Directed selection of MIP-1 alpha neutralizing CCR5 antibodies from a phage display human antibody library

The seven trans-membrane chemokine receptor CCR-5 is a coreceptor for macrophage tropic HIV-1 strains. CCR-5 responds to a number of chemokines, including macrophage inflammatory protein (MIP)-1 alpha. We describe the use of MIP-1 alpha in a biotin tyramine-mediated proximity selection to guide the selection of CCR-5-specific phage antibodies from a large phage display human library. Proximity based selections resulted in a population of antibodies recognizing CCR-5 on primary CD4+ lymphocytes, none of which blocked MIP-1 alpha binding to cells. The selected population of phage antibodies were subsequently used as guide molecules for a second phase of selection that was carried out in the absence of MIP-1 alpha. This generated a panel of CCR-5-binding antibodies, of which around 20% inhibited MIP-1 alpha binding to CD4+. The single chain Fvs (scFv) generated by this step-back selection procedure also inhibited MIP-1 alpha-mediated calcium signaling.     

The cortisol response to psychological stress in temporomandibular dysfunction

Recombinant antibody fragments can be produced in large quantities using bacterial expression systems and could potentially be useful for the generation of biofilters for the selective removal of viral particles from fluids. A human single chain-Fv antibody library, derived from synthetic repertoires of germ line VH-gene segments rearranged in vitro and paired to a single light chain (Nissim et al., 1994, EMBO J., 13, 692-698), has recently been used to isolate hundreds of different binding specificities by panning with antigen. Antibodies from this library typically have affinities in the 10(6)-10(7) M-1 range. Occasionally, better binders are isolated but at other times the affinities recovered are poor. In the latter situation binding cannot be detected with soluble antibodies, but only by high-avidity display of multiple copies of antibodies on phage. By panning with human cytomegalovirus (HCMV)-coated immunotubes, we have isolated a number of antibody clones from this library that bound to the antigen only if displayed on the filamentous phage, but not in soluble form. One of these clones was selected for an affinity maturation procedure, achieved by combinatorial mutagenesis of the complementarity determining region 3 (CDR3) of the antibody light chain, followed by selection of the resulting library for HCMV binding. By this means, we were able to isolate a number of binders, some of which exhibited specific HCMV binding in soluble form. The clone that gave the strongest ELISA signal was expressed in bacteria, purified in solution, characterised using a novel capture methodology with surface plasmon resonance detection on a BIAcore instrument and used for the production of an immunofilter for the removal of HCMV form human serum. The filter removed more than 99% of applied HCMV in 10 min circulation time, while the amount of HCMV retained non-specifically in a cartridge derivatised with a non-specific antibody was less than 10% under similar conditions.     

Construction of a semisynthetic antibody library using trinucleotide oligos

A semisynthetic antibody library composed of single chain Fv fragments (scFv) was constructed by replacing the heavy chain CDR3 region of a human scFv by a random sequence of eight amino acids using trinucleotide codons. After cloning into a phage display vector, an antibody library was generated with a complexity of 8 x 10(8) independent clones. The library was screened for binders to dinitrophenol, fluorescein isothiocyanate and 3-nitro-4-hydroxy-5-iodophenylacetic acid. scFv antibodies that specifically bound the antigen were obtained in each case.     

Comparative mapping of cloned human and murine antithyroglobulin antibodies: recognition by human antibodies of an immunodominant region

Antibodies (Ab) to thyroglobulin (Tg) are common in patients with autoimmune thyroid diseases, but it is currently unclear how Tg Ab are involved in the pathology of autoimmune thyroid disease. We have previously reported the isolation of immunoglobulin G (IgG)kappa and IgGlambda Fab from phage display combinatorial libraries from the cervical lymph node of a single Hashimoto's thyroiditis patient with a high anti-Tg titer. Sequence analysis of these Fab indicated a restricted heavy chain usage with a nonrestricted light chain usage, with Fab inhibiting the binding of patient Tg Ab by between 39% and 79%. Comparative mapping of nine each of these IgGkappa and IgGlambda Fab, and the patient serum from whom the Fab were derived, is described here, using a panel of 10 murine monoclonal antibodies (Mab) to human thyroglobulin (hTg). The Fab interacted principally with mAb defining the overlapping antigenic domains I and IV, previously characterized as the region recognized by the majority of patient serum Tg Ab. Tg Ab from serum of the patient from whom the Fab were derived were also directed at this region, suggesting that the Fab are representative of the Tg Ab present in this patient.     

Familial brain wave patterns: study of a 12-sib family

To identify peptide-specific antibodies which define sperm surface antigens, hybridomas were derived from the splenocytes of mice immunized with swollen human spermatozoa which had been subjected to limited proteolytic cleavage under reducing conditions prior to immunization. A total of 13.7% of the hybrid clones secreted antibodies which reacted with deglycosylated human seminal plasma glycoproteins when screened by an ELISA. A monoclonal antibody, designated mAb 4A8 sp., specifying a peptide epitope of human epididymal and a sperm surface glycoprotein was selected which inhibited human sperm-egg binding in a dose-dependent manner, and totally blocked sperm penetration in vitro. This inhibition did not result from an effect of the antibody on the motility of spermatozoa, nor was it due to premature induction of the acrosome reaction. Exclusion of oligosaccharide chains by chemical hydrolysis with trifluoromethane sulphonic acid (TFMS), enzymatic degradation and binding of lectins, did not abrogate the reactivity of mAb 4A8 to the cognate epitope whereas antibody binding was precluded upon digestion with proteolytic enzymes. In Western immunoblots of human seminal plasma glycoproteins, the antigen presented as a set of immunoreactive polypeptides, a major glycoprotein of M(r) 78 kDa and less prominent bands of M(r) 56 and 44 kDa. Immunocytochemical staining of a number of human reproductive and somatic tissues revealed strong immunostaining of the luminal epithelium of the epididymis as well as of spermatozoa in the lumen. Immunolocalization to the plasma membrane of ejaculated human spermatozoa was demonstrated by immunofluorescence, although on undigested spermatozoa the antigen epitope was less accessible. Upon capacitation the antigen persisted on the sperm surface and was present on the head of capacitated acrosome-intact spermatozoa. The pronounced peripheral immunostaining of the sperm head was accentuated after DTT/trypsin treatment, implicating the dynamic accessibility of the epitope on the plasma membrane of capacitated spermatozoa. It is suggested that the protein in question appears on the sperm membrane as a consequence of its modification in the epididymis (insertion and processing), and may be involved in the processes leading to sperm attachment and interaction with the human zona pellucida.     

Selection of recombinant anti-HuD Fab fragments from a phage display antibody library of a lung cancer patient with paraneoplastic encephalomyelitis

Antibodies against the HuD antigen expressed in small-cell lung cancer (SCLC) cross-react with proteins expressed in neurons of the central and peripheral nervous system and are associated with paraneoplastic encephalomyelitis and sensory neuropathy (PEM/SN). We isolated anti-HuD Fab fragments from an antibody phage display library that was constructed from mRNA of a metastatic lymph node from a patient with SCLC and Pem/SN. Fab GLN495 recognized HuD and other related proteins (HuC and Hel-N1, or Hu antigens) in immunoblots of these recombinant proteins and in immunohistochemical and Western blot analysis of SCLC and neurons. Fab GLN495 inhibited up to 75% of the anti-Hu antibodies of the patient from which it was derived, suggesting that recognizes a dominant epitope in the polyclonal anti-Hu antibody response. Fab GLN495 also competed with anti-Hu sera from most but not all patients with PEM/SN, indicating that the same epitope is recognized by a large subgroup of patients. Human monoclonal anti-HuD antibodies may be useful in diagnosis of HuD expressing tumors and in clarifying the autoimmune etiology of PEM/SN. This study, the first to demonstrate that tumor specific recombinant antibodies can be isolated from metastatic lymph node tissue, shows that this approach may be generally applicable to isolate human antibodies against tumor specific antigens.     

Generation of a panel of related human scFv antibodies with high affinities for human CEA

BACKGROUND: A human single chain Fv (scFv) specific for human carcinoembryonic antigen (CEA) has been isolated from a 2.0 x 10(9) phage display library from unimmunised human donors. The dissociation constant of the scFv has been measured by surface plasmon resonance (SPR) and found to be 7.7 x 10(-9) M, with an off-rate component of 6.2 x 10(-3) s-1. In order to investigate directly whether increased affinity leads to improved targeting of CEA-positive tumours, this scFv has been affinity matured by both targeted mutagenesis of the CDRs of heavy and light chains, and by light chain shuffling. STUDY DESIGN: A partial randomisation scheme, biased towards amino acids commonly found as somatic mutations of germline antibody sequences, was used for directed diversification of VH and VL CDR3s. Diversification of the entire VL region was also introduced by light chain shuffling of the parental anti-CEA scFv. Selection of the mutagenised repertoires was carried out to enrich for antibodies with a reduced koff. RESULTS: Sequencing the selected clones identified a number of amino acid changes in the VH CDR3, one of which gave a four-fold reduction in koff. Stringent selection of the light chain shuffled library resulted in several clones with a two- to three-fold reduction in koff. It has been possible to combine the selected changes from both mutagenesis approaches by using the mutagenised heavy chain and a light chain derived by shuffling to give a human scFv with a dissociation constant for human CEA of 6.0 x 10(-10) M. CONCLUSION: A panel of human anti-CEA scFvs has been generated with differing dissociation constants for antigen, which will allow the correlation between tumour targeting efficiency in relation to binding affinity to be assessed directly. The scFv panel will be valuable in the optimisation of human antibodies for immunotherapy.     

Generation and application of type-specific anti-heparan sulfate antibodies using phage display technology. Further evidence for heparan sulfate heterogeneity in the kidney

Detailed analysis of various heparan sulfate (HS) species is seriously hampered by a lack of appropriate tools, such as antibodies. We adopted phage display technology to generate anti-HS antibodies. A "single pot" semisynthetic human antibody phage display library was subjected to four rounds of selection on HS from bovine kidney using panning methodology. Three different phage clones expressing anti-HS single chain variable fragment antibodies (HS4C3, HS4D10, and HS3G8) were isolated, with an amino acid sequence of the complementarity-determining region 3 of GRRLKD (VH3 gene, DP-38), SLRMNGCGAHQ (VH3 gene, DP-42), and YYHYKVN (VH1 gene, DP-8), respectively. The antibodies react with HS and heparin, but not with DNA or other glycosaminoglycans. Kd values for HS are about 0.1 microM. The three antibodies react differently toward various HS preparations and show different staining patterns on rat kidney sections, indicating recognition of different HS molecules. This also holds for two described mouse anti-HS IgMs (JM403 and 10E4; both generated by conventional hybridoma technique) and indicates the presence of at least 5 different HS species in the kidney. O- and N-sulfation are important for binding of HS to HS4C3 and HS3G8. The three single chain antibodies, but not JM403, block a basic fibroblast growth factor binding site of HS. It is concluded that phage display technology presents a powerful technique to generate antibodies specific for HS epitopes. This is the first time this technique has been successfully applied to obtain directly antibodies to (poly)saccharides.     

Anti-bull sperm monoclonal antibodies: I. Identification of major antigenic domains of bull sperm and manifestation of interspecies cross-reactivity

The overall objective of this series of experiments is to generate immunological markers that may elucidate bull sperm surface changes in vitro. Here we report the initial experiments of the study, involving the production and characterization of monoclonal antibodies (mAbs) again bull sperm. BALB/c mice were immunized with phosphate-buffered saline (PBS)-washed whole bull sperm, and their spleen cells were fused with NS-1 myeloma cells in two separate cell fusion experiments, resulting in the generation of 15 mAbs. The mAbs were specific to antigens of either the posterior tail or the head regions of bull sperm and detected five major domains of antigen localization in the bull sperm (apical crescent, equatorial band, principal acrosomal, whole head, and posterior tail). Eleven of the 13 head-specific mAbs recognized intra-acrosomal antigens, whereas 2 mAbs recognized antigens that were localized in the plasma membrane. One mAb specific to the tail region was of the IgM class; the remaining 14 mAbs were of the IgG class. They were all sperm specific, with no cross-reactivity to bovine oocytes or to any of the 12 bovine somatic tissues tested. The mAbs were not species specific, however, because 11, 10, 2, and 1 of the 15 mAbs cross-reacted with sheep, pig, mouse, and human sperm, respectively. None of the mAbs cross-reacted with rooster sperm. The cognate antigens of the 11 tested mAbs were of testicular origin, but several of them showed enhanced binding to epididymal sperm. In western blot analysis, 3 of the 13 mAbs tested identified more than one protein band (40-200 kDa). Seven others recognized proteins of > or = 200 kDa, whereas three mAbs recognized no proteins.     

Diversity of the blocking effects of antisperm antibodies on fertilization in human and mouse

The blocking effects of complement-dependent sperm immobilizing antibodies in the sera of infertile women and monoclonal antisperm antibodies against humans and mice on fertilization were investigated. The hemizona assay (HZA) and sperm penetration assay (SPA) were used to study the inhibitory effects of sera from 22 infertile patients positive for sperm immobilizing antibodies. Use of these tests allowed us to differentiate whether the antibody blocked sperm-zona pellucida tight binding and/or sperm penetration into the ooplasm. The zona pellucida penetration assay (ZPA) was also used to study the effects of four monoclonal antibodies (mAbs) on human sperm penetration into the zona pellucida. Seven mAbs against murine spermatozoa were tested for their inhibitory effects on in-vitro fertilization (IVF) and HZA in mice. Of 22 patient sera with sperm immobilizing antibodies, 21 (95.5%) inhibited HZA attachment and penetration, whereas this did not occur in any of 13 patient sera without these antibodies. However, 19 of 22 (86.4%) patient sera with sperm immobilizing antibodies and eight of 13 (61.5%) patient sera without these antibodies inhibited the SPA. Two (2C6, 1G12) of four mAbs against human spermatozoa showed strong inhibitory effects in all the assays (HZA, ZPA and SPA). One mAb (3B10) did not inhibit HZA but blocked ZPA and SPA. Another mAb (H6-3C4) seemed to have no inhibitory effects on fertilization. Two (Vx 5 and Vx 8) of seven mAbs against murine spermatozoa inhibited IVF in mice but did not block mouse HZA. These findings suggest that antisperm antibodies block fertilization at specific stages. Some of them may inhibit sperm capacitation and thus prevent all processes of fertilization that follow. Some other antibodies may not affect capacitation and sperm binding to zona pellucida but inhibit the acrosome reaction, followed by the blocking of sperm penetration through zona pellucida and ooplasm.     

Can active immunization redirect an anti-IgE immune response?

We used as a template a mouse monoclonal antibody against IgE to isolate peptides from random peptide phage display libraries. Thereby, two types of peptides were isolated that corresponded to two different epitopes on the human IgE molecule. These peptides, also called mimotopes, seem to be a suitable tool in conjunction with carriers to induce an autoimmune response with a beneficial effect in humans, because the originally used template antibody is capable of neutralizing IgE, is nonanaphylactogenic, and inhibits IgE synthesis. The vaccination approach is further supported by the fact that we were capable of isolating anti-idiotypic antibodies from antibody phage display libraries against the template antibody. These anti-idiotypic antibodies were inhibited by both of the isolated IgE mimotopes. Thus, active vaccination with defined IgE mimotopes may represent a follow-up drug for the presently used anti-IgE antibodies.     

Regionalization and redistribution of membrane phospholipids and cholesterol in mouse spermatozoa during in vitro capacitation

Fracture-label, surface-replica, and routine freeze-fracture techniques were used in combination with phospholipase A2-colloidal gold (PLA2-CG) and filipin as probes to study changes in the distribution of phospholipids and cholesterol, respectively, in morphologically defined plasma membrane domains of mouse spermatozoa during in vitro capacitation. In noncapacitated spermatozoa, quantitative analysis revealed that the fractured plasma membrane overlying the equatorial segment carried the highest PLA2-CG labeling density. The next highest labeling densities were found in the anterior acrosome region and the post-acrosomal region. On the external surface of the plasma membrane revealed by surface replicas, a uniform distribution of PLA2-CG was confined mainly to the acrosomal region of the head. The plasma membrane of the sperm tail had a relatively low labeling density for PLA2-CG. In freeze-fracture replicas of filipin-treated spermatozoa, the labeling density of filipin/sterol complexes (FSCs) was high in the plasma membrane over the acrosomal region where the FSCs were uniformly distributed. The postacrosomal region was weakly labeled. After in vitro capacitation, the densities of PLA2-CG and FSCs were significantly reduced in the fractured plasma membrane of the sperm head and the middle piece of the tail. However, surface replicas revealed an increased PLA2-CG labeling on the external surface of the plasma membrane covering the postacrosomal region, the middle piece, and the principal piece. Another major change detected in capacitated spermatozoa was the presence of small aggregates and patches of elevated, membrane-associated particles on the surface-replicated plasma membrane in the upper portion of the postacrosomal domain. Here the PLA2-CG labeling density was found to be higher than in noncapacitated spermatozoa. These results provide new information with respect to the reorganization and redistribution of phospholipids in specific regions of the plasma membrane during capacitation and provide further support for the concept that removal or loss of antifusigenic sterol from the sperm plasma membrane constitutes an important step of the capacitation process.     

Chimeric macaque/human Fab molecules neutralize simian immunodeficiency virus

A collection of simian immunodeficiency virus (SIV) neutralizing recombinant Fab fragments was generated using the combinatorial antibody library approach. Functional antibody fragments efficiently expressed in Escherichia coli were identified only in the form of chimeric macaque heavy chain gamma 1 and human light chain kappa. The gamma 1 and kappa chains were derived from a clinically healthy long-term surviving SIVsm-infected cynomolgus macaque and from an asymptomatic HIV-2 seropositive individual, respectively. The combinatorial library was constructed on the surface of filamentous phage using the pComb3 phagemid vector and screened against purified SIVsm surface glycoprotein (gp148). Twelve chimeric clones reacting with the antigen were isolated. Six of these clones showed a pronounced neutralizing activity against SIVsm with effects at concentrations of 0.01-0.1 micrograms/ml. All neutralizing Fab fragments were clonally unrelated as demonstrated by nucleic acid sequencing. These potent neutralizing reagents will be used for prophylactic and therapeutic immune intervention of lentivirus infection in macaques and to map neutralizing determinants of SIV.     

Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris

We have applied the combinatorial immunoglobulin library and phage display technologies to generate monoclonal rabbit single-chain Fv (scFv) antibody fragments specific for recombinant human leukemia inhibitory factor (rhLIF). The B cell immunoglobulin repertoire of an immunized rabbit was immortalized by the combinatorial cloning of the rearranged variable domains of light (VL) and heavy (VH) chains. Affinity selection of the library displaying the rabbit antibody domains on the phage surface resulted in the isolation of phage encoding scFv antibodies which specifically bind to the antigen. We utilized the methylotrophic yeast Pichia pastoris for high level secretion of soluble and functional scFv antibody fragment. More than 100 mg/L of pure and functional rabbit anti-rhLIF scFv antibody was obtained directly from the P. pastoris culture supernatant by one-step affinity chromatography.     

Cloning and sequence analysis of kappa and gamma cynomolgus monkey immunoglobulin cDNAs

One gamma heavy chain and 10 kappa light chain cynomolgus monkey (Macaca fascicularis) immunoglobulin cDNAs have been cloned and sequenced. Comparisons of the variable (V) regions to human antibody sequences have revealed extensive identity, exhibiting 93% at the amino acid level for the VH framework regions, and 88-99% for the V kappa frameworks. Identification of very few cynomolgus monkey-specific framework region residues suggests a role for cynomolgus monkey antibodies as donators of variable regions to chimeric monoclonal antibodies for utilisation in human therapy with human constant (C) regions. The cynomolgus monkey C kappa region exhibited 83% amino acid identity to its human counterpart, and the C gamma region was 95, 93, 95, and 95% similar to the human C gamma 1, C gamma 2, C gamma 3, and C gamma 4 regions, respectively. Evolutionary analysis of the C gamma genes, using the silent molecular clock, suggests that the divergence between cynomolgus monkey and human occurred before the time at which the ancestral gamma gene diverged into the multiple isotypes observed in humans.     

Induction of capacitation in human spermatozoa in vitro by an endometrial sialic acid-binding protein

A Ca(2+)-dependent sialic acid-binding protein (SABP) of human endometrium, which specifically bound to human sperm head plasma membrane in vitro, was found to increase the percentage motility and acrosome-reacted pattern of uncapacitated spermatozoa. The protein was synthesized in the endometrium and secreted into the uterine fluid. This intra-uterine factor, which is apparently advantageous in vitro in inducing human sperm capacitation, may play a significant role in promoting the post-release maturation of ejaculated spermatozoa by enhancing 45Ca uptake into spermatozoa by a pathway which is insensitive to calcium-channel blockers. However, the 45Ca uptake could be enhanced on exposure to the divalent cation ionophore A23187 and inhibited in the presence of the calmodulin inhibitor trifluoperazine. The SABP also induces an increase in intracellular Ca2+ in spermatozoa, as seen by FURA-2 AM studies. Furthermore, overlay studies show human SABP to be a Ca(2+)-binding protein. The data presented here suggest that SABP induces in-vitro sperm capacitation and the subsequent acrosome reaction by increasing intracellular Ca2+ concentration.     

Diverse Fab specific for acetylcholine receptor epitopes from a myasthenia gravis thymus combinatorial library

The muscle weakness in myasthenia gravis (MG) is caused by heterogeneous high-affinity IgG autoantibodies to the nicotinic acetylcholine receptor (AChR), a complex ion channel glycoprotein. These antibodies are clearly responsible for reducing AChR numbers at the neuromuscular junction in myasthenia; however, the origins, diversity, specificity and pathogenicity of individual antibodies have not yet been established. We have cloned and characterized four different AChR-specific Fab from an MG patient's thymus by screening an IgG1/kappa gene combinatorial lambda phage library with soluble human AChR labeled with [125I] alpha-bungarotoxin. Unlike most previously cloned human antibodies, all four Fab immunoprecipitated soluble human muscle AChR. Two Fab strongly inhibited binding of mAb to the main immunogenic region on the alpha subunits and one Fab bound to an epitope on the fetal-specific gamma subunit. In sensitivity and fine specificity, these Fab resembled the anti-AChR antibodies found in many MG patients, including the donor. The closest germline counterparts for their heavy chains were in VH families 1, 3 and 4; however, there were many differences consistent with an antigen-driven response of diverse B cell clones. The combinatorial approach holds promise for further analysis of human autoantibodies.     

Changes in the sialylglycoconjugate distribution on the human sperm surface during in-vitro capacitation: partial purification of a 20 kDa sialylglycoprotein of capacitated spermatozoa

Changes in the distribution of sialylglycoconjugates on the surface of uncapacitated and in-vitro capacitated human spermatozoa were studied by means of two sialic acid specific lectins (Maackia amurensis agglutinin and Sambucus nigra agglutinin). On the uncapacitated sperm surface, sialylglycoconjugates were found to be localized from the post-acrosomal region of the sperm head to the tail middle piece, whereas after in-vitro capacitation these molecules were only found in a small area of the post-acrosomal region. The surface of capacitated human spermatozoa was also investigated by specifically radiolabelling its terminal sialic acid residues. A 20 kDa glycoprotein, which was partially purified by anion-exchange chromatography, was the main component of the sialylglycoconjugate pattern after in-vitro capacitation.     

Elucidation of a common structure of selective fibrinogen receptor antagonists

In a prospective, blind study, we have examined the relationship among the expression of human sperm surface progesterone receptors, the ability to undergo a mannose-stimulated acrosome reaction and the rate of fertilization in vitro. Individual aliquots of motile spermatozoa were surface-labelled with progesterone and/or mannose-fluoresceinated ligands. Spontaneous acrosome loss and the increase in acrosome reactions following exposure of spermatozoa to mannose ligands were assessed using rhodaminated Pisum sativum agglutinin. Progesterone fluoresceinated ligand binding was observed to occur in two patterns: (i) a uniform distribution of labelling over the acrosome cap (pattern II), and (ii) labelling limited to the equatorial and postacrosomal regions of the human sperm head (pattern III). A conversion of pattern II to pattern III binding was observed and was associated with the acrosome reaction. Pattern III binding was highly correlated with both fertilization potential and the ability to undergo a mannose-stimulated acrosome reaction (P < 0.001). In contrast, normal sperm mannose receptor expression was seen in five men whose abnormal progesterone receptor expression/function and inability to acrosome react after mannose treatment were correlated with their reduced fertility in vitro. In conclusion, surface progesterone receptor aggregation enhances the mannose ligand-stimulated acrosome reaction. Such detection of defective sperm surface progesterone receptor expression/function may be useful in the evaluation and management of male infertility.