Lars Soegaard Nielsen

Kinetic, Affinity, and Diversity Limits of Human Polyclonal Antibody Responses against Tetanus Toxoid

Due to technical limitations, little knowledge exists on the composition of Ag-specific polyclonal Ab responses. Hence, we here present a molecular analysis of two representative human Ab repertoires isolated by using a novel single-cell cloning approach. The observed genetic diversity among tetanus toxoid-specific plasma cells indicate that human polyclonal repertoires are limited to the order of 100 B cell clones and hypermutated variants thereof. Affinity and kinetic binding constants are log-normally distributed, and median values are close to the proposed affinity ceilings for positive selection. Abs varied a million-fold in affinity but were restricted in their off-rates with an upper limit of 2 × 10−3 s−1. Identification of Abs of high affinity without hypermutations in combination with a modest effect of hypermutations on observed affinity increases indicate that Abs selected from the naive repertoire are not only of low affinity but cover a relatively large span in affinity, reaching into the subnanomolar range.

Single-Batch Production of Recombinant Human Polyclonal Antibodies

We have previously described the development and implementation of a strategy for production of recombinant polyclonal antibodies (rpAb) in single batches employing CHO cells generated by site-specific integration, the SympressTM I technology. The SympressTM I technology is implemented at industrial scale, supporting a phase II clinical development program. Production of recombinant proteins by site-specific integration, which is based on incorporation of a single copy of the gene of interest, makes the SympressTM I technology best suited to support niche indications. To improve titers while maintaining a cost-efficient, highly reproducible single-batch manufacturing mode, we have evaluated a number of different approaches. The most successful results were obtained using random integration in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. This new expression process is termed the SympressTM II technology. Here we describe proof-of-principle data demonstrating the feasibility of the SympressTM II technology for single-batch rpAb manufacturing using two model systems each composed of six target-specific antibodies. The compositional stability and the batch-to-batch reproducibility of rpAb produced by the ECHO cells were at least as good as observed previously using site-specific integration technology. Furthermore, the new process had a significant titer increase.

Reduced Susceptibility of Recombinant Polyclonal Antibodies to Inhibitory Anti-Variable Domain Antibody Responses

The immunogenicity of therapeutic Abs is a concern as anti-drug Abs may impact negatively on the pharmacodynamics and safety profile of Ab drugs. The factors governing induction of anti-drug Abs are not fully understood. In this study, we describe a model based on mouse-human chimeric Abs for the study of Ab immunogenicity in vivo. Six chimeric Abs containing human V regions and mouse C regions were generated from six human anti-Rhesus D Abs and the Ag-binding characteristics of the parental human Abs were retained. Analysis of the immune response toward the individual chimeric Abs revealed the induction of anti-variable domain Abs including anti-idiotypic Abs against some of these, thereby demonstrating the applicability of the model for studying anti-drug Ab responses in vivo. Immunization of BALB/c, C57, and outbred NMRI mice with a polyclonal composition consisting of all six chimeric Abs demonstrated that the immunogenicity of the individual Abs was haplotype dependent. Chimeric Abs, which were nonimmunogenic when administered individually, did not become immunogenic as part of the polyclonal composition, implying the absence of epitope spreading. Ex vivo Ab-binding studies established a clear correlation between the level of immunogenicity of the Abs comprised in the composition and the impact on the pharmacology of the Abs. These analyses demonstrate that under these conditions this polyclonal Ab composition was generally less susceptible to blocking Abs than the respective mAbs.

Targeting Three Distinct HER2 Domains with a Recombinant Antibody Mixture Overcomes Trastuzumab Resistance.

HER2 plays an important role in the development and maintenance of the malignant phenotype of several human cancers. As such, it is a frequently pursued therapeutic target and two antibodies targeting HER2 have been clinically approved, trastuzumab and pertuzumab. It has been suggested that optimal inhibition of HER2 is achieved when utilizing two or more antibodies targeting nonoverlapping epitopes. Superior clinical activity of the trastuzumab plus pertuzumab combination in metastatic breast cancer supports this hypothesis. Because trastuzumab and pertuzumab were not codeveloped, there may be potential for further optimizing HER2 targeting. The study herein evaluated functional activity of anti-HER2 antibody combinations identifying optimal epitope combinations that provide efficacious HER2 inhibition. High-affinity antibodies to all four extracellular domains on HER2 were identified and tested for ability to inhibit growth of different HER2-dependent tumor cell lines. An antibody mixture targeting three HER2 subdomains proved to be superior to trastuzumab, pertuzumab, or a combination in vitro and to trastuzumab in two in vivo models. Specifically, the tripartite antibody mixture induced efficient HER2 internalization and degradation demonstrating increased sensitivity in cell lines with HER2 amplification and high EGFR levels. When compared with individual and clinically approved mAbs, the synergistic tripartite antibody targeting HER2 subdomains I, II, and IV demonstrates superior anticancer activity. Mol Cancer Ther; 14(3); 669–80. ©2015 AACR.

Human antibody repertoires.

Symplex is an antibody discovery technology that identifies fully human antigen-specific antibody repertoires directly from plasma cells. The technology utilizes reverse transcription and overlap extension polymerase chain reaction performed on single-cell-sorted plasma cells, whereby the heavy- and light-chain cognate pairing of the antibodies is maintained. The isolated antibodies from a plasma cell donor reflect the diversity, affinity, and selectivity of the donor antibody repertoire, making the technology an ideal tool for identifying drug leads and studying the development of human antibody repertoires.

Efficient Expression from One CMV Enhancer Controlling Two Core Promoters

The human CMV promoter/enhancer is one of the strongest promoters for recombinant protein expression in mammalian cells, making the promoter very popular for production of recombinant antibodies. We used an antibody vector design where the antibody heavy and light chain genes were transcribed from a promoter complex consisting of two promoters arranged divergently with the 5′ ends of the promoters in close proximity. However, when two identical CMV promoters constituted this promoter complex, the antibody expression observed was lower than expected based on the strength of the individual promoters. To optimize expression we prepared truncated promoter complexes where only one CMV enhancer controlled the initiation of transcription from two divergent minimal CMV core promoters. Antibody expression from the truncated promoter complexes was analyzed both when transiently transfected and upon stable site-specific integration into a CHO DG44 derived cell line. The data showed that it was possible for one enhancer to drive the expression of two core promoters. However, efficient expression from both divergent core promoters was seen only when the unique region upstream of the CMV enhancer was removed. Notably, a 12-fold increase in expression was found from the best of the truncated promoter complexes after stable site-specific integration when compared to the full-length double CMV promoter complex.

Recombinant antibody mixtures; optimization of cell line generation and single-batch manufacturing processes

Background Recombinant antibody mixtures represent an important new class of antibody therapeutics as demonstrated by the increasing amount of literature showing that combinations of two or more antibodies show superiority compared to monoclonal antibodies (mAbs) for treatment of cancer and infectious diseases [1-5]. Sym004, composed of two antibodies targeting non-overlapping epitopes of the epidermal growth factor receptor (EGFR) act in a synergistic manner to induce an efficient internalization of EGFR leading to subsequent degradation and exhibit superior anticancer efficacy as demonstrated in several preclinical in vivo models [5]. At Symphogen A/S, we have developed an expression platform, SympressTM, for cost-efficient production of antibody mixtures. The antibody mixtures are produced using a single-batch manufacturing approach where a polyclonal working cell bank (pWCB) prepared by mixing the individual stable cell lines producing all the desired antibodies is used as seed material for a bioreactor process [6]. By using a single-batch approach the CMC development costs of antibody mixtures are comparable to costs for monoclonal antibodies. However, the single-batch manufacturing approach raises questions with regard to control of composition ratios, compositional stability and robustness of the cell banking procedure. Here, we present experimental data addressing these key questions and demonstrate that mixtures of recombinant antibodies can be produced under predictable, reproducible and stable conditions using the SympressTM technology.