Software Technology for Neuropilin antagonists

Invented by Ryan J. Watts, Yan Wu, Genentech Inc

The Genentech Inc invention works as follows

Background for Neuropilin antagonists

Development and maintenance of a vascular network is essential for many physiological as well as pathological processes. A sufficient blood supply is essential for active growth tissues, such as tumors and embryos. Pro-angiogenic substances, which stimulate new blood vessel formation and maintain existing ones via a process commonly known as angiogenesis, help them meet this need. Vascular formation is an orderly biological process that involves many steps. Hanahan, D. Science 277:48-50 (1997); Hogan, B. L. & Kolodziej, P. A. Nature Reviews Genetics. 3:513-23 (2002); Lubarsky, B. & Krasnow, M. A. Cell. 112:19-28 (2003).

It is now known that angiogenesis plays a role in the pathogenesis a number of disorders. These include solid tumors and metastasis as well as atherosclerosis, retrolental fibrillary, hemangiomas and chronic inflammation. Folkman et al., J. Biol. Chem., 267:10931-10934 (1992); Klagsbrun et al., Annu. Rev. Physiol. 53:217-239 (1991); Garner A., “Vascular diseases?”, In: Pathobiology of Ocular Disease. A Dynamic Approach, Garner A., Klintworth G K, eds., 2nd Edition (Marcel Dekker, NY, 1994), pp 1625-1710.

Angiogenesis is crucial in the case of tumor development. It provides nourishment and energy for growth and metastasis. Folkman et al., Nature 339:58 (1989). Neovascularization gives tumor cells a proliferative advantage and growth advantage over normal cells. A tumor can start as one aberrant cell. It can only proliferate to a few cubic meters due to distance from capillary beds. And it can remain?dormant’. It can remain dormant for long periods of time without undergoing further growth or dissemination. Some tumor cells switch to an angiogenic phenotype in order to activate endothelial cell proliferation and maturation. These blood vessels allow for continued growth and dissemination of metastatic tumor cells. A correlation was observed between the density of microvessels within tumor sections and patient survival in breast carcinoma as well as other types of cancer. Weidner et al., N. Engl. J. Med 324:1-6 (1991); Horak et al., Lancet 340:1120-1124 (1992); Macchiarini et al., Lancet 340:145-146 (1992). Although the exact mechanisms of the angiogenic switch are not known, it is believed that tumor mass neovascularization is caused by the combination of many angiogenesis stimulators or inhibitors (Folkman 1995, Nat Med 1(1), 27-31).

The process of vascular growth is tightly controlled. A significant number of molecules, most commonly secreted by surrounding cells, have been demonstrated to regulate EC differentiation and proliferation, migration, coalescence into cord-like structures, and thereby, vascular development. VEGF, for example, has been identified to be the key factor in stimulating angiogenesis as well as inducing vascular permeability. Ferrara et al., Endocr. Rev. 18:4-25 (1997). 18:4-25 (1997). VEGF is also a key mediator in neovascularization, which can be associated with tumors or intraocular diseases. Ferrara et al., Endocr. Rev. supra. Berkman et. al., J. Clin. Invest. 91:153-159 (1993); Brown and al., Human Pathol. 26:86-91 (95); Brown and al., Cancer Res. 53:4727-4735 (1993); Mattern et al., Brit. J. Cancer 73:931-934 (1996); Dvorak et al., Am. J. Pathol. 146:1029-1039 (1995).

Also the concentrations of VEGF within eye fluids are strongly correlated with the presence of active proliferation blood vessels in patients suffering from ischemia-related retinopathies or diabetics.” Aiello et al., N. Engl. J. Med. 331:1480-1487 (1994). Studies have also shown that VEGF is found in choroidal neovascular cells in patients with AMD. Lopez et al., Invest. Ophthalmol. Vis. Sci. 37:855-868 (1996).

During the development of the nervous systems, neurons send out cable-like Axons to move over long distances to reach their targets. Review by Carmeliet & Tessier Lavigne, Nature 436:193-200. A highly mobile sensory structure known as the growth cone is located at the tip of a growing anxon. The growth cone senses and assesses the spatial environment and selects the correct track to extend towards its final target. It performs dynamic cycles of filopodial extensions extension and retraction.

Over the last decade, significant progress has been made in understanding axon guide mechanisms. Review by Dickson, Science 298:1959-60. There are four types of guidance cues: repellents, attractants, and matrix-associated. They can act at a short range or at a longer range (i.e. cell- or matrix-associated), or both. There are four main families of axon guidance molecular families: netrins (semaphorins), ephrins (ephrins) and slits. See review by Huber et al (2003) Annu Rev Neurosci 26:509-63.

The semaphorins (Sema), also known as collapsins belong to a large group of membrane-associated, secreted proteins that are phylogenetically conserved. The semaphorins are capable of mediating attractive and repulsive axon guidance events in neural development. Raper (2000) Curr Opin Neurobiol 10:88-94. All thirty known semaphorins share a conserved Nterminal Sema domain containing approximately 500 amino acids. Semaphorins are divided into eight families based on their structural similarities and origin species. See Semaphorin Nomenclature Committee (1999), Cell 97.551-552 for more information.

The neuropilin family (NRP) is composed of two homologous protein, neuropilin-1 and neuropilin-2. NRP1 was identified first as a type 1130-kDa transmembrane protein expressed in the growth cones for growing axons. Expression cloning was used to identify NRP2. Fujisawa & Kitsukawa (1998) Curr Opin Neurobiol 8. 587-592. The NRPs have been shown to be receptors for subsets of semaphorins (class 3 semaphorins). NRPs could be non-signaling coreceptors with plexins, another semaphorin receptor group.

NRPs were initially thought to be a mediator for axon guidance. However, they have been shown to play crucial roles in vascular development. Carmeliet (2005). It is an isoform-specific VEGF receptor expressed on tumor cells and endothelial cell. This discovery has prompted considerable research into the role of NRPs within vascular and tumor biology. Soker et al (1998) Cell 92:735-745; Klagsbrun et al (2002) Adv Exp Med Biol 515:33-48. Numerous genetic studies have shown that Nrp1 is essential for vascular morphogenesis. The loss of Nrp1 function can lead to vascular remodeling and branching defects. This phenotype can be further enhanced if Nrp2 function is lost. Kawasaki et al. (1999) Development 126:4895-4902; Takashima et al. (2002) Proc Natl Acad Sci USA 99:3657-3662. These results indicate that Nrp1 and Nrp2 might have overlapped functions early in development. The expression of each Nrp differs in that Nrp1 is expressed in the arteries and Nrp2 is found in the veins and lymphatics. Yuan et al (2002) Development 129:4797-4806; Herzog et al. (2001) Mech Dev 109:115-119. It is notable that the loss of Nrp2 function only impairs lymphatic growth.

Nrp1 is expressed in many cell types during development. The role of vascular Nrp1 has been addressed by the generation of an EC specific knock-out. This resulted in similar vascular defects as those in the null allele. Gu et al. Dev Cell 5, pp. 45-57. This study showed, however, that Sema3A binding with NRP1 was not necessary for vascular development. Another study found defects in the guidance of endothelial tips cells in the developing hindbrain in Nrp1KO embryos. Gerhardt et al. (2004) Dev Dyn 231:503-509.

Despite extensive research on NRP1’s role as a vascular developer, it is not clear if NRP1 acts solely via the VEGF Receptor 2 pathway (VEGFR2), as an enhancer of VEGF binding to VEGFR2 thereby for VEGFR2 Signaling or via a signaling pathway that is independent of VEGFR2 or a combination of both.

Recombinant DNA technology can make monoclonal antibodies. Monoclonal antibodies have been widely used, especially those derived from rodents. However, nonhuman antibodies can be antigenic to humans. This problem has been solved by the art of creating?chimeric? Antibodies in which a nonhuman antigen binding domain is coupled with a human constantdomain (Cabilly and al., U.S. Pat. No. 4,816,567). To tailor the chimeric antigen for use in complement-dependent and antibody-dependent cell cytotoxicity (ADCC), the isotype of human constant domain could be chosen. Humanized antibodies were created for several antigens. They are substantially smaller than the intact human variable domain and have been replaced at certain regions with the sequence from non-human species. This is done to reduce the use of heterologous sequences within antibodies. Rodent residues were substituted for human antibodies in some cases. Humanized antibodies are usually human antibodies that have some complementarity-determining region (CDR), and possibly some framework (FR) residues. These residues are then replaced by similar sites in rodent antibodies. Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239, 1534-1536

Preclinical studies in nonhuman mammals to assess the effectiveness and/or toxicity of therapeutic antibodies to humans are required before administering them to patients. These studies should ensure that the antibodies tested are capable of reacting with high potency against a target antigen from the host animal, such as a mouse or nonhuman primate.

Phage technology has been a powerful tool in the generation and selection of novel proteins that bind with a ligand such as an antigen. Large libraries of protein variants can quickly be created using the technique of phage presentation. The sequences that bind with a target antigen have high affinity and can be quickly sorted. The nucleic acids that encode variant polypeptides are fused with a sequence of nucleic acids that encode a viral coat protein such as the gene III or VIII proteins. Monovalent phage display system have been created in which the nucleic acids sequence encoding the polypeptide or protein is fused with a nucleic acids sequence encoding a part of the geneIII protein. Lowman and Wells (1991), Methods: A Companion To Methods in Enzymology 3:105. Monovalent phage display systems use gene fusion at low levels. Wild type gene III proteins are also used to preserve the infectivity of the particles. Many patents have disclosed methods for generating peptide libraries, and screening those libraries. No. 5,723,286, U.S. Pat. No. 5,432,018, U.S. Pat. No. 5,580,717, U.S. Pat. No. No. No. 5,498,530).

The development of antibody phage display library was made possible by the demonstration of peptide expression on the surface and functional antibody fragments being expressed in the periplasm of E. coli. Smith et al. (1985) Science 228.1315; Skerra, Pluckthun (1988 Science 240.1038). There are many ways to create libraries of antigen binding polypeptides or antibodies. One way is to alter a single gene with random DNA sequences, or by cloning several related genes. U.S. Pat. describes methods for displaying antigen binding fragments or antibodies using phage display. Nos. Nos. 5,750,373, 5,733,743, 6,969,108 5,837,242, 5,969.108, 5,969.108, 6,172.197, 5,580.717 and 5,658,727. The library is then screened to identify antibodies and antigen binding proteins that have the desired characteristics.

Phage technology offers many advantages over traditional hybridoma and other recombinant methods to prepare antibodies with desired characteristics. This technology makes it possible to create large libraries of diverse sequences of antibodies in a shorter time frame and without using animals. The preparation of hybridomas and humanized antibodies can take several months. Additionally, because no immunization is necessary, phage antibodies libraries can be created for antigens that are toxic or low in antigenicity (Hogenboom (1988), Immunotechniques 4-20). You can also use phage antibody libraries to identify and generate new therapeutic antibodies.

It is crucial to be able to isolate high affinity antibodies from phage display libraries. This is essential in the development of therapeutic antibodies. The library size, efficiency in production and diversity are all factors that affect the isolation of high affinity antibodies. See, for e.g., Knappik et al. (1999) J. Mol. Biol. 296:57. Inefficiency in production, improper folding of antigen binding proteins and stop codons can reduce the size of the library. If the antigen binding domain or antibody is not folded correctly, it can inhibit expression in bacterial cells. Mutating specific CDR residues or the surface of the constant/variable interface can improve expression. (Deng et al. (1994) J. Biol. Chem. 269:9533, Ulrich et al. (1995) PNAS, 92:11907-11911; Forsberg et al. (1997) J. Biol. Chem. 272:12430). When antibody phage libraries in bacterial cells are made, the sequence of the frame region is important in order to ensure proper folding.

To isolate high affinity antibodies, it is important to have a varied library of antigen binding proteins or antibodies. There are many ways to generate libraries with diversification in limited CDRs. Tomlinson, Nature Biotech, 2000. 18:989-994. CDR3 regions are important in part because it is often found that they participate in antigen binding. The CDR3 regions of the heavy chain can vary in their size, sequence, and structural conformation.