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Therapeutic AntibodiesTherapeutic antibodies are a key driving force in the biotechnology industry thanks to their ability to safely and effectively target a very broad range of diseases, including cancer, autoimmune disorders and infectious diseases. Global sales reached $38B in 2009, and the market continues to grow (1). However, making antibodies to the next set of therapeutic targets, e.g., GPCRs and other multipass membrane proteins, requires highly detailed approaches that are not routinely established. mAbs to GPCRs useful in flow cytometry and functional assays are difficult and expensive to make. Recently a series of papers were published demonstrating the lack of specificity and performance of existing GPCR mAbs (2-6). Paramount to GPCR antibody discovery are selection of the target site, the immunogen, antigen, and screening assays. Immunization is key to eliciting antibodies that recognize membrane-dependent structural epitopes made of very small extracellular domains that frequently have high sequence homology with mice and rabbits and elicit weak antibody responses. A diverse set of screening antigens (cells), and assays are required to demonstrate specific performance including binding, killing, and functional activity. Critical bottlenecks include:

• The ability to generate antibodies against multi-pass membrane proteins.

• The ability to generate panels of mAbs to diverse epitopes including novel epitopes.

• The cost and time required to deploy current technologies to find the specified antibody.

The mAb Discovery Engine to Transmembrane TargetsSDIX has developed technologies comprising an antibody engine (mAb discovery engine) for discovery of antibodies to transmembrane proteins like GPCRs.The mAb discovery engine generates panels of diverse antibodies in terms of sequence, somatic hypermutation, epitopes, and performance.The mAb discovery engine is a platform that is

capable of routinely generating antibody candidates that are diverse, functional and novel.

The platform includes DNA immunization technology (vectors, response modifiers, protocols), high throughput flow cytometry, cell constructs, screening assays including functional analyses, antibody gene sequencing and expression, and epitope mapping. mAb discovery engine projects include polyclonal serum evaluation, selection of responding animals, fusions, and mAb screening, and may include significant additional effort in terms of immunogen vectors, immunized mice, immune response modifiers and protocols, screening strategies, and antibody performance characterization.

GPCRs are difficult antibody targets because their 3D conformation is membrane-dependent. Functional proteins working on the surfaces of cells are highly conformationally dependent. The mAb discovery engine DNA immunization presents a high fidelity representation of the target to the animal’s immune system and stimulates a significant response that is comprised of antibodies with unique, affinity-matured sequences recognizing multiple epitope specificities and having diverse function.

mAb Discovery Engine Data

Panels of mAbs were generated using the mAb discovery engine to 3 multi-span membrane proteins; CXCR4, ADORA2A, and CD20 (Figure 1).

Figure 1. mAb discovery engine target proteins.

mAb Discovery EngineMonoclonal Antibodies to Transmembrane Proteins

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ImmunizationMice were immunized using mAb discovery engine protocols including DNA immunization, transfected cells, adjuvants, and immune response modifiers designed to elicit high specific activity antibody responses to native transmembrane proteins expressed endogenously on the surfaces of host cells.

Figure 2. Evaluation of mAb discovery engine DNA immunization with cell-based and virus-like particle (VLP) immunizations.

Polyclonal Antibody AnalysisPolyclonal antisera from immunized mice were evaluated by flow cytometry using positive and control transfected cells. Figure 2 shows a comparison of the amount of polyclonal antibody made in response to mAb discovery engine DNA immunization, cells alone, and virus-like particles (VLP). mAb discovery engine DNA immunization elicits more specific antibody than cell and VLP immunizations.

Table 1 shows the number of animals with antibody responses following mAb discovery engine immunization with 13 transmembrane protein targets, including 9 GPCRs. Animals producing antibody were identified for all 13 proteins. Antibodies were made to human proteins having high homology with mouse. While only

13% of mice immunized for ADORA2A produced antibody, mAbs were isolated successfully from antibody-producing animals (Figure 11).

Table 1. Polyclonal mouse antibody response to 13 transmembrane protein targets. High human/mouse homology is a potential risk limiting existing antibody development platforms.

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Monoclonal Antibody DiscoveryThe performance of mAbs isolated using the mAb discovery engine was characterized and compared to existing benchmark therapeutic antibodies.

Antibodies expressed by cells taken from immunized mice were analyzed for binding to transfected cells in flow cytometry and exhibited unique titration curves (Figure 3). Multiple mAbs exhibited greater binding than benchmark antibodies.

mAb gene sequences demonstrated that the majority of the antibodies isolated are unique (Figure 4).

Figure 3. Titration of mAbs with CXCR4 transfected HEK 293 cells. Rx designates data from existing therapeutic benchmark antibodies.

Figure 4. Antibody gene sequence diversity of mAb discovery engine mAb panels.

The numbers of somatic hypermutations (SHM) in the variable region of the mAbs were compared with a set of benchmark therapeutic mAbs. The results indicate that the mAb discovery engine antibodies have undergone similar affinity maturation compared to the benchmark therapeutic antibodies (Figure 5).

Figure 5. Somatic hypermutation (SHM) of mAbs.

Figure 6. Epitope map illustrating CXCR4 extracellular regions and 8 diverse mAb epitope specificities.

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Epitope Mapping Demonstrates Antibody Diversity and Unique ReactivityMost currently known CXCR4 mAbs are directed primarily to ECL2 (7). Transfected cells expressing mutant forms of transmembrane proteins were used to map amino acid residues of extracellular loops involved in mAb binding. Reactivity patterns demonstrated mAb discovery engine CXCR4 mAbs recognize all extracellular domains with 8 different epitope specificities (Figure 6). Similarly, most existing CD20 mAbs fall into two epitope classes (rituximab-like and ofatumumab-like) (8). mAb discovery engine CD20 mAbs recognize 9 epitope specificities (Figure 7), including rituximab and ofatumumab-like specificities. Both CXCR4 and CD20 mAbs were discovered having previously described specificities. Importantly, mAbs with novel specificities were also isolated.

Figure 7. Epitope diversity of mAb discovery engine CD20 mAbs.

Figure 8. Functional activity of mAb discovery engine CXCR4 mAbs compared to benchmark mAbs.

Figure 9. Direct cell killing (apoptosis) by mAb discovery engine CD20 mAbs.

Panels of mAb discovery engine-generated mAbs were tested in functional assays vs. benchmark antibodies (CXCR4, Figure 8; CD20, Figure 9). Multiple mAb discovery engine-generated mAbs exhibit better functional performance than existing benchmark mAb controls.

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Generation of mAbs to GPCR ADORA2AADORA2A is a GPCR (9, 10) with very small extracellular loops, multiple post-translational modifications, and 83% sequence homology between human and mouse (Figure 10).

Figure 11 shows reactivity of 12 mAb discovery engine-generated ADORA2A mAbs to transfected cells in flow cytometry.

Figure 10. Schematic illustration of GPCR ADORA2A structure highlighting the small extracellular loops and post-translational modifications. Figure 11. Reactivity of 12 mAb discovery

engine-generated mAbs with transfected cells expressing ADORA2A in flow cytometry (red, black - positively transfected cells; blue - negative and control cells).

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References

1. Trent P. Munro, Stephen M. Mahler, Edwin P.Huang, David Y. Chin and Peter P.Gray. Bridging the gap: Facilities and technologies for development of early stage therapeutic mAb candidates. (2011) mAbs 3:5,440-452

2. N. Hamdani and J. van der Velden. Lack of specificity of antibodies directed against human beta-adrenergic receptors. (2009) Naunyn-Schmied Arch Pharmacol 379:403–407 DOI 10.1007/s00210-009-0392-1

3. Gitte Jositsch, Tamara Papadakis, Rainer V. Haberberger, Miriam Wolff, Jürgen Wess, and Wolfgang Kummer. Suitability of muscarinic acetylcholine receptor antibodies for immunohistochemistry evaluated on tissue sections of receptor gene-deficient mice. (2009) Naunyn-Schmiedeberg’s Arch Pharmacol 379:389–395 DOI 10.1007/s00210-008-0365-9

4. Brian C. Jensen, Philip M. Swigart, and Paul C. Simpson. Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific. (2009) Naunyn-Schmied Arch Pharmacol 379:409–412 DOI10.1007/s00210-008-0368-6

5. Serena Bodei, Nicola Arrighi, PierFranco Spano, and Sandra Sigala. Should we be cautious on the use of commercially available antibodies to dopamine receptors? (2009) Naunyn-Schmied Arch Pharmacol 379:413–415 DOI 10.1007/s00210-008-0384-6

6. Xiaoying Lu and Tamas Bartfai. Analyzing the validity of GalR1 and GalR2 antibodies using knockout mice. (2009) Naunyn-Schmied Arch Pharmacol 379:417–420 DOI 10.1007/s00210-009-0394-z

7. Sven Jähnichen, etal., CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells. (2010) PNAS 107:20565-20570

8. Ezogelin Oflazoglu and Laurent P. Audoly. Evolution of anti-CD20 monoclonal antibody therapeutics in oncology. (2010) mAbs 2:1, 14-19

9. György Haskó*, etal., Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. (2008) Nat. Rev. Drug Discovery 7:759-770

10. Kenneth A. Jacobson and Zhan-Guo Gao. Adenosine receptors as therapeutic targets. (2006) Nat. Rev. Drug Discovery 5:247-264

11. Alastair D. G. Lawson. Antibody-enabled small-molecule drug discovery. (2012) Nat. Rev. Drug Discovery 11:519-525

SummaryThe mAb discovery engine was used to generate monoclonal antibodies to 3 multi-pass transmembrane proteins. The antibodies generated bind to native proteins on living cells in flow cytometry, and exhibit cell killing and functional modulation of GPCR activity. Panels of mAbs were developed for all 3 targets with minimum numbers of fusions. The antibody genes were sequenced and shown to be unique. Sequence analysis demonstrated that mAb discovery engine mAbs have similar levels of SHM (affinity maturation) compared to benchmark therapeutic antibodies. Epitope mapping demonstrated diverse specificities

within the antibody panels. Functional assays further demonstrated diversity and performance. Importantly, mAbs with novel specificities were isolated to all 3 targets.

These data underscore the value of the mAb discovery engine to generate first-in-class and best-in-class antibodies against important and challenging therapeutic targets.

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