Kevin Stuart Johnson

A fully human antibody neutralising biologically active human TGFβ2 for use in therapy

Phage display provides a methodology for obtaining fully human antibodies directed against human transforming growth factor-beta (TGFbeta) suitable for the treatment of fibrotic disorders. The strategy employed was to isolate a human single chain Fv (scFv) fragment that neutralises human TGFbeta2 from a phage display repertoire, convert it into a human IgG4 and then determine its TGFbeta binding and neutralisation properties and its physical characteristics. Several scFv fragments binding to TGFbeta2 were isolated by panning of an antibody phage display repertoire, and subsequent chain shuffling of the selected V(H) domains with a library of V(L) domains. The three most potent neutralising antibodies were chosen for conversion to IgG4 format. The IgG4 antibodies were ranked for their ability to neutralise TGFbeta2 and the most potent, 6B1 IgG4, was chosen for further characterisation. 6B1 IgG4 has a high affinity for TGFbeta2 with a dissociation constant of 0.89 nM as determined using the BIAcore biosensor and only 9% cross-reactivity with TGFbeta3 (dissociation constant, 10 nM). There was no detectable binding to TGFbeta1. 6B1 IgG4 strongly neutralises (IC50 = 2 nM) the anti-proliferative effect of TGFbeta2 in bioassays using TF1 human erythroleukaemia cells. Similarly, there was strong inhibition of binding of TGFbeta2 to cell surface receptors in a radioreceptor assay using A549 cells. 6B1 IgG4 shows no detectable cross-reactivity with related or unrelated antigens by immunocytochemistry or ELISA. The 6B1 V(L) domain has entirely germline framework regions and the V(H) domain has only three non-germline framework amino acids. This, together with its fully human nature, should minimise any potential immunogenicity of 6B1 IgG4 when used in therapy of fibrotic diseases mediated by TGFbeta2.

An Antibody Fragment from a Phage Display Library Competes for Ligand Binding to the Low Density Lipoprotein Receptor Family and Inhibits Rhinovirus Infection

Recently antibodies with a wide range of binding specificities have been isolated from large repertoires of antibody fragments displayed on filamentous phage, including those that are difficult to raise by immunization. We have used this approach to isolate an antibody fragment against chicken very low density lipoprotein (VLDL) receptor. It binds to the receptor with good affinity (Kaff = 2 × 108M−1) as measured by plasmon surface resonance, and competes for binding of natural ligands (vitellogenin, VLDL, and receptor-associated protein). The antibody also binds to other members of the low density lipoprotein (LDL) receptor family including rat LDL receptor and human and rat low density lipoprotein receptor-related protein (LRP/α2MR), and it competes for binding of receptor-associated protein to LRP/α2MR. Moreover, the antibody fragment inhibits infection of human fibroblasts deficient in LDL-R but expressing LRP/α2MR by human rhinovirus. Binding of the antibody is abolished upon reduction of the receptors and is strictly Ca2+ dependent. The phage antibody thus recognizes the ligand binding site(s) of several members of the LDL receptor family, in contrast to antibodies produced by hybridoma technology.

Human antibody engineering: Current Opinion in Structural Biology 1993, 3:564–571

Abstract The most recent developments in manipulation and selection of antibody genes indicate that the next generation of antibody-based products, particularly those destined for human therapy, could be created entirely in vitro.

CD3× anti-nitrophenyl bispecific diabodies: Universal immunotherapeutic tools for retargeting T cells to tumors†

We developed a universal recombinant bispecific molecule (BiMol) that is capable of redirecting cytotoxic T cells to tumor cells via tagged anti‐tumor ligands such as antibody fragments or cytokines. A recombinant bispecific diabody with binding specificities for the CD3 molecule on T cells as well as for the hapten nitrophenyl (NIP) was produced. This bispecific molecule is capable of redirecting cytotoxic T cells to kill a series of malignant cells, including B cell lymphoma, Hodgkins lymphoma, and colon carcinoma via NIP‐conjugated ligands to tumor‐associated antigens. Cytotoxic activity of the diabody was found to be comparable to tetradoma‐derived bispecific antibodies with similar specificities. Our findings demonstrate that universal CD3×anti‐NIP diabodies could be used for T cell based cellular immunotherapy in a variety of human malignancies. Additionally, these bispecific molecules allow fast and economic testing of tumor‐associated antigens on malignant cells for their potential use as immunotherapeutic target structures if corresponding hapten‐conjugated antibodies or ligands are available. Int. J. Cancer 82:700–708, 1999.

Fab-arm exchange

123 beta1 chain integrin) bispecific antibodies mediate transport of JCV into the central nervous system (CNS) to cause PML. Not only have they failed to provide supporting experimental data but they offer no substantiating evidence from the literature. The mechanism by which JCV-VLA4 bispecifics might mediate transfer of virus to the CNS is presumably based on the following assumptions. First, sufficient amounts of anti-JCV IgG4 should be present in the circulation to form JCV-VLA4 bispecifics. Labrijn et al.1 do not report data on potential detection of JCV-VLA4 bispecific antibodies in natalizumab-treated patients, nor do they predict what the chances of formation of these would be. In a recent study, Egli et al.8 measured the prevalence of JCV infection and replication in 400 healthy donors. They report the IgG seroprevalence for JCV to be 58%. However, it is not further specified what proportion of the JCV IgG was IgG4. But assuming that IgG4 anti-JCV will be present in a proportion of healthy donors, JCV-VLA4 bispecifics are only expected to exist transiently, given the dynamic nature of Fab-arm exchange2. Second, if JCV-VLA4 bispecifics were “mediating the capture of JCV,” as postulated by Labrijn et al.1, free viral particles would need to be in the circulation or in tissues. Egli et al.8 report that JCV DNA could not be detected in any of the 400 blood samples from healthy donors. In addition, Iacobaeus et al.9 analyzed the cerebrospinal fluid, cerebrospinal fluid cells and blood from 217 patients with multiple sclerosis (MS) and 212 controls for detection of JCV DNA. They reported a low copy number of JCV DNA in only four samples (two MS and two controls), none in the other 425. These four individuals had no sign or symptom of PML nor did they develop the disease during follow-up. The combined publications by Egli et al.8 and Iacobaeus et al.9 thus demonstrate that free virus particles could not be detected in plasma or cerebrospinal fluid samples of the majority of healthy donors and MS patients. This is in line with previous studies that report residence of JCV in the kidney in an asymptomatic as created by van der Neut Kolfschoten et al.2 and confirmed by Labrijn et al.1, is of substantial value for antibody development companies who choose IgG4 as the preferred subclass for their products. We therefore agree with the conclusion that mutations that prevent Fab-arm exchange in vivo should be considered when designing therapeutic IgG4. In a Perspectives article that accompanied the original Fab-arm exchange paper by van der Neut Kolfschoten et al.2 in Science, Burton and Wilson5 rightly drew attention to the significance of the work. They concluded by stating, “[In instances where wildtype IgG4 molecules have been used in clinical trials] the possibility that Fab arm exchange could contribute to adverse effects in IgG4 therapy[6,7] should be explored immediately,” thereby referring to papers presenting the cytokine storm precipitated by TGN1412 (ref. 6) or the occurrence of progressive multifocal leukoencephalopathy (PML) in natalizumab-treated patients7. This concluding statement introduced an alleged link between wild-type IgG4 and adverse events. Labrijn et al.1 refer to exactly this statement in the last part of their letter where they recite that the potential exchange with preexisting IgG4 with undesired specificity raises the possibility that Fab-arm exchange could have contributed to some of the adverse events reported for wild-type IgG4. It is regrettable that, in contrast to Burton and Wilson’s5 suggestion, Labrijn et al.1 do not further explore the possibility that Fabarm exchange could have contributed to either the occurrences of PML or cytokine storm experimentally. Instead, Labrijn et al.1 briefly summarize the current theory of reduced immune surveillance, which plausibly explains the occurrence of PML in natalizumab-treated patients, but go on to counter that JC virus (JCV)–very late activation antigen 4 (VLA4; or alpha4 To the Editor: In a recent Letter, Labrijn et al.1 reported that therapeutic wild-type IgG4s engage in Fab-arm exchange with endogenous human IgG4 in vivo. The work presented embellishes a theme that was revived by van der Neut Kolfschoten et al.2, who confirmed in an excellent paper previous hypotheses and findings3,4, that is, that IgG4s are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half molecule) with a heavylight chain pair from another molecule, resulting in bispecific antibodies. Whereas van der Neut Kolfschoten et al.2 suggest that future studies should address the contribution of IgG4 Fab-arm exchange to in vivo activity of therapeutic monoclonal IgG4 antibodies, Labrijn et al.1 demonstrate that Fab-arm exchange between natalizumab (Tysabri) and endogenous human IgG4 can indeed be observed in blood samples from natalizumab-treated individuals. It is unfortunate that Labrijn et al.1 do not address the intriguing suggestion raised by van der Neut Kolfschoten et al.2, and no data are presented on how the phenomenon of Fab-arm exchange may affect the therapeutic activity of therapeutic wild-type IgG4. Instead, Labrijn et al.1 repeat what was already postulated by van der Neut Kolfschoten et al.2; that is, that Fab-arm exchange could have biological consequences in that the binding to the cognate antigen could change in time from an avidity to an affinity interaction, thereby possibly decreasing binding strength and changing homologous cross-linking to non-cross-linking behavior. Indeed, this is a significant take-home message and it contributes to the overall knowledge of an important subclass of therapeutic antibodies. The awareness of Fab-arm exchange and the subsequent undesired introduction of unpredictability for human immunotherapy, Fab-arm exchange c o r r e s p o n d e n c e