Crystallization and preliminary X-ray diffraction study of a chimaeric Fab′ fragment of antibody binding tumour cells

doi:10.1016/0022-2836(91)90656-Q

Journal of Molecular Biology

Volume 219, Issue 4, 20 June 1991, Pages 603-604

https://doi.org/10.1016/0022-2836(91)90656-Q Get rights and content

Abstract

Crystals have been obtained of a chimaeric Fabt′ fragment that binds to a tumour-associated mucin-like glycoprotein TAG72. The Fab′ fragment comprises the variable heavy and light-chain domains of a murine monoclonal antibody, B72.3, coupled to human γ4 and κ constant regions. The crystals are orthorhombic and belong to the space group P2₁2₁2₁, with unit cell dimensions $a = 67.9 A ̊, b = 94.2 A ̊ and c = 208.8 A ̊$ . Diffraction to 2.6 Å resolution was observed using synchrotron radiation. Despite the acute radiation sensitivity of the crystals a full native data set has been collected using the Weissenberg camera at the Photon Factory synchrotron. These data will be used for molecular replacement calculations in an attempt to elucidate the structure of this chimaeric Fab′ fragment.

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Crystallization and structure determination of an autoimmune anti-poly(dT) immunoglobulin Fab fragment at 3.0 Å resolution
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B.W. Matthews
Solvent content of protein crystals
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D.R. Rose et al.
Preliminary crystal structure of an Fab specific for a Salmonella O-polysaccharide antigen
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Pharmacokinetics of the monoclonal antibody B72.3 and its fragments labelled with either 1251 or 1111n
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A spectrum of monoclonal antibodies reactive with human mammary tumor cells

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Cited by (24)

The Fab conformations in the solution structure of human immunoglobulin G4 (IgG4) restrict access to its Fc region implications for functional activity
2014, Journal of Biological Chemistry
Citation Excerpt :
A total of 20,000 conformationally randomized full sized IgG4(Ser222) models were created by joining molecular structures for the IgG4 Fab and Fc regions. A homology model for human IgG4 was constructed from crystal structures for Fab B72.3 and human IgG1 (Protein Data Bank codes 1BBJ and 1HZH) (36, 37) as described previously (20). Four sets of 5000 randomized IgG4 models were created using different hinge lengths and compositions.
Human IgG4 antibody shows therapeutically useful properties compared with the IgG1, IgG2, and IgG3 subclasses. Thus IgG4 does not activate complement and shows conformational variability. These properties are attributable to its hinge region, which is the shortest of the four IgG subclasses. Using high throughput scattering methods, we studied the solution structure of wild-type IgG4(Ser²²²) and a hinge mutant IgG4(Pro²²²) in different buffers and temperatures where the proline substitution suppresses the formation of half-antibody. Analytical ultracentrifugation showed that both IgG4 forms were principally monomeric with sedimentation coefficients s_20,w⁰ of 6.6–6.8 S. A monomer-dimer equilibrium was observed in heavy water buffer at low temperature. Scattering showed that the x-ray radius of gyration R_g was unchanged with concentration in 50–250 mm NaCl buffers, whereas the neutron R_g values showed a concentration-dependent increase as the temperature decreased in heavy water buffers. The distance distribution curves (P(r)) revealed two peaks, M1 and M2, that shifted below 2 mg/ml to indicate concentration-dependent IgG4 structures in addition to IgG4 dimer formation at high concentration in heavy water. Constrained x-ray and neutron scattering modeling revealed asymmetric solution structures for IgG4(Ser²²²) with extended hinge structures. The IgG4(Pro²²²) structure was similar. Both IgG4 structures showed that their Fab regions were positioned close enough to the Fc region to restrict C1q binding. Our new molecular models for IgG4 explain its inability to activate complement and clarify aspects of its stability and function for therapeutic applications.
Module based antibody engineering: A novel synthetic REDantibody
2011, Journal of Immunological Methods
Citation Excerpt :
In essence replacing the CH1 and CL domains of Fab with a single FP domain. Here we report the design, assembly, production and characterisation of a VH–RFP–VL–His-tag (REDantibody) molecule, where monomeric red fluorescent protein (mRFP) from Discosoma (Campbell et al., 2002) is inserted as a rigid linker between the VH and VL domains of three recombinant distinct antibodies, anti-carbohydrate antibodies B72.3 (Brady et al., 1991), CA19.9 (Koprowski et al., 1979) and 4D5-8 anti-p185HER2 (Eigenbrot et al., 1993). The resulting recombinant molecules are characterised by SDS–PAGE, size exclusion chromatography, spectrophotometry, surface plasmon resonance and by utility in immunofluorescence detection of Trypanosoma cruzi epimastigotes by confocal microscopy to demonstrate that the two functionalities are retained, i.e., binding affinity and optical properties.
We describe the facile generation of a stable recombinant antibody with intrinsic red fluorescent properties for qualitative and potentially quantitative immunofluorescence analysis. The REDantibody based on the X-ray crystallographic structures of the anti-sialyl-Tn antibody B72.3 and 3D model of the monomeric red fluorescent protein was designed to retain optimal spatial geometry between the C- and N-termini of the V_H and V_L chains respectively to mimic the domains interface pairing in antibody Fab fragments and to incorporate the red fluorescent protein as a bridging scaffold. The model was further validated by assembling a REDantibody based on CA19.9 the anti-sialylated Lewis (Le)^a blood group antigen and 4D5-8 the anti-p185^HER2 antibodies. The chimeric heavy and light chains containing red fluorescent protein as a bridge were correctly processed and secreted into Escherichia coli periplasm for assembly and disulphide bond formation, further analysis revealed the molecules to be exclusively monomers. Purified anti-glycan proteins were used for an immunofluorescent analysis of Trypanosoma cruzi epimastigotes, and the anti-p185^HER2 used to determine the binding properties. The REDantibody platform facilitates rapid generation of scFv chimeras that could be used for screening antibodies against cell surface markers. Furthermore, such modular assembly should permit the interchange of binding sites and of fluorophores to create robust panels of coloured antibodies.
Site-directed immobilization of immunoglobulin G on 3-aminopropyltriethoxylsilane modified silicon wafer surfaces
1999, Materials Science and Engineering C
Immobilization and distribution properties of the polyclonal antibodies to hepatitis B surface antigen (anti-HBsAg) onto 3-aminopropyltriethoxylsilane (APTES) modified silicon wafer surfaces under two different kinds of coupling conditions were investigated using atomic force microscopy (AFM). Enzyme immunoassay (EIA) and X-ray photoelectric spectroscopy (XPS) were also used as the complimentary techniques to quantify a NH₂-terminated siloxane monolayer and a antibody layer. Anti-HBsAg antibodies were site-directly immobilized onto the APTES modified surface: (1) in acetate buffer of pH 5.2 for 3 days at 4°C and (2) in acetate buffer of pH 5.2 containing 5 mM NaBH₄ for 12 h, via aldehydes generated on the carbohydrate side chains at the C-terminal of IgG using periodate (NaIO₄)-oxidized reactions.
Controlled site-directed assembly of antibodies by their oligosaccharide moieties onto APTES derivatized surfaces
1999, Journal of Colloid and Interface Science
A convenient and efficient method for the site-directed incorporation of aldehydes generated on the oligosaccharide moieties at the C-terminal of immunoglobulin (IgG) using NaIO₄oxidation reaction is explored as a means of ensuring controlled assembly of IgG antibodies onto aminopropyltriethoxylsilane (APTES) derivatized silicon wafer surfaces. The orientation and antigen binding capacity (AgBC) of site-directly assembled IgG antibodies on derivatized surfaces were investigated using atomic force microscopy (AFM) and enzyme immunoassay (EIA), respectively. A major difference in preferential orientation is observed when the incubation of derivatized surfaces with oxidized IgG molecules is compared in two different kinds of buffer solutions. We obtained the stable and homogeneous IgG layer without loss of the AgBC on the APTES derivatized surface using the controlled incubation condition.
The discrimination of IgM and IgG type antibodies and Fab′ and F(ab) <inf>2</inf> antibody fragments on an industrial substrate using scanning force microscopy
1996, Ultramicroscopy
We have previously employed scanning force microscopy (SFM) to study antibody-antigen molecular interactions on microtiter wells used for enzyme linked immunosorbant assays (ELISA). Here we demonstrate the ability of SFM to image and discriminate different types of antibody and antibody fragments bound to an ELISA well surface. The samples studied include a type IgG antibody with a proportion of bound IgM and two-dimensional films of whole IgG antibody, and Fab' and F(ab)₂ antibody fragments. Molecular resolution is achieved in each case despite the size of substrate features exceeding most of the molecular dimensions observed. Analysis of the data shows that the SFM overestimates molecular dimensions by an approximately constant amount, which is proposed to principally result from the effects of a finite probe size and not from deformation of the molecular species due to the imaging forces employed.
Crystal structure of a chimeric Fab′ fragment of an antibody binding tumour cells
1992, Journal of Molecular Biology
The crystal structure of a chimeric Fab′ fragment of a monoclonal antibody is presented. The Fab′ comprises the murine light chain and heavy chain variable domains of the carcinoma-binding antibody B72.3 fused to the constant domain of human κ, and the first constant domain and hinge domain of human γ4, respectively. A model for the Fab′ has been determined by molecular replacement and refined to a resolution of 3·1 Å with an R-factor of 17·6%. The additional residues that distinguish a Fab′ from a Fab fragment are seen to be disordered in the crystals. The H3 hypervariable loop is short and adopts a sharp hairpin turn in a conformation that results from an interaction between the lysine side-chain of H93 and the main-chain carbonyl group of H96. The remaining hypervariable loops display conformations similar to those predicted from the canonical structures approach, although loop H2 is apparently displaced by a salt-bridge formed between H55 Asp and the neighbouring H73 Lys. These and other features of the structure likely to be important in grafting the hypervariable loops to an otherwise human framework are discussed.

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Journal of Molecular Biology

Abstract

References (10)

Crystallization and structure determination of an autoimmune anti-poly(dT) immunoglobulin Fab fragment at 3.0 Å resolution

J. Biol. Chem

Solvent content of protein crystals

J. Mol. Biol

Preliminary crystal structure of an Fab specific for a Salmonella O-polysaccharide antigen

J. Mol. Biol

Pharmacokinetics of the monoclonal antibody B72.3 and its fragments labelled with either 1251 or 1111n

Cancer Res

A spectrum of monoclonal antibodies reactive with human mammary tumor cells

Cited by (24)

The Fab conformations in the solution structure of human immunoglobulin G4 (IgG4) restrict access to its Fc region implications for functional activity

Module based antibody engineering: A novel synthetic REDantibody

Site-directed immobilization of immunoglobulin G on 3-aminopropyltriethoxylsilane modified silicon wafer surfaces

Controlled site-directed assembly of antibodies by their oligosaccharide moieties onto APTES derivatized surfaces

The discrimination of IgM and IgG type antibodies and Fab′ and F(ab) <inf>2</inf> antibody fragments on an industrial substrate using scanning force microscopy

Crystal structure of a chimeric Fab′ fragment of an antibody binding tumour cells

Journal of Molecular Biology

CommunicationCrystallization and preliminary X-ray diffraction study of a chimaeric Fab′ fragment of antibody binding tumour cells

Abstract

J. Biol. Chem

J. Mol. Biol

J. Mol. Biol

Pharmacokinetics of the monoclonal antibody B72.3 and its fragments labelled with either 1251 or 1111n

Cancer Res

A spectrum of monoclonal antibodies reactive with human mammary tumor cells

Communication
Crystallization and preliminary X-ray diffraction study of a chimaeric Fab′ fragment of antibody binding tumour cells