid generation of domain specific single-chain Fv antibodies (scFvs). This system was validated using the I-domain of alpha2 integrin as a model. The alpha2beta1 integrin, which is expressed on many cell types, is the dominant collagen attachment receptor on platelets, functioning in close interplay with the collagen signalling receptor glycoprotein VI. A novel set of I-domain specific antibodies was obtained by a DNA vaccination/V gene repertoire cloning approach. Mice were first immunised with a DNA vaccine in which the alpha2 I-domain is expressed as a fusion protein with fragment C of tetanus toxoid (FrC-TT). Then the heavy and kappa light chain variable gene repertoires were rescued from immune splenocytes using antibody phage display. A total of four alpha2 I-domain specific scFvs were isolated by selection on recombinant I-domain or native platelet alpha2beta1 integrin. Characterisation of the scFvs indicated that they recognised distinct epitopes that had profound differences in accessibility between native and recombinant I-domain. Our data suggest DNA immunisation and phage display represent versatile alternatives to protein immunisation and hybridoma-fusion techniques for the isolation of recombinant antibody reagents. This approach will be particularly useful for the generation of domain or splice-variant specific antibodies that recognise native protein.

Chimpanzee Fab Fragments and a Derived Humanized Immunoglobulin G1 Antibody That Efficiently Cross-Neutralize Dengue Type 1 and Type 2 Viruses

Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative to vaccines for prevention of illness caused by dengue viruses (DENV) and other flaviviruses, including the West Nile virus. In a previous study, repertoire cloning to recover Fab fragments from bone marrow mRNA of chimpanzees infected with all four DENV serotypes (dengue virus serotype 1 [DENV-1] to DENV-4) was described. In that study, a humanized immunoglobulin G1 (IgG1) antibody that efficiently neutralized DENV-4 was recovered and characterized. In this study, the phage library constructed from the chimpanzees was used to recover Fab antibodies against the other three DENV serotypes. Serotype-specific neutralizing Fabs were not identified. Instead, we recovered DENV-neutralizing Fabs that specifically precipitated the envelope protein and were cross-reactive with all four DENV serotypes. Three of the Fabs competed with each other for binding to DENV-1 and DENV-2, although each of these Fabs contained a distinct complementarity determining region 3 (CDR3)-H sequence. Fabs that shared an identical or nearly identical CDR3-H sequences cross-neutralized DENV-1 and DENV-2 at a similar high 50% plaque reduction neutralization test (PRNT(50)) titer, ranging from 0.26 to 1.33 microg/ml, and neutralized DENV-3 and DENV-4 but at a titer 10- to 20-fold lower. One of these Fabs, 1A5, also neutralized the West Nile virus most efficiently among other flaviviruses tested. Fab 1A5 was converted to a full-length antibody in combination with human sequences for production in mammalian CHO cells. Humanized IgG1 1A5 proved to be as efficient as Fab 1A5 for cross-neutralization of DENV-1 and DENV-2 at a titer of 0.48 and 0.95 microg/ml, respectively. IgG1 1A5 also neutralized DENV-3, DENV-4, and the West Nile virus at a PRNT(50) titer of approximately 3.2 to 4.2 microg/ml. This humanized antibody represents an attractive candidate for further development of immunoprophylaxis against DENV and perhaps other flavivirus-associated diseases.

Identification of chimpanzee Fab fragments by repertoire cloning and production of a full-length humanized immunoglobulin G1 antibody that is highly efficient for neutralization of dengue type 4 virus.

A safe and effective dengue vaccine is still not available. Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative for the prevention of dengue virus infection. Fab monoclonal antibodies to dengue type 4 virus (DENV-4) were recovered by repertoire cloning of bone marrow mRNAs from an immune chimpanzee and analyzed for antigen binding specificity, V(H) and V(L) sequences, and neutralizing activity against DENV-4 in vitro. Fabs 5A7, 3C1, 3E4, and 7G4 were isolated from a library constructed from a chimpanzee following intrahepatic transfection with infectious DENV-4 RNA. Fabs 5H2 and 5D9, which had nearly identical V(H) sequences but varied in their V(L) sequences, were recovered from a library constructed from the same chimpanzee after superinfection with a mixture of DENV-1, DENV-2, and DENV-3. In radioimmunoprecipitation, Fab 5A7 precipitated only DENV-4 prM, and Fabs 3E4, 7G4, 5D9, and 5H2 precipitated DENV-4 E but little or no prM. Fab 3E4 and Fab 7G4 competed with each other for binding to DENV-4 in an enzyme-linked immunosorbent assay, as did Fab 3C1 and Fab 5A7. Fab 5H2 recognized an epitope on DENV-4 that was separate from the epitope(s) recognized by other Fabs. Both Fab 5H2 and Fab 5D9 neutralized DENV-4 efficiently with a titer of 0.24 to 0.58 micro g/ml by plaque reduction neutralization test (PRNT), whereas DENV-4-neutralizing activity of other Fabs was low or not detected. Fab 5H2 was converted to full-length immunoglobulin G1 (IgG1) by combining it with human sequences. The humanized chimpanzee antibody IgG1 5H2 produced in CHO cells neutralized DENV-4 strains from different geographical origins at a similar 50% plaque reduction (PRNT(50)) titer of 0.03 to 0.05 micro g/ml. The DENV-4 binding affinities were 0.42 nM for Fab 5H2 and 0.24 nM for full-length IgG1 5H2. Monoclonal antibody IgG1 5H2 may prove valuable for passive immunoprophylaxis against dengue virus in humans.

Natural anti-Fc εRI α autoantibodies may interfere with diagnostic tests for autoimmune urticaria

IgG autoantibodies against the α-chain of the high affinity IgE receptor are claimed to play a pathogenetic role in autoimmune urticaria. The best methods for detection of functional autoantibodies are currently the autologous serum skin test and the basophil histamine release assay. A simplified and feasible screening test would facilitate the diagnosis of autoimmune urticaria. Here we offer an explanation for the difficulties in establishing a screening test for autoantibodies directed against the α-chain of the high affinity IgE receptor in autoimmune urticaria. Identical autoantibodies in chronic urticaria patients and healthy donors belonging to the natural autoantibody repertoire were found by sequence analysis of anti- α-chain autoantibodies isolated by repertoire cloning from antibody libraries. These natural autoantibodies bound to the receptor and triggered histamine release but only if IgE was previously removed from the receptor. Diagnostic assays used for detection of antibodies directed against the IgE receptor may require signal comparison with and without the artificial removal of IgE, immune complexes, and complement in order to avoid false positive or negative results. After IgE removal diagnostic tests will detect natural autoantibodies against the high affinity IgE receptor regardless of whether they are pathogenic or not.

Molecular expression systems for anti-DNA antibodies—1

Molecular expression systems can be used to produce whole antibodies or antibody fragments. The properties of these expression products can be tested in assays of binding or pathogenicity. Expression systems can be used to produce large quantities of antibodies which are already well-characterized, to produce new antibodies by repertoire cloning, or to produce slight modifications in the sequences of antibodies by mutagenesis prior to expression. This paper reviews the ways in which these methods have been used to study the structure and function of human and murine anti-DNA antibodies. A consistent finding, from experiments using a range of different expression methods and antibodies, is that sequence motifs including arginine residues play a major role in binding to DNA. These motifs can be present on either the heavy or the light chain, but are particularly reported in V(H)CDR3.

Genetic analysis of autoantibodies in idiopathic thrombocytopenic purpura reveals evidence of clonal expansion and somatic mutation

Although idiopathic thrombocytopenic purpura (ITP) is the most common autoimmune hematologic disorder, little is known about the associated autoantibodies on a molecular level. Consequently, diagnostic assays and therapy for ITP lack specificity. To avoid technical limitations imposed by B-cell immortalization methods, we used repertoire cloning (Fab/phage display) to clone platelet autoantibodies and examine the relation between immunoglobulin (Ig) gene usage, clonality, and antigen specificity. Phage display libraries were constructed from splenocytes from 2 patients with chronic ITP, and competitive cell-surface selection was used to isolate several dozen unique IgG platelet-specific autoantibodies. Platelet-reactive Fabs in both patients were associated almost exclusively with rearrangements of a single Ig heavy-chain variable-region gene (V(H)3-30), despite an apparent diversity of antigen specificities. Comparative analysis of platelet-reactive Fab Ig gene rearrangements from each patient suggested that they evolved from a restricted number of B-cell clones through somatic mutation with high replacement-to-silent mutation ratios. Although V(H)3-30-encoded heavy chains were found with light chains encoded by several different Ig genes, molecular repairing experiments showed exquisite restriction on the specific heavy- and light-chain pairings that permitted platelet reactivity. Together, these data suggest that the development of platelet-reactive antibodies associated with ITP is driven by an encounter with diverse platelet antigens through the clonal expansion of B cells using genetically restricted and highly specific combinations of heavy- and light-chain gene products. The extraordinarily high usage of the V(H)3-30 heavy-chain gene in these patients has implications for the pathogenesis, diagnosis, and management of chronic ITP.

Recombinant monoclonal antibody technology

With the development of murine hybridoma technology over a quarter century ago, the ability to produce large quantities of well-characterized monoclonal antibody preparations revolutionized diagnostic and therapeutic medicine. For many applications in transfusion medicine, however, the production of serological reagents in mice has certain biological limitations relating to the difficulty in obtaining murine monoclonal antibodies specific for many human blood group antigens. Furthermore, for therapeutic purposes, the efficacy of murine-derived immunoglobulin preparations is limited by the induction of anti-mouse immune responses. Technical difficulties inherent in human hybridoma formation have led to novel molecular approaches that facilitate the isolation and production of human antibodies without the need for B-cell transformation, tissue culture, or even immunized individuals. These technologies, referred to as ‘repertoire cloning’ or ‘Fab/phage display’, involve the rapid cloning of immunoglobulin gene segments to create immune libraries from which antibodies with desired specificities can be selected. The use of such recombinant methods in transfusion medicine is anticipated to play an important role in the development and production of renewable supplies of low-cost reagents for diagnostic and therapeutic applications.

Conditional autoantibodies in urticaria patients: a unifying hypothesis.

Chronic urticaria may be characterized by conditional autoantibodies against the alpha-chain of the high-affinity receptor for IgE (FcepsilonRI). These autoantibodies are termed conditional as they only recognize unoccupied FcepsilonRI. The same conditional reactivity pattern has also been found in sera of atopic and normal healthy donors. Any condition resulting in accessibility of FcepsilonRI will render these autoantibodies anaphylactogenic. This finding offers a unifying hypothesis for the manifestation of different forms of urticaria. Non-immunologic triggers may thereby influence directly or indirectly the number of accessible FcepsilonRI allowing the conditional autoantibodies to induce urticaria symptoms.