Molecular analysis of heavy chain-only antibodies of Camelus bactrianus.

In this work, IgG content and structures of antigen-binding domains and hinge regions of different IgG subtypes of Camelus bactrianus were analyzed in detail for the first time. Our data demonstrate that C. bactrianus contains a very large amount of heavy chain-only antibodies that can be used as a source of VHH domain-containing molecules. Despite some minor sequence differences identified in this study, C. bactrianus VHH domains possess principally the same unique features as those of C. dromedarius and the llama. These features are important for developing an efficient phage display-based antibody selection technology. We conclude that C. bactrianus is a very suitable animal to raise an immune response that serves as a source to identify antigen-specific VHHs selected after phage display.

Camelid immunoglobulins and nanobody technology.

It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from other species, these special antibodies are devoid of light chains and are composed of a heavy-chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma-genes. An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.

The piglet as a model for B cell and immune system development.

The ability to identify factors responsible for disease in all species depends on the ability to separate those factors which are environmental from those that are intrinsic. This is particularly important for studies on the development of the adaptive immune response of neonates. Studies on laboratory rodents or primates have been ambiguous because neither the effect of environmental nor maternal factors on the newborn can be controlled in mammals that: (i) transmit potential maternal immunoregulatory factors in utero and (ii) are altricial and cannot be reared after birth without their mothers. Employing the newborn piglet model can address each of these concerns. However, it comes at the price of having first to characterize the immune system of swine and its development. This review focuses on the porcine B cell system, especially on the methods used for its characterization in fetal studies and neonatal piglets. Understanding these procedures is important in the interpretation of the data obtained. Studies on neonatal piglets have (a) provided valuable information on the development of the adaptive immune system, (b) lead to important advances in evolutionary biology, (c) aided our understanding of passive immunity and (d) provided opportunities to use swine to address specific issues in veterinary and biomedical research and immunotherapy. This review summarizes the history of the development of the piglet as a model for antibody repertoire development, thus providing a framework to guide future investigators.

Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains.

The antigen-binding site of antibodies from vertebrates is formed by combining the variable domains of a heavy chain (VH) and a light chain (VL). However, antibodies from camels and llamas are an important exception to this in that their sera contain, in addition, a unique kind of antibody that is formed by heavy chains only. The antigen-binding site of these antibodies consists of one single domain, referred to as VHH. This article reviews the mutations and structural adaptations that have taken place to reshape a VH of a VH-VL pair into a single-domain VHH with retention of a sufficient variability. The VHH has a potent antigen-binding capacity and provides the advantage of interacting with novel epitopes that are inaccessible to conventional VH-VL pairs.

Combinatorial Design of a Nanobody that Specifically Targets Structured RNAs.

Recent advances in transcriptome sequencing and analysis have revealed the complexity of the human genome. The majority (≈ 98%) of cellular transcripts is not translated into proteins and represents a vast, unchartered world of functional non-coding RNAs. Most of them adopt a well-defined three-dimensional structure to achieve their biological functions. However, only very few RNA structures are currently available which reflects the challenges associated with RNA crystallization. Nevertheless, these structures would represent a critical step in understanding functions of non-coding RNAs and their molecular mechanisms in the cell. The overall goal of this study is to develop an innovative and versatile tool to facilitate the functional study and crystallization of structured RNAs (stRNAs). In this work, we have engineered an antibody fragment from camelid heavy-chain antibody (nanobody) able to specifically bind with low nanomolar affinity to stRNA, while no binding could be detected for single-stranded DNA/RNA, double-stranded DNA/RNA or a negatively charged protein. However, this nanobody recognizes different and non-related stRNAs, this observation suggests that it binds to an epitope shared by these stRNAs. Finally, our data also show that the binding of the nanobody does not alter the secondary structure content of the stRNA as well as its unfolding/refolding processes during heat treatment. This work constitutes a successful proof of concept demonstrating that nanobodies can be engineered to recognize RNA-related epitopes.

A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops.

BACKGROUND: Camelid serum contains a large fraction of functional heavy-chain antibodies - homodimers of heavy chains without light chains. The variable domains of these heavy-chain antibodies (VHH) have a long complementarity determining region 3 (CDR3) loop that compensates for the absence of the antigen-binding loops of the variable light chains (VL). In the case of the VHH fragment cAb-Lys3, part of the 24 amino acid long CDR3 loop protrudes from the antigen-binding surface and inserts into the active-site cleft of its antigen, rendering cAb-Lys3 a competitive enzyme inhibitor. RESULTS: A dromedary VHH with specificity for bovine RNase A, cAb-RN05, has a short CDR3 loop of 12 amino acids and is not a competitive enzyme inhibitor. The structure of the cAb-RN05-RNase A complex has been solved at 2.8 A. The VHH scaffold architecture is close to that of a human VH (variable heavy chain). The structure of the antigen-binding hypervariable 1 loop (H1) of both cAb-RN05 and cAb-Lys3 differ from the known canonical structures; in addition these H1 loops resemble each other. The CDR3 provides an antigen-binding surface and shields the face of the domain that interacts with VL in conventional antibodies. CONCLUSIONS: VHHs adopt the common immunoglobulin fold of variable domains, but the antigen-binding loops deviate from the predicted canonical structure. We define a new canonical structure for the H1 loop of immunoglobulins, with cAb-RN05 and cAb-Lys3 as reference structures. This new loop structure might also occur in human or mouse VH domains. Surprisingly, only two loops are involved in antigen recognition; the CDR2 does not participate. Nevertheless, the antigen binding occurs with nanomolar affinities because of a preferential usage of mainchain atoms for antigen interaction.

Canonical antigen-binding loop structures in immunoglobulins: more structures, more canonical classes?

Grafting the antigen-binding loops onto a human antibody scaffold is a widely used technique to humanise murine antibodies. The success of this approach depends largely on the observation that the antigen-binding loops adopt only a limited number of canonical structures. Identification of the correct canonical structure is therefore essential. Algorithms that predict the main-chain conformation of the hypervariable loops using only the amino acid sequence often provide this information. Here, we describe new canonical loop conformations for the hypervariable regions H1 and H2 as found in single-domain antibody fragments of dromedaries or llama. Although the occurrence of these new loop conformations was not predicted by the algorithms used, it seems that they could occur in human or mouse antigen-binding loops. Their discovery indicates that the currently used set of canonical structures is incomplete and that the prediction algorithms should be extended to include these new structures.

A DNA sequence for positioning chromatosomes.

We have analysed the sequences of 280 chromatosomal DNA molecules. In approximately half the clones, a short DNA sequence of the preferred form NGGR is located at one, but not both, of the termini of the cloned DNA. We show that the clones lacking this signal possess a substantially stronger rotational positioning signal than those that contain it. These results indicate that the sequence organisation of chromatosomal DNA is asymmetric with respect to the midpoint and imply that the sequence NGGR may play a direct role in positioning chromatosomes in condensed chromatin.

DNA sequence organization in chromatosomes.

The chromatosome is a structural unit of chromatin which contains a histone octamer and one linker histone molecule (H5, H1a or H1b in chicken erythrocytes) bound to 168 bp of DNA (= core particle DNA extended by 22 bp). We have cloned and sequenced 280 DNA fragments of 163 to 173 bp in length isolated from chicken erythrocyte chromatosomes. We have analysed both this set and a subset of 171 clones whose lengths varied between 166 and 170 bp. The periodic modulation of the frequency of occurrence of trinucleotide sequences is neither as regular nor as pronounced for core particle DNA, even for the trinucleotide ApApA/TpTpT. Nevertheless for this trinucleotide the congruence of the preferential and avoided locations between the two sets is remarkable. We conclude that the rotational positioning of the DNA bound to the histone octamer is essentially the same in chromatosomes and nucleosome core particles. This conclusion is confirmed by Fourier analysis, which shows that within the central 145 bp the average rotational periods of about 10-20 bp and phases are very similar to those in core DNA. However the amplitude of the modulations is less, indicating that the binding of the linker histone can overcome to a certain extent the sequence-dependent bending preferences of DNA. Although the rotational signals are largely conserved the sequence organization of the chromatosomal DNA differs in significant ways from that of core particle DNA. The major difference is the preferential occurrence of short G+C-rich instead of A+T-rich sequences at the midpoint of the binding site. The second apparent difference is the exclusion of short oligo(dA).(dT) tracts from positions about 40 to 43 bp on either side of the midpoint in chromatosomal, but not in core particle DNA. Finally, we show that particular base-steps are preferentially enriched close to the termini of chromatosomal DNA and propose that these sequences may constitute one of the two DNA binding sites for the bivalent globular domain of histones H5 or H1. The implications for the positioning of GH5 relative to the path of the DNA superhelix are discussed.

Formation, stability and core histone positioning of nucleosomes reassembled on bent and other nucleosome-derived DNA.

DNA originating from chicken erythrocyte mononucleosomes was cloned and sequenced. The properties of nucleosome reconstruction were compared for two cloned inserts, selected on account of their interesting sequence organization, length and difference in DNA bending. Cloned fragment 223 (182 base-pairs) carries alternatively (A)3-4 and (T)4-5 runs approximately every ten base-pairs and is bent; cloned fragment 213 (182 base-pairs) contains a repeated C4-5ATAAGG consensus sequence and is apparently not bent. Our experiments indicate the preference of the bent DNA fragment 223 over fragment 213 to associate in vitro with an octamer of histones under stringent conditions. We provide evidence that the in vitro nucleosome formation is hampered in the case of fragment 213, whereas the reconstituted nucleosomes were equally stable once formed. For the correct determination of the positioning of the histone octamer with regard to the two nucleosome-derived cloned DNA sequences, the complementary use of micrococcal nuclease, exonuclease III and DNase I is a prerequisite. No unique, but rotationally related, positions of the histone octamer were found on these nucleosome-derived DNA fragments. The sequence-dependent anisotropic flexibility, as well as intrinsic bending of the DNA, resulting in a rotational setting of the DNA fragments on the histone core, seems to be a strong determinant for the allowed octamer positions, Exonuclease III digestion indicates a different histone-DNA association when oligo(d(C.G)n) stretches are involved. The apparent stagger near oligo(d(A.T)n) stretches generated by DNase I digestion on reconstituted nucleosome 223 was found to be inverted from the normal two-base 3' overhang to a two-base 5' overhang. Two possibilities of the oligo(d(A.T)n) minor groove location relative to the histone core are envisaged to explain this anomaly in stagger.

The specific variable domain of camel heavy-chain antibodies is encoded in the germline.

The variable domains of the functional heavy-chain antibodies (VHHs) discovered in camels are related to the human VH subgroup III. They are nevertheless clearly distinguishable from the VHs of conventional four-chain immunoglobulins by the presence of important amino acid substitutions, located in the solvent-exposed surface normally covered by the variable domain of the light chain. The analysis of an unrearranged dromedary DNA library revealed that the specific VHH gene with its characteristic amino acid substitutions is encoded in the germline. Therefore, it is concluded that the VHHs do not arise through an ontogenic process of somatic hypermutation. The presence of putative DNA recombination signals that are more prevalent in the camel VHH, compared to the VH germline gene, might play a role in the formation and efficient expansion of the VHH repertoire.

Degenerate interfaces in antigen-antibody complexes.

In most of the work dealing with the analysis of protein-protein interfaces, a single X-ray structure is available or selected, and implicitly it is assumed that this structure corresponds to the optimal complex for this pair of proteins. However, we have found a degenerate interface in a high-affinity antibody-antigen complex: the two independent complexes of the camel variable domain antibody fragment cAb-Lys3 and its antigen hen egg white lysozyme present in the asymmetric unit of our crystals show a difference in relative orientation between antibody and antigen, leading to important differences at the protein-protein interface. A third cAb-Lys3-hen lysozyme complex in a different crystal form adopts yet another relative orientation. Our results show that protein-protein interface characteristics can vary significantly between different specimens of the same high-affinity antibody-protein antigen complex. Consideration should be given to this type of observation when trying to establish general protein-protein interface characteristics.

Loss of splice consensus signal is responsible for the removal of the entire C(H)1 domain of the functional camel IGG2A heavy-chain antibodies.

The molecular basis for the absence of the C(H)1 domain in naturally occurring heavy-chain antibodies of the camelids was assessed by determining the entire Camelus dromedarius gamma2a heavy-chain constant gene. The organization of the camel gamma2a constant heavy-chain gene obtained from a liver genomic library appears to be typical of all other mammalian gamma genes sequenced to date. It contains the switch, CH1, hinge, CH2, CH3, M1 and M2 exons. In contrast to the case in mouse and human heavy chain diseases, the camel gamma2a gene shows no major structural defect, and its equivalent CHI exon is intact. However, sequence analysis has revealed that the splicing site, immediately after the CH1 exon, is defective due to point mutations, especially the G(+1) to A(+1) transversion seems to be detrimental. It is concluded that the loss of the splice consensus signal is responsible for the removal of the entire CH1 domain in camel gamma2a heavy-chain immunoglobulins. Additionally, a closer analysis of the hinge exon suggests the possible involvement of transposons in the genetic variation of mammalian Cgamma hinges.

Comparison of llama VH sequences from conventional and heavy chain antibodies.

Forty different PCR clones encoding a llama variable heavy chain domain were analysed. The majority of these clones are derived from heavy-chain antibody cDNA in which the entire CH1 exon is absent. It appears from the amino acid within the VHH framework 1 and 3 that all the llama clones belong to the VH III family. However, the individual llama VHH sequences differ more substantially from each other than expected for members of the same family. Several remarkable amino acid substitutions in the framework 2 hinder the proper association of the VL. However, they lay the foundation for the secretion from the endoplasmic reticulum and good solubility behaviour of llama H2 antibodies. The repertoire of the llama VHHs may be extensive due to the presence of a long CDR3-loop, often constrained by a disulfide bridge and the occurrence of H1 and H2 loop conformations not yet encountered in mice or human VHs. The variability plot of the amino acids in the VHH shows that the first hypervariable region coincides with the structural H1 loop in contrast to the situation found in mice and man where the CDR1 and H1 are slightly offset. We propose that the amino acids of the llama H1 loop participate actively in the antigen binding. All these observations are characteristic for the llama VHHs of the homodimeric heavy-chain H2 antibodies, but are not maintained in the llama clones from conventional heterotetrameric H2L2 immunoglobulins.

Putative regulatory sequences for the transcription of mini-exons in Trypanosoma brucei as revealed by S1 sensitivity.

The 35-nucleotide (nt) mini-exon found at the 5' end of most Trypanosoma brucei mRNAs is encoded as part of a tandem 1.35-kb repeat in genomic DNA. We cloned this DNA and identified an S1-sensitive site in supercoiled plasmids containing mini-exon repeats. This site is situated on a poly(dA-dT) stretch that is variable in length in different copies of repeat. Poly(dA-dT) is capable of forming abnormal DNA helix configurations, some of which are induced by supercoiling. The S1 site may have a role in regulation of mini-exon transcription.

Selection and identification of single domain antibody fragments from camel heavy-chain antibodies.

Functional heavy-chain gamma-immunoglobulins lacking light chains occur naturally in Camelidae. We now show the feasibility of immunising a dromedary, cloning the repertoire of the variable domains of its heavy-chain antibodies and panning, leading to the successful identification of minimum sized antigen binders. The recombinant binders are expressed well in E. coli, extremely stable, highly soluble, and react specifically and with high affinity to the antigens. This approach can be viewed as a general route to obtain small binders with favourable characteristics and valuable perspectives as modular building blocks to manufacture multispecific or multifunctional chimaeric proteins.

The regulated expression of an intrabody produces a mutant phenotype in Drosophila.

Intrabodies show great promise for controlling gene expression. As an initial attempt to evaluate the intrabody technology in Drosophila, the gene poxn was used as target. Transgenic flies harboring different anti-Poxn scFv genes integrated into various chromosomes were obtained. In one transformant, a phenocopy resembling the hypomorphic poxn-phenotype was produced in embryos and larvae following induction of expression of alpha-Poxn2 intrabody. The antisense approach was used as control. Parameters that can affect the success of intrabody technology are described.

Isolation and characterization of single-chain Fv genes encoding antibodies specific for Drosophila Poxn protein.

The usefulness of intrabodies as specific inhibitors of gene function has been extensively demonstrated in cell culture assays. However, very few experiments have been conducted with intrabodies expressed in whole organisms. To evaluate the intrabody technology in Drosophila, we focused on poxn protein, since its effects can be easily studied. We purified the recombinant poxn protein. We next isolated three single-chain variable fragments (scFv) which specifically recognize poxn protein. Two scFvs, designated alpha-Poxn2 and alpha-Poxn4, react with both denatured and native Poxn with half maximal inhibition values of 100 nM and 40 nM, respectively. The alpha-Poxn5 scFv also recognizes denatured Poxn but either does not recognize native Poxn or its half maximal inhibition value for native Poxn is high.

Single domain antibodies: comparison of camel VH and camelised human VH domains.

The antigen binding sites of conventional antibodies are formed primarily by the hypervariable loops from both the heavy and the light chain variable domains. Functional antigen binding sites can however also be formed by heavy chain variable domains (VH) alone. In vivo, such binding sites have evolved in camels and camelids as part of antibodies, which consist only of two heavy chains and lack light chains. Analysis of the differences in amino acid sequence between the VHs of these camel heavy chain-only antibodies and VH domains from conventional human antibodies helped to design an altered human VH domain. This camelised VH proved, like the camel VH, to be a small, robust and efficient recognition unit formed by a single immunoglobulin (Ig) domain. Biochemical, structural and antigen binding characterisation properties of both camel VH domains and camelised human VH domains suggest that these can compete successfully with single chain variable domain (Fv) fragments from conventional antibodies in many applications. Of special importance in this respect is the use of such VH domains as enzyme inhibitors, for which they seem to be better suited than Fv fragments. This function appears to be closely related to their often very long third hypervariable loop, which is central for antigen recognition in their binding sites.

Chromosome rearrangement in Leishmania mexicana M379.

Circular extrachromosomal elements were observed in a variety of Leishmania species. We show here that two lines originating from the same isolate have been found to contain a circular DNA molecule of 26.6 kb and a linear chromosome of about 250 kb, respectively, which share a homology of more than 20 kb. The circular DNA molecule and its related region on the linear chromosome were cloned and their restriction maps compared. This investigation reveals information about chromosome rearrangement in L. mexicana M379. Further examination will enable us to understand the nature of chromosome rearrangement such as circularization or linearization.