In vivo clearing of idiotypic antibodies with antiidiotypic antibodies and their derivatives

At immunolocalization of experimental tumors, idiotypic monoclonal antibodies, such as TS1 against cytokeratin 8, can be used to carry and deposit in vivo terapeutics in the tumor. These carriers also remain in the circulation and may cause negative side-effects in other tissues. In this report, several derivatives of the antiidiotypic antibody alphaTS1 were produced and tested for their clearing capacity of the idiotypic carrier antibody TS1. Intact monoclonal alphaTS1, scFv of a alphaTS1 and alphaTS1 Fab'2 and fragments were produced by recombinant technology or by cleavage with Ficin. The scFv was tailored by use of the variable domain genes of the light and heavy chain from the hybridoma clone in combination with a (Gly4Ser)3-linker, followed by expression in E. coli. When tested for clearing capacity, the intact divalent antiidiotypic IgG was found to be the most efficient. The divalent and the monovalent Fab fragment also demonstrated significant clearing, but lower than the intact antiidiotypic IgG. The alphaTS1 scFv antibody when injected separately was not found to clear the idiotype, but could do so when preincubated with the idiotype. Rapid excretion and in vivo instability of this low molecular weight antibody fragment may be the major reasons. Similar results were obtained when the system was reversed and the 131I-labeled antiidiotype IgG was cleared with the idiotype fragment. It is concluded that both intact antiidiotypic IgG, and Fab'2 fragments are able to clear the idiotypic antibodies. The experimental data support the conclusion that the Fc parts from both the idiotype and the antiidiotype may contribute to this elimination.     

A monoclonal antibody and an enzyme immunoassay for human Ala-IL-8(77)

We used a relatively small library of 5520 randomly generated single 15-mer peptides prepared by SPOT synthesis as an array of 28.5x19.0 cm to identify epitopes for three distinct monoclonal antibodies, namely anti-p24 (human immunodeficiency virus (HIV)-1) monoclonal anibody (mab) CB4-1, anti-interleukin-10 (IL-10) mab CB/RS/13, and anti-transforming growth factor alpha (TGFalpha) mab Tab2. Initially identified peptide ligands mostly had very low affinities for the antibodies with dissociation constants around 10(-4) M. Subsequent identification of residues critical for the antibody interactions involved complete L-amino acid substitutional analyses. Several substitutions resulted in analogs with dissociation constants in the low micromolar and high nanomolar range. Specifically binding peptides with key residue patterns matching the wild-type epitopes were identified for all three antibodies. In addition, for antibody CB4-1 mimotopes that showed no homology to the known epitope were selected. Our results suggest that a very limited library diversity, although far from covering the entire sequence repertoire, can suffice to rapidly and economically select peptidic antibody epitopes and mimotopes.     

Splenic γδ T cell subsets can be separated by a novel mab specific for two CD45 isoforms

CD45 isoforms have been identified in a variety of different species and mab against various isoforms have been instrumental to define cellular subsets. In the process of generating novel mab against chicken γδ T cells two mab with specificity for CD45 were identified and characterized. The analysis of the chicken CD45 genomic structure suggested three exons being involved in alternative splicing. We cloned and expressed the full length CD45 isoform and three shorter isoforms. While the 7D12 mab reacted with all of these isoforms, the 8B1 mab selectively reacted with two short isoforms lacking either exons 3 and 5 or exons 3, 5 and 6. As expected, the reactivity of 7D12 included all leukocyte subsets, also including thrombocytes. In contrast, the 8B1 mab only reacted with lymphocytes and monocytes. 8B1 expression was found on almost all blood αβ T cells, while a γδ T cell subset and virtually all B cells lacked 8B1 reactivity. The fraction of 8B1^- αβ and γδ cells was larger in splenocytes as compared to PBL and there was also a population of 8B1⁺ splenic B cells. CD3 stimulation of splenic T cells resulted in upregulation of the 8B1 antigen on all T cells. Three-color immunofluorescence revealed differences in CD28 expression between the 8B1⁺ and 8B1¯ γδ T cell subsets with a higher CD28 expression level on 8B1¯ cells. The CD28 antigen was upregulated upon stimulation of the cells with IL-2 and IL-12. This novel mab will be a useful tool to further analyze chicken γδ T cells in more detail.     

Variability of the immunoglobulin light chain in the Siberian sturgeon, Acipenser baeri

All sturgeon VL segments isolated in this study belong to a single family, VLI, which can be divided into two subfamilies. Of the 79 cDNA clones isolated, 76 belong to the larger subfamily, VLIa, and only 3 clones constitute the smaller subfamily, VLIb. To evaluate variability, the Shannon entropy was estimated for each individual amino acid position, and to facilitate comparisons of variability between species the mean entropy of the CDR regions was calculated. In such a comparison, the sturgeon was found to have CDR1 and CDR3 variability approaching those found in mouse and clawed frog, but showed very low variability for CDR2. Amino acid position 50 does however display variability in the range of mouse and clawed frog. It is further confirmed that the sturgeon has numerous J segments, but that the junctional diversity does not contribute greatly to the diversity of the light chain. Comparisons of cDNA clones and a genomic VL segment indicate that the VL undergoes changes, particularly in the CDR regions, in a manner that can be explained by somatic hypermutation and/or gene conversion.     

T cell receptor CDR3 loop length repertoire is determined primarily by features of the V(D)J recombination reaction

The third complementarity-determining region (CDR) of the TCR alpha and beta chains forms loops that engage amino acid residues of peptides complexed with MHC. This interaction is central to the specific discrimination of antigenic-peptide-MHC complexes by the TCR. The TCRbeta chain CDR3 loop is encoded by the Dbeta gene segment and flanking portions of the Vbeta and Jbeta gene segments. The joining of these gene segments is imprecise, leading to significant variability in the TCRbeta chain CDR3 loop length and amino acid composition. In marked contrast to other pairing antigen-receptor chains, the TCR beta and alpha chain CDR3 loop size distributions are relatively narrow and closely matched. Thus, pairing of TCR alpha and beta chains with relatively similar CDR3 loop sizes may be important for generating a functional repertoire of alpha beta TCR. Here we show that the TCRbeta chain CDR3 loop size distribution is minimally impacted by TCRbeta chain or alpha beta TCR selection during thymocyte development. Rather, this distribution is determined primarily at the level of variable-region gene assembly, and is critically dependent on unique features of the V(D)J recombination reaction that ensure Dbeta gene segment utilization.     

Targeting and subsequent selection of somatic hypermutations in the human V kappa repertoire.

The number and distribution of nucleotide substitutions in human VkappaJkappa genes were examined using a PCR technique that analyzed nonproductive and productive rearrangements amplified from genomic DNA of individual B cells. The results indicate that the mutational mechanism introduces replacement (R) mutations comparably throughout the length of the VkappaJkappa rearrangement, but tends to target specific triplets. Moreover, hotspots of mutational activity were identified in complementarity determining regions (CDR). A marked increase in the frequency of R mutations in CDR was noted when productive were compared to nonproductive rearrangements, indicating that these were selected into the expressed repertoire. Of note, amino acids encoded by codons adjacent to hotspots of mutation were also positively selected implying that similar regions were targeted for hypermutation and subsequent selection. In contrast to the distribution of CDR mutations, R mutations in the framework (FR) regions tended to be eliminated from productive VkappaJkappa rearrangements, implying that the somatic hypermutational machinery frequently introduced amino acid changes that were deleterious to the structural integrity of the kappa chain protein. The difference in the ratio of R to silent mutations in CDR and FR in the expressed repertoire, therefore, reflects the summation of positive selection of R mutations in the CDR and the elimination of R mutations in the FR. The data indicate that the balance between targeted mutation of VkappaJkappa rearrangements and subsequent selection and elimination governs the pattern of mutations manifest within the expressed kappa repertoire.     

Macropodid Herpesvirus 1 Encodes Genes for Both Thymidylate Synthase and ICP34.5

Macropodid herpesvirus 1 (MaHV-1) is an unclassified alphaherpesvirus linked with the fatal infections of kangaroos and other marsupials. During the characterisation of the internal repeat region of MaHV-1, an open reading frame (ORF) encoding for thymidylate synthase (TS) gene was identified and completely sequenced. Southern blot analysis confirmed the presence of two copies of the TS gene in the MaHV-1 genome as expected. Computer analysis of the TS ORF showed it was 948 nucleotides in length. A putative polyadenylation signal was identified 17–22 bp inside the ORF implying a minimal or absent 3 untranslated region. The predicted polypeptide was 316 amino acid residues in length and contained the highly conserved motifs for folate binding and F-dUMP binding, typical of all TS enzymes. Interestingly, MaHV-1 TS polypeptide had highest similarity to the human TS polypeptide (81%) compared to the TS polypeptides of other herpesviruses (72–75%). Immediately upstream of the TS gene, a second ORF of 510 bp, encoding a polypeptide with 170 amino acid residues, was identified. The carboxyl domain of this MaHV-1 polypeptide shared 68% similarity to a 59 amino acid motif of human herpesvirus 1 ICP34.5, identifying it as the MaHV-1 ICP34.5 homologue. This is the first report of a herpesvirus that encodes for both TS and ICP34.5.     

Amino acid sequence of the variable region of heavy chain in immunoglobulin (Mot) having unusual papain cleavage sites

An IgGl(lambda) Mot myeloma protein showed a unique susceptibility toward papain digestion. The Fab fragment of Mot was more digestible with papain than the Fc fragment. This phenomenon was found to occur by unusual cleavage of the Fd fragment with papain. Determination of the complete primary structure of the V region of the H chain of Mot identified two papain cleavage sites in the second complementarity-determining region (CDR). Amino acid sequence of the cleavage sites was Ser(55)-Asp-Asp-Argdecrease-Thr-Thr-Tyr-Gly-Pro-Argdecrease- Ser-Gln- (decrease = cleavage site). In the vicinity of these cleavage sites, many hydrophilic and polar residues are present and the predicted secondary structure near these cleavage sites suggested that this region was exposed on the surface of the molecule, and that the unusual papain cleavage of the IgG Mot might be caused by a unique conformation of the molecule, making it highly susceptible to enzyme digestion.     

Molecular signatures of antinuclear antibodies-contributions of heavy chain CDR residues

A database of the Ig heavy chains of 143 anti-ssDNA, 103 anti-dsDNA and 23 anti-nucleosome antinuclear antibodies (ANAs) was constructed, with no clonal overlap, gleaning from published literature. In comparison to the Kabat database of antibodies (N>3600), ANAs (total=269) demonstrated several significant changes, particularly in the incidence of charged or polar residues, in their CDR regions. In particular, anti-dsDNA ANAs differed significantly from anti-ssDNA ANAs in having (a) more 'D' residues at H31 and more 'Y' residues at H33, in CDR1, (b) significantly different distributions of charged or polar residues at H53, H55 and H56 of CDR2, and (c) more 'R' residues at H95-H100 of CDR3. Whereas, the differences in CDR1 and CDR3 are likely to characterize anti-dsDNA ANAs encoded by all VH families, the sequence differences in CDR2 are likely to be VH family specific. Finally, among anti-dsDNA ANAs, there was an enrichment of VH1/J558 germline genes (notably, VH 45.21.1), which bear germline-encoded amino acid residues in their CDR regions that may potentially facilitate nuclear antigen binding. This ANA heavy chain database thus constitutes a useful resource for analyzing the molecular requirements for nuclear antigen reactivity.     

Affinity maturation of a V(H)H by mutational hotspot randomization

V(H)Hs from naive libraries have dissociation constants (K(D)s) in the low micromolar range and thus, for most antibody applications, their intrinsic affinities need to be improved significantly. Non-targeted in vitro affinity maturation approaches based on indiscriminate randomization of complementarity-determining region (CDR) residues or random mutagenesis of conventional antibody variable domains have been shown to improve the affinity of recombinant antibodies by 450- to over 6000-fold. A different, targeted approach based on selective randomization of CDR codons containing AGY/RGYW nucleotide mutational hotspots i.e., "hotspot codons", also promises to be very efficient for improving antibody affinities. Here we employed the latter approach for improving the affinity of PTH22, a parathyroid hormone (PTH)-derived peptide-specific V(H)H that was isolated from a naive llama phage display library. A PTH22 mutant ribosome display library was constructed by randomizing nine CDR2 and CDR3 hotspot codons. The affinity improvement of the lead binder was 30-fold, which seems somewhat low in view of the large number of randomized hotspot codons. Nucleotide sequence analyses of PTH22 and 23 naive V(H)Hs suggested that many AGY/RGYW mutational hotspots are not affinity mutational hotspots but play a role in V(H)H solubility, structure, and deletion/insertion events. Our results indicate that the mutagenesis approach described here is beneficial in terms of yielding moderate increases in affinity while fine-tuning physical properties of an antibody.     

Sequence, specificity and crystallization of an oestrone-3-glucuronide antibody (3910).

We describe the specificity profile and V region sequences of a high-affinity monoclonal antibody (mAb), 3910, directed against oestrone-3-glucuronide (E3G). Inhibition studies show that the D-ring is critical for steroid specificity, while the glucuronic acid attached to the A ring is required for high binding affinity, suggesting that both 'ends' of the E3G ligand are recognized. The VH domain is encoded by a gene from the VH7183 family, while VL appears to be encoded by the Vk5.1 gene (kappa II subgroup) with a deletion of six residues from complementarity-determining region-1 (CDR1). The VH CDR3 is 10 amino acid residues in length, of which D/N contributes five residues. Comparison of VH CDR of 3910 with those of mAb against progesterone (DB3) and digoxin (26-10, 40-50), for which crystal structures have been determined, suggests that aromatic side chains are important for E3G binding and that tyrosine residues H50, H97 and H100 may interact with the ligand. The Fab fragment of 3910 has been crystallized in its native and steroid (E3G and oestriol-3-glucuronide) complexed forms. An X-ray diffraction data set to 3 A resolution has been collected for the native Fab.     

Amyloidosis Related to a λ IV Immunoglobulin Light Chain Protein

Amyloid subunit proteins related to the lambda IV subgroup of immunoglobulin light chains have not been previously reported. We have determined the amino acid sequence of an AL amyloid protein BAK and shown that it has the structure typical of lambda IV light chain proteins. This protein, which was isolated from the spleen of a patient with AL amyloidosis, has 111 residues in the variable domain and also includes the first tryptic peptide of the constant domain for a total of 130 residues. Comparison of the primary structure of this protein with the only other completely characterized lambda IV protein (SH) reveals that they are highly homologous with only one amino acid change in FR1, two changes in FR2, and one change in FR3. The CDR regions also show few changes, with only three in CDR1, one in CDR2, and five in CDR3. To test the hypothesis that the formation of AL amyloid is related to changes in the FR regions which could affect molecular aggregation, the structure of BAK was compared with the myeloma protein SH with respect to the presumed tertiary structure. Only limited amino acid substitution was found in the surface positions that might affect intradimer and interdimer aggregation. These included an isoleucine for leucine change at position 43 and phenylalanine for valine at 45, which may affect intradimer interaction and a change of histidine to asparagine at position 67.     

Improvement in affinity and HIV-1 neutralization by somatic mutation in the heavy chain first complementarity-determining region of antibodies triggered by HIV-1 infection

We assessed the impact of somatic hypermutation in the framework region 1 (FR1) and complementarity-determining region 1 (CDR1) of three clonally-related heavy chains from the human monovalent antigen-binding fragments Fab S19, S8 and S20 on gp120 binding and HIV-1 neutralization capacity. Nucleotide changes were introduced in the heavy chains to revert single and multiple amino acid residues, and two Fab libraries were constructed with the same light chain to express equivalent amounts of parental and reverted phage Fab. We studied the contribution of each amino acid replacement to antigen binding by calculating the frequency of phage Fab retrieval after competitive library selection on gp120. Whereas mutations in FR1 had no effect on antigen binding, somatic replacements in the CDR1 of the heavy chain (HCDR1) appeared to produce significant changes. In S19 HCDR1, somatic mutation of residue 32 reduced gp120 binding. In Fab S20, the Arg(30) and Asp(31) somatically replaced residues in HCDR1 improved antigen binding. Both of these residues are necessary to increase Fab binding to gp120; reversion of either residue alone results in a decrease in binding. The impact of these two replacements was confirmed by the greater neutralization capacity of S20 compared to the other Fab. Molecular modeling of S20 HCDR1 suggests that Arg(30) and Asp(31) are the main interaction sites for gp120, increasing antibody affinity and promoting the enhanced neutralization ability of S20. These findings are consistent with a gp120-driven process, supporting a role for affinity maturation and intraclonal evolution of HIV-1 neutralizing antibodies.     

Identification of a novel bluetongue virus 1-specific B-cell epitope using a monoclonal antibody against the VP2 protein

Bluetongue virus (BTV) VP2 is an important antigenic protein that can be used for the differential diagnosis of different BTV serotypes. Here, we generated a serotype-specific monoclonal antibody (mab) against BTV1. A series of peptides synthesized based on the amino acid sequence of BTV1 VP2 were screened to define ¹¹⁵AQPLKVGL¹²² as the minimal linear peptide epitope recognized by mab 4B6. Using an immunofluorescence assay (IFA), we found that mab 4B6 reacted strongly with BTV1, but did not react with other BTV serotypes (BTV2-24). The 4B6 will serve as a novel reagent in the development of diagnostic tests for BTV1 infection.     

Microheterogeneity in the recognition of a HLA-DR2-restricted T cell epitope from a meningococcal outer membrane protein

The trimolecular interaction of T cell receptor (TcR), antigen and major histocompatibility complex (MHC) class II was analyzed using a panel of HLA-DR2-restricted T cell clones recognizing the 49-61 region of a meningococcal class I outer membrane protein (OMP). The clones, all CD3⁺CD4⁺CD8^?TcRct/β⁺, were selected by restimulation with the synthetic peptide OMP(49-61), which contains an immunodominant T helper determinant. Using a series of peptides that were sequentially truncated from the N or C terminus, four different epitope fine-specificity patterns were identified. Furthermore, each clone was found to exhibit a distinct recognition pattern for a panel of 20 single-residue substitution analogues of the minimal epitope OMP(50-58). Most substitutions that were not tolerated in the nonamer were allowed when the analogues were prepared departing from the native peptide OMP(49-61). Obviously, the residues outside the minimal epitope contribute to stabilization of the trimolecular complex. These findings suggest that defining the minimal size of T cell determinants may be of limited value. By performing proliferation competition assays putative MHC and TcR contact residues were identified in the peptide. Most likely, He 51 and Phe 54 act as MHC-anchoring residues, whereas Asp 53 represents a critical TcR contact residue for all of the clones. MHC anchoring may be provided by other residues as well, since He 51 and Phe 54 can be substituted by conservative residues [as OMP(50-58) and OMP(49-61) analogues] and with Ala [as OMP(49-61) analogues only]. Some evidence was found for interaction of particular side chains at other positions with TcR molecules, but this contribution was not equally important for all clones. Apparently, the clonotypic TcR can see a single epitope in different ways in the context of the same MHC restriction element. Since most clones use different V_α and V_β genes (which encompass the putative MHC-binding regions first and second complementarity-determining regions, CDR1 and CDR2) different modes of interaction with the HLA-DR2 molecule indeed are likely to occur.     

Structural and mutational analyses of a CD8alphabeta heterodimer and comparison with the CD8alphaalpha homodimer

The crystal structure of a recombinant mouse single chain CD8alphabeta ectodomains at 2.4 A resolution reveals paired immunoglobulin variable region-like domains with a striking resemblance to CD8alphaalpha in size, shape, and surface electrostatic potential of complementarity-determining regions (CDR), despite <20% sequence identity between the CD8alpha and CD8beta subunits. Unlike the CD8alpha subunit(s) in the heterodimer or homodimer, the CDR1 loop of CD8beta tilts away from its corresponding CDR2 and CDR3 loops. Consistent with this observation, independent mutational studies reveal that alanine substitutions of residues in the CDR1 loop of CD8beta have no effect on CD8alphabeta coreceptor function, whereas mutations in CD8beta CDR2 and CDR3 loops abolish CD8alphabeta coreceptor activity. The implications of these findings and additional CD8alpha mutational studies for CD8alphabeta- versus CD8alphaalpha-MHCI binding are discussed.     

Predicting interactions between T cell receptors and MHC-peptide complexes

Conserved interactions between T cell receptors (TCRs) and major histocompatibility complex (MHC) proteins with bound peptide antigens are not well understood. In order to gain a better understanding of the interaction modes of human TCR variable (V) regions, we have performed a structural analysis of the TCRs bound to their MHC-peptide ligands in human, using the available structural models determined by X-ray crystallography. We identified important differences to previous studies in which such interactions were evaluated. Based on the interactions found in the actual experimental structures we developed the first rule-based approach for predicting the ability of TCR residues in the complementarity-determining region (CDR) 1, CDR2, and CDR3 loops to interact with the MHC-peptide antigen complex. Two relatively simple algorithms show good performance under cross validation.     

Llama heavy-chain V regions consist of at least four distinct subfamilies revealing novel sequence features

In addition to conventional antibodies (Abs), camelids possess Abs consisting of only heavy chains. The variable domain of such a heavy-chain Ab (VHH) is fully capable of antigen (Ag) binding. Earlier analysis of 47 VHHs showed sequence features unique to VHH domains. These include the presence of characteristic amino acid substitutions in positions which, in conventional VH domains are involved in interdomain interactions, and the presence of a long third complementarity-determining region (CDR3) which is frequently constrained by an interloop disulphide bond. Here, we describe a large (152) set of Lama glama VHH cDNAs. Based on amino acid sequence similarity, these and other published camelid VHHs were classified into four subfamilies. Three subfamilies are absent in dromedaries, which have been the primary source of VHHs thus far. Comparison of these subfamilies to conventional VH regions reveals new features characteristic of VHHs and shows that many features earlier regarded as characteristic of VHHs in general are actually subfamily specific. A long CDR3 with a concomitant putative additional disulphide bond is only observed in two VHH subfamilies. Furthermore, we identified new VHH-characteristic residues at positions forming interdomain sites in conventional VH domains. The VHH subfamilies also differ from each other and conventional VH domains in the canonical structure of CDR1 and CDR2, mean CDR3 length, and amino acid residue variability. Since different VHH-characteristic residues are observed in all four subfamilies, these subfamilies must have evolved independently from classical VH domains.     

Identical VHD and DJH Junctions in Monoclonal Antibodies Derived in Response to Dextran B512 Could be the Result of Developmental Selection

We describe here the CDR3s of a collection of monoclonal antibodies (MoAb) with specificity for the carbohydrate dextran B512 produced in the mouse strain C57BL/6. In spite of the postulated mechanisms for variability in this region, a high proportion of these monoclonals displayed identical V_HD (24/30) and DJ_H (21/30) junctions and 21 of them were identical in the whole CDR3. These 21 independently generated identical CDR3s could be ordered in eight groups indicating that not a particular CDR3, but instead the mechanism for generating identical junctions was preserved. Two of the CDR3s in this study were found to be identical to the CDR3 of the monoclonal B1-8 produced in C57BL/6 in response to proteins bearing the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). This and other parameters support the notion that the generation of identical junctions could be independent of antigenic selection. We also report here the association between J_H usage and amino acid (aa) residues at the V_HD and DJ_H junctions. Since these MoAb were generated in response to dextran B512, immunoglobulin conformation has to be compatible with antigen binding. Nevertheless, no aa residue of CDR3 could be directly related to antigen binding. We postulate therefore, that the observed selection of CDR3s could be directed to the production of variable regions with protein configuration most suitable with immunoglobulin folding and may occur prior to antigenic selection. Selection for junctional residues in relation to J_H usage and the generation of identical CDR3s are probably different events. Possible genetic mechanisms operating for CDR3 construction and/or selection by cellular ligands are discussed.