Eduardo A. Padlan

Phosphocholine binding immunoglobulin Fab McPC603. An X-ray diffraction study at 2.7 A.

The crystal structure of the Fab of McPC603, a phosphocholine-binding mouse myeloma protein, has been refined at 2.7 A resolution by a combination of restrained least-squares refinement and molecular modeling. The overall structure remains as previously reported, with an elbow bend angle between the variable and constant modules of 133 degrees. Some adjustments have been made in the structure of the loops as a result of the refinement. The hypervariable loops are all visible in the electron density map with the exception of three residues in the first hypervariable loop of the light chain. A sulfate ion occupies the site of binding of the phosphate moiety of phosphocholine.

The structural and genetic basis for expression of normal and latent VHa allotypes of the rabbit

The immunoglobulin heavy chain variable regions of the rabbit are unusual in having genetically controlled, serologically detectable alternative forms, the VHa allotypes, as well as minor VH allotypes of the x, y and w groups. New insights into the probable structural basis for the VHa allotypes have come from re-examination of earlier protein sequence data in the light of newly deduced protein sequences derived from sequencing cloned cDNAs and genomic DNAs encoding VH regions. Here we review this sequence information, and define the allotype-correlated differences at seven positions in framework region 1 and 10 positions in framework region 3 that may lead to the serologically detectable allotypic determinants (allotopes). Most alternative amino acids at allotype-correlated positions can be derived from each other by single-base changes. Thus somatic mutations and/or gene conversion-like events must be considered along with other serological and genetic explanations for various reported observations of the production of latent VHa allotypes. The proximity of rabbit VH genes (approximately 3 kb apart) might enhance the likelihood of conversion-like events in both germline and somatic cells.

On the specificity of antibody/antigen interactions: phosphocholine binding to McPC603 and the correlation of three-dimensional structure and sequence data.

Refined three-dimensional structures of McPC603 Fab and the complex with phosphocholine permit a detailed assessment of the residues crucial to determining the antibody specificity. Correlation with sequence data suggests that the structure of the binding site is highly conserved in immunoglobulins with phosphocholine-binding specificity. There is suggestive evidence that coupling of somatic mutations occurs to preserve antigen-binding specificity. The immune response is characterized by specificity and diversity. While each antibody appears to be specific for a single antigen, the immune response can generate up to 10(9) different specificities. In order to understand, at the molecular level, the nature of the interaction between antibody and antigen, it is necessary to have a high-resolution three-dimensional picture of the complex. Today it is possible to investigate antibody/antigen interactions directly by the crystallographic analysis of hybridoma products [5, 10]; in the past, structural studies were limited to myeloma proteins which, in some cases, could be shown to complex to certain haptens. Of the four Fab structures that have been determined by X-ray diffraction, only two have been demonstrated to bind hapten in the crystal. They are Fab NEW, which was shown to bind a vitamin K1 derivative [1] and McPC603, which binds to phosphocholine [6,9]. During the last few years, the McPC603 Fab structure has been refined at 2.7 A resolution and the complex of McPC603 Fab with phosphocholine has been refined independently at 3.1 A. In this communication, we make a comparative analysis of the sequences of a number of mouse phosphocholine-binding immunoglobulins based on the refined structure of the phosphocholine-binding site in McPC603.

Generation and characterization of a novel single-gene-encoded single-chain immunoglobulin molecule with antigen binding activity and effector functions

Monoclonal antibody (MAb) CC49 is a murine IgG1 that reacts with tumor-associated glycoprotein (TAG)-72, a pancarcinoma antigen. Clinical trials using radiolabeled CC49 for diagnostic imaging have demonstrated specific localization of more than 90% of carcinomas. The feasibility of adopting in vivo gene inoculation methods for antibody-based immunotherapy requires introduction and expression of two genes, encoding immunoglobulin (Ig) heavy and light chains, in a single cell to generate a functional antibody. To circumvent the problems inherent in this approach, we have constructed a single-gene encoding a single-chain immunoglobulin (SCIg) that, unlike previously developed SCIgs, contains all IgG domains. To construct the novel SCIg, the carboxyl end of the constant region of the chimeric (c) CC49 kappa chain is joined, via a 30 residue Gly-Ser linker peptide, to the amino terminus of the CC49 heavy chain. To our knowledge, neither a linker peptide this long nor a linkage between the constant light (C(L)) and variable heavy domains has been reported previously. Transfectomas developed by introducing the expression construct of the amplifiable gene in dihydrofolate reductase-deficient Chinese hamster ovary (CHO dhfr-) cells secrete a 160 kDa homodimeric molecule, SCIgcCC49. The in vitro antigen binding properties of SCIgcCC49 are comparable to those of cCC49 and SCIgcCC49deltaC(H)1, a single-chain Ig deficient in constant heavy chain-1 (C(H)1) and C(L) domains. The antibody-dependent cellular cytotoxicity (ADCC) of SCIgcCC49 and cCC49 were also comparable. This single-gene approach for generating an immunoglobulin molecule may facilitate in vivo gene inoculation as well as ex vivo transfection of patients cultured tumor-infiltrating lymphocytes for immunotherapy protocols for a variety of diseases, including cancer.

A model of the Fc of immunoglobulin E

A model of the Fc of human IgE was constructed using the known three-dimensional structure of IgG Fc. On the basis of amino acid sequence homology, the CE2, CE3 and CE4 domains were modelled after CG3, CG2 and CG3, respectively. The mode of association of the CG2 and of the CG3 pairs of domains was assumed for the CE3 and the CE4 pairs, respectively; the CE2 pair of domains was assembled such that they interact like the CG3 domains. An asymmetric linkage is favored for the two inter-epsilon chain disulfide bridges, i.e. the bonds were assumed to be between non-homologous cysteines. The atomic interactions in the interface of the CE2:CE2 and CE4:CE4 domain pairs were computed, and predictions are made on the solvent accessibility of the individual residues in the fragment. The model can be useful in the study of regions that may be involved in the interaction of IgE with Fc(epsilon) receptors.

Antibody Fab assembly: The interface residues between CH1 and CL

The effective assembly of an antibody molecule requires the proper association of the light and heavy chains, namely the tight, canonical association of VH with VL, and of CH1 with CL. In this paper the interaction of CH1 is examined by looking at the degree of conservation of residues in the interface between CH1 and CL, where CH1 can belong to any of the heavy chain classes, and CL can be either lambda or kappa. The three-dimensional structures of four antibody Fabs have been examined to see which are the significant interacting residues and to see whether they also correspond to the conserved residues in the different classes. It was found that there are a few hydrophobic residues buried in the interface which make numerous contacts with residues of the other chain and which remain invariant, or else are highly conserved. Around the periphery of the interface there are numerous interacting residues that have appreciable variability. Within the interface there is a cavity, the function of which may be to permit some changes in the central interface residues while still preserving the same relative orientation of CH1 and CL.

Kappa chain structure from a crystallized murine Fab': role of joining segment in hapten binding.

Abstract The entire variable region sequence of the kappa chain from M603, a phosphorylcholine(PC)-binding myeloma protein has been determined. This structure is of particular interest in that the Fab fragment from M603 has been crystallized and its three-dimensional structure determined. Analysis of the light-chain amino acid sequence has permitted identification of specific residues involved in light-heavy chain and light chain-hapten interactions. Position 96 (Leu) was found to be a hapten-contacting amino acid. This residue is not encoded in the variable region gene but rather in the ‘joining’ gene which encodes amino acids 96–108. When the M603 sequence was compared to that of a second PC-binding myeloma protein M 167, these structures were found to differ by 31% in their framework segments and 62% in complementarity-determining regions. Both light chains have Leu at position 96 and use the same joining segment sequence as apparently does a third PC-binding protein H8. These results suggest that Leu at position 96 and this particular joining segment sequence may be required in PC-binding antibodies.

A humanized antibody with specificity for hepatitis B surface antigen

A murine monoclonal antibody H67 was characterized for the binding specificity, which showed that H67 recognizes a disulfide-bond-dependent conformational epitope of common a antigenic determinant on the hepatitis B surface antigen. The result suggested that this antibody may have the potential of replacing hepatitis B immune globulin in the prevention of hepatitis B virus (HBV) infection. Therefore, we have constructed the humanized antibody HuS10 by grafting the complementarity determining regions and some framework amino acid residues of H67 onto the most homologous human antibody variable regions, 21/28 for heavy chain variable region and B1 and J kappa 2 for light chain variable region, followed by combining with human constant regions C gamma 1 and C kappa. The affinity of the HuS10 was the same as that of the H67, 8 x 10(8) x 10(8)M-1, and the HuS10 neutralized the in vitro infection of adult human hepatocyte primary culture by adr or ayw subtype of HBV. The neutralization assay showed that the HuS10 had approximately 2,000-times higher specific activity than commercially available polyclonal HBIG. These results suggest that the humanized antibody will be useful in the prevention or treatment of HBV infection.

Crystalline monoclonal antibody Fabs complexed to hen egg white lysozyme.

The Fab of a monoclonal anti-lysozyme antibody (HyHEL-10) has been crystallized as the free Fab and as the Fab-antigen complex. Crystals have also been grown of the antigen complex of the Fab of another monoclonal anti-lysozyme antibody (HyHEL-9), which recognizes a different binding surface of lysozyme. All three crystals diffract to at least 3 A resolution and are suitable for X-ray diffraction studies.

Quantitation of the immunogenic potential of protein antigens

A procedure to quantitate the immunogenic potential of protein antigens is presented. It is hypothesized that the intrinsic immunogenicity of an accessible region on the protein arises from the overall structural effect of the presence of the particular assemblage of amino acid residues in the given region. Structural parameters previously derived by Grantham [Science 185, 862-864 (1974)] to differentiate the various amino acids are assigned to each residue position. At each point chosen on the molecule, an av. value is computed due to all residues within 8.5 A of this point; it is proposed that this local average is proportional to the immunogenic potential of the region centered at this point. The method can be used to locate the immunodominant regions of a molecule and to compare the antigenicity of related molecules. Test calculations on hen egg-white lysozyme, sperm whale myoglobin and horse cytochrome c show that segments in these molecules, that have been shown in immunochemical studies to possess antigenic activity, are predicted by this method to be immunodominant.