Lutz Riechmann

REMISSION INDUCTION IN NON-HODGKIN LYMPHOMA WITH RESHAPED HUMAN MONOCLONAL ANTIBODY CAMPATH-1H

A genetically reshaped human IgG1 monoclonal antibody (CAMPATH-1H) was used to treat two patients with non-Hodgkin lymphoma. Doses of 1-20 mg daily were given intravenously for up to 43 days. In both patients lymphoma cells were cleared from the blood and bone marrow and splenomegaly resolved. One patient had lymphadenopathy which also resolved. These effects were achieved without myelosuppression, and normal haemopoeisis was restored during the course of treatment, partially in one patient and completely in the other. No antiglobulin response was detected in either patient. CAMPATH-1H is a potent lympholytic antibody which might have an important use in the treatment of lymphoproliferative disorders and additionally as an immunosuppressive agent.

Affinity improvement of single antibody VH domains: residues in all three hypervariable regions affect antigen binding

BACKGROUND Through antibody engineering, immunoglobulins can be tailored for their particular application. In this respect, small recognition units are desired for the targeting of antigens in obstructed locations like solid tumors. OBJECTIVES To design efficient, minimum size recognition units, heavy chain variable regions (VH) had previously been modified for the use as antigen specific, single domain antibody fragments. To develop a rational approach to improve affinity, antigen binding is investigated here by analysing the effect of randomisations of CDR1 and 2 residues in VH domains specific for hapten and protein ligands. STUDY DESIGN Randomised repertoires were displayed on phage and affinity selected to improve and analyse antigen binding. Affinities of newly selected VH domains were determined in their soluble format to assess the role of modified residues in binding. RESULTS In four of five randomisation experiments, a new VH with an improved antigen affinity compared to the primary VH was selected. Dissociation constants decreased from 160 nM to 25 nM or 47 nM (CDR1 or CDR2 randomisation of an anti-Ox VH), from 300 nM to 31 nM (CDR2 randomisation of an anti-NIP VH) and from 3.1 microM to 1.6 microM (CDR2 randomisation of an anti-lysozyme VH). CONCLUSIONS Thus the affinity of VH domains can be improved after site specific, secondary randomisations in CDR1 and CDR2, phage display and antigen selection. As differences in the CDR3 sequences had formed the only difference between the primary VH domains used in this study, the effect of CDR1 and CDR2 mutations of affinity is consistent with a participation of all three CDRs in antigen binding by single VH domains.

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.

‘Camelising’ human antibody fragments: NMR studies on VH domains

A human heavy chain variable domain (VH) was expressed in bacteria for structural analysis by NMR spectroscopy. NMR analysis was initially impossible due to the short transverse proton relaxation time of the VH, probably caused by aggregation through the exposed interface naturally in contact with the light chain. The relaxation time was improved to normal values when this interface was mutated to mimic heavy chains of camel antibodies naturally devoid of light chains and through the use of the detergent CHAPS. Assignment of NMR signals will now be possible after isotopic labeling. Implications for the design of VH domains as minimum size immunoreagents are outlined.

The C-Terminal Domain of TolA Is the Coreceptor for Filamentous Phage Infection of E. coli

Filamentous bacteriophages infecting gram-negative bacteria display tropism for a variety of pilus structures. However, the obligatory coreceptor of phage infection, postulated from genetic studies, has remained elusive. Here we identify the C-terminal domain of the periplasmic protein TolA as the coreceptor for infection of Escherichia coli by phage fd and the N-terminal domain of the phage minor coat protein g3p as its cognate ligand. The neighboring g3p domain binds the primary receptor of phage infection, the F pilus, and blocks TolA binding in its absence. Contact with the pilus releases this blockage during infection. Our findings support a sequential two-way docking mechanism for phage infection, analogous to infection pathways proposed for a range of eukaryotic viruses including herpes simplex, adenoviruses, and also lentiviruses like HIV-1.

Expression of an antibody Fv fragment in myeloma cells

The antigen binding site on antibodies is fashioned by loops at the tips of the beta-sheet framework of both heavy and light chain variable domains. A heterodimer of both variable domains (Fv fragment), incorporating loops from an anti-lysozyme antibody, was expressed and secreted from myeloma cells in good yield (8 mg/l in supernatant from roller bottles), and shown to bind lysozyme. The two subunits were found to be in dynamic equilibrium but are overwhelmingly associated at neutral pH. The small size of Fv fragments (25 x 10(3) Mr) make them attractive for structural studies, in vivo imaging, and therapy.

A conserved infection pathway for filamentous bacteriophages is suggested by the structure of the membrane penetration domain of the minor coat protein g3p from phage fd.

BACKGROUND . Gene 3 protein (g3p), a minor coat protein from bacteriophage fd mediates infection of Escherichia coli bearing an F-pilus. Its N-terminal domain (g3p-D1) is essential for infection and mediates penetration of the phage into the host cytoplasm presumbly through interaction with the Tol complex in the E. coli membranes. Structural knowledge of g3p-D1 is both important for a molecular understanding of phage infection and of biotechnological relevance, as g3p-D1 represents the primary fusion partner in phage display technology. RESULTS . The solution structure of g3p-D1 was determined by NMR spectroscopy. The principal structural element of g3p-D1 is formed by a six-stranded beta barrel topologically identical to a permutated SH3 domain but capped by an additional N-terminal alpha helix. The presence of structurally similar domains in the related E. coli phages, lke and 12-2, as well as in the cholera toxin transducing phage ctxφ is indicated. The structure of g3p-D1 resembles those of the recently described PTB and PDZ domains involved in eukaryotic signal transduction. CONCLUSIONS . The predicted presence of similar structures in membrane penetration domains from widely diverging filamentous phages suggests they share a conserved infection pathway. The widespread hydrogen-bond network within the beta barrel and N-terminal alpha helix in combination with two disulphide bridges renders g3p-D1 a highly stable domain, which may be important for keeping phage infective in harsh extracellular environments.

AN ANTIBODY VH DOMAIN WITH A LOX-CRE SITE INTEGRATED INTO ITS CODING REGION : BACTERIAL RECOMBINATION WITHIN A SINGLE POLYPEPTIDE CHAIN

Bacterial lox‐Cre recombination within a single antibody VH domain was achieved through integration of a loxP site into its coding sequence. The 5′ half of the VH gene, in which the H2 loop was replaced by a mutant loxP site, was fused to geneIII in an ‘acceptor’ fd‐phage vector containing also a wild type loxP site. With a ‘donor’ plasmid vector harbouring the 3′ half of the VH gene flanked by the same, differing loxP sites it recombined into a full‐length VH with the loxP site‐H2 loop. This VH was purified from bacterial periplasm, where it folded into a typical immunoglobulin domain. The system allows the generation of large VH repertoires using lox‐Cre recombination.

IMPROVING THE ANTIGEN AFFINITY OF AN ANTIBODY FV-FRAGMENT BY PROTEIN DESIGN:

The affinity of an antibody for its ligand 2-phenyloxazolone was improved by protein design. For the design two-dimensional nuclear magnetic resonance spectroscopy, protein engineering and molecular modelling were used in an interactive scheme. Initially the binding site was localized with the help of transferred nuclear Overhauser enhancement signals from two, site specifically assigned tyrosine side-chains in the complementarity-determining regions of the antibody to the ligand 4-glycyl-2-phenyloxazolone. On their basis the hapten was placed into a model of the Fv-fragment built according to the principles of canonical antibody structures. From the model, unfavourable contacts between hapten and an aspartyl side-chain in complementarity-determining region 3 of the heavy chain were predicted. Substitution of the aspartyl residue by alanine resulted in a threefold increase in affinity of the antibody Fv-fragment for two hapten derivatives when compared with the wild-type. Nuclear magnetic resonance analysis of the improved Fv-fragment revealed an interaction between the alpha-carbon proton of alanyl residue with the ligand, which was not seen for the aspartyl residue. This interaction is not entirely in accordance with the model, which predicts an interaction between the side-chain of this residue and the hapten. However, it shows that by combined use of nuclear magnetic resonance analysis and molecular modelling, a residue that is critical for antigen binding was identified, whose mutation allowed the design of an improved antibody combining site.

Backbone assignment, secondary structure and Protein A binding of an isolated, human antibody VH domain

SummaryAntibody heavy chain variable domains (VH) lacking their light chain domain (VL) partner are prime candidates for the design of minimum-size immunoreagents. To obtain structural information about isolated VH domains, a human VH was labelled with 15N or doubly labelled with both 15N and 13C and was studied by heteronuclear nuclear magnetic resonance spectroscopy. Most (90%) of the 1H and 15N main-chain signals were assigned through two-dimensional TOCSY and NOESY experiments on the unlabelled VH and three-dimensional heteronuclear multiple quantum correlation TOCSY and NOESY experiments on the 15N-labelled VH. Four short stretches of the polypeptide chain could only be assigned on the basis of three-dimensional HNCA and HN(CO)CA experiments on the 13C-/15N-labelled protein. Long-range interstrand backbone NOEs suggest the presence of two adjacent β-sheets formed by altogether nine antiparallel β-strands. 3JHNHCα coupling constants and the location of slowly exchanging backbone amides support this interpretation. The secondary structure of the isolated VH is identical to that of heavy chain variable domains in intact antibodies, where VH domains are packed against a VL domain. The backbone assignments of the VH made it possible to locate its Protein A binding site. Chemical shift movements after complexing with the IgG binding fragment of Protein A indicate binding through one of the two β-sheets of the VH. This β-sheet is solvent exposed in intact antibodies. The Protein A binding site obviously differs from that on the Fc portion of immunoglobulins and is unique to members of the human VHIII gene subgroup.