Bodo Brocks

The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies.

This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody repertoire was analyzed in-depth, and individual CDR libraries were designed and generated for each CDR and each antibody family. Trinucleotide mixtures were used to synthesize the CDR libraries in order to ensure a high quality within HuCAL GOLD, and a beta-lactamase selection system was employed to eliminate frame-shifted clones after successive cloning of the CDR libraries. With these methods, a large, high-quality library with more than 10 billion functional Fab fragments was achieved. By using CysDisplay, the antibody fragments are displayed on the tip of the phage via a disulfide bridge between the phage coat protein pIII and the heavy chain of the antibody fragment. Efficient elution of specific phages is possible by adding reducing agents. HuCAL GOLD was challenged with a variety of different antigens and proved to be a reliable source of high-affinity human antibodies with best affinities in the picomolar range, thus functioning as an excellent source of antibodies for research, diagnostic, and therapeutic applications. Furthermore, the data presented in this article demonstrate that CysDisplay is a robust and broadly applicable display technology even for high-throughput applications.

Antifibrotic effects of a tissue inhibitor of metalloproteinase-1 antibody on established liver fibrosis in rats.

Liver fibrosis is characterized by increased synthesis, and decreased degradation, of extracellular matrix (ECM) within the injured tissue. Decreased ECM degradation results, in part, from increased expression of tissue inhibitor of metalloproteinase‐1 (TIMP‐1), which blocks matrix metalloproteinase (MMP) activity. TIMP‐1 is also involved in promoting survival of activated hepatic stellate cells (HSCs), a major source of ECM. This study examined the effects of blocking TIMP‐1 activity in a clinically relevant model of established liver fibrosis. Rats were treated with carbon tetrachloride (CCl4), or olive oil control, for 6 weeks; 24 days into the treatment, the rats were administered a neutralizing anti–TIMP‐1 antibody derived from a fully human combinatorial antibody library (HuCAL), PBS, or an isotype control antibody. Livers from CCl4‐treated rats exhibited substantial damage, including bridging fibrosis, inflammation, and extensive expression of smooth muscle α‐actin (α‐SMA). Compared to controls, rats administered anti–TIMP‐1 showed a reduction in collagen accumulation by histological examination and hydroxyproline content. Administration of anti–TIMP‐1 resulted in a marked decrease in α‐SMA staining. Zymography analysis showed antibody treatment decreased the activity of MMP‐2. In conclusion, administration of a TIMP‐1 antibody attenuated CCl4‐induced liver fibrosis and decreased HSC activation and MMP‐2 activity. (HEPATOLOGY 2004.)

In vitro affinity maturation of human GM-CSF antibodies by targeted CDR-diversification.

The mammalian immune system applies somatic hypermutation to select for antibodies with improved dissociation rates in vivo up to an intrinsic limit, previously termed as affinity ceiling. However, for certain therapeutic applications it may be desirable to further improve antibody affinities beyond that limit. In this study the selection of antibodies specific for the pro-inflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) from the HuCAL GOLD human antibody library is described. In order to increase affinity and also functional activity, in vitro affinity maturation of a pool of lead Fab candidates was carried out. CDR-L3 and parallel CDR-H2 diversification using trinucleotide consensus cassettes were followed by the combination of optimized CDR-L3 and CDR-H2 leading to a 5000-fold improved affinity finally reaching a K(D) of 400 fM. Cytokine neutralizing potential of MOR04357 was evaluated in a TF-1 proliferation assay. Along with affinity optimization a 2000-fold increase in potency was observed compared to the parental antibody. Due to species cross-reactivity MOR04357 also blocks rat GM-CSF induced proliferation of FDCP-1 cells. Receptor inhibition studies showed that MOR04357 prevents the interaction of GM-CSF with the GM-CSF receptor alpha chain. As a consequence this leads to a blockade in signal transduction as measured by abolished STAT5 phosphorylation in the presence of GM-CSF and antibody. Due to its pro-inflammatory role GM-CSF has been implicated in the pathophysiology of inflammatory diseases like rheumatoid arthritis or asthma. Based on the mode of action described herein MOR04357 shows favourable antibody features as a potential drug candidate.

A TNF receptor antagonistic scFv, which is not secreted in mammalian cells, is expressed as a soluble mono- and bivalent scFv derivative in insect cells

Single chain antibodies (scFv) are usually produced in E. coli, but generation of certain scFv derivatives, such as complex fusion proteins or glycosylated forms of scFv is restricted to eukaryotic expression systems. We investigated the production of soluble mono- and bivalent single chain antibodies (scFv) in eukaryotic cells and describe a cassette vector system for mammalian and baculovirus expression which is compatible with an established vector system for bacterial expression and phage display selection of scFvs. The applied model scFv was derived from a murine antibody (H398) against human tumor necrosis factor receptor 1 (TNFR60), known to be a potent antagonist of TNF action in its monomeric form and a potential therapeutic agent for treatment of TNF-mediated diseases. Surprisingly, the monomeric scFv form of H398 (scFv H398) is expressed but not secreted in different mammalian cells. In contrast, in insect cells using recombinant baculovirus, a monovalent scFv H398 and a bivalent scFv fusion protein with an human IgG1 Fc region were expressed and secreted with correctly processed signal sequence. Concerning the influence of valency of the model Ab and its derivatives on antigen binding affinity and neutralisation of TNF activity, we found that the mono- and bivalent form of scFv H398 possesses the same characteristics as proteolytically produced Fab H398 and original mAb H398, respectively. Furthermore, fusion of the Ig Fc protein to scFv H398 increase the in vitro half-life at 37 degrees C. We conclude that the described cassette vectors readily allow the eukaryotic expression of mono- and bivalent scFv derivatives to analyse the influence of valency of scFv molecules on antigen binding and biological activity.

Human antibody derivatives against the fibroblast activation protein for tumor stroma targeting of carcinomas.

The fibroblast activation protein (FAP) is selectively expressed on activated fibroblasts of the tumor stroma on more than 90% of lung, breast and colon carcinomas. The high prevalence and abundance of FAP+ stroma make it a promising target for in vivo diagnosis and therapy of a variety of carcinomas. We describe the humanization of the murine FAP‐specific MAb, F19, which has already been clinically used for in vivo diagnostic purposes. Using phage display technology and human V‐repertoires, VL and VH regions of F19 were replaced by analogous human V‐regions while retaining the original HCDR3 sequence in order to maintain F19 epitope specificity. The resulting human single‐chain fragments of immunoglobulin variable regions (scFv 34, scFv 18) showed affinities of 6 nM on cell membrane‐bound FAP. scFv 34 was expressed as a bivalent minibody (Mb 34). The antigen‐binding characteristics of Mb 34 were comparable to the parental and a complementarity‐determining region (CDR)‐grafted version of F19. This was revealed by binding competition studies, FACS analyses and immunohistochemistry on various tumor samples including breast, colon and lung carcinomas. Importantly, compared with the CDR‐grafted humanized scFv version of F19, the V‐regions of the selected human scFv 34 showed sequence identity with the parental antibody (Ab) only over the short, 15‐amino acid long HCDR3. Thus, a largely reduced xenoantigenic potential is expected. These human Ab derivatives are suitable to develop novel therapeutic concepts with broad applicability for a wide variety of histological carcinomas based on tumor stroma targeting.

Characterization and screening of IgG binding to the neonatal Fc receptor

The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from degradation and increases the serum half-life of IgG, thereby contributing to a higher concentration of IgG in the serum. Because altered FcRn binding may result in a reduced or prolonged half-life of IgG molecules, it is advisable to characterize Fc receptor binding of therapeutic antibody lead candidates prior to the start of pre-clinical and clinical studies. In this study, we characterized the interactions between FcRn of different species (human, cynomolgus monkey, mouse and rat) and nine IgG molecules from different species and isotypes with common variable heavy (VH) and variable light chain (VL) domains. Binding was analyzed at acidic and neutral pH using surface plasmon resonance (SPR) and biolayer interferometry (BLI). Furthermore, we transferred the well-accepted, but low throughput SPR-based method for FcRn binding characterization to the BLI-based Octet platform to enable a higher sample throughput allowing the characterization of FcRn binding already during early drug discovery phase. We showed that the BLI-based approach is fit-for-purpose and capable of discriminating between IgG molecules with significant differences in FcRn binding affinities. Using this high-throughput approach we investigated FcRn binding of 36 IgG molecules that represented all VH/VL region combinations available in the fully human, recombinant antibody library Ylanthia®. Our results clearly showed normal FcRn binding profiles for all samples. Hence, the variations among the framework parts, complementarity-determining region (CDR) 1 and CDR2 of the fragment antigen binding (Fab) domain did not significantly change FcRn binding.

From EST to IHC: human antibody pipeline for target research

We have developed a method for the high-level expression of expressed sequence tags (ESTs) as inclusion bodies in Escherichia coli by C-terminal fusion to the N1-domain of g3p of filamentous phage M13. Soluble fusion protein is obtained by an efficient refolding procedure. We have applied such protein preparations to the selection of human antibody fragments from phage-displayed HuCAL libraries. For all fusion proteins tested in this study, HuCAL antibodies could be generated which specifically detect, e.g. in immunohistochemistry, the maternal full-length protein corresponding to the protein fragment. This expression technology, in combination with the automated HuCAL antibody generation (AutoCAL), has proven to be useful for the rapid, high-throughput generation of high-quality human antibodies against EST-encoded protein fragments for target research.

Fab Mor03268 Triggers Absorption Shift of a Diagnostic Dye Via Packaging in a Solvent-Shielded Fab Dimer Interface

Molecular interactions between near-IR fluorescent probes and specific antibodies may be exploited to generate novel smart probes for diagnostic imaging. Using a new phage display technology, we developed such antibody Fab fragments with subnanomolar binding affinity for tetrasulfocyanine, a near-IR in vivo imaging agent. Unexpectedly, some Fabs induced redshifts of the dye absorption peak of up to 44 nm. This is the largest shift reported for a biological system so far. Crystal structure determination and absorption spectroscopy in the crystal in combination with microcalorimetry and small-angle X-ray scattering in solution revealed that the redshift is triggered by formation of a Fab dimer, with tetrasulfocyanine being buried in a fully closed protein cavity within the dimer interface. The derived principle of shifting the absorption peak of a symmetric dye via packaging within a Fab dimer interface may be transferred to other diagnostic fluorophores, opening the way towards smart imaging probes that change their wavelength upon interaction with an antibody.

An automated immunoassay for early specificity profiling of antibodies

Antibody-based therapeutics are of great value for the treatment of human diseases. In addition to functional activity, affinity or physico-chemical properties, antibody specificity is considered to be one of the most crucial attributes for safety and efficacy. Consequently, appropriate studies are required before entering clinical trials. High content protein arrays are widely applied to assess antibody specificity, but this commercial solution can only be applied to final therapeutic antibody candidates because such arrays are expensive and their throughput is limited. A flexible, high-throughput and economical assay that allows specificity testing of IgG or Fab molecules during early discovery is described here. The 384-well microtiter plate assay contains a comprehensive panel of 32 test proteins and uses electrochemiluminescence as readout. The Protein Panel Profiling (3P) was used to analyze marketed therapeutic antibodies that all showed highly specific binding profiles. Subsequently, 3P was applied to antibody candidates from early discovery and the results compared well with those obtained with a commercially available high content protein chip. Our results suggest that 3P can be applied as an additional filter for lead selection, allowing the identification of favorable antibody candidates in early discovery and thereby increasing the speed and possibility of success in drug development.

Construction of scFv from Hybridoma by Two-Step Cloning

Despite the fact that a growing number of recombinant antibodies has been isolated from phage display libraries, still many antibody specificities are available from hybridoma cell lines. Here, a method is presented to obtain the genetic information for the antigen binding part of the antibody from hybridoma cells, and to assemble it into a functional bacterially expressed fusion protein (scFv fragment). To achieve this, vectors have been constructed which combine the two variable regions (Vh and Vl) with a peptide linker to yield an scFv fragment. The genetic information for Vh and Vl is amplified from hybridoma cells using the polymerase chain reaction (PCR) with antibody specific primers.