Variable Domains Of Light Chains Research Articles

Changes in antibody binding and functionality after humanizing a murine scFv anti-IFN-α2: From in silico studies to experimental analysis

The structural and dynamic changes introduced during antibody humanization continue to be a topic open to new contributions. For this reason, the study of structural and functional changes of a murine scFv (mu.scFv) anti-rhIFN-α2b after humanization was carried out. As it was shown by long molecular dynamics simulations and circular dichroism analysis, changes in primary sequence affected the tertiary structure of the humanized scFv (hz.scFv): the position of the variable domain of light chain (VL) respective to the variable domain of heavy chain (VH) in each scFv molecule was different. This change mainly impacted on conformation and dynamics of the complementarity-determining region 3 of VH (CDR-H3) which led to changes in the specificity and affinity of humanized scFv (hz.scFv). These observations agree with experimental results that showed a decrease in the antigen-binding strength of hz.scFv, and different capacities of these molecules to neutralize the in vitro rhIFN-α2b biological activity. Besides, experimental studies to characterize antigen-antibody binding showed that mu.scFv and hz.scFv bind to the same antigen area and recognize a conformational epitope, which is evidence of docking results. Finally, the differences between these molecules to neutralize the in vitro rhIFN-α2b biological activity were described as a consequence of the blockade of certain functionally relevant amino acids of the cytokine, after scFv binding. All these observations confirmed that humanization affected the affinity and specificity of hz.scFv and pointed out that two specific changes in the frameworks would be responsible.

Al amyloidosis with cardiac involvement: cardiotoxicity of aggregation prone peptides deriving from variable domains of immunoglobulin light chains

Abstract Background/Introduction Light chain (AL) amyloidosis is an uncommon malignancy manifested by systemic extracellular deposition of immunoglobulin light chain fibrils. The cardiac phenotype is characterised by ventricular wall thickening and stands as the most prominent cause of morbidity and mortality. Although, it has been established that the circulating light chains directly impair cardiomyocyte function, the cytotoxic effect of specific amyloidogenic peptides that may appear due to excessive cleavage of light chains remains unspecified. Purpose In the present work, we aimed to detect amyloidogenic “hot-spots” on the variable domains of light chains associated with cardiac AL amyloidosis (IGLV1-44 and IGLV3-01) or inferior outcomes (IGLV6-57) and define their cytotoxic effect in vitro. Methods At first, we used the curated database ALBase and we performed a multiple sequence alignment of the IGLV1-44, IGLV3-01 and IGLV6-57 inputs that derived only from patients with AL amyloidosis. “Aggregation-prone” hot-spots in the conserved amino acid sequences were identified with the aid of AMYLPRED2, a tool which combines 11 independent computational methods and provides a consensus result of potent amyloidogenic regions. Five peptides were rationally selected and synthetically produced in order to be tested in vitro. The amyloidogenic properties of the peptides were evaluated with Transmission Electron Microscopy and Congo red staining, while the rate of fibril formation at lower concentrations was monitored with Thioflavin T and confirmed with Scanning Electron Microscopy. In order to assess the cytotoxic effect of the non-polymerized peptides, H9C2 cells were incubated with the peptides for 24 hours at 200μg/mL and 100μg/mL and cell death was determined by lactate dehydrogenase release assay. Results Interestingly, sequence alignment on the variable domains of cardiac related light chains revealed the presence of several conserved domains in patients with AL amyloidosis. The chosen peptides were proven to be amyloidogenic suggesting that the variable domains share common amyloidogenic cores. Treatment of H9C2 cells with the peptides at 200μg/mL led to significant reduction in cell viability compared to vehicle treated cells (p<0.001). Two of the peptides deriving from the IGLV6-57 and IGLV3-01 significantly increased cell death at 100μg/mL (p<0.01 and p<0.001 respectively). During the 24h treatment the tested peptides comprised of soluble species and not amyloid fibrils suggesting that monomeric and oligomeric intermediates are highly toxic. Conclusion We discovered five novel amyloidogenic prone regions of cardiac related variable domains that are associated with cellular toxicity and could be exploited for targeted therapeutic interventions. Funding Acknowledgement Type of funding source: None

Isolation and purification of recombinant immunoglobulin light chain variable domains from the periplasmic space of Escherichia coli.

Immunoglobulin light chain amyloidosis is the most common form of systemic amyloidosis. However, very little is known about the underlying mechanisms that initiate and modulate the associated protein aggregation and deposition. Model systems have been established to investigate these disease-associated processes. One of these systems comprises two 114 amino acid light-chain variable domains of the kappa 4 IgG family, SMA and LEN. Despite high sequence identity (93%), SMA is amyloidogenic in vivo, but LEN adopts a stable dimer, displaying amyloidogenic properties only under destabilising conditions in vitro. We present here a refined and reproducible periplasmic expression and purification protocol for SMA and LEN that improves on existing methods and provides high yields of pure protein (10-50mg/L), particularly suitable for structural studies that demand highly concentrated and purified proteins. We confirm that recombinant SMA and LEN proteins have structure and dimerization capabilities consistent with the native proteins and employ fluorescence to probe internalization and cellular localization within cardiomyocytes. We propose periplasmic expression and simplified chromatographic steps outlined here as an optimized method for production of these and other variable light chain domains to investigate the underlying mechanisms of light chain amyloidosis. We show that SMA and LEN can be internalised within cardiomyocytes and were observed to localise to the perinuclear area, assessed by confocal microscopy as a possible mechanism for underlying cytotoxicity and pathogenesis associated with amyloidosis.

Why Would a Monomeric Ig Domain Form Amyloid Fibers?

Antibody light chains are composed of two domains, variable and constant, that associate with heavy chains to form functional antibodies. In a collection of B-cell diseases, an excess of light chains is produced. These light chains form various types of aggregates, amongst which are amyloid fibers. In this case a lethal multi-organ disease called light chain amyloidosis, or AL, ensues. Recombinant variable light chain domains, corresponding to proteins isolated from patients with AL, are structurally indistinguishable from those of healthy donors. Many happen to be less stable thermodynamically, leading to a hypothesis linking poor stability to amyloidogenesis. The native state will not aggregate into fibers, so an unfolded intermediate of unknown conformation has been proposed as the fiber building block.We propose that monomeric amyloidogenic Ig domains may form amyloid fibers by combining any of the following non-exclusive attributes: a) low thermodynamic stability, which favors local or global denaturation; b) low structural diversity in the conformational ensembles sampled upon denaturation, which allows for more frequent population of aggregation-prone monomers; c) sets of non-native interactions that selectively stabilize aggregation-prone monomers; d) multiple viable polymerization surfaces, to maximize the probability for seeding and extension of fibers; and e) stable protofibers that can live long enough to progress to fibers.We test the relative relevance of these five contributions for a set of four light chain variable Ig domains belonging to class Δ6a, one of the most common in AL, by performing MD simulations under native and high temperature conditions, docking of putative unfolding intermediates, and MD of the protofibers. For the most amyloidogenic variant derived form a patient (WIL), preliminary data suggest that it uses all five attributes.Acknowledgements: computing at KanBalam (UNAM) and Centro Nacional de Supercomputo (IPICyT); funding from CONACyT 133294.

Exploring the Effect of Mutations on the Conformational Landscape of Amyloidogenic Antibody Fragments

Antibodies are composed of heavy chains and light chains. In Light Chain Amyloidosis (AL), antibody-secreting cells export only the light chains. These light chains are prone to misfold, forming amyloid fibers, which get deposited in various organs, leading to organ disfunction and patient death. The transition from the native state to the amyloid fiber requires partial or total denaturation. The structures of monomeric precursors to the oligomeric nuclei, or of those assembled at the ends of the fibers, are unknown. There is an inverse correlation between the stability of the native state and the speed of fiber formation, suggesting that lower stability allows the population of “excited states” of the native ensemble, some of which could be amyloid fiber precursors. Starting from the crystal structure of a variable light chain domain belonging to class 6a (one of the most common in clinical cases of AL), we generated point mutants that eliminate charges (R24G and D52A) or a proline (P7S). These mutants destabilize the native state, and speed up fiber formation (for R24G and P7S). We carried out MD simulations at three temperatures (298, 398 and 498K), to explore the effect of these mutations on the conformational landscape. We found many metastable unfolding intermediates, which have eluded experimental detection because their fluorescence is indistinguishable from that of the native state. A common early unfolding intermediate exposes strand D, which has a high potential for fiber formation according to ZipperDB. Those variants with a higher speed of fiber formation expose this area with greater frequency.We are thankful for computer resources at: Centro Nacional de Supercomputo, IPICyT; Kan Balam, UNAM; Sputnik, IBT-UNAM; Orion, FC and CIQ-UAEM.Funding: CONACYT 102182 and 133294.

Amyloid Fiber Precursors in Native and Denaturing MD Simulations of an IG Light Chain Domain

The native structure of proteins is generally necessary for function. Mutations and changes in the environment can lead to altered conformations that are prone to aggregation, leading to the formation of amorphous or fibrillar aggregates, which are associated with diseases such as systemic amyloidoses. Precursors for these aggregates can come from any point in the folding route for the protein, and there is a direct correlation between poor protein stability and its potential to form fibers. Immunoglobulin variable light chain domains are prominent in primary systemic amyloidoses; in particular, class 6a appears in ∼40% of clinical cases, despite being expressed only in ∼2% of B-cells under normal conditions. 6aJL2 is a prototype for these protein domains, containing the germline sequence of the VIa gene fused to the lambda J2 segment, built to explore the properties of this particular class. An allotypic variant, containing the R24G mutation, is even more susceptible to fibril formation, and is also less stable. Both forms are two-state folders. Starting from the crystal structure of 6aJL2, we carry out MD simulations of the original 6aJL2 protein and the R24G variant under native conditions (298K) and under strong denaturing conditions (573K), to explore the native basin and the unfolding pathway of these proteins, in order to identify possible precursors for the formation of fibers. We compare the results of the simulations to available circular dichroism, fluorescence and NMR data. Preliminary data provide a rationale for the lower stability of R24G, due to the loss of both stacking and H-bond interactions. Analysis of both native and denaturing simulations suggest that unfolding for both variants starts at a proline-rich loop and makes the N-terminus of the protein available for seeding the amyloid.

General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold

Nanobodies, single-domain antigen-binding fragments of camelid-specific heavy-chain only antibodies offer special advantages in therapy over classic antibody fragments because of their smaller size, robustness, and preference to target unique epitopes. A Nanobody differs from a human heavy chain variable domain in about ten amino acids spread all over its surface, four hallmark Nanobody-specific amino acids in the framework-2 region (positions 42, 49, 50, and 52), and a longer third antigen-binding loop (H3) folding over this area. For therapeutic applications the camelid-specific amino acid sequences in the framework have to be mutated to their human heavy chain variable domain equivalent, i.e. humanized. We performed this humanization exercise with Nanobodies of the subfamily that represents close to 80% of all dromedary-derived Nanobodies and investigated the effects on antigen affinity, solubility, expression yield, and stability. It is demonstrated that the humanization of Nanobody-specific residues outside framework-2 are neutral to the Nanobody properties. Surprisingly, the Glu-49 --> Gly and Arg-50 --> Leu humanization of hallmark amino acids generates a single domain that is more stable though probably less soluble. The other framework-2 substitutions, Phe-42 --> Val and Gly/Ala-52 --> Trp, are detrimental for antigen affinity, due to a repositioning of the H3 loop as shown by their crystal structures. These insights were used to identify a soluble, stable, well expressed universal humanized Nanobody scaffold that allows grafts of antigen-binding loops from other Nanobodies with transfer of the antigen specificity and affinity.

Highly specific inhibition of C1q globular-head binding to human IgG: A novel approach to control and regulate the classical complement pathway using an engineered single chain antibody variable fragment

We sought to specifically regulate the binding of human C1q, and thus the activation of the first complement component, via the construction of a single chain antibody variable binding region fragment (scFv) targeting the C1q globular heads. Here we describe details of the construction, expression and evaluation of this scFv, which was derived from a high-affinity hybridoma (Qu) specific for the C1q globular heads. The scFv was comprised of the Qu variable heavy chain domain (VH) linked to the Qu variable light chain domain (VL) and was termed scFv-QuVHVL. When mixed with either purified C1q or with human serum as a source of C1, scFv-QuVHVL bound to C1q and competitively restricted the interaction of C1q or C1 with immobilized IgG or with IgG1 antibody-coated cells, and prevented the activation of native C1 in human serum as determined by analyses of C1-mediated C4 deposition and fluid-phase C4 conversion. However scFv-QuVHVL could be manipulated to become a C1 activator when it was irreversibly immobilized onto microtiter ELISA plates, prior to contact with human serum complement. This functional dichotomy can be a useful tool in selectively elucidating, differentiating, inducing or inhibiting specific roles of human C1q and the classical complement pathway in complement-mediated physiological processes. We project that once fully humanized, fluid-phase scFv-QuVHVL could become a useful therapeutic in limiting inadvertent host tissue damage elicited by the classical complement pathway.

Functional Intracellular Antibody Fragments Do Not Require Invariant Intra-domain Disulfide Bonds

Intracellular antibody fragments that interfere with molecular interactions inside cells are valuable in investigation of interactomes and in therapeutics, but their application demands that they function in the reducing cellular milieu. We show here a 2.7-Å crystal structure of intracellular antibody folds based on scaffolds developed from intracellular antibody capture technology, and we reveal that there is no structural or functional difference with or without the intra-domain disulfide bond of the variable domain of heavy chain or the variable domain of light chain. The data indicate that, in the reducing in vivo environment, the absence of the intra-domain disulfide bond is not an impediment to correction of antibody folding or to interaction with antigen. Thus, the structural constraints for in-cell function are intrinsic to variable single-domain framework sequences, providing a generic scaffold for isolation of functional intracellular antibody single domains.

Construction of a human functional single‐chain variable fragment (scFv) antibody recognizing the malaria parasitePlasmodium falciparum

Falciparum malaria is one of the most deadly and profound human health problems around the tropical world. Antimalarial drugs are now considered to be a powerful treatment; however, there are drugs currently being used that are resistant to Plasmodium falciparum parasites spreading in different parts of the world. Although the protective immune response against intraerythrocytic stages of the falciparum malaria parasite is still not fully understood, immune antibodies have been shown to be associated with reduced parasite prevalence. Therefore antibodies of the right specificity present in adequate concentrations and affinity are reasonably effective in providing protection. In the present study, VH (variable domain of heavy chain) and VL (variable domain of light chain) were isolated from human blood lymphocytes of P. falciparum in one person who had high serum titre to RESA (ring-infected erythrocyte surface antigen). Equal amounts of VH and VL were assembled together with universal linker (G4S)3 to generate scFvs (single-chain variable fragments). The scFv antibodies were expressed with a phage system for the selection process. Exclusively, an expressed scFv against asynchronous culture of P. falciparum-infected erythrocytes was selected and characterized. Sequence analysis of selected scFv revealed that this clone could be classified into a VH family-derived germline gene (VH1) and Vkappa family segment (Vkappa1). Using an indirect immunofluorescence assay, we could show that soluble expressed scFv reacted with falciparum-infected erythrocytes. The results encourage the further study of scFvs for development as a potential immunotherapeutic agent.

Development of a Human Light Chain Variable Domain (V L) Intracellular Antibody Specific for the Amino Terminus of Huntingtin via Yeast Surface Display

Intracellular antibodies (intrabodies) provide an attractive means for manipulating intracellular protein function, both for research and potentially for therapy. A challenge in the isolation of effective intrabodies is the ability to find molecules that exhibit sufficient binding affinity and stability when expressed in the reducing environment of the cytoplasm. Here, we have used yeast surface display of proteins to isolate novel scFv clones against huntingtin from a non-immune human antibody library. We then applied yeast surface display to affinity mature this scFv pool and analyze the location of the binding site of the mutant with the highest affinity. Interestingly, the paratope was mapped exclusively to the variable light chain domain of the scFv. A single domain antibody was constructed consisting solely of this variable light chain domain, and was found to retain full binding activity to huntingtin. Cytoplasmic expression levels in yeast of the single domain were at least fivefold higher than the scFv. The ability of the single-domain intrabody to inhibit huntingtin aggregation, which has been implicated in the pathogenesis of Huntington's disease (HD), was confirmed in a cell-free in vitro assay as well as in a mammalian cell culture model of HD. Significantly, a single-domain intrabody that is functionally expressable in the cytoplasm was derived from a non-functional scFv by performing affinity maturation and binding site analysis on the yeast cell surface, despite the differences between the cytoplasmic and extracellular environment. This approach may find application in the development of intrabodies to a wide variety of intracellular targets.

Building novel binding ligands to B7.1 and B7.2 based on human antibody single variable light chain domains

Ligands specific for B7.1 (CD80) and B7.2 (CD86) have applications in disease indications that require inhibition of T-cell activity. As we observed significant sequence and structural similarity between the B7-binding ligand, cytotoxic T-lymphocyte associated protein-4 (CTLA-4), and antibody variable light chain domains (VLs), we have explored the possibilities of making novel B7 binding molecules based on single VL domains.We first describe the “rational” design and construction of a VL/CTLA-4 hybrid molecule in which we have grafted both the CDR1 and CDR3-like loops of CTLA-4 onto a single VL light chain, at sites determined by sequence and structure-based alignment. This molecule was secreted as a soluble product from Escherichia coli, but did not show any binding to B7.1 and B7.2. In a second approach we constructed a VL library in which human VL genes derived from B-cells were spiked with the CDR3-like loop of CTLA-4 and further diversified by DNA shuffling. This library was displayed on phage, and after selection gave B7.1 binding ligands which competed with CTLA-4. In order to evaluate the possible general utility of VL domains as binding ligands, we have constructed a non-biased VL library. From this DNA-shuffled human VL library we have selected single VL domains specific for B7.1, B7.2 or human IgG. Two B7.1-specific VL ligands and one B7.2-specific VL ligand showed competition with CTLA-4. One candidate VL domain-specific for B7.1 was affinity matured by simultaneous randomisation of all CDR loops using DNA shuffling with degenerate CDR-spiking oligonucleotides. From this library, a single VL domain with affinity of 191 nM for B7.1 was obtained, which also showed binding to B7.1 in situ. This VL had mutations in CDR1 and CDR3, indicating that antigen recognition for this single VL is most likely mediated by the same regions as in the VL domain of whole antibodies.The B7.1 and B7.2-specific VL domains described in this study may form the basis of a new family of immunomodulatory recombinant molecules. Furthermore, our studies suggest that it is feasible to create specific single VL domains to diverse targets as is the case for single VH domains.

Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts

MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly4Ser)3 linker to a variable light-chain (VL) domain (ĸ chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 Å resolution by molecular replacement with an Rcryst of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a β-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the β-sheet of the VL domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts

MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

Multivalent Fvs: characterization of single-chain Fv oligomers and preparation of a bispecific Fv.

Single-chain Fv proteins are known to aggregate and form multimeric species. We report here that these molecules represent a new class of molecular assembly, which we have termed multivalent Fvs. Each binding site in a multivalent Fv comprises the variable light-chain (VL) domain from a single-chain Fv, and the variable heavy-chain (VH) domain from a second single-chain Fv. Each single-chain Fv in a multivalent Fv is part of two binding sites. We have characterized the multivalent forms of the 4-4-20, CC49 and B6.2 sFvs. The degree of multivalent Fv formation is linker-dependent. Multivalent Fvs cannot form in the absence of an intact linker. Multivalent Fvs can be stabilized by their antigen. The conversion between different forms of the multivalent Fvs can be catalyzed by disassociating agents such as 0.5 M guanidine hydrochloride with 20% ethanol. Multivalent Fvs have significantly different stabilities depending on the specific variable domains from which they are constructed. Two models have been proposed for the structure of a multivalent Fv. We have tested each model by attempting to produce a heterodimer from the anti-fluorescein 4-4-20 and anti-tumor CC49 variable regions. We successfully produced a 4-4-20/CC49 heterodimer that comprises two mixed sFvs. The first mixed sFv is composed of the 4-4-20 VL domain, a 12 residue linker and the CC49 Vh domain. The second mixed sFv is composed of a CC49 VL domain, a 12 residue linker and the 4-4-20 VH domain. The 4-4-20/CC49 heterodimer bound both fluorescein and the tumor-associated glycoprotein-72 antigen. These results support a VH/VL 'rearrangement' model in which each variable domain of a multivalent Fv binding site comes from a different polypeptide chain.