A recombinant bispecific single-chain fragment variable specific for HLA class II and Fc alpha RI (CD89) recruits polymorphonuclear neutrophils for efficient lysis of malignant B lymphoid cells.

Bispecific Abs offer new perspectives for cancer immunotherapy. In this study, we describe a recombinant bispecific single-chain fragment variable (bsscFv) directed against Fc alpha RI (CD89) on polymorphonuclear neutrophils (PMNs) or monocytes/macrophages and HLA class II on lymphoma target cells. Fc alpha RI and HLA class II-directed single-chain fragment variable (scFv) fragments were isolated from phage display libraries, established from the hybridomas A77 and F3.3, respectively. The two scFv molecules were connected with a 20 aa flexible linker sequence. After expression in SF21 insect cells and chromatographic purification, the bispecific molecule showed specific binding to both Ags at K(D) values of 148 +/- 42 nM and 113 +/- 25 nM for the anti-Fc alpha RI and anti-HLA class II scFv components in the bsscFv, respectively. In Ab-dependent cytotoxicity assays with PMNs as effectors and a series of lymphoma-derived cell lines (ARH-77, RAJI, REH, NALM-6, RS4;11), the bsscFv was significantly more cytotoxic than the parental murine IgG1 and its chimeric IgG1 derivative. When targeting primary tumor cell isolates from six patients with B cell malignancies, the killing capacity of the (Fc alphaRI x HLA class II) bsscFv compared favorably to conventional HLA class II mAb. Importantly, the cell lines NALM-6 and RS411, as well as two primary tumor cell isolates, were exclusively lysed by the bsscFv. To our knowledge, this is the first report of an Fc alpha RI-directed bsscFv effectively recruiting PMNs for redirected cytotoxicity against human B cell malignancies. Our data show that an (Fc alpha RI x HLA class II) bsscFv is an interesting candidate for further engineering of small, modular immunopharmaceuticals.

Transgenic expression of single-chain anti-CTLA-4 Fv on beta cells protects nonobese diabetic mice from autoimmune diabetes.

T cell-mediated immunodestruction of pancreatic beta cells is the key process responsible for both the development of autoimmune diabetes and the induction of rejection during islet transplantation. In this study, we investigate the hypothesis that transgenic expression of an agonistic, membrane-bound single-chain anti-CTLA-4 Fv (anti-CTLA-4 scFv) on pancreatic beta cells can inhibit autoimmune processes by selectively targeting CTLA-4 on pathogenic T cells. Strikingly, transgenic expression of anti-CTLA-4 scFv on pancreatic beta cells significantly protected NOD mice from spontaneous autoimmune diabetes. Interestingly, local expression of this CTLA-4 agonist did not alter the diabetogenic properties of systemic lymphocytes, because splenocytes from transgenic mice or their nontransgenic littermates equally transferred diabetes in NOD/SCID recipients. By analyzing the T cell development in anti-CTLA-4 scFv/Th1/Th2 triple transgenic mice, we found that beta cell-specific expression of CTLA-4 agonist did not affect the development of Th1/Th2 or CD4(+)CD25(+) regulatory T cells. Most strikingly, islets from transgenic mice inhibited T cell response to immobilized anti-CD3 in a T cell-islet coculture system, suggesting a trans-mediated inhibition provided by transgenic islets. Finally, transgenic islets implanted in diabetic recipients survived much longer than did wild-type islets, indicating a therapeutic potential of this genetically modified islet graft in autoimmune diabetes.

Glycosylation of a VH residue of a monoclonal antibody against alpha (1----6) dextran increases its affinity for antigen.

We have observed that antidextran hybridomas with potential N-linked glycosylation sites in VH have higher affinity for polymeric dextran and for isomaltoheptaose than those lacking potential glycosylation sites. In these studies we have used gene transfection and expression techniques to verify that the carbohydrate addition sites in VH were used. The carbohydrate of the VH region was accessible for binding by the lectin Con A. By ELISA analysis it was demonstrated that the aKa of the antibody for dextran was influenced by the presence of carbohydrate in VH, with the aglycosylated antibody having an aKa 15-fold lower than its untreated counterpart. The aKa for antigen of antibodies that contain carbohydrate only in their constant region was unaffected by lack of carbohydrate. Thus, not only the amino acid sequence of the variable region but also its carbohydrate moieties can determine the magnitude of the antigen-antibody interaction.

Idiotypic self binding of a dominant germline idiotype (T15). Autobody activity is affected by antibody valency.

We have previously described (1-3) an IgM antibody that binds to PC, expresses the T15 idiotype, and binds also to itself or T15 if insolubilized. Because of the simultaneous presence of complementary idiotopes and paratopes this type of antibody has been termed autobody. The self binding involves the antigen-binding site because the F(ab')2 fragment of T15, PC, and no other haptens inhibit the self binding. DNA sequence analysis of 11E7-1 using primer extension cDNA sequencing showed that the variable sequences of H and L chains of 11E7-1 are identical to the germline sequence of the prototype T15 idiotype. Furthermore, monomeric and dimeric T15 IgA were shown to bind to insolubilized T15 and other T15+ antibodies including 11E7-1. Thus, the self-binding activity is an inherent property of the T15 germline sequence. The self binding is highly dependent on the polymeric state of the binding antibody since the IgM pentamer of 11E7-1 is about three fold more effective than the T15 dimer and 50 times more than the T15 monomer. These data suggest that the self-binding activity of a germline-encoded idiotype may play an important role in the biology of its expression, and more specifically, may be responsible for the establishment of its dominant expression.

Analysis of the structural correlates for antibody polyreactivity by multiple reassortments of chimeric human immunoglobulin heavy and light chain V segments.

Polyreactive antibodies (Abs) constitute a major proportion of the early Ab repertoire and are an important component of the natural defense mechanisms against infections. They are primarily immunoglobulin M (IgM) and bind a variety of structurally dissimilar self and exogenous antigens (Ags) with moderate affinity. We analyzed the contribution of Ig polyvalency and of heavy (H) and light (L) chain variable (V) regions to polyreactivity in recombinatorial experiments involving the VH-diversity(D)-JH and V kappa-J kappa gene segments of a human polyreactive IgM, monoclonal antibody 55 (mAb55), and those of a human monoreactive anti-insulin IgG, mAb13, in an in vitro C gamma l and C kappa human expression system. These mAbs are virtually identical in their VH and V kappa gene segment sequences. First, we expressed the VH-D-JH and V kappa-J kappa genes of the IgM mAb55 as V segments of an IgG molecule. The bivalent recombinant IgG Ab bound multiple Ags with an efficiency only slightly lower than that of the original decavalent IgM mAb55, suggesting that class switch to IgG does not affect the Ig polyreactivity. Second, we coexpressed the mAb55-derived H or kappa chain with the mAb13-derived kappa or H chain, respectively. The hybrid IgG Ab bearing the mAb55-derived H chain V segment paired with the mAb13-derived kappa V segment, but not that bearing the mAb13-derived H chain V segment paired with the mAb55-derived kappa V segment, bound multiple Ags, suggesting that the Ig H chain plays a major role in the Ig polyreactivity. Third, we shuffled the framework 1 (FR1)-FR3 and complementarity determining region 3 (CDR3) regions of the H and kappa chain V segments of the mAB55-derived IgG molecule with the corresponding regions of the monoreactive IgG mAb13. The mAb55-derived IgG molecule lost polyreactivity when the H chain CDR3, but not the FR1-FR3 region, was replaced by the corresponding region of mAb13, suggesting that within the H chain, the CDR3 provides the major structural correlate for multiple Ag-binding. This was formally proved by the multiple Ag-binding of the originally monoreactive mAb13-derived IgG molecule grafted with the mAb55-derived H chain CDR3. The polyreactivity of this chimeric IgG was maximized by grafting of the mAb55-derived kappa chain FR1-FR3, but not that of the kappa chain CDR3.(ABSTRACT TRUNCATED AT 400 WORDS)

Peptidomimics of antigen are present in variable region of heavy and light chains of anti-idiotypic antibody and function as surrogate antigen for perpetuation of immunological memory.

Peptide antigens composed of relevant B cell and T cell epitopes, capable of inducing protective immune response against the whole pathogen, are potentially safe, alternative vaccine antigens for prevention of wide range of diseases. Here, we show that short peptides derived from internal image sequences of anti-idiotypic antibody (peptidomimics) can function as both B and T cell epitopes and perpetuate antigen specific immunological memory. We have sequenced the variable regions of heavy and light chains of the anti-idiotypic antibody specific to rinderpest virus hemagglutinin protein and predicted T cell epitopes in these sequences by an immuno-informatics approach. We have studied the interaction of these epitopes with MHC class I by in vitro assays and in silico analysis by molecular modeling of the idiopeptide-MHC complexes as well as antigen-derived peptide-MHC complexes. The functional capacity of anti-idiotypic antibody derived peptides to stimulate antigen specific T cells in vitro was tested. The ability of peptidomimics to proliferate the immune splenocytes in vitro was 10 times more when compared with that of a control peptide taken from the constant region of immunoglobulin. Similarly three- to fivefold more amounts of IL-2 and IFN-gamma were secreted by immune splenocytes in response to in vitro re-stimulation with peptidomimics. Further, we have provided evidence for the generation of antibodies against peptidomimics in memory response generated on antigen or anti-idiotypic antibody immunizations. In summary, our experiments suggest that peptidomimics are generated in the body after antigen immunization and may have important roles in vivo in regulating antigen specific immunological memory.

Functional mapping and single chain construction of the anti-cytokeratin 8 monoclonal antibody TS1.

The anti-cytokeratin (CK) 8 monoclonal antibody (mab) TS1 has been shown to efficiently bind to CK8 expressed in carcinomas in vivo. The anti-idiotypic antibody of TS1, alphaTS1, can be used to regulate the tumor:non-tumor ratio of TS1 by clearing non-tumor binding TS1 from the circulation. If the interaction of TS1 to CK8 and alphaTS1 is fully understood, mutations can be used to improve the tumor:non-tumor ratio. A scFv was made of the mab TS1 and residues earlier identified by Erlandsson et al. as important for the interaction with both its antigen CK8 and its anti-idiotype alphaTS1, were mutated to alanine or amides and expressed in E. coli. The effects of the mutations were studied by ELISA and residues important for the interactions to both CK8 and alphaTS1 were identified as mainly tyrosines, charged residues, a serine and a tryptophan. Altogether, nine amino acid residues in TS1 were found to be important in the interaction to alphaTS1 and six residues for the interaction to CK8. Important residues, clustered together in the modelled protein, were identified as residues from CDR 3 of the heavy chain and the unexpected participation of a residue in CDR 2 of the light chain. Some of the important residues are likely to be hotspots. Hotspots constitute a few residues in an interaction that contribute most to the binding, energetically. Amino acid residues in hotspots often cluster together in the center of the interaction interface, but can also be spread out to the periphery. The hotspots are often surrounded by hydrophobic patches, which are seen in the modelled TS1 protein used in this study. Amino acid residues that increased the affinity when mutated were also identified for both interactions. These residues are likely to be located outside the interacting interface. It can from this study be concluded that it is wise to precede the mutational procedure with experiments that can give guidelines for the selection of which amino acid residues to mutate. If the guidelines from the chemical modifications from Erlandsson et al. not had been used, this study would have left some residues unmutated and thereby missed important information.

Structure-function relationships of the variable domains of monoclonal antibodies approved for cancer treatment.

Due to their exquisite specificity for a given epitope on the target antigen, recombinant monoclonal antibodies (rmAb) can deliver "targeted therapy" in oncology. This review focuses on the structural bases of "antigen specificity" to aid clinical researchers and pharmacologists in managing these new drugs. The fine structure of the Fv (Fragment variable) module (combination of VH and VL domains) from the five unconjugated antibodies currently approved for cancer treatment, namely rituximab, cetuximab, alemtuzumab, trastuzumab and bevacizumab, is presented and analysed. Co-crystal and functional studies are reviewed to define rmAb residues contributing to antigen binding site (paratope)-epitope interfaces. The genetic origin of these recombinant monoclonal antibodies, determined through the IMGT/3Dstructure-DB database and IMGT/V-QUEST (http://imgt.cines.fr), is presented, allowing the evaluation of homologies between antibodies and their closest germline human counterparts and hence their possible immunogenicity. Overall, the IMGT standards appear as a first and crucial step in the evaluation of recombinant antibodies.

The role of interface framework residues in determining antibody V(H)/V(L) interaction strength and antigen-binding affinity.

While many antibodies with strong antigen-binding affinity have stable variable regions with a strong antibody heavy chain variable region fragment (V(H))/antibody light chain variable region fragment (V(L)) interaction, the anti-lysozyme IgG HyHEL-10 has a fairly strong affinity, yet a very weak V(H)/V(L) interaction strength, in the absence of antigen. To investigate the possible relationship between antigen-binding affinity and V(H)/V(L) interaction strength, a novel phage display system that can switch two display modes was employed. We focused on the two framework region 2 regions of the HyHEL-10 V(H) and V(L), facing each other at the domain interface, and a combinatorial library was made in which each framework region 2 residue was mixed with that of D1.3, which has a far stronger V(H)/V(L) interaction. The phagemid library, encoding V(H) gene 7 and V(L) amber codon gene 9, was used to transform TG-1 (sup+), and the phages displaying functional variable regions were selected. The selected phages were then used to infect a nonsuppressing strain, and the culture supernatant containing V(H)-displaying phages and soluble V(L) fragment was used to evaluate the V(H)/V(L) interaction strength. The results clearly showed the existence of a key framework region 2 residue (H39) that strongly affects V(H)/V(L) interaction strength, and a marked positive correlation between the antigen-binding affinity and the V(H)/V(L) interaction, especially in the presence of a set of particular V(L) residues. The effect of the H39 mutation on the wild-type variable region was also confirmed by a SPR biosensor as a several-fold increase in antigen-binding affinity owing to an increased association rate, while a slight decrease was observed for the single-chain variable region.

Antibody-mediated Hsp70 protein therapy.

Intracellular Hsp70 provides cytoprotection against a variety of stressful stimuli, and an effective means of increasing intracellular Hsp70 levels could prove beneficial in the prevention and treatment of a variety of human diseases. A novel protein transduction domain consisting of the single chain Fv fragment of an anti-DNA antibody known to penetrate into living cells and tissues, mAb 3E10, has recently been used to deliver functional proteins to cells. The ability of the single chain Fv fragment to deliver Hsp70 into living cells was tested by generating an Fv-Hsp70 fusion protein. Fv-Hsp70 was produced as a secreted protein in both COS-7 cells and the methylotropic yeast strain Pichia pastoris and was shown capable of penetrating into COS-7 cells and primary rat cortical neurons. Pre-treatment with Fv-Hsp70 protected both COS-7 cells and primary rat cortical neurons against subsequent exposure to hydrogen peroxide. These results provide the first evidence that the Fv fragment of mAb 3E10 is capable of delivering proteins to neurons and indicate its potential in the development of Hsp70 protein therapy.

[Inhibitory effect of anti-type IV collagenase intrabody on invasiveness of human pulmonary giant cell carcinoma PG cells in vitro].

OBJECTIVE: To explore the inhibitory effects of endoplasmic reticulum-retained intrabody on the secretion of type IV collagenase and the invasion of human pulmonary giant cell carcinoma PG cells in vitro. METHODS: Two expression plasmids were constructed, pcDNA3.1-CP.scFv and pcDNA3.1-ER.scFv encoding cytoplasm-retained and endoplasmic reticulum-retained single chain antibodies against the type IV collagenase, respectively. The intracellular antibody genes were transfected into the human pulmonary giant cell carcinoma PG cells. Western blot was performed to detect the expression of pcDNA3.1-CP.scFv and pcDNA3.1-ER.scFv. Gelatin zymography was performed to detect seretion of type IV collagenase in PG cells and Matrigel assay was employed for determination of the cell invasiveness. RESULTS: Both of cytoplasm-retained and endoplasmic reticulum-retained introbodies, CP.scFv and ER.scFv, were expressed in PG cells. ER.scFv, significantly inhibited the secretion of type IV collegenase. As shown, matrix metalloproteinase 9 and matrix metalloproteinase 2 were inhibited by 85.7% and by 51.2%, respectively. However, CP.scFv did not show such inhibitory effect. The ER.scFv encoding gene-transfected PG cells were much less invasive than parental or vector control cells, the inhibition rate was 76.3% (P < 0.05), whereas CP.scFv encoding gene-transfected PG cells showed no reduction in invasiveness. CONCLUSION: Those findings demonstrate that endoplasmic reticulum (ER)-retained intracellular antibody technology may selectively abrogate the activity of type IV collagenase in the protein trafficking and secretory pathway and effectively inhibit tumor cell invasion in vitro. Anti-type IV collagenase intrabody may be further used in cancer gene therapy.

The 5' noncoding region and virulence of poliovirus vaccine strains.

All three live, attenuated vaccine strains of poliovirus contain important attenuation determinants in a short conserved sequence in the 5' noncoding region. Evidence suggests these act by weakening a secondary-structural element critical for the unusual mechanism of translational initiation of picornaviruses, in which ribosomes bind directly to a site far downstream of the 5' end. Understanding the molecular basis of attenuation may allow novel vaccine strains to be designed.

Diabetes in NOD mice does not require T lymphocytes expressing V beta 8 or V beta 5.

The incidence of destructive pancreatic infiltrates and overt diabetes in animal models of insulin-dependent (type I) diabetes mellitus can be greatly reduced by inactivating or eliminating most T lymphocytes early in life. Because of theoretical and practical concerns about inducing long-term pan-T-lymphocyte inactivation for prevention or treatment of type I diabetes in humans, we hoped that more selective suppression of only the diabetogenic T lymphocyte population might be possible. To this end, two groups suggested that diabetogenic subpopulations of T lymphocytes in NOD mice could be identified by the protein sequence of their T-lymphocyte receptors. This assertion was based on experimental elimination of candidate T-lymphocyte subpopulations in two different short-term models of diabetes induction in NOD mice. For these experiments, identification and elimination of T-lymphocyte subsets were accomplished with monoclonal antibodies that bind specifically to the variable region of the beta-chain (V beta) of the T-lymphocyte antigen receptor and divide the T-lymphocyte pool of the NOD mouse into approximately 20 V beta subsets. To test the relationship between the two T-lymphocyte V beta subsets implicated in these studies and pancreatic beta-cell destruction in unmanipulated animals, both T-lymphocyte subpopulations identified were genetically eliminated from NOD-derived mice by introduction of a mutant T-lymphocyte receptor V beta gene, from which these sequences are genomically deleted. Histological evidence of severe beta-cell destruction and overt diabetes was found in mice homozygous for the deleted V beta gene, indicating that neither V beta gene segment identified in previous studies is required for diabetogenesis in unmanipulated diabetes-prone mice.

Anti-beta 2-glycoprotein-I antibodies in scFv format.

Phage display was introduced almost 20 years ago. It has been used to produce large amounts of diverse proteins, to analyze protein-ligand interactions, to improve the affinity of proteins for their binding receptors, and to characterize antibody binding sites. The recombinant version of the antibody Fv is termed single-chain variable fragment (scFv). Many large phage libraries have been developed that have yielded antibodies to several hundred antigens, but only 5 human anti-beta2-glycoprotein-I and three anti-prothrombin antibodies in scFV have been so far characterized. Antibodies to beta2GP-I thus generated show 92-94% homology with their nearest germ line genes. Their mutations frequently appear to be independent of antigen. Two anti-prothrombin antibodies show strong crossreactivity with beta2GP-I. Four mouse anti-beta2GP-I scFV show less binding properties than their original counterparts, but had the same capacity of inducing experimental antiphospholipid syndrome. This pathogenicity appears to reside in the V(H)DJ(H)C(H) region of the scFv since the V(H)DJ(H)C(H) regions of pathogenic scFV combined with irrelevant V(L) J(L)C(L) regions retained their pathogenicity while the opposite failed to do so.

Neutralizing human anti-B-cell-activating factor of the TNF family (BAFF) scFv selected from phage antibody library.

Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. We now report the isolation by phage display of human single-chain antibody fragment (scFv) anti-BAFF. After four rounds of panning against BAFF, thirty-two out of 92 phage clones displayed BAFF binding activity. One of the positive clones, designated F8, bound to BAFF with relatively high affinity and neutralized BAFF bioactivity in vitro. F8 clone was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography (IMAC). The purified scFv recognized BAFF with the affinity constant (K(aff)) of 2.5 x 10(7)M(-1) without cross-reaction to APRIL. In addition to binding, the purified scFv could does-dependently inhibit BAFF-induced mouse spleen B lymphocyte proliferation. Together with its fully human mature, F8 scFv may have therapeutic implications in therapy of autoimmune disorders mediated by BAFF.

Limits on heavy chain junctional diversity contribute to the recurrence of an antibody variable region.

Antibody V region structural diversity in the mouse is generated, in part, by the combinatorial joining of different gene segments, as well as by the "imprecision" of these joining events. The same two gene segments can be joined at different locations, and nucleotides can be deleted or added de novo to the segment junction. While it is clear that such junctional processes are a major contributor to V region diversity, the mechanisms that generate this diversity are poorly understood. Here I present sequences in the VH-D-JH region of 34 VH genes that are composed of the same three VH gene segments. In combination with a single V kappa-J kappa pair, these VH genes encode a family of V regions that are recurrently expressed in the immune response of A/J mice to p-azophenylarsonate (Ars). The germline sequences of the three constituent gene segments for these VH genes are known, making it possible to determine the origin of the nucleotides in junctional regions. An examination of the frequency and type of nucleotides present in these regions provides insight into the properties of the segment joining mechanism. In addition, the data suggest that recurrent expression of the anti-Ars V regions which these VH genes partially encode is due not only to antigenic selection, but to the high probability with which these VH genes are formed during B cell differentiation.

The function of conserved amino acids in or near the complementarity determining regions for related antibodies to Cryptococcus neoformans glucuronoxylomannan.

Most monoclonal antibodies (mAbs) to Cryptococcus neoformans glucuronoxylomannan (GXM) antigen (Ag) are grouped as Class II based on usage of V(H)7183, Vkappa5.1, J(H)2, and Jkappa1 gene elements. Comparative analysis of 43 Class II mAbs revealed conservation of I51, G54, and D61 in heavy-chain variable region (V(H)) complementarity determining region 2 (CDR2), and R95 and D96 in CDR3. Furthermore, position 100b (Kabat numbering scheme) in CDR3 always had an aromatic amino acid (aa) and F was found at position 100c in 96% of mAbs. The function of these conserved residues for binding to GXM and peptide mimetics, and idiotype (Id) structure, was investigated using site-directed mutagenesis. In addition, we mutated W36 and V37 in the second framework. Mutations W36A, Y100bA, and F100cA interfered with antibody (Ab) secretion, but not assembly, and cytoplasmic Ab bound to GXM and Id mAbs. In contrast, mutations V37A, I51A, G54A, and D61A did not affect assembly, secretion, or binding to GXM. Mutating the R95-D96 motif in CDR3 to DR, DD, RR, RA, AD, KD, HD, RE, RN or AA revealed that the positive charge at position 95 was essential for binding GXM, whereas the negative charge at position 96 could be substituted for a non-charged aa. Our results: (1) extend the concept that CDR3 diversity is essential for Ag and Id specificity to a polysaccharide-binding Ab; (2) show that aa conservation in CDRs does not imply a requirement for Ag binding; (3) establish a role for W36 in secretion; and (4) demonstrate that aa motifs used for binding GXM and peptide mimetics can differ.

Analysis of the structural diversity of monoclonal antibodies to cyclosporine.

The immunosuppressive cyclic undecapeptide cyclosporine (Cs) represents a useful model for studying the molecular basis of antibody-antigen interactions. The three-dimensional structure of the Cs molecule is known and a large panel of monoclonal antibodies (mAbs) to Cs has been well characterized by cross-reactivity studies with numerous Cs analogs. In the present study, the sequences of the variable regions of seven mAbs to Cs were determined and a striking relationship was found between the expressed variable region genes and the Cs recognition pattern. An analysis of the length and hydrophobic content of the hypervariable regions and sequence similarities suggested that the heavy chain plays a major role in Cs recognition. Different fine specificities were observed for mAbs exhibiting identical light chains, while two antibodies differed by only a single amino acid located in the heavy chain. The presence of a duplication of 12 nucleotides within the heavy chain third hypervariable region of two antibodies suggests the existence of an additional mechanism for creating antibody diversity.

Cloning of a single-chain variable fragment (scFv) switching active plasminogen activator inhibitor-1 to substrate.

Increased levels of plasminogen activator inhibitor-1 (PAI-1) are a well-known risk for cardiovascular diseases. A significant number of investigations are aimed at lowering plasma levels of PAI-1 to enhance endogenous fibrinolysis. We have recently generated monoclonal antibodies that neutralize PAI-1 activity by switching the inhibitory conformation to a substrate conformation. However, intact murine antibodies have quite some disadvantages for therapeutic use in man. In the current study, we describe the construction of a smaller antibody fragment derived from a monoclonal antibody (MA-8H9D4) with PAI-1 neutralizing properties. The cDNAs encoding the variable domains of the heavy and light chain were amplified, linked and cloned into a phagemid vector. Resulting clones were expressed as a single-chain variable fragment (scFv, VH-(Gly4Ser)3-VL) on the surface of a phage and selected for binding to PAI-1. Subsequently, a positive phage was used for the production of soluble scFv-8H9D4. Following purification, the characteristics of the scFv-8H9D4 were compared to those of the original MA-8H9D4. The scFv inhibited PAI-1 activity to a similar extent as MA-8H9D4 and by a similar mechanism, i.e., induction of a conformational switch. Thus, this smaller antibody fragment, exhibiting the same properties as the parent molecule may constitute a useful starting point for the design of PAI-1 neutralizing therapeutics.

Phylogeny of immunoglobulin structure and function. XIV. Peptide map and amino acid composition studies of shark antibody light chains.

Light chains from antibodies to the streptococcal A-variant carbohydrate from individual nurse sharks were compared by peptide maps of tryptic digests and amino acid compositions. Although the amino acid compositions of the different chains were quite similar, considerable differences as well as similarities were demonstrable on peptide maps. The peptide maps were interpreted as indicating that shark L chains likely have constant and variable regions as seen in the immunoglobulins of higher animals. Furthermore the unique peptides characteristic of different L chains support the hypothesis that nurse sharks, as a species, possess a relatively large number of different L chain amino acid sequences which are compatible with antibody binding sites to the streptococcal antigen. Hence the repetoire of nurse shark antibody combining sites to this antigen appears to be quite extensive.