Elevating the Value of Monoclonal Using Mapping Differentiating mechanism of action and enhancing intellectual property Soma S.R. Banik, Xiaoxiang Deng, and Benjamin J. Doranz Epitope information is increasingly requirement showing that an inventor is ers who are reasonably ‘skilled in the easily predicted from sequence being used to understand the mechanism ‘in possession’ of the invention. Epitope art’. Claims for mAbs with linear and can be obtained only from immuni- of action of therapeutic monoclonal anti- mapping data is being used to meet these can be subject to obviousness zation with a large and complex immu- bodies (mAbs), as well as to overcome requirements for patents. For challenges, since the target antigen’s nogen. Thus, data that the mounting challenges of patenting example, epitope data has been central in sequence and the use of corresponding demonstrates targeting of a them. Differentiating mAbs by their dis- high profile cases such as Biogen v. short linear peptides for immunization conformational epitope strengthens tinct mechanisms of action helps select GlaxoSmithKline (differentiating are now deemed to constitute routine assertions of non-obviousness in a the best lead mAb(s) for development epitopes for CD20 mAbs fulfills novelty, practice. mAb’s patent claims. and aids in regulatory filings. To protect Federal Circuit, 2013) and Amgen v. By contrast, mAbs targeting con- Epitope mapping data are particu- the intellectual property (IP) of such Sanofi (high resolution epitopes for mul- formational epitopes are more likely to larly valuable for later-generation mAbs, mAbs, epitope data is being used to sup- tiple PCSK9 mAbs fulfills written be considered non-obvious, since they allowing clear demonstration that a mAb port patent novelty, non-obviousness, description, District Court of Delaware, recognize a complex three-dimensional is novel, not “anticipated” and distinct and satisfy the recently tightened written 2017). binding site formed by association of from “prior art.” The demonstration of description requirement for broad anti- For an invention to be non-obvious, non-sequential regions of a protein. This novelty has grown increasingly impor- body claims. High resolution epitope it should not be readily conceived by oth- type of conformational epitope is not tant as the therapeutic mAb field be- maps allow differentiation between mAbs, including those that have compet- ing binding sites. Applying a mapping A. Epitopes differentiate mAb MoA B. Epitope maps strengthen IP technology that provides epitopes at Novel: new epitope amino acid resolution, even for MoA 1: receptor binding site conformational epitopes against difficult Non-obvious: conformational epitope targets, can increase the breadth and Written description: amino acid resolution strength of mAb patents. MoA 2: fusion loop

Epitope Mapping Informs MoA Trastuzumab MoA 3: GP cleavage New epitope Epitope Epitope mapping helps to differen- tiate candidate mAbs that have the most MoA 4: GP rearrangement therapeutically promising mechanism of Pertuzumab action (MoA). An epitope in a defined epitope functional region of a protein informs Ebola GP HER2 the mechanism by which the mAb affects protein function, an important Figure 1. Epitope mapping differentiates MoAs and strengthens IP claims. (A) Mechanisms by which mAbs neutralize component of regulatory filings. For the Ebola virus glycoprotein (GP) have been defined by epitope mapping (MoAs #1-4). Each mAb can neutralize infec- tivity by inhibiting one of a number of steps essential for infectivity: mAb-receptor interaction, membrane fusion, proteo- example, mAbs can block a ligand, lytic cleavage, or transmembrane structural rearrangement. Select combinations of mAbs with different MoAs have led inhibit protein-protein dimerization, or to highly protective cocktails of therapeutic antibodies. (B) The amino acid binding site for a novel epitope (Fynomer C12, prevent a conformational change orange residues) strengthens IP claims by establishing novelty (because the epitope is distinct from other epitopes on required for function. Thus, knowledge HER2, such as those for Trastuzumab and Pertuzumab), non-obviousness (because the epitope forms a conformational of a mAb’s binding location helps to epitope of discontinuous residues). It also fulfills written description requirements by identifying the specific amino acids clarify exactly how binding modulates energetically required for its binding. target function, and which mAbs might have the most value. For lead selection, choosing mAbs with diverse MoA’s pro- A. Shotgun Mutagenesis Epitope Mapping >95% success rate, vides unique therapeutic candidates that 500+ epitopes mapped each act on the target differently. Create Library Measure Binding Map Residues Defining MoA is also important for M Q V R T S R P C.... M A V R T S R P C.... Figure 2. Shotgun Muta- selecting which components to combine s t

n M Q A R T S R P C.... genesis Epitope Mapping a in therapeutic cocktails, which are t

u M Q V A T S R P C.... provides high resolution, M increasingly being used to treat diseases M Q V R A S R P C.... n conformational epitopes a where escape mutations can thwart the c M Q V R T A R P C.... with >95% success. (A) The S

a .... efficacy of a monotherapy. For example, l M Q V R T S A P C epitopes of over 500 mAbs A an experimental three-mAb cocktail M Q V R T S R A C.... have been mapped using (ZMapp™) was used during the recent One mutant per well S h o t g u n M u t a g e n e s i s , Ebola virus outbreak in West Africa, and Comprehensive Expression in human cells Conformational epitopes including epitopes that are new inhibitory mAbs have since been mutagenesis on complex proteins components of complex pro- identified. Understanding where each teins and conformation- component mAb binds relative to the dependent mAbs. Shotgun B. Epitope Mapping Technologies Mutagenesis uses high- functional regions of the protein pro- Conformational High Complex Energetic throughput alanine scanning vides a rationale for choosing the most High Speed Epitopes Reessolluttiion Proteins Hot-Spots and expression in human synergistic combination of mAbs, each cells to identify critical resi- with a unique epitope and MoA for virus Co-Crystallography dues for binding. (B) Shot- inhibition (Figure 1A). ü ü gun Mutagenesis has been Epitope Binning used to elucidate epitopes Epitope Data Strengthens IP Claims ü ü at amino acid resolution, identify which amino acids Peptide Scanning For a patent to be allowed, the ü ü form the energetic hot-spots invention must be shown to be useful, of binding, and rapidly map Shotgun Mutagenesis dozens of conformational non-obvious, and novel. Patents must Epitope Mapping additionally fulfill a written description ü ü ü ü ü mAbs simultaneously.

SEPTEMBER 1, 2017 | GENengnews.com | Genetic Engineering & Biotechnology News comes more competitive, with multiple genus of mAbs that bind to the epitope gen-deuterium exchange, and epitope epitopes, pinpointing those which are companies developing distinct mAbs residues that Amgen identified. Sanofi’s binning can be fast and efficient for lin- energetically critical for binding. that target the same protein. For exam- competitor mAb Praluent® (alirocumab) ear epitopes. These techniques, however, Because it offers high resolution ple, the cancer target HER2 has been recognizes residues overlapping those of cannot provide amino acid resolution epitopes even for conformationally com- successfully targeted by two FDA Amgen’s, forcing Sanofi to defend itself and cannot always be applied to plex proteins and epitopes, Shotgun approved mAbs, trastuz umab against Amgen’s patents in court. While conformational epitopes or complex Mutagenesis Epitope Mapping has been (Herceptin ®) and pertuz umab the case remains contested, it is clear proteins. used to differentiate the MoAs of lead (Perjeta®). The developers of an addi- that the detailed epitope residues and the To provide a robust and rapid solu- mAbs entering the clinic as well as to tional molecule (Fynomer C12) were number of mAbs epitope mapped in this tion for comprehensive site directed establish defensible IP claims by show- able to claim the novelty of its epitope case have defined the legal breadth of mutagenesis, Integral Molecular ing that the mAbs are novel and non- in a new patent by demonstrating that patent protection. (www.integralmolecular.com) has devel- obvious and fulfill written description this epitope was distinct from those of oped Shotgun Mutagenesis Epitope requirements. existing mAbs and therefore was not Epitope Mapping Techniques Mapping, a technology that works Antibody drug discovery is a previously described by prior art that Add Value directly in human cells and combines resource intensive process in terms of (Figure 1B). comprehensive alanine mutagenesis, both time and money. For the success of The written description A number of technologies are used expression, and detection (Figure 2). the most promising mAbs, epitope map- requirement of an antibody patent spec- to map epitopes, with varying degrees of This technique has been used to map ping elevates the value of mAb drug ifies that an inventor must adequately relevance for identifying MoA and pro- over 500 epitopes to date, with a success candidates by providing data to secure describe an invention to demonstrate tecting IP. Each technique has its merits, rate of greater than 95%. Epitopes suc- their IP and differentiate their therapeu- that the inventor is in ‘possession’ of and in many cases, multiple approaches cessfully mapped include confor- tic MoAs. the invention. A successful written are utilized depending on a mAb’s stage mationally complex epitopes on soluble description can also be used to support in research and development. For exam- proteins, membrane proteins (including broad claims around the epitopes of a ple, co-crystallography and cryo-electron 1TMs, G protein-coupled receptors, group (or ‘genus’) of mAbs. The microscopy enable direct visualization transporters, ion channels, and viral breadth of such epitope claims was of the interaction between the antigen proteins), and complex epitopes depen- recently demonstrated by Amgen’s and mAb, but these techniques are still dent on multiple subunits or specific Integral Molecular US8829165 and US8859741 patents limited. They cannot always be applied conformational states. [email protected] claiming the detailed epitope residues to complex proteins. They usually The success of this technique is 215.966.6061 require substantial time and resources. based on assessing the binding of mAbs for mAbs against the target PCSK9 for www.integralmolecular.com Amgen’s Repatha® (evolocumab). And they do not identify which residues to conformationally intact proteins Because detailed epitopes for several within a binding footprint are driving the expressed in their native form directly representative mAbs were included, energetics of the interaction. within live human cells. This approach Amgen’s allowed patents cover a broad Similarly, peptide scanning, hydro- provides single amino-acid resolution

Integral Molecular’s Membrane Protein Solutions

For more information please visit www.integralmolecular.com

SEPTEMBER 1, 2017 | GENengnews.com | Genetic Engineering & Biotechnology News