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.

Biomek 3000: the workhorse in an automated accredited forensic genetic laboratory.

We have implemented and validated automated protocols for a wide range of processes such as sample preparation, PCR setup, and capillary electrophoresis setup using small, simple, and inexpensive automated liquid handlers. The flexibility and ease of programming enable the Biomek 3000 to be used in many parts of the laboratory process in a modern forensic genetics laboratory with low to medium sample throughput. In conclusion, we demonstrated that sample processing for accredited forensic genetic DNA typing can be implemented on small automated liquid handlers, leading to the reduction of manual work as well as increased quality and throughput.

Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation.

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.

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 x 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.

Isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing.

The humoral immune system in higher vertebrates is unique in its ability to generate highly diverse antibody responses against most pathogens as well as against certain malignancies. Several technologies have been developed to exploit this vast source of potentially therapeutic antibodies, including hybridoma technology, phage display and yeast display. Here, we present a novel, high-throughput technology (the Symplex Technology) for rapid direct cloning and identification of human antigen-specific high-affinity antibodies from single antibody-producing cells of immune individuals. The utility of the technology was demonstrated by isolation of diverse sets of unique high-affinity antibodies against tetanus toxoid and influenza virus from immunized volunteers. Hence, the Symplex Technology is a new method for the rapid isolation of high-affinity antibodies directly from humans.