Lars Toleikis

Bi-specific Aptamers Mediating Tumor Cell Lysis

Antibody-dependent cellular cytotoxicity plays a pivotal role in antibody-based tumor therapies and is based on the recruitment of natural killer cells to antibody-bound tumor cells via binding of the Fcγ receptor III (CD16). Here we describe the generation of chimeric DNA aptamers that simultaneously bind to CD16α and c-Met, a receptor that is overexpressed in many tumors. By application of the systematic evolution of ligands by exponential enrichment (SELEX) method, CD16α specific DNA aptamers were isolated that bound with high specificity and affinity (91 pm–195 nm) to their respective recombinant and cellularly expressed target proteins. Two optimized CD16α specific aptamers were coupled to each of two c-Met specific aptamers using different linkers. Bi-specific aptamers retained suitable binding properties and displayed simultaneous binding to both antigens. Moreover, they mediated cellular cytotoxicity dependent on aptamer and effector cell concentration. Displacement of a bi-specific aptamer from CD16α by competing antibody 3G8 reduced cytotoxicity and confirmed the proposed mode of action. These results represent the first gain of a tumor-effective function of two distinct oligonucleotides by linkage into a bi-specific aptamer mediating cellular cytotoxicity.

Generation and analysis of the improved human HAL9/10 antibody phage display libraries.

BackgroundAntibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library.ResultsThe naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×1010 independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations.ConclusionThe highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

Rise and Fall of an Anti-MUC1 Specific Antibody

Background So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate. Results A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10−10 M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells. Conclusions Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these “best in class” binding parameters, the therapeutic success of this antibody was prevented by the target biology.

Cloning Single-Chain Antibody Fragments (ScFv) from Hyrbidoma Cells

Despite the rising impact of the generation of antibodies by phage display and other technologies, hybridoma technology still provides a valuable tool for the generation of high-affinity binders against different targets. But there exist several limitations of using hybridoma-derived antibodies. The source of the hybridoma clones are mostly rat or mouse B-lymphocytes. Therefore a human-anti-mouse or human-anti-rat antibody response may result in immunogenicity of these antibodies. This leads to the necessity of humanization of these antibodies where the knowledge of the amino acid sequence of the proteins is inalienable. Furthermore, additional in vitro modifications, e.g., affinity maturation or fusion to other proteins, are dependent on cloning of the antigen-binding domains.Here we describe the isolation of RNA from hybridoma cells and the primers that can be used for the amplification of VL and VH as well as the cloning of the antibody in scFv format and its expression in Escherichia coli.

A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display

There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics.

Therapeutic antibodies and antibody fusion proteins.

For the past few decades, antibodies have been anticipated as ideal molecules for the therapy of cancer and infectious disease. Their elnergence as an attractive therapy is the result of the evolution of monoclonal antibody technology froln fully mouse to fully human antibodies. Today, a large number of antibody-based clinical trials are now well under way, ranging from cancer, autoimmune disease, viral infection, allograft rejection~ asthma, stroke, and glaucoma surgery, to name just a few. These trials constitute approxinlately 30% of all clinical trials registered by the Food and Drug Adnlinistration (FDA) in the USA (not including vaccine and gene therapy). Thus, antibodies account for one of the largest groups of biotechnology-derived molecules in clinical trials., and have an estimated potentiallnarket of several billion US dollars. This is a big difference to the status of therapeutic Inonoclonal antibodies seen in the early 19905, when failing clinical trials, toxicity, and expensive production did not favour the production and Inarkeling ofantibodies. When the first nlonoclonal Inouse antibodies entered elinical studies in the early 1980s, they were considered as the stepping-stones for the treatment of cancer. The development of hybridonla technology and the steady advances in monoclonal antibody (MAb) production revitalized the initial concept of Ehrlich concerning the existence of specific cancer

Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates

Spontaneous isopeptide bond formation, a stabilizing posttranslational modification that can be found in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here we adapted this technology to establish a novel modular antibody labeling approach which is based on isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy allows the attachment of a reporting cargo of interest to an antibody scaffold by fusing it chemically to KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody with SpyTag. This strategy was successfully used to engineer site-specific antibody-drug conjugates (ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of antibody conjugates based on peptide-tags that have minimal effects on protein structure and function, thus expanding the toolbox of site-specific antibody conjugation.

Balancing Selectivity and Efficacy of Bispecific Epidermal Growth Factor Receptor (EGFR) × c-MET Antibodies and Antibody-Drug Conjugates.

Bispecific antibodies (bsAbs) and antibody-drug conjugates (ADCs) have already demonstrated benefits for the treatment of cancer in several clinical studies, showing improved drug selectivity and efficacy. In particular, simultaneous targeting of prominent cancer antigens, such as EGF receptor (EGFR) and c-MET, by bsAbs has raised increasing interest for potentially circumventing receptor cross-talk and c-MET-mediated acquired resistance during anti-EGFR monotherapy. In this study, we combined the selectivity of EGFR × c-MET bsAbs with the potency of cytotoxic agents via bispecific antibody-toxin conjugation. Affinity-attenuated bispecific EGFR × c-MET antibody-drug conjugates demonstrated high in vitro selectivity toward tumor cells overexpressing both antigens and potent anti-tumor efficacy. Due to basal EGFR expression in the skin, ADCs targeting EGFR in general warrant early safety assessments. Reduction in EGFR affinity led to decreased toxicity in keratinocytes. Thus, the combination of bsAb affinity engineering with the concept of toxin conjugation may be a viable route to improve the safety profile of ADCs targeting ubiquitously expressed antigens.

Generation of human bispecific common light chain antibodies by combining animal immunization and yeast display

Bispecific antibodies (bsAbs) pave the way for novel therapeutic modes of action along with potential benefits in several clinical applications. However, their generation remains challenging due to the necessity of correct pairings of two different heavy and light chains and related manufacturability issues. We describe a generic approach for the generation of fully human IgG-like bsAbs. For this, heavy chain repertoires from immunized transgenic rats were combined with either a randomly chosen common light chain or a light chain of an existing therapeutic antibody and screened for binders against tumor-related targets CEACAM5 and CEACAM6 by yeast surface display. bsAbs with subnanomolar affinities were identified, wherein each separate binding arm mediated specific binding to the respective antigen. Altogether, the described strategy represents a combination of in vivo immunization with an in vitro selection method, which allows for the integration of existing therapeutic antibodies into a bispecific format.

Engineering bispecific antibodies with defined chain pairing

Bispecific IgG-like antibodies can simultaneously interact with two epitopes on the same or on different antigens. Therefore, these molecules facilitate novel modes of action, which cannot be addressed by conventional monospecific IgGs. However, the generation of such antibodies still appears to be demanding due to their specific architecture comprising four different polypeptide chains that need to assemble correctly. This review focusses on different strategies to circumvent this issue or to enforce a correct chain association with a focus on common-chain bispecific antibodies.