Patent Application
20230118983
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety.
Wnt signaling pathways are critical for embryonic development and tissue homeostasis in adults. Wnt signaling is initiated when a Frizzled (FZD) receptor on the cell surface membrane binds with a Wnt ligand. Wnt ligands are secreted growth factors that regulate various cellular processes such as proliferation, differentiation, survival and migration. Nineteen Wnt ligands exist in humans that interact with a network of ten Frizzled cell surface receptors (FZD) and one of several co-receptors that guide the selective engagement of different intracellular signaling branches (Wodarz, A. and Nusse, R. Annu. Rev. Cell Dev. Biol. 14, 59-88 (1998); Angers, S and Moon, R. T., transduction. Nat. Rev. Mol. Cell Biol. 10, 468-477 (2009)). FZDs have conserved structural features including seven hydrophobic transmembrane domains and a cysteine-rich ligand-binding domain. FZDs are known to function in three distinct signaling pathways, known as the Wnt planar cell polarity (PCP) pathway, the canonical Wnt/β-catenin pathway, and the Wnt/calcium pathway. The presence of Wnt co-receptors is also required to direct the differential engagement of the intracellular signaling cascades listed above. For example, Wnt ligands bind to a Frizzled receptor and a member of the low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) co-receptor family to activate the Wnt/β-catenin pathway, or with a receptor tyrosine kinase-like orphan receptors 1 and 2 (ROR1/2), related to receptor tyrosine kinase (RYK) or protein tyrosine kinase 7 (PTK7) co-receptor to activate alternate β-catenin-independent signaling pathways.
Wnt ligands are universally important for the control of tissue stem cells self-renewal and regulation of many progenitor cell populations, but the hydrophobicity and sensitive tertiary structure of Wnt proteins makes their biochemical purification challenging and their use in vitro and in vivo inefficient. Described herein are tetravalent binding antibody molecules that activate a Wnt signaling pathway and methods for their use.
Described herein are tetravalent binding antibody molecules that activate a Wnt signaling pathway and methods for their use. The tetravalent binding antibody molecules bind to both an FZD receptor, e.g., Frizzled Class Receptor 1 (FZD1), Frizzled Class Receptor 2(FZD2), Frizzled Class Receptor 3 (FZD3), Frizzled Class Receptor 4 (FZD4), Frizzled Class Receptor 5 (FZD5), Frizzled Class Receptor 6 (FZD6), Frizzled Class Receptor 7 (FZD7), Frizzled Class Receptor 8 (FZD8), Frizzled Class Receptor 9 (FZD9), or Frizzled Class Receptor 10 (FZD10) and a Wnt co-receptor, e.g., LRP5 or LRP6 (LRP5/6), thereby activating a Wnt signaling pathway. In an embodiment, the tetravalent binding antibody molecules bind to both a FZD4 receptor and LRP5 and/or LRP6 and activate the Wnt/β-catenin signaling pathway. The tetravalent binding antibody molecules of this invention are also referred herein as “FZD Agonists”, Frizzled and LRP5/6 Agonist (FLAg), and in some embodiments as “ANTs”.
The tetravalent binding antibody molecules include an Fc domain comprised of CH2 and CH3 domains or fragment thereof comprising the CH3 domain, and a first bivalent binding domain that interacts with one or more FZD receptor, e.g., one or more of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10, and a second bivalent binding domain that binds a WNT co-receptor, e.g., LRP5 or LRP6, wherein the FZD binding domain is linked to one terminus of the Fc domain and the co-receptor binding domain is linked to the other terminus of the Fc domain. Thus, the binding domain for the FZD receptor and the binding domain for the WNT co-receptor are not directly linked rather they are separated by the Fc domain, or fragment thereof comprising the CH3 domain.
The Fc domain of the FZD Agonists may be an Fc domain of an immunoglobulin with or without effector function. The immunoglobulin may be an IgG, e.g., an IgG1. In an embodiment of this invention the tetravalent binding antibody molecule comprises two polypeptides containing an Fc region that dimerize via the intrinsic ability of the Fc region in each polypeptide to dimerize or via a knob-in-holes configuration within the Fc. Thus, the Fc dimer may be a heterodimer or a homodimer. Methods for dimerizing peptides via a knob-in-hole configuration are described in WO2018/026942, inventors Van Dyk et al., Carter P. (2001) J. Immunol. Methods 248, 7-15; Ridgway et al. (1996) Protein Eng. 9, 617-621; Merchant, et al. (1998) Nat. Biotechnol. 16, 677-681, and; Atwell et al., (1997) J. Mol. Biol. 270, 26-35, all incorporated herein by reference.
In an embodiment, each of the binding domains of the FZD Agonists described herein are bivalent and each may be monospecific, having two binding sites for the same epitope of an FZD receptor, e.g., FZD4, or Wnt co-receptor, e.g. LRP5/6, or bispecific having two binding sites with each site binding a different epitope on an FZD or Wnt co-receptor, e.g., a Wnt1 binding (domain E1-E2 within the extracellular domain of LRP5/6) site and a Wnt3 binding site (domain E3-E4 within the extracellular domain of LRP5/6) within the LRP5/6 co-receptor. In an embodiment, the LRP5/6 binding domain binds to a Wnt3A site (domain E3-E4) on LRP5 and binds to a Wnt3A site (domain E3-E4) on LRP6.
In embodiments of this invention the FZD binding domain linked to the Fc domain of the FZD Agonist comprises one or more immunoglobulin heavy-chain variable domain (VH) fragments and/or one or more immunoglobulin light-chain variable domain (VL) fragments that bind the FZD, e.g., FZD4. In an embodiment of this invention the FZD binding domain may comprise Fabs, a diabody or single chain variable fragments (scFv) single-domain antibody fragments, e.g., VHH, or combinations thereof that bind to the same or different epitopes on the FZD.
In an embodiment of this invention the VHs and/or VLs of the FZD binding domain binds FZD4 or FZDS and comprise the light chain CDRs and the heavy chain CDRs of a FZD4 or FZDS binding antibody of Table 1, Table 2, or Table 6, and/or comprise light chain CDRs and heavy chain CDRs that are 50%, 55%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the CDRs of an FZD4 antibody of Table 1, Table 2 or Table 6, and still retain binding to the FZD4 or FZDS receptor. For example, in an embodiment of the invention, the FZD binding domain may comprise a first heavy chain (CDR-H1), a second heavy chain (CDR-H2), and/or a third heavy chain (CDR-H3), wherein the VH that binds FZD may comprise CDR-H1 of SEQ ID NO: 24, SEQ ID NO: 365, or SEQ ID NO: 893, a CDR-H2 of SEQ ID NO: 51, SEQ ID NO: 61, SEQ ID NO: 462, or SEQ ID NO: 894 and/or CDR-H3 of SEQ ID NO: 79, SEQ ID NO: 90, SEQ ID NO: 484, or SEQ ID NO: 895 and a first light chain (CDR-L1), a second light chain (CDR-L2), and/or a third light chain (CDR-L3), wherein the VL that binds FZD may comprise CDR-L1 of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 12, a CDR-L2 of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 12 and/or a CDR-L3 of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 285, or SEQ ID NO: 896.
In an embodiment of this invention the co-receptor (LRP5/6) binding domain linked to the Fc domain of the FZD Agonist comprises one or more immunoglobulin heavy-chain variable domain (VH) fragments and/or one or more immunoglobulin light-chain variable domain (VL) fragments that bind to the Wnt co-receptor, e.g., LRP5 and/or LRP6. For example, in an embodiment of the invention, the LRP binding domain may comprise a first heavy chain (CDR-H1), a second heavy chain (CDR-H2), and/or a third heavy chain (CDR-H3), wherein the VH that binds LRP may comprise a CDR-H1 of SEQ ID NO: 527, SEQ ID NO: 528, SEQ ID NO: 536, SEQ ID NO: 716, or SEQ ID NO: 720, a CDR-H2 of SEQ ID NO: 552, SEQ ID NO: 553, or SEQ ID NO: 566, SEQ ID NO: 785, or SEQ ID NO: 791 and/or a CDR-H3 of SEQ ID NO: 584, SEQ ID NO: 585, SEQ ID NO: 586 or SEQ ID NO: 603, SEQ ID NO: 856 or SEQ ID NO: 862 CDR-H3 and a first light chain (CDR-L1), a second light chain (CDR-L2), and/or a third light chain (CDR-L3), wherein the VL that binds LRP may comprise CDR-L1 of SEQ ID NO: 1, a CDR-L2 of SEQ ID NO: 2, or SEQ ID NO: 491 and/or a CDR-L3 of SEQ ID NO: 130, SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 510, SEQ ID NO: 623 or SEQ ID NO: 665.
In an embodiment of this invention, the Wnt co-receptor binding domain is bivalent and may comprise a diabody, or may comprise a Fab, a single chain variable fragment (scFv) or a single domain antibody fragments (VHH) or combinations thereof for binding to the same or different epitopes on the co-receptor. In embodiments of this invention the VHs and VLs of the Wnt coreceptor binding domain comprise the light chain CDRs and/or the heavy chain CDRs of a LRP5 and/or LRP6 binding antibody of Table 3, Table 4 or Table 6, or comprise light chain CDRs and/or heavy chain CDRs that are 50%, 55%, 60%, 75%. 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the CDRs of an LRP5 and/or LRP6 antibody of Table 3, Table 4 or Table 6, and still retain binding to the LRP5 and/or LRP6 co-receptor. In an embodiment of this invention the Wnt co-receptor binding domain linked to the Fc domain of the FZD Agonists described herein comprises a diabody, formed by two peptides each peptide comprising a heavy-chain variable domain (VH or VH domain) linked to a light-chain variable domain (VL or VL domain) wherein the VH and the VL from one peptide pair with the VL and VH of the other peptide forming the diabody. In this configuration, the binding domain has two binding sites that bind to the Wnt co-receptor, e.g., LRP5 or LRP6. The diabody may be monospecific binding the same site on the co-receptor or may be bispecific (bs) binding two different sites on the co-receptor. By using a knobs-in-holes Fc configuration, the peptides comprising the VH and VL linked to Fc regions, can be non-identical but will still pair to form a bispecific binding domain capable of binding to two different sites on the Wnt co-receptor (e.g. LRP5 or LRP6).
The peptides forming the diabodies, the VHH, the scFv, and the Fabs that form the binding domains may be derived from an antibody selected for its binding to a desired target, a “source antibody”. For the FZD binding domain, the “FZD source antibody” may be an antibody that binds to one or more of the FZD receptor(s), e.g., one or more of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10, and antagonizes Wnt signaling or inhibits Wnt binding to the given FZD receptor(s). Alternatively, the FZD source antibody may be an antibody that binds to the FZD receptor(s) without antagonizing Wnt signaling or without inhibiting Wnt binding to the FZD receptor Likewise, for the co-receptor binding domain, the “co-receptor source antibody” may be an antibody that binds to the Wnt co-receptor, e.g., LRP5/6, and antagonizes Wnt signaling or inhibits Wnt binding to the Wnt co-receptor. Alternatively, the co-receptor source antibody may be an antibody that binds to a co-receptor, e.g., LRP5/6, without antagonizing Wnt signaling or without inhibiting Wnt binding to the co-receptor.
In an embodiment of this invention the FZD binding domain of the FZD Agonist may bind specifically to a specific FZD, e.g., FZD4, with a higher affinity than to other FZDs, i.e., FZD1, FZD2, FZD3, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10, or may be pan-specific, binding to one or more other members of the FZD receptor family. In an embodiment the FZD binding domain binds specifically to one FZD with an affinity greater than 10-fold over the binding to any other Frizzled family member.
In an embodiment of this invention the FZD Agonist binds to FZD4, a “FZD4 Agonist”. The FZD4 binding domain of the FZD4 Agonist may bind specifically to FZD4, binding with a higher affinity to FZD4 over other FZDs, or may be pan-specific, binding to FZD4 and one or more other members of the FZD receptor family, e.g., Frizzled Class Receptor 1 (FZD1), Frizzled Class Receptor 2(FZD2), Frizzled Class Receptor 3 (FZD3), Frizzled Class Receptor 5 (FZD5), Frizzled Class Receptor 6 (FZD6), Frizzled Class Receptor 7 (FZD7), Frizzled Class Receptor 8 (FZD8), Frizzled Class Receptor 9 (FZD9), or Frizzled Class Receptor 10 (FZD10). In an embodiment the FZD binding domain binds specifically to FZD4 with an affinity greater than 10-fold over any other Frizzled family member listed above.
In an embodiment of this invention the FZD Agonist binds to FZD5, a “FZD5 Agonist.” The FZD5 binding domain of the FZD5 Agonist may bind specifically to FZD5, binding with a higher affinity to FZD5 over other FZDs, or may be panspecific, binding to FZD5 and one or more other members of the FZD receptor family, e.g., FZD1, FZD2, FZD3, FZD4, FZD6, FZD7, FZD8, FZD9, or FZD10. In an embodiment the FZD binding domain binds specifically to FZD5 with an affinity greater than 10-fold over any other Frizzled family member listed above.
In an embodiment of the FZD Agonists of this invention the Wnt co-receptor binding domain is a monospecific bivalent LRP5/6 co-receptor binding domain and binds to a single epitope on the LRP5 and/or LRP6 co-receptor, e.g., an epitope of the LRP5 and/or LRP6 coreceptor that binds to Wnt1 (E1-E2 domain of LRP5 or LRP6) or binds Wnt3a (E3-E4 domain of LRP5 or LRP6). In an embodiment of this invention the co-receptor binding domain is a bispecific bivalent LRP5/6 binding domain that binds to two epitopes within the LRP5 and/or LRP6 co-receptor extracellular domain, e.g., the co-receptor binding domain interacts with the Wnt1 (E1-E2) and Wnt3 (E3-E4) epitopes of the LRP5 and/or LRP6 co-receptor. In an embodiment of this invention the co-receptor binding domain is a bispecific bivalent binding domain that binds to an extracellular domain of LRP5 and LPR6, e.g., the domain interacts with the Wnt1 (E1-E2) epitope of the LRP5 co-receptor and the Wnt1 (E1-E2) epitope of the LRP6 co-receptor LRP5, or the domain interacts with the Wnt3a (E3-E4) epitope of the LRP5 co-receptor and the Wnt3a (E3-E4) epitope of the LRP6 co-receptor or alternatively the domain interacts with a Wnt1 (E1-E2) epitope or LRP5 co-receptor and a Wnt3a (E3-E4) epitope of LPR6 co-receptor or vis versa.
Various formats of tetravalent binding antibody molecules described herein are depicted in
We previously reported multivalent binding molecules comprising an Fc domain, a FZD binding domain and a Wnt co-receptor (LRP5/6) binding domain on opposite termini of the Fc domain, e.g., a molecule having a FZD4 diabody linked one terminus of an Fc domain and a LRP5/6-binding diabody linked to the other terminus of the Fc domain, see PCT/IB2019/051174 inventors Angers et al. and PCT/IB2020/055463 inventors Angers et al., both incorporated in their entirety by reference.
It has been reported that Wnt-βcatenin signaling, specifically through activation of FZD4, is important for vasculature development and for adult vasculature homeostasis. More specifically, it is critical for barrier function at the blood-retina and blood-brain barriers (BRB and BBB). Defects in FZD4 signaling can lead to endothelial cell permeability defects and genetic mutations within this pathway are known to lead to vascular defects (e.g. Norrie disease, FEVR). At the blood-retina barrier, the extracellular ligand Norrin predominantly activates a FZD4-TSPAN12-LRP5 complex to regulate endothelial cell-cell interactions, barrier functions and permeability (Wang et al. (2012) Norrin/Frizzled4 signaling in retinal vascular development and blood brain barrier plasticity. Cell. 151:1332-1344). At the blood-brain barrier the secreted Wnt7a/b growth factor chiefly activates the FZD4-GPR124-LRP6 receptor complex (Chang et al., (2017). GPR124 is essential for blood-brain barrier integrity in central nervous system disease. (Nat. Med. 23: 450-460). The FZD4 Agonists described herein, e.g., the configurations having a diabody binding domain for a LRP5/6 and an FZD4 binding domain comprised of two Fab fragments that bind FZD4, wherein the binding domains are on opposite termini of an Fc domain, produce a particularly stable and homogenous molecule with an unexpectedly high level of Wnt-βcatenin signaling pathway activation in endothelial cells that translates into increased barrier function and decreased vascular permeability (
This invention also includes methods for using the FZD Agonists described herein. Described herein are methods to activate a Wnt signaling pathway, e.g., the Wnt/β-catenin signaling pathway, using the tetravalent binding antibody molecules of this invention, which are contemplated to promote the proximity of FZD receptors and Wnt co-receptors, e.g., one or more of FZD1, FZD2, FZD3, FZD4, FZDS, FZD6, FZD7, FZD8, FZD9, and FZD10 receptors and LRP5 and/or LRP6 co-receptors, on a cell wherein binding by the FZD Agonists to both FZD receptor(s) and the LRP5 and/or LPR6 co-receptor(s) activates the Wnt signaling pathway.
Blood-retina barrier (BRB) formation and retinal angiogenesis require βcatenin signaling induced by the ligand norrin (NDP [Norrie disease protein]), the receptor FZD4, co-receptor LRP5, and the TSPAN12 (tetraspanin 12). As such, an aspect of this invention is a method for promoting and/or maintaining retinal vasculature barrier function and angiogenesis by treating eye tissue, e.g., retinal tissue, with an effective amount of a tetravalent FZD4 Agonists of this invention.
Also, an aspect of this invention is a method for promoting, restoring and/or maintaining the BRB and BBB functions by treating the BRB or BBB vasculature with an effective amount of a tetravalent FZD4 Agonist described herein. A further aspect of this invention is a method for treating a subject having a disorder or condition characterized by defective retinal or brain angiogenesis characterized by reduced endothelial cell barrier function leading to vascular leakage by administering to such subject an effective amount of a FZD4 Agonists of this invention. A further aspect of this invention is a FZD4/LRP5 tetravalent binding antibody molecule or pharmaceutical composition for use in the treatment or prevention of a disorder or condition characterized by defective retinal or brain angiogenesis and/or characterized by reduced endothelial cell barrier function and/or vascular leakage. A further aspect of this invention is a method of treating or preventing a disorder or condition characterized by defective retinal or brain angiogenesis and/or reduced endothelial cell barrier function and/or vascular leakage comprising administering to a person in need thereof a therapeutically effective amount of a FZD4/LRP5 tetravalent binding antibody molecule described herein. A further aspect of the invention is the use of a FZD4/LRP5 tetravalent binding antibody molecule for the manufacture of a medicament for the treatment or prevention of a disorder or condition characterized by defective retinal or brain angiogenesis and/or reduced endothelial cell barrier function and/or vascular leakage. Such disorders or conditions include ocular disorders, including but are not limited to disorders of the retina or macula. Such disorders of the retina or macula include, but are not limited to diabetic retinopathy, retinopathy of prematurity, Coats' disease, FEVR, Norrie disease, macular degeneration, diabetic macular edema, and pediatric vitreoretinopathies. Additional disorders or conditions included in embodiments of this invention include but are not limited to Alzheimer's disease, epilepsy, multiple sclerosis, ischemia, and stroke.
An embodiment of this invention includes methods for producing vascularized cerebral organoids by promoting the barrier function of the vasculature network throughout the organoids, and thereby mimicking blood-brain-barrier function using an effective amount of a tetravalent FZD4 Agonist described herein.
Also, an embodiment of this invention is a method of treating a subject suffering from a gastrointestinal disorder, including a subject having inflammation of all or part of the intestines, also known as inflammatory bowel disease, by administering to such subject an effective amount of a pharmaceutical composition of this invention, e.g., a composition comprising a FZD5 Agonist. Examples of inflammatory bowel disease include, but are not limited to, Crohn's disease, and ulcerative colitis.
Also, an embodiment of this invention are methods for directing differentiation of iPS or other pluripotent stem cells (PSCs) towards various lineages by culturing these cells in the presence of an effective amount of a tetravalent binding antibody molecule of this invention.
Also described herein are methods for making the tetravalent binding antibody molecules of this invention.
The modular aspects of this invention allow for mixing and matching binding domains derived from FZD-binding antibodies and LRP5/6-binding antibodies on opposite termini of the Fc domain to generate a tetravalent binding antibody molecule that can engage a FZD-LRP5/6 co-receptor complexes to selectively activate Wnt signaling. The modularity and effectiveness of the tetravalent binding antibody molecules for activating Wnt signaling pathways described herein contrasts with the Wnt surrogates described in the prior art that consists of monovalent FZD and Wnt co-receptor binding ligands, or FZD and Wnt co-receptor binding ligands wherein the binding ligands are not attached to opposite ends of an Fc domain.
dark gray=LRP5
CH3 domains of the Hole construct Fc region and Knob construct Fc region. The various domains of the tetravalent molecules, VL, VH, CH1, CH2, CH3, CL1 and Fc, are joined via linkers, e.g., peptide linkers.
Described herein are tetravalent binding antibody molecules comprising an Fc domain, with or without effector function, a bivalent FZD binding domain and a bivalent LRP-binding domain, wherein the binding domains are attached to opposite ends of the Fc domain. In an embodiment, the FZD binding domain is attached to the carboxy terminus of the Fc region and the LRP co-receptor binding domain is attached to the amino terminus of the Fc domain. Alternatively, the FZD binding domain is attached to the amino terminus of the Fc region and the co-receptor binding domain is attached to the carboxy terminus of the Fc domain. The binding domains may be attached directly to the Fc domain or attached to the Fc domain via a linker. The FZD binding domain may bind to one or to more than one FZD receptor, i.e., one or more of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10. In an embodiment of the invention the FZD binding domain is bivalent and comprises a diabody or comprises a scfv, a VHH fragment, or an Fab fragment or combinations thereof that bind FZD, and the co-receptor binding domain is bivalent and comprises a diabody or a VHH fragment, an Fab, or a scFv or combinations thereof that bind the LRP5/6 co-receptor. In an embodiment of the invention the FZD binding domain is attached to the carboxy-terminus of the Fc domain and comprises two scfv, two VHH fragments, two Fab fragments or a diabody that bind FZD, and the co-receptor binding domain attached to the amino terminus of the Fc domain comprises a diabody, two VHH fragments or two scFvs that binds to the LRP5/6 co-receptor. When attached to the carboxy terminus of the Fc domain the FZD-binding Fabs are linked to the CH3 of the Fc domain via the Fab variable heavy region or variable light region. In other embodiments the FZD binding domain is attached to the amino terminus of the Fc domain and is comprised of two Fabs and the LRP5/6 co-receptor binding domain is attached to the carboxy terminus of the Fc domain and is comprised of a diabody or two scFvs that bind the co-receptor.
An embodiment of this invention is a tetravalent binding antibody molecule in a Diabody-Fc-scFv format having (i) an Fc domain, (ii) a LRP5/6 co-receptor binding domain that comprises a bispecific diabody that binds two different sites on the co-receptor, e.g., a Wnt1 (E1-E2) site on LRP5/6, and a Wnt3 site (E3-E4) on LRP5/6, wherein the diabody is attached to the amino terminus of the Fc domain and (iii) a FZD binding domain, attached to the carboxy terminus of the Fc domain comprising two scFv fragments each binding FZD. The scFv may be specific for the FZD, or may be pan-specific, binding to the FZD and one or more other FZD.
An embodiment of this invention is a tetravalent binding antibody molecule in an IgG-Diabody format comprising (i) an Fc domain, (ii) an N-terminal binding domain for a FZD, comprising two FZD-binding Fabs and (ii) a C-terminal binding domain for a LRP5 and/or LRP6 co-receptor, comprising a LRP5/6 coreceptor-binding diabody. This FZD Agonist in the IgG-Diabody format comprises,
(1) a first and second heavy chain monomer, wherein each heavy chain monomer comprises a single-chain polypeptide comprising from N-terminus to C-terminus:
The first and second heavy chain monomers dimerize via their Fc regions, or fragments thereof. The linker between the VH and VL that bind the LRP5/6 is of a length that promotes the pairing of the VH and VL of the first heavy chain monomer with the VL and VH of the second heavy chain monomer thereby forming a LRP5/6 co-receptor binding diabody. The FZD-binding Fabs are formed by the pairing of each heavy chain monomer with a light chain monomer such that the VH that binds FZD4 and CH1 of each of the heavy chain monomer, pairs with the VL that binds FZD4 and CL1 of the light chain monomers. In this IgG-Diabody format, the Fabs form the FZD4-binding domain on the N-terminus of the Fc domain and the diabody forms the co-receptor-binding domain on the C-terminus of the Fc domain. The Fabs may be specific for one FZD, e.g., FZD4 or FZD5, or may be pan-specific, binding to more than one FZD, e.g., to FZD4 and/or FZD5, and in some cases more FZD. The Fc regions may dimerize via a knob-in-hole configuration. Methods for dimerizing peptides via a knob-in-hole configuration are described in WO2018/026942, inventors Van Dyk et al., Carter P. (2001) J. Immunol. Methods 248, 7-15; Ridgway et al. (1996) Protein Eng. 9, 617-621; Merchant, et al. (1998) Nat. Biotechnol. 16, 677-681, and; Atwell et al., (1997) J. Mol. Biol. 270, 26-35. The Fc regions may be Merrimack (knob chain: Q347M, Y349F, T350D, T366W and L368M; hole chain: 53541, E357L, T366S, L368A and Y407V), Merchant (knob chain: T366W; hole chain: T336S, L368A and Y407V) or Merchant S:S (Merchant mutations with additional S354C variant in the knob chain and Y349C in the hole chain). The Fc regions may also contain mutations that alter their effector function, e.g., the Fc region may have attenuated effector functions due to amino acid mutations, e.g., DANG variants and LALAPS variants.
Although in
Also an embodiment of this invention is a tetravalent binding antibody molecule in the Diabody-Fc-Fab format comprising (i) an Fc domain, (ii) an N-terminal binding domain comprising a diabody that binds to the co-receptor, e.g., LRP5 and/or LRP6 co-receptor and (ii) a C-terminal binding domain comprising two Fab that bind to one or more FZD, e.g., FZD4 or FZD5. This FZD Agonist in the Diabody-Fc-Fab format comprises,
(1) a first and second heavy chain monomer, wherein each heavy chain monomer comprises a single-chain polypeptide comprising, from N-terminus to C-terminus:
The first and second heavy chain monomers dimerize via the Fc regions or fragments thereof and a bivalent LRP5/6-binding diabody is formed by the pairing of the VH domain and VL domain that bind LRP5/6 of the first heavy chain monomer with the VL domain and VH domain that bind LRP5/6 of the second heavy chain monomer. The two FZD-binding Fabs are formed by the pairing of each heavy chain monomer with a light chain monomer such that the VL that binds the FZD and the CL1 of a light chain monomer pairs with the VH that binds the FZD and the CH1 of each of the heavy chain monomers. In this Diabody-Fc-Fab format, the diabody forms the LRP5/6 co-receptor binding domain on the amino terminus of the tetravalent molecule and the two Fabs form the FZD binding domain on the C-terminus of the tetravalent binding antibody molecule. The Fc regions may dimerize via a knob-in-hole configuration.
In an embodiment of the invention is a tetravalent binding antibody molecule comprising a bivalent, bispecific LRP5 binding domain, wherein
In an embodiment of the invention the first heavy chain monomer comprises
In an embodiment of the invention the second heavy chain monomer comprises
In an embodiment of the invention the third and fourth light chain monomers comprise a VL that binds FZD4 comprising CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2 and CDR-L3 of SEQ ID NO: 12, wherein the third and fourth light chain monomers comprise a sequence which has at least 70% identity, such as 75% identity, such as 80% identity, such as 85% identity, such as 90% identity, such as 91% identity, such as 92% identity, such as 93% identity, such as 94% identity, such as 95% identity, such as 96% identity, such as 97% identity, such as 98% identity, such as 99% identity, such as 100% identity to SEQ ID NO: 909 or 952. Methods for dimerizing peptides via a knob-in-hole configuration are described in WO2018/026942, inventors Van Dyk et al., Carter P. (2001) J. Immunol. Methods 248, 7-15; Ridgway et al. (1996) Protein Eng. 9, 617-621; Merchant, et al. (1998) Nat. Biotechnol. 16, 677-681, and; Atwell et al., (1997) J. Mol. Biol. 270, 26-35. The Fc regions may be Merrimack (knob chain: Q347M, Y349F, T350D, T366W and L368M; hole chain: S354I, E357L, T366S, L368A and Y407V), Merchant (knob chain: T366W; hole chain: T336S, L368A and Y407V) or Merchant S:S (Merchant mutations with additional S354C variant in the knob chain and Y349C in the hole chain). The Fc regions may also contain mutations that alter their effector function, e.g., the Fc region may have attenuated effector functions due to amino acid mutations, e.g., DANG variants, LALA and LALAPS variants. In an embodiment of the invention the Fc regions of the heavy chain monomers described previously comprise Merrimack knob-in-hole mutations and DANG amino acid mutations. In an embodiment of the invention the Fc regions of the heavy chain monomers described previously comprise Merrimack knob-in-hole mutations and LALAPS amino acid mutations. In an embodiment of the invention the Fc regions described previously of the heavy chain monomers comprise Merchant knob-in-hole mutations and LALAPS amino acid mutations. In an embodiment of the invention the Fc regions of the heavy chain monomers described previously comprise Merchant S:S knob-in-hole mutations and LALAPS amino acid mutations.
In an embodiment of the invention the polypeptides comprising monomer chains further comprise a signal peptide. In an embodiment of the invention the polypeptides comprising monomer chains do not comprise a signal peptide. The signal peptide may have been cleaved from the immature chain to produce the mature chain.
Although in
In an embodiment of this invention the binding moiety of the FZD binding domain is derived from an antibody, or an antibody fragment, that binds specifically to one FZD, e.g. FZD4 or FZD5, or is pan-specific interacting with a specific FZD, e.g. FZD4 or FZD5, and one or more additional FZD receptors (an FZD source antibody), and the co-receptor binding domain comprises a binding moiety that is derived from an antibody or antibody fragment that binds to a LPR5 and/or LRP6 (a LRP5/6 coreceptor source antibody). In an embodiment of the invention the FZD-binding antibodies bind to an extracellular cysteine rich domain (CRD) of the FZD receptor. The antibody that binds FZD may be an antibody that binds the FZD receptor and antagonizes Wnt signaling or inhibits binding of a Wnt ligand to the FZD receptor. The antibody that binds FZD may be an antibody that binds the FZD receptor without antagonizing or inhibiting binding of a Wnt ligand to the FZD receptor. The antibody that binds FZD may be an antibody that binds FZD and enhances Wnt signaling. The antibody that binds the LRP5/6 co-receptor may be an antibody that binds the LRP5/6 co-receptor and antagonizes Wnt signaling or inhibits binding of a Wnt ligand to the co-receptor, or the antibody that binds the LRP5/6 co-receptor may be an antibody that binds the co-receptor without antagonizing Wnt or Norrin signaling or inhibiting binding of a Wnt or Norrin ligand to the co-receptor.
In an embodiment of this invention the LRP5/6 co-receptor binding domain binds to a single epitope on a co-receptor, e.g., an epitope that binds to the Wnt1 (E1-E2) or Wnt3 (E3-E4) interacting domain of LRP5/6. In an embodiment of this invention the LRP5/6 co-receptor binding domain binds to two epitopes within the co-receptor, e.g., a paratope that binds to the Wnt1 (E1-E2) interacting epitope and a paratope that binds to Wnt3 (E3-E4) epitope of LRP5/6. In an embodiment of this invention the multivalent binding molecule comprises a Fc domain, wherein the Fc domain is the Fc domain of an immunoglobulin or a fragment thereof comprising the CH3 domain. In an embodiment of the invention the immunoglobulin is an IgG. In an embodiment of this invention the IgG is an IgG1.
In an embodiment of this invention the LRP5/6 binding domain comprises a diabody comprising two peptides each comprising a heavy chain variable domain (VH) that binds to LRP5/6 linked to a light-chain variable domain (VL) that binds LRP5/6 wherein the binding domain is formed by pairing of the VH and the VL from one peptide to the VL and VH of the other peptide thereby forming the LRP5/6 binding domain.
In the tetravalent binding antibody molecules of this invention both of the binding domains are bivalent and one or both of the bivalent binding domains may be bispecific for the respective FZD receptor, e.g., FZD4 or FZD5, or LRP5/6 co-receptor. For example, the binding molecule may comprise an FZD binding domain that is bivalent and monospecific (each binding site binding to the same epitope) and the LRP 5/6 binding domain is bivalent and bispecific, binding to two different epitopes (the Wnt1 (E1-E2) and Wnt3 (E3-E4) sites on the LRP5/6 ectodomain). In an embodiment of this invention both binding domains are bivalent and bispecific, each binding domain binding to two different epitopes on their respective target FZD receptor or LRP 5/6 co-receptor.
The VH and VL domains of the FZD binding domain of the tetravalent molecules of this invention may comprise the three light chain CDRs and three heavy chain CDRs of a FZD source antibody, e.g. the FZD4 or FZD5, binding antibodies of Table 1, Table 2 or Table 6, or three light chain CDRs and three heavy chain CDRs that are at least 50%, at least 55%, at least 60%, at least 75, at least. 80%, at least 85%, at least 90%, at least at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the CDRs of the FZD source antibody, e.g., the FZD4 antibodies of Table 1, Table 2 or Table 6, and still retain binding to the FZD or FZD5 receptor bound by the source antibody.
The VH and VL domains of the LRP5/6 co-receptor binding domain of the tetravalent molecules of this invention may comprise the three light chain CDRs and three heavy chain CDRs of an LRP5/6 co-receptor source antibody, e.g., the LRP5/6 binding antibodies of Table 3, Table 4 or Table 6, or three light chain CDRs and three heavy chain CDRs that are at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH and VL of the Wnt co-receptor source antibody, e.g., the LRP5/6 binding antibodies of Table 3, Table 4 or Table 6, and still bind to the LRP5/6 co-receptor.
In an embodiment of this invention the FZD binding domain of the tetravalent binding molecule of this invention binds FZD4 (an FZD4 Agonist) or FZD5 (FZD5 Agonist) or FZD4 and/or FZD5 and one or more other FZDs (a pan-FZD Agonist) and comprises the CDR-H1, CDR-H2 and CDR-H3 and the CDR-L1, CDR-L2 and CDR-L3 of the antibodies of Table 1, Table 2 or Table 6, or CDRs that are at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the CDR-H1, CDR-H2 and CDR-H3 and CDR-L1, CDR-L2 and CDR-L3 of the antibodies of Table 1, Table 2 or Table 6, and still bind to FZD4 or FZD5, and the LRP5/6 binding domain of the FZD4 Agonist or FZD5 Agonist or pan-FZD Agonist comprises the CDR-H1, CDR-H2 and CDR-H3 and CDR-L1, CDR-L2 and CDR-L3 of the antibodies of Table 3, Table 4 or Table 6 or the CDRs are at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the CDR-H1, CDR-H2 and CDR-H3 and CDR-L1, CDR-L2 and CDR-L3 of the antibodies in Table 3, Table 4 or Table 6, and still bind to LRP5 or LRP6.
In an embodiment, the tetravalent binding antibody molecule's FZD binding domain does not comprise a diabody, scFv, or Fab comprising the three heavy chain CDRs or three light chain CDRs of the FZD4-binding antibody 5044 in combination with a Wnt co-receptor binding domain comprising a diabody, scFv, or Fab comprising the three heavy chain CDRs and three light chain CDRs of LRP6-binding antibody 2542 and/or antibody 2539. In an embodiment, the tetravalent binding molecule does not comprise a diabody, scFv, or Fab, comprising the three heavy chain CDRs and three light chain CDRs of the FZD4-binding antibody 5027 in combination with a Wnt co-receptor binding domain comprising a diabody, scFv, or Fab comprising the three heavy chain CDRs and three light chain CDRs of LRP6-binding antibody 2542 and/or antibody 2539.
An embodiment of the invention is a polypeptide comprising a chain monomer of the tetravalent binding antibody molecule of the invention.
In an embodiment of the invention is a polypeptide comprising the first heavy chain monomer of the binding antibody molecule of the invention. In a further embodiment, the polypeptide comprises a sequence which has at least 70% identity, such as 75% identity, such as 80% identity, such as 85% identity, such as 90% identity, such as 91% identity, such as 92% identity, such as 93% identity, such as 94% identity, such as 95% identity, such as 96% identity, such as 97% identity, such as 98% identity, such as 99% identity, such as 100% identity to any sequence selected from SEQ ID NOs: 908, 921 to 928, 937, 940 and 941.
In an embodiment of the invention is a polypeptide comprising the second heavy chain monomer of the binding antibody molecule of the invention. In a further embodiment, the polypeptide comprises a sequence which has at least 70% identity, such as 75% identity, such as 80% identity, such as 85% identity, such as 90% identity, such as 91% identity, such as 92% identity, such as 93% identity, such as 94% identity, such as 95% identity, such as 96% identity, such as 97% identity, such as 98% identity, such as 99% identity, such as 100% identity to any sequence selected from SEQ ID NOs: 929 to 936 and 944 to 951 In an embodiment of the invention is a polypeptide comprising a light chain monomer of the binding antibody molecule of the invention. In a further embodiment, the polypeptide comprises a sequence which has at least 70% identity, such as 75% identity, such as 80% identity, such as 85% identity, such as 90% identity, such as 91% identity, such as 92% identity, such as 93% identity, such as 94% identity, such as 95% identity, such as 96% identity, such as 97% identity, such as 98% identity, such as 99% identity, such as 100% identity to SEQ ID NO: 909 or 952.
Also, an embodiment of this invention are the nucleic acid molecules encoding the tetravalent binding molecules described herein. An embodiment of this invention are the nucleic acid molecules encoding the polypeptides of the tetravalent binding molecules described herein comprising the heavy chain and light chain CDRs set forth in Tables 1, 2, 3, 4, 6. Also an embodiment of this invention are the nucleic acid molecules that encode the polypeptides of the tetravalent binding molecules, e.g., FZDS Agonists or FZD4 Agonists, of
In an embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain monomer of the tetravalent binding antibody molecule of the invention. In a further embodiment, the nucleic acid molecule comprises a sequence which has at least 70% identity, such as 75% identity, such as 80% identity, such as 85% identity, such as 90% identity, such as 91% identity, such as 92% identity, such as 93% identity, such as 94% identity, such as 95% identity, such as 96% identity, such as 97% identity, such as 98% identity, such as 99% identity, to any one of SEQ ID NOs: 1030 to 1061. In a further embodiment, the nucleic acid comprises any one of SEQ ID NOs: 1030 to 1061. In a further embodiment the nucleic acid molecule consists of any one of SEQ ID NOs: 1030 to 1061.
In an embodiment the nucleic acid encodes a polypeptide comprising a light chain monomer of the tetravalent binding antibody molecule of the invention. In a further embodiment, the nucleic acid molecule comprises a sequence which has at least 70% identity, such as 75% identity, such as 80% identity, such as 85% identity, such as 90% identity, such as 91% identity, such as 92% identity, such as 93% identity, such as 94% identity, such as 95% identity, such as 96% identity, such as 97% identity, such as 98% identity, such as 99% identity, to SEQ ID NO: 1062 or 1063. In a further embodiment, the nucleic acid molecule comprises SEQ ID NO: 1062 or 1063. In a further embodiment, the nucleic acid molecule consists of SEQ ID NO: 1062 or 1063.
In an embodiment of the invention the nucleic acid encodes the first heavy chain monomer, the second heavy chain monomer and the third and fourth light chain monomers of the tetravalent binding antibody molecule of the invention.
In an embodiment of the invention is a set of one or more polynucleotides wherein each polynucleotide encodes at least one of the monomer chains of the tetravalent binding antibody molecule of the invention, such that all chains of said tetravalent binding antibody molecule are encoded. In a further embodiment of the invention, the set of one or more polynucleotides encodes two chains of the tetravalent binding antibody molecule. In a further embodiment of the invention, the set of one or more polynucleotides encodes three chains of the tetravalent binding antibody molecule. In a further embodiment of the invention, the set of one or more polynucleotides encodes four chains of the tetravalent binding antibody molecule.
In an embodiment of the invention the nucleic acid molecules encode polypeptides of the invention further comprising a signal peptide. In an embodiment of the invention the nucleic acid molecules encode polypeptides of the invention which do not comprise a signal peptide. As used in this invention, the term “vector” refers to a nucleic acid delivery vehicle or plasmid that can be engineered to contain a nucleic acid molecule, e.g., a nucleic acid sequence encoding the tetravalent binding antibody molecules described herein. The vector that can express protein when inserted with a polynucleotide is called an expression vector.
Vectors can be inserted into the host cell by transformation, transduction, or transfection, so that the carried genetic substances can be expressed in the host cell. Vectors are well known to the technical personnel in the field, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1 derived artificial chromosome (PAC); phage such as λphage or M13 phage and animal viruses etc. Animal viruses may include but not limited to, reverse transcriptase virus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e. g. herpes simplex virus), chicken pox virus, baculovirus, papilloma virus, and papova virus (such as SV40). A vector can contain multiple components that control expression of the tetravalent binding antibody molecules described herein, including but not limited to, promoters, e.g., viral or eukaryotic promoters, e.g., a CMV promoter, signal peptides, e.g., TRYP2 signal peptide, transcription initiation factor, enhancer, selection element, and reporter gene. In addition, the vector may also contain replication initiation site(s). In an embodiment of this invention, the vector comprises a nucleic acid encoding a heavy chain of the tetravalent binding antibody molecule of the invention. In an embodiment, the vector comprises a nucleic acid encoding a light chain of the tetravalent binding antibody molecule of the invention. In an embodiment, the vector comprises nucleic acids encoding two heavy chain sequences and one light chain sequence. An embodiment of the invention is a set of one or more vectors which collectively comprise the set of one or more polynucleotides described previously, such that all chains of the tetravalent binding antibody molecule of the invention are encoded in the set of vectors.
As used in this invention, the term “host cell” refers to cells that can import expression cassettes and vectors, including but not limited to, prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast and Aspergillus, insect cells such as S2 drosophila cells and Sf9, or animal cells, including human cells, e.g., fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells. An embodiment of this invention is a host cell expressing a vector of the invention. An embodiment of this invention is a process for the production of a tetravalent binding antibody molecule of the invention using a vector.
An embodiment of this invention is a pharmaceutical composition comprising a FZD Agonist or a nucleic acid molecule, expression cassette, vector, a set of nucleic acid molecules or a set of vectors encoding a FZD Agonist described herein and a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may further comprise an additional agent, e.g., a second therapeutic antibody e.g. an anti-VEGF antibody (aflibercept, ranibizumab and bevacizumab), a growth factor, e.g., VEGF, or an agent that activates a Wnt pathway, e.g., the small molecule CHIR99021, a Norrin or R-Spondin, or a nucleic acid molecule, expression cassettes and vectors that encode the agent. The pharmaceutical composition may consist of or consist essentially of a FZD Agonist, or a nucleic acid molecule, an expression cassette or vector encoding an FZD Agonist described herein, and a pharmaceutically acceptable diluent, carrier or excipient. Suitable carriers, diluents and excipients, and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution may be e.g., from about 5 to about 8, from about 5 to 7.5 or from about 6 to 7. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the agonist, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of the FZD Agonists being administered.
This invention also includes methods for using the FZD Agonists, described herein. An embodiment of this invention is a method for activating a Wnt signaling pathway in a cell, comprising contacting a cell having an FZD receptor and a LRP5/6 co-receptor, with a tetravalent binding antibody molecule of this invention that binds the FZD, e.g., FZD4, and the LRP5/6 in an amount effective to activate Wnt signaling. It has been reported that the Norrin-FZD4 pathway plays a role in retinal angiogenesis (see Wang et al. Cell. 2012; 151(6):1332-1344; Braunger B M, Tamm E R. Adv Exp Med Biol. 2012; 723:679-683; Ohlmann A, Tamm E R. Prog Retin Eye Res. 2012; 31(3):243-257 and; Ye et al. Trends Mol Med. 2010; 16(9):417-425). Signaling through Norrin-FZD4 pathway is necessary for development and maintenance of retinal vasculature. Mutations affecting genes of this pathway may result in several vitreoretinopathies, such as Norrie Disease, Familial Exudative Vitreoretinopathy (FEVR), and Pseudoglioma and Osteoporosis Syndrome. Additionally, Retinopathy of Prematurity (ROP) has been associated with mutations in this Norrin-FZD4 pathway, and Wnt-pathway mutations have been reported in Coats Disease and Persistent Fetal Vasculature (PFV). FZD4 signaling activated by Norrin and/or WNT7A/B pathway is also associated with CNS blood brain barrier development and homeostasis. Genetic ablation of the Norrin, FZD4, LRP5, LRP6 and the co-receptor Tetraspanin-12 (Tspan-12) result in defective angiogenesis and barrier disruption in the retinal and/or cerebellar vessels (Cho et al. (2017) Neuron 95, 1056-1073; Zhou et al., (2014) J Clin Invest 124:3825-3846). Thus, a functional Wnt signaling system plays a key fundamental role in the development of a sufficient vascular and neural network in the eye and retina to support vision and in the CNS to support BBB development and homeostasis.
An aspect of this invention is a method for promoting and/or maintaining retinal vasculature by treating eye tissue, e.g., retinal tissue, with an effective amount of a pharmaceutical compositions comprising the tetravalent antibody molecules of this invention, e.g., tetravalent antibody molecules that binds FZD4 and LRP5/6, a FZD4 Agonists, having the structures illustrated in
A further aspect of this invention is a method for treating a subject having a disorder characterized by vascular leakage, particularly retinal vascular leakage, and/or endothelial cell leakage, and disorders characterized by reduced retinal or brain endothelial cell barrier functions or a compromised BBB or BRB, e.g., diabetic retinopathy, retinopathy of prematurity, Coat's disease, FEVR, Norrie disease, macular degeneration, diabetic macular edema, and pediatric vitreoretinopathies, by administering to such subject an effective amount of a pharmaceutical compositions of this invention, e.g., a composition comprising a FZD4 Agonist having the structures depicted in
The FZD4 Agonists of this invention particularly the FZD4 Agonist in the diabody-Fc-Fab format comprising two Fab fragments forming the FZD4 binding domain on the carboxy terminal of the Fc receptor and a binding domain for LRP5 and/or LRP6 composed of a diabody on the amino terminal of the Fc domain, e.g., as illustrated in
A further aspect of the invention is a method for treating a subject having inflammation of all or part of the intestines, also known as inflammatory bowel disease, by administering to such subject an effective amount of a pharmaceutical composition of this invention, e.g., a composition comprising a FZD5 Agonist. Examples of inflammatory bowel disease include, but are not limited to, Crohn's disease, and ulcerative colitis. An effective amount of such composition is an amount sufficient to reduce, ameliorate, eliminate, or treat the inflammation. A subject in need thereof includes a subject having inflammation of the mucosal of the gastrointestinal tract. The methods disclosed herein may be practiced to reduce inflammation (e.g., inflammation associated with IBD or in a tissue affected by IBD, such as gastrointestinal tract tissue, e.g., small intestine, large intestine, or colon), activate WNT signaling, or reduce any of the histological symptoms of IBD (e.g., those disclosed herein).
The FZD Agonists of the present invention may be administered systemically or locally, e.g., by injection (e.g. subcutaneous, intravenous, intraperitoneal, intrathecal, intraocular, intravitreal, etc.), implantation, topically, or orally. Depending on the route of administration, the FZD Agonists may be coated in a material to protect the agonists from conditions that may inactivate the agonists. The tetravalent binding antibody molecules described herein may be dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier. In addition, the composition comprising the FZD Agonists can contain excipients, such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc. An extensive listing of excipients that can be used in such a composition, can be, for example, taken from A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The tetravalent binding antibody molecules can also be administered together with immune stimulating substances, such as cytokines.
An embodiment of this invention includes a method for deriving cerebral organoids with a vascular network exhibiting barrier functions by using the tetravalent antibody molecules described herein. The tetravalent binding antibody molecules described herein that activate FZD4 signaling are envisioned to promote barrier function within endothelial cells cultured with cerebral organoids and thereby promoting angiogenesis.
An embodiment of this invention includes a method for directed differentiation of multipotent or pluripotent stem cells (PSC) or induced pluripotent stem (iPS) cells comprising culturing the cells under conditions suitable for directed differentiation wherein said culturing conditions further comprise an effective amount of a tetravalent binding antibody molecule described herein. Studies in mouse and human PSCs have identified specific approaches to the addition of growth factors, including Wnt, which can induce PSC differentiation into different lineages. Methods for directed differentiation of PSCs comprising the activation of Wnt signaling are known in the art see e.g. Lam et al. (2014) Semin Nephol 34(4); 445-461; Yucer et al. (Sep. 6, 2017) Scientific Reports 7, Article number 10741. It is contemplated that the FZD Agonists, e.g. FZD4 Agonists, described herein can be used in an amount sufficient to effect activation of Wnt signaling pathways to direct differentiation of the PSCs to certain mesodermal lineages such as cardiomyocytes (cite Yoon et al. FZD4 Marks Lateral Plate Mesoderm and Signals with NORRIN to Increase Cardiomyocyte Induction from Pluripotent Stem Cell-Derived Cardiac Progenitors. Stem Cell Reports. 2018 January; 10(1):87-100. DOI: 10.1016/j.stemcr.2017.11.008.PMID: 29249665).
An embodiment of this invention is a method for enhancing tissue regeneration in a subject in need thereof by activating Wnt signaling in such subject by administering to the subject in need thereof an effective amount of a FZD Agonists described herein.
An embodiment of this invention includes a method for promoting endothelial cell barrier functions in eye tissue, e.g., retinal tissue, in a subject in need thereof, by administering an effective amount of a tetravalent binding molecule of this invention that binds FZD4 and LPR5/6, an FZD4 Agonist. In a particular embodiment the FZD4 Agonist of this invention that binds to FZD4 and a binding domain that binds to LRP5 or/and LRP6 has a diabody-Fc-Fab structure depicted in
A subject as used herein may be any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, horses, cows, dogs, cats, rodents, and the like. The subject may be a fetus. Typically, the subject is human.
Effective dosages and schedules for administering the FZD Agonists and nucleic acids that encode them described herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of such FZD Agonists that must be administered will vary depending on, for example, the subject who will receive the antibody, the route of administration, the particular type of FZD Agonists used and other drugs being administered. Guidance in selecting appropriate doses for FZD Agonists is found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone, eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith, Antibodies in Human Diagnosis and Therapy, Haber, eds., Raven Press, New York (1977) pp. 365-389. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect, e.g., promote endothelial cell barrier functions, vascular homeostasis, or enhance Wnt signaling. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, gender and the extent of the disease or disorder, in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. While individual needs vary, determination of optimal ranges of effective amounts of the vector is within the skill of the art.
Also, an aspect of this invention is a method for making the tetravalent binding antibody molecules described herein. The amino acid sequences of FZD receptors, e.g. FZD4, and the Wnt co-receptors LRP5/6, and nucleotide sequences encoding FZD receptors and the Wnt co-receptors LRP5/6, as well as antibodies and libraries of antibodies that bind FZD, e.g., FZD4, or the Wnt co-receptors LRP5/6, are readily available or can be generated using methods well known in the art (see e.g., U.S. publication no. 2015/0232554, inventors Gurney et al. and US publication no. 2016/0194394, inventors Sidhu et al. and US 20190040144, inventors Pan et al.; U.S. publication no. 2017/0166636, inventors Wu et al.; U.S. publication no. 2016/0208018, inventors Chen et al.; U.S. publication no. 2016/0053022, inventors Macheda et al.; U.S. publication no. 2015/031293, inventors Damelin et al.). And a variety of methods are known in the art for generating and screening such phage display libraries for antibodies, and antibody fragments, scFv, Fab, VL, and VH possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004), all incorporated herein by reference. In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360, all incorporated herein by reference. Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.
In an embodiment of this invention a tetravalent binding antibody molecule in a diabody-Fc-scFv format comprising a LRP5/6 coreceptor binding domain comprising LRP5/6-binding diabody and an FZD-binding domain comprising two FZD-binding scFvs is generated by,
(a) selecting an Fc domain having a C-terminus and an N-terminus
(b) identifying an antibody that binds to an FZD receptor (the “FZD source antibody”), and
(c) identifying an antibody that binds LRP 5/6 co-receptor (“coreceptor source antibody” or “LRP 5/6 source antibody”),
(d) generating a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide monomer comprising
In an embodiment of this invention, the tetravalent binding antibody molecule has two FZD-binding Fabs, e.g., FZD4-binding Fabs, linked to one terminus of the Fc domain and two LRP5/6-binding scFvs or a LRP5/6-binding diabody linked to the other terminus of the Fc domain and is generated by,
(a) identifying the light chain complementary determining regions (CDR-L1, CDR-L2, and CDR-L3) and/or heavy chains complementary determining regions (CDR-H1, CDR-H2, and CDR-H3) of an antibody that binds to the FZD, e.g., FZD4 or FZDS, (the “FZD source antibody”) and
(b) identifying the CDR-L1, CDR-L2, and CDR-L3 and/or the CDR-H1, CDR-H2, and CDR-H3 of one or more antibodies that binds to LRP5 or LRP6, (the “LRP5/6 source antibody”),
(c) generating a nucleic acid molecule encoding a “heavy chain” polypeptide comprising
The FZD source antibody may be an antibody that binds specifically to one FZD, e.g., FZD4, or is a pan-specific antibody binding FZD, e.g., FZD4 or FZD5, and one or more other FZD receptors and antagonizes Wnt signaling or inhibits Wnt binding to the receptor.
Alternatively, the FZD source antibody may be an antibody that binds specifically to one FZD, e.g., FZD4 or FZD5, or is a pan-specific antibody binding one FZD, e.g., FZD4 or FZD5, and one or more other FZD receptors without antagonizing Wnt signaling or inhibiting Wnt binding to the receptor. The LRP source antibody may be an antibody that binds specifically to LRP5/6, or is panspecific binding to LRP5/6 and to one or more of the Wnt co-receptors, and antagonizes Wnt signaling or inhibits Wnt binding to the co-receptor.
Alternatively, the LRP5/6 source antibody may be an antibody that binds to the LRP 5/6 co-receptor, or is panspecific binding to LRP5/6 and to one or more of the Wnt co-receptors, without antagonizing Wnt signaling or inhibiting Wnt binding to the LRP5/6 co-receptor.
The FZD source antibody may be an antibody fragment that binds the FZD receptor, e.g., an Fab, a VL or VH. The light chain and heavy chain CDRs, the VH and/or VL in the FZD binding domain of the FZD Agonists may be identical to the CDRs, the VH and/or VL of the FZD source antibody or may be at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the CDRs, VH or VL of the source antibody and still retain binding to the FZD receptor. The CDRs, the VH and/or VL in the FZD binding domain of the FZD Agonists may be identical to the CDRs, the VH and/or VL of a FZD4-binding or FZD5-binding antibody of Table 1, Table 2 or Table 6, or may be at least 50%, at least 55%, at least 60%, 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the CDRs, VH or VL of a FZD4-binding or FZD5-binding antibody of Table 1 or Table 2 or Table 6 and still retain binding to the FZD receptor.
Likewise, the Wnt co-receptor source antibody may be an antibody fragment, e.g. an Fab, a VL or a VH, that binds the LRP co-receptor, e.g., LRP5/6. The light chain CDRs and heavy chain CDRs, the VH and/or VL in the Wnt co-receptor binding domain of the FZD4 Agonists may be identical to the CDRs, the VH and/or VL of the Wnt co-receptor source antibody or may be at least at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the CDRs, VHs or VLs of the source antibody and still retain binding to the LRP co-receptor. The light chain CDRs and heavy chain CDRs, the VH and/or VL in the LRP5/6 binding domain of the FZD Agonists may be identical to the light chain CDRs and heavy chain CDRs, the VH and/or VL of a LRP-binding antibody of Table 3, Table 4 or Table 6 or may be at least at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light chain CDRs and heavy chain CDRs, VH or VL of a LRP-binding antibody of Table 3, Table 4 or Table 6 and still retain binding to the LRP co-receptor. In an embodiment of this invention, two polypeptides of the tetravalent binding antibody molecule dimerize via knob-in-hole configuration of their Fc sequences. The tetravalent binding antibody molecules of this invention may be generated by dimerizing two polypeptides in a “knob-in-hole” configuration. The knob-in-hole configuration increases the modularity of this invention by facilitating the association of peptides that comprise binding moieties that bind different epitopes on a FZD receptor or LRP5/6 co-receptor or to epitopes on different members of the FZD receptor or co-receptor family, see e.g.,
Without wishing to be bound by theory, it is contemplated that the tetravalent binding antibody molecules of this invention facilitate the interaction of a FZD receptor and an LRP5/6 co-receptor on a cell by promoting their proximity and stabilizing conformations of the receptor proteins that are favorable for activating Wnt signaling pathways. Another embodiment of this invention is a method for facilitating the interaction of a FZD receptor and an LRP5/6 co-receptor on a cell thereby activating a Wnt signaling pathway in the cell comprising, a) selecting an Fc domain, or fragment thereof comprising a CH3 domain, having a C-terminus and an N-terminus b) linking a first bivalent binding domain, which binds the FZD receptor, on one terminus of the Fc domain and linking a second bivalent binding domain, which binds to the Wnt co-receptor, on the other terminus of the Fc domain thereby forming a tetravalent binding antibody molecule; c) contacting said tetravalent binding antibody molecule with the cell expressing said FZD receptor and Wnt co-receptor under conditions wherein the FZD receptor and co-receptor both bind to the tetravalent binding antibody molecule thereby activating the Wnt signaling pathway. The Wnt co-receptor binding domain and FZD binding domain are bivalent and each comprise a VL and/or a VH, or VHH domain and one or both of the binding domains may be monospecific. In an embodiment of the invention one or both the Wnt co-receptor binding domain and FZD binding domain are bispecific. In an embodiment of the invention the Wnt co-receptor binding domain is bivalent and bispecific. The FZD binding domain may comprise a scFv that binds FZD, a VHH that binds FZD, or an Fab that binds FZD, or combinations thereof, or a diabody that binds FZD. The Wnt co-receptor binding domain may comprise a scFv that binds the LRP5/6 co-receptor, a VHH that binds LRP5/6, an Fab that binds the LRP5/6 co-receptor, or combinations thereof, or a diabody that binds the LRP5/6 co-receptor. In an embodiment of the invention the FZD binding domain comprises two FZD-binding Fabs and the Wnt co-receptor binding domain comprises a bispecific bivalent diabody that binds LRP5/6 on two different epitopes.
The tetravalent binding antibody molecules of this invention initiate the Wnt signaling pathway(s) that are stimulated by the FZD-co-receptor complexes, e.g., the β-catenin pathway stimulated by FZD-LRP5/6 complexes. Wnt ligands function by promoting the clustering of FZD receptors with co-receptors. Without wishing to be bound by theory, it is contemplated that the FZD Agonists described herein bind both the FZD receptor and its LRP5/6 co-receptor thereby forming a complex that mimics the binding of a Wnt molecule to the FZD receptor and LRP 5/6 co-receptor(s), which in turn activates Wnt signaling pathways, the Wnt β-catenin pathway.
An embodiment of this invention is a method for activating a Wnt signaling pathway comprising contacting a cell expressing a FZD receptor and its LRP5/6 co-receptor with an effective amount of the FZD Agonists of this invention comprising a FZD binding domain and a LRP5/6 co-receptor binding domain.
The FZD Agonists of this invention may be made recombinantly, e.g., by Gibson assembly (see Gibson et al. (2009) Nature Methods 6 (5): 343-345 and Gibson DG. (2011) Methods in Enzymology 498: 349-361), or the molecules may be made synthetically e.g., using commercial synthetic apparatuses, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface.
The binding domains of the FZD Agonists may be linked to the Fc domain via a linker. In some embodiments, adjacent VH and VL domains may be attached to each other via a peptide linker. In some embodiments adjacent constant domains and variable domains are attached via a peptide linker. The linker may be, e.g. a polypeptide linker, or a non-peptidic linker. In some embodiments the constant domains and variable domains of the FZD Agonists are attached to the Fc domain via a peptide linker. Suitable linkers are well known in the art, e.g., an XTEN linker (see WO2013120683, inventors Schellenberger et al.) In some embodiments, the peptide linker comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or at least 100 amino acids. In some embodiments, the peptide linker is between 1 to 100, 5 to 75, 1 to 50, 5 to 50, 1 to 30, 1 to 25, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 1-10 or 1-5 amino acids in length. The modular aspects of this invention allow for mixing and matching of binding domains derived from antibodies that bind to FZD receptor or antibodies that bind LRP5/6 co-receptor on the opposite termini of the Fc domain to generate a tetravalent binding antibody molecule that can engage FZD receptor—LRP5/6 co-receptor complexes to activate Wnt signaling.
The Fc domain of the FZD Agonists, with or without the linker, is of a length and flexibility that allows for the tetravalent binding antibody molecule of this invention to bind both the FZD receptor and its LRP5/6 co-receptor thereby stabilizing receptor conformations that are compatible with activation of downstream Wnt signaling pathways. In an embodiment of this invention the Fc domain, or fragment thereof comprising the CH3 domain, with or without the linker is greater than 100 amino acids spanning up to 300 Å, greater than 125 amino acids spanning up to 375 Å, greater than 150 amino acids spanning up to 450 Å, greater than 175 amino acids spanning up to 525 Å, or greater than 300 amino acids spanning up to 900 Å. Preferably the Fc domain is about 200 amino acids to about 300 amino acids in length. As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the peptide” includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.
An “affinity matured” antibody or “maturation of an antibody” refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent or source antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen or to other desired properties of the molecule.
By “comprising” it is meant that the recited elements are required in the composition/method/kit, but other elements may be included to form the composition/method/kit etc. within the scope of the claim. For example, a composition comprising tetravalent binding antibody molecules is a composition that may comprise other elements in addition to the tetravalent binding antibody molecules, e.g. functional moieties such as polypeptides, small molecules, or nucleic acids bound, e.g. covalently bound, to the tetravalent binding antibody molecules; agents that promote the stability of the tetravalent binding antibody molecule composition, agents that promote the solubility of the tetravalent binding antibody molecule composition, adjuvants, etc. as will be readily understood in the art, with the exception of elements that are encompassed by any negative provisos.
By “consisting essentially of”, it is meant a limitation of the scope of composition or method described to the specified materials or steps that do not materially affect the basic and novel characteristic(s) of the subject invention. For example, a tetravalent binding antibody molecule “consisting essentially of” a disclosed sequence has the amino acid sequence of the disclosed sequence plus or minus about 5 amino acid residues at the boundaries of the sequence based upon the sequence from which it was derived, e.g. about 5 residues, 4 residues, 3 residues, 2 residues or about 1 residue less than the recited bounding amino acid residue, or about 1 residue, 2 residues, 3 residues, 4 residues, or 5 residues more than the recited bounding amino acid residue.
By “consisting of”, it is meant the exclusion from the composition, method, or kit of any element, step, or ingredient not specified in the claim. For example, a tetravalent binding antibody molecule “consisting of” a disclosed sequence consists only of the disclosed amino acid sequence.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector functions, e.g., binding Fc receptors and activation of antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Methods for dimerizing peptides via a knob-in-hole configuration are described in WO2018/026942, inventors Van Dyk et al., Carter P. (2001) J. Immunol. Methods 248, 7-15; Ridgway et al. (1996) Protein Eng. 9, 617-621; Merchant, et al. (1998) Nat. Biotechnol. 16, 677-681, and; Atwell et al., (1997) J. Mol. Biol. 270, 26-35. The Fc regions may be Merrimack (knob chain: Q347M, Y349F, T350D, T366W and L368M; hole chain: S354I, E357L, T366S, L368A and Y407V), Merchant (knob chain: T366W; hole chain: T336S, L368A and Y407V) or Merchant S:S (Merchant mutations with additional S354C variant in the knob chain and Y349C in the hole chain). The Fc regions may also contain mutations that alter their effector function, e.g., the Fc region may have attenuated effector functions due to amino acid mutations, e.g., DANG variants and LALAPS variants. Methods are well known in the art for mitigating antibody effector function, including for example amino acid substitutions in the Fc regions, e.g., the N297G and D265A, N297G (DANG) variants, L234A, L235A, P331S (LALAPS), LALAPS Merchant, LALAPS Merchant S-S (Merchant A. M. et al Nature Biotechnol 1998 vol 16 p 677-681) variants, or L234A, L235A, P329G (LALA-PG) substitutions, see e.g., Lo et al. “Effector Attenuating Substitutions that Maintain Antibody Stability and Reduce Toxicity in Mice. The Journal of Biological Chemistry Vol. 292, No. 9, pp. 3900-3908, Mar. 3, 2017, incorporated herein by reference. In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG1, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.
Three highly divergent stretches within each of the heavy chain variable domain, VH or VH domain, and light chain variable domain, VL or VL domain, referred to as complementarity determining regions (CDRs), are interposed between more conserved flanking stretches known as “framework regions”, or “FRs”. Thus, the term “FR” refers to amino acid sequences which are naturally found between, and adjacent to, CDRs in immunoglobulins. A VH domain typically has four FRs, referred to herein as VH framework region 1 (FR1), VH framework region 2 (FR2), VH framework region 3 (FR3), and VH framework region 4 (FR4). Similarly, a VL domain typically has four FRs, referred to herein as VL framework region 1 (FR1), VL framework region 2 (FR2), VL framework region 3 (FR3), and VL framework region 4 (FR4). In an antibody molecule, the three CDRs of a VL domain (CDR-L1, CDR-L2 and CDR-L3) and the three CDRs of a VH domain (CDR-H1, CDR-H2 and CDR-H3) are disposed relative to each other in three-dimensional space to form an antigen-binding site within the antibody variable region. The surface of the antigen-binding site is complementary to a three-dimensional surface of a bound antigen. The amino acid sequences of VL and VH domains may be numbered, and CDRs and FRs therein identified/defined, according to the Kabat numbering system (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) or the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM (IMGT numbering system; Lefranc et al., 2003, Development and Comparative Immunology 27:55-77), both incorporated herein by reference. One of ordinary skill in the art would possess the knowledge for numbering amino acid residues of a VL domain and of a VH domain, and identifying CDRs and FRs therein, according to a routinely employed numbering system such as the IMGT numbering system, the Kabat numbering system, and the like.
The term “antibody” as referred to herein includes whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chain thereof. A “whole antibody” or full-length refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region or domain (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL or CL1. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term “antigen-binding portion” or “antigen-binding fragment” of an antibody (or simply “antibody portion” or “antibody fragment”), as used herein, refers to one or more fragments, portions or domains of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) an Fab fragment, a monovalent fragment consisting of the VL, VH, CL1 and CH1 domains; (ii) an F(ab′)2 fragment, a bivalent fragment comprising two F(ab)′ fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al. (1989) Nature 241:544-546), which consists of a VH domain; and (vi) an isolated complementary determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single contiguous chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed (see e.g., Holliger et al. (1993) PNAS. USA 90:6444-6448).
“Diabodies,” or sometimes referred to herein as “Dia,” as used herein are dimeric antibody fragments. In each polypeptide of the diabody, a heavy-chain variable domain (VH) is linked to a light-chain variable domain (VL) but unlike single-chain Fv fragments, the linker between the VL and VH is too short for intramolecular pairing and as such each antigen-binding site is formed by pairing of the VH and VL of one polypeptide with the VH and VL of the other polypeptide. Diabodies thus have two antigen-binding sites, and can be monospecific or bispecific. (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123; Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5) incorporated herein by reference.
As used herein an “effective amount” of an agent, e.g., the tetravalent binding antibody molecules or a pharmaceutical composition comprising the molecules, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, stabilizes one or more characteristics of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. In some embodiments, the amount of a FZD Agonists administered to the subject is in the range of about 0.001 mg/kg to 10 mg/kg, 0.5 mg/kg to about 10 mg/kg, or about 0.5 mg/kg to about 1 mg/kg of the subject's body weight. For example, in some embodiments the FZD4 Agonist may be applied to the eye in an amount of, e.g., about 0.02-1.5 mg, about 0.05-1.0 mg, or about 0.1-0.5 mg per eye. As used herein, the term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or fragment thereof, or a T-cell receptor. The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is ≤10 μM; e.g., ≤100 nM, preferably ≤10 nM and more preferably ≤1 nM.
The constant region of immunoglobulin molecules is also called the fragment crystallizable region, the “Fc region” or “Fc domain.” The Fc domain is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains and the Fc domains of IgGs bear a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity. In an embodiment of the invention the Fc domain of the tetravalent binding antibody molecule is engineered such that it does not target the cell that binds the tetravalent binding antibody molecule for ADCC or CDC-dependent death. In an embodiment of the invention the Fc domain of the tetravalent binding antibody molecule is a peptide dimer in a knob-in-hole configuration. The peptide dimer may be a heterodimer.
The terms “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
“LRP”, “LRP proteins” and “LRP receptors” is used herein to refer to members of the low density lipoprotein receptor-related protein family. These receptors are single-pass transmembrane proteins that bind and internalize ligands in the process of receptor-mediated endocytosis. LRP proteins LRP5 (e.g., LRP5: NP_002326.2) and LRP6 (e.g., LRP6: NP_002327.2) are included in a Wnt receptor complex required for activation on the Wnt-Bcatenin signaling pathway. See also, for human/mouse LRP5 and LRP6: https://www.uniprot.org/uniprot/O075197, https://www.uniprot.org/uniprot/Q91VN0, https://www.uniprot.org/uniprot/O075581, https://www.uniprot.org/uniprot/088572.
The term “polypeptide fragment” as used herein refers to a polypeptide that has an amino terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence deduced, for example, from a full-length cDNA sequence.
As used herein the term “paratope” includes the antigen binding site in the variable region of an antibody that binds to an epitope.
“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv and other antibody fragments, see James D. Marks, Antibody Engineering, Chapter 2, Oxford University Press (1995) (Carl K. Borrebaeck, Ed.).
“Single-domain antibody” (sdAb), or “nanobody”, is an antibody fragment consisting of a single monomeric variable antibody domain. “VHH” or “VHH fragment” as used herein refers to a human VH that has been engineered to be independent of the light chain (Nilvebrant et al. Curr Pharm Des. (2016) 22(43):6527-6537; Barthelemy et al., Journal of Biological Chemistry (2007) 283:3639-3654).
The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., slowing or arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
The ability of the tetravalent binding antibody molecules of this invention to activate Wnt signaling can be confirmed by a number of assays. The tetravalent binding antibody molecules of this invention typically initiate a reaction or activity that is similar to or the same as that initiated by the FZD receptor's natural ligand. The tetravalent binding antibody molecules of this invention activates the Wnt signaling pathways, e.g., the canonical Wnt-βcatenin signaling pathway. As used herein, the term “activates” refers to a measurable increase in the intracellular level of a Wnt signaling pathway, e.g., the Wnt-βcatenin signaling pathway, compared with the level in the absence of a FZD Agonist of the invention.
Various methods are known in the art for measuring the level of Wnt-βcatenin activation. These include but are not limited to assays that measure: Wnt-βcatenin target gene expression; LEF/TCF reporter gene expression (such as TopFLASH, superTopFLASH, pBAR); βcatenin stabilization; LRP5/6 phosphorylation; Dishevelled phosphorylation; Axin translocation from cytoplasm to cell membrane and binding to LRP5/6. The canonical Wnt-βcatenin signaling pathway ultimately leads to changes in gene expression through the transcription factors TCF1, TCF7L1, TCF7L2 and LEF1. The transcriptional response to Wnt activation has been characterized in a number of cells and tissues. As such, global transcriptional profiling by methods well known in the art can be used to assess Wnt-βcatenin signaling activation.
Changes in Wnt-responsive gene expression are generally mediated by TCF and LEF transcription factors. A TCF reporter assay assesses changes in the transcription of TCF/LEF controlled genes to determine the level of Wnt-βcatenin signaling. A TCF reporter assay was first described by Korinek, V. et al., 1997. Also known as TOP/FOP this method involves the use of three copies of the optimal TCF motif CCTTTGATC, or three copies of the mutant motif CCTTTGGCC, upstream of a minimal c-Fos promoter driving luciferase expression (pTOPFLASH and pFOPFLASH, respectively) to determine the transactivational activity of endogenous βcatenin/TCF. A higher ratio of these two reporter activities (TOP/FOP) indicates higher βcatenin/TCF activity. A newer and more sensitive version of this reporter is called pBAR and contains 12 repeats of the TCF motifs (Biechele and Moon, Methods Mol Biol. 2008; 468:99-110, PMID: 19099249).
General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., CSH Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998).
Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
A. FZD4 Antibodies from Affinity Matured Libraries of FZD4-Binding Antibody 5027 and 5044; FZD5 Antibodies from Affinity Matured Libraries of FZD5-Binding Antibody 2919 and 2928.
Affinity matured libraries of known FZD4-binding antibodies 5027 and 5044 and known FZD5-binding antibodies 2919 and 2928 were prepared using routine methods, essentially as described in US publication no. 2016/0194394, inventors Sidhu et al., see also Persson et al. J. Mol. Biol., 2013 Feb. 22; 425(4):803-11 https://pubmed.ncbi.nlm.nih.gov/23219464/, both incorporated herein in their entirety by reference.
The 6 CDRs of the heavy chain (CDR-H1, CDR-H2 and CDR-H3) and light chains (CDR-L1, CDR-L2 and CDR-L3) of antibodies 5044, 5027, 2919, and 2928 antibodies isolated from the affinity matured libraries are set forth in Table 1 and Table 2.
Single point ELISAs were performed on 96-well Maxisorp plates coated with the extracellular domains (ECDs) of human FZD4 protein in the presence or absence of a saturating concentration of 5027 diabody-Fc (a diabody comprising the VL and VH of 5027 linked to an Fc domain). The plates were incubated with monoclonal Fab-phage followed by incubation with horseradish peroxidase (HRP)-conjugated anti-M13 antibody. Wells were subsequently washed 8 times followed by incubations with 3,3,′5,5′-tetramethylbenidine/H2O2 peroxidase (TMB) substrate for 5-10 min. The reaction was stopped by adding 1M H3PO4 and the absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. The results of the assay are depicted in
B. Epitope Mapping of Lead FZD4 Antibodies.
ELISA assays were performed in 384-well Maxisorp plates coated with FZD4 ECD wild-type (FZD4) or mutant FZD4 proteins (FZD_swap1-18) that replace segments of the FZD4 ECD with corresponding regions from FZDS. The plates were incubated with 10 nM IgG known to bind specifically to FZD4, i.e., 5044 and 5027, or to be panspecific, i.e., 5016 (binds FZD4, FZDS, and other FZD receptors), followed by incubation with horseradish peroxidase (HRP)-conjugated anti-Kappa light chain antibody. Phosphate buffered saline (PBS) and IgG 4275 which does not bind FZD4 or FZDS were used as controls. The wells were washed 6 times followed by incubations with 3,3,′5,5′-tetramethylbenidine/H2O2 peroxidase (TMB) substrate for 3-5 min. The reaction was stopped by adding 1M H3PO4 and the absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader, see
C. Characterization of FZD4 IgG.
FZD4-binding full length IgGs were expressed via transient transfection in an Expi293 cell culture system, essentially as described in Tao et al., Tailored tetravalent antibodies potently and specifically activate Wnt/Frizzled pathways in cells, organoids and mice. Elife. 2019 Aug. 27; 8:e46134. doi: 10.7554/eLife.46134; PMID: 31452509. and purified via Protein A affinity chromatography. Briefly, cells were grown to a density of approximately 2.5×106 cells/ml in Expi293 Expression Media (Gibco) in baffled cell culture flasks and transfected with the appropriate vectors using FectoPRO transfection reagent (Polyplus-transfection) using standard manufacture protocols (ThermoFisher). Expression was allowed to proceed for 5 days at 37° C. and 8% CO2 with shaking at 125 rpm. After expression, cells were removed by centrifugation and protein was purified from the conditioned media using Protein A Sepharose (GE Healthcare). Purified protein was buffer exchanged into either PBS or a formulated stabilization buffer (36.8 mM citric acid, 63.2 mM Na2HPO4, 10% trehalose, 0.2 M L-arginine, 0.01% Tween-80, pH 6.0) for storage. Proteins concentrations were determined by absorbance at 280 nm and purity was confirmed by SDS-PAGE analysis. Expression titers were determined as mg of purified protein per liter of mammalian cell culture. Size exclusion chromatography (SEC) results in Table A below are defined as “−”: evidence of multiple peaks on SEC trace, <50% monomeric species; “+”: >50% monomeric species, delayed retention time (>14 min.); “++”: >90% of major peak at/near expected retention time for a monomeric IgG. Standard retention time was determined by comparison to Trastuzumab.
Trac ID corresponds to the antibody number in Table 1 and Table 2.
D. Size-Exclusion Chromatography Analysis and ELISA Specificity Measurements of the FZD4 IgGs.
Twenty micrograms of the FZD4 binding IgGs were separated over an AdvanceBio SEC, 300 Å, 2.7 μm, 4.6×300 mm column in a mobile phase of PBS using an Agilent Bio-Inert HPLC. Protein elution was monitored using absorbance at 280 nM. The results are presented in
ELISA specificity measurements of the FZD4 antibodies were determined against FZD1 and FZD10, the two FZD family member most closely related to FZD4. ELISA assays were performed in 384-well Maxisorp plates coated with FZD ECD wild-type or mutant proteins at a concentration of 1 μg/ml and excess binding sites were blocked with 0.5% BSA. The plates were incubated with 10 nM of the FZD4 binding IgGs followed by incubation with horseradish peroxidase (HRP)-conjugated anti-Kappa light chain antibody. The wells were washed 6 times followed by incubations with 3,3,′5,5′-tetramethylbenidine/H2O2 peroxidase (TMB) substrate for 3-5 min. The reaction was stopped by adding 1M H3PO4 and the absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. The results are presented in
E. Identification of the CDRs of the FZD4 or FZD5 binding antibodies.
The amino acid sequences of the CDRs of the FZD4-binding and FZD5-binding immunoglobulins are set forth in Tables 1 and 2. The CDRs were identified according to the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM (IMGT numbering system; Lefranc et al., 2003, Development and Comparative Immunology 27:55-77), and annotated as described in Persson et al. J Mol Biol. 2013 Feb. 22; 425(4):803-11, both incorporated herein by reference.
A. Phage Clonal ELISA of Synthetic Antibodies Targeting LRP5 and LRP6.
Single point ELISAs were performed on 96-well Maxisorp plates coated with the ECDs of mouse LRP5-his protein or human Fc and blocked with BSA (0.5%). The plates were incubated with monoclonal Fab-phage, or VH-phage and titers >109 phage/ml followed by incubation with horseradish peroxidase (HRP)-conjugated anti-M13 antibody. The wells were washed 8 times followed by incubations with 3,3,′5,5′-tetramethylbenidine/H2O2 peroxidase (TMB) substrate for 5-10 min. the reaction was stopped by adding 1M H3PO4 and the absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. The results are presented in
Single point ELISAs were performed on 96-well Maxisorp plates coated with the ECDs of human LRP6-Fc protein chimeras. The plates were incubated with the monoclonal Fab-phage, or VH-phage and titers >109 phage/ml followed by incubation with horseradish peroxidase (HRP)-conjugated anti-M13 antibody. The wells were washed 8 times followed by incubations with 3,3,′5,5′-tetramethylbenidine/H2O2 peroxidase (TMB) substrate for 5-10 min. the reaction was stopped by adding 1M H3PO4 and the absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. The results are presented in
B. Identification of the CDRs of the Synthetic Antibodies Targeting LRP5 and LRP6.
The CDRs of the LRP5-binding and LRP6-binding immunoglobulins set forth in Tables 3 and 4 were identified according to the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM (IMGT numbering system; Lefranc et al., 2003, Development and Comparative Immunology 27:55-77) and annotated as described in Persson et al. J Mol Biol. 2013 Feb. 22; 425(4):803-11, both incorporated herein by reference.
A. We generated various formats of tetravalent binding antibody molecules comprising pan-FZD and LRP5/6 antibody fragments, e.g., scFv, diabodies and Fab, on either end of an Fc domain, see Table 5 and
These various of tetravalent binding antibody molecules comprising pan-specific FZD and LRP5/6 antibody fragments were tested in a TOPFLASH assay to monitor beta catenin-mediated gene reporter activity. Proteins were compared against the native ligand Wnt3a. Assays were performed by plating TOPFLASH cells to ˜70% confluency in a 96-well tissue culture treated plate. Agonists were diluted in DMEM to provide a final assay concentration of 0.046 nM-100 nM and cells were treated overnight at 37° C. under 5% CO2. Luciferase expression was quantified using the Dual-Luciferase Reporter Assay System (Promega) in 96-well black plates in accordance with the manufacturer's instructions. Briefly, HEK293T cells were transduced with lentivirus coding for the pBARls reporter (Biechele and Moon in Wnt Signaling: Pathway Methods and Mammalian Models, E. Vincan, Ed. (Humana Press, Totowa, N.J., 2008), pp. 99-110) and with Renilla Luciferase as a control to generate a Wnt-βcatenin signaling reporter cell line. 1-2×103 cells in 120 μl were seeded in each well of 96-well plates for 24 hours prior to transfection or stimulation. The following day, FZD Agonists or Ab protein was added, and following 15-20 hours of stimulation, cells were lysed and luminescence was measured in accordance with the dual luciferase protocol (Promega) using an Envision plate reader (PerkinElmer). For the FZD4 Agonist assay, FZD4 cDNA was transfected for 6 hours prior to adding the FZD Agonist. For the Wnt inhibition assays, Wnt1 was introduced by cDNA transfection or WNT3A protein was applied for 6 hours prior to the addition of Ab protein. All assays were repeated at least three times. The results are presented in Table 5. As shown in Table 5, each of the tetravalent formats activate FZD signaling to differing degrees when clustering FZD4 and LRP5. These formats were also evaluated for stability, homogeneity and yield production from Expi293 (
B. Diabody-Fc-Fab format FZD4 Agonists.
FZD Agonists having a bispecific LRP5-binding diabody and a FZD4 binding domain comprising FZD4-binding Fabs (FZD4 Agonists), a FZD5 binding domain comprising FZD5-binding Fabs (FZD5 Agonists), or a FZD binding domain that binds multiple FZD (pan-FZD Agonist) were generated using a knob-in-holes system. Briefly, the constructs were generated by chemical synthesis (Twist Biosciences) or by standard molecular biology techniques in a mammalian expression vector (pSCSTa). Diabody constructs were arranged in a VH-VL manner with a short (GGGGS (SEQ ID NO: 886)) linker linking the VH and VL to favor intermolecular pairing. For bispecific diabody arrangements, the variable domains for paratopes A and B, respectively, were arranged as VH(A)-VL(B) on the Hole Fc chain and VH(B)-VL(A) on the Knob Fc chain to facilitate proper paratope formation. Diabodies were fused to the N-terminus of an optimized knob-in-holes heterodimeric Fc (Ridgway et al. Protein Eng. 9, 617-621 (1996) via a GGGGSGGGGSEPKSS linker (SEQ ID NO: 890). The Fc region also contains the effector-null mutations D278A and N314G (Kabat numbering), corresponding to D655A/N297G (EU numbering). Fab domains were fused to the C-terminus of the heterodimeric Fc via a GGGSGGGSGGGSGGGSGSTG linker (SEQ ID NO: 891). Directly to this linker was fused the N-terminus of the Fab VH domain followed by CH1, terminating at T238 (Kabat numbering). This Fab pairs with a standard kappa light chain which was cloned as described above. For all constructs, the entire coding region was cloned into a mammalian expression vector in frame with the secretion signal peptide.
In addition, Diabody-fc-Fab formats were constructed as VH-x-VL-y-[human IgG1 Fc]-z-VH where linkers are x=GGGGS (SEQ ID NO: 886), y=GGGGSGGGGSEPKSSDKTHT (SEQ ID NO: 892), and z=GGGSGGGSGGGSGGGSGSTG (SEQ ID NO: 891). Diabody domains were arranged in a VH-VL orientation with the variable domains separated by a short GGGGS linker (SEQ ID NO: 886), which favors intermolecular association between VH and VL domains and thus favors diabody formation. Further, the Fc region may exhibit attenuated effector functions due amino acid mutations to N297G and D265A (DANG) variants or L234A, L235A, P331S (LALAPS) variants, and with the Fc region further comprising knob-in-hole heterodimerization variants Merrimack, Merchant or Merchant S:S.
B. IgG-Diabody Format FZD4 Agonists.
FZD Agonists having two FZD-binding Fabs forming an N-terminal binding domain and a bispecific LRP5/6 binding diabody forming the C-terminal binding domain and an Fc domain were generated using a knob-in-holes system. IgG-Diabody proteins were constructed as VH-[human IgG1 Fc]-y-VH-x-VL where linkers are x=GGGGS (SEQ ID NO: 886) and y=GGGSGGGSGGGSGGGSGSTG (SEQ ID NO: 891).
C. FZD Agonists are Highly Specific for FZD4, Bind with High Specificity and are Stable in Solution.
Using biolayer interferometry (BLI) we have found that FZD4 Agonists described herein are highly specific for FZD4 over other FZD receptors. Recombinant FZD ECD proteins were immobilized on BLI sensors. The FZD4 Agonists in the Diabody-Fc-Fab format, having a LRP5 binding domain comprised of a diabody that is bivalent and bispecific for LRP5 and a FZD4 binding domain comprised of two FZD4 binding Fab fragments, were tested at a concentration of 100 nM in a buffer of PBS+0.05% Tween-20 and 1% BSA for binding to the ECD proteins. The results are presented in
The FZD4 Agonists also did not recognize common non-specific antigens. The FZD4 Agonists were tested at 100 nM for binding to a panel of antigens essentially as described in Monquet et al. “Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation” Nature 2010 Sep. 30; 467(7315):591-5(PMC3699875), and Jain et al., “Biophysical properties of the clinical-stage antibody landscape” Proc Natl Acad Sci 2017 Jan. 31; 114(5):944-949. (PMC5293111). Controls in the assay included CM0199, a diabody-Fc-diabody format FZD agonist that recognizes FZD4 and LRP5 and immunoglobulin 6606, which is an IgG that is particularly prone to non-specific binding in this assay. The results are presented in
The FZD4 Agonists comprising binding domains for FZD4 and LRP5 bind both FZD4 and LRP5 with high affinity. The apparent affinity of the FZD4 Agonists for recombinant ECD of FZD4 were determined by biolayer interferometry essentially as described in Elife. 2019 Aug. 27; 8: e46134. Briefly, BLI assays were performed using an Octet HTX instrument (ForteBio). For measuring binding to antigen, FZD-Fc proteins were captured on AHQ BLI sensors (18-5001, ForteBio) to achieve a BLI response of 0.6-1 nm and remaining Fc-binding sites were saturated with human Fc (009-000-008, Jackson ImmunoResearch). FZD-coated or control (Fc-coated) sensors were transferred into 100-0.1 nM tetravalent FZD agonist in assay buffer (PBS, 1% BSA, 0.05% Tween20) and association was monitored for 300 s. Sensors were then transferred into assay buffer and dissociation was monitored for an additional 300 s. Shake speed was 1000 rpm and temperature was 25° C. The results are presented in Table 7.
The FZD4 Agonists were also analyzed by SEC as compared to trastuzumab IgG. The results are presented in
The FZD4 Agonists are also stable in solution. Purified FZD4 Agonists, ANT16, ANT18, ANT20, ANT21 and ANT 36 were resuspended to 1 mg/ml (except for ANT18, which was resuspended at 0.34 mg/ml) in 10 mM Histidine, 140 mM NaCl, 0.9% sucrose, pH 6 and stored either at 4° C. or 40° C. for a period of 6 days. Samples were removed at various time points, centrifuged to remove precipitated protein and residual protein concentration was measured. The results are presented in Tables 8 and 9.
On Day 6, the amount of FZD4-specific binding sites remaining in the samples were quantified using BLI. Analysis by differential scanning fluorimetry showed that the FZD4 Agonists having a Diabody-Fc-Fab formats with an LRP-binding diabody on the N-terminal of the Fc domain and two FZD4-binding Fabs on the C-terminal of the Fc domain, have thermal denaturation profiles similar to that of Trastuzumab. IgGs generally display two peaks in a thermal stability assay, the first corresponding to CH2, the later to the Fab domain and CH3, see
The FZD4 Agonists were also assayed for induction of the beta-catenin target gene AXIN2 in a mouse endothelial cell line (bEND3.1) and were shown to induce transcription in a concentration dependent manner. These results are presented in
EXAMPLE 4. The FZD4 agonist was assayed for its ability to oppose the effect on cell junction disassembly and increased permeability mediated by VEGF, a cytokine released during tissue hypoxia. VEGF treatment of bEND3.1 cells leads to junction disassembly as seen by loss of plasma membrane staining of CLDN3, CLDN5 and ZO-1. Co-treatment of cells with VEGF and the FZD4 agonist leads to a near-complete rescue of this effect (
A) Immunofluorescence of ZO-1 (green)/CLDN3(red) and ZO-1 (green)/CLDN5(red) localization on bEnd.3 cell junctions. bEnd.3 cells were treated or not with 30 nM of F4L5.13 (aka CM0199) and Norrin in the presence or absence of VEGF (100 ng/ml) for 1h. DAPI (blue) stain the nucleus. B) Transendothelial permeability was determined by measuring the passage of FITC-dextran through the bEnd.3 monolayer. Passage of FITC-dextran was measured after bEnd.3 treatment with VEGF (100 ng/ml) and F4L5.13 (30 nM) alone or simultaneously or upon pre-treatment with VEGF for 1h followed by F4L5.13 treatment for 1h. Error bar indicate SEM, n=5. The results are presented in
Single point ELISAs were performed on 96-well Maxisorp plates coated with the ECDs of human FZD5 protein in the presence or absence of a saturating concentration of 2919 IgG. The plates were incubated with the monoclonal Fab-phage followed by incubation with horseradish peroxidase (HRP)-conjugated anti-M13 antibody. Wells were subsequently washed 8 times followed by incubations with 3,3,′5,5′-tetramethylbenidine/H2O2 peroxidase (TMB) substrate for 5-10 min. the reaction was stopped by adding 1M H3PO4 and the absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. The results are presented in
Single point ELISAs were performed on 96-well Maxisorp plates coated with the ECDs of human FZD2, FZD5, or FZD8 protein. The plates were incubated with the monoclonal Fab-phage followed by incubation with horseradish peroxidase (HRP)-conjugated anti-M13 antibody. Wells were subsequently washed 8 times followed by incubations with 3,3,′5,5′-tetramethylbenidine/H2O2 peroxidase (TMB) substrate for 5-10 min. the reaction was stopped by adding 1M H3PO4 and the absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. The results are presented in
TOPFLASH HEK293 cells were treated overnight with varying concentrations of FZD agonist or a non-targeting control molecule (CM0156) and TCF/LEF-driven luciferase expression was measured using a standard luciferase assay. Both molecules are able to activate FZD-mediated luciferase expression in a concentration-responsive manner. ANT9, which is able to bind to 7 of the 10 FZD receptor subtypes produces a higher maximal activation signal than the FZD5-specific ANT59. The results are presented in
In Vivo Experiments
DSS Induced Colitis Model
In
Histology
For histological analysis, harvested tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections of 5 μm were stained with haematoxylin and eosin (H&E). Images were captured using a Nikon Eclipse microscope (
Organoid Culture and Viability Measurement
Small intestine crypts were harvested from 8-week-old, female, C57BL/6 mice and cultured as previously described (O'Rourke et al., 2016). Organoid cultures were passaged and embedded in 25 μl Growth Factor Reduced Matrigel (Corning, 356231) and plated in triplicates in a 48-well plate. Organoid cultures were treated with DMSO, 1 μM LGK974, 1 μM LGK974+50% WNT3A conditioned media, 1 μM LGK974+30 nM Pan-FLAg, 1 μM LGK974+30 nM FZD2-FLAg, 1 μM LGK974+30 nM FZD4-FLAg, 1 μM LGK974+30 nM FZDS-FLAg, 1 μM LGK974+30 nM FZD7-FLAg. Treatments were prepared in 250 μl of complete media, added to each well on day of passaging and changed every 2-3 days. At the endpoint (7 days), 150 μl Cell Titer-Glo3D (Promega) was added to 150 μl media in each well. Organoids were lysed on a rocking platform for 30 min at room temperature. The luminescence reading was measured in duplicates for 20 μl lysate from each well on the Envision multilabel plate reader. The average luminescence reading for each condition was normalized to the control condition to calculate relative viability (
A. Transient expression of 8 ANT39 variants. A series of eight ANT39 variants (
B. Eight ANT39 variants, produced at 2:1:3 DNA ratio, were analysed by SEC-HPLC purity after purification using Protein A Sepharose at 280 nm wavelength. Briefly, samples were loaded on to a Protein A column (POROS®A 20 m Column, Stainless Steel, 2.1 mm×30 mm, 0.1 mL) at neutral pH, where the samples are bound to the Protein A ligands and retained on the column. Then the retained antibodies are eluted with an acidic eluent (100 mM Glycine, 150 mM NaCl, pH 2.5) and detected by UV absorbance at 280 nm. The concentration of the sample is quantified by external standard method. Size exclusion chromatography was performed on an Agilent UPLC system with a SEC column (Waters Acquity BEH 150×4.6 mm, 1.7 μm). The sampler temperature was set to 5±3° C. and the column oven temperature was set as 25±3° C. The mobile phase was 50 mM PB, 300 mM NaCl, pH 6.8±0.1 and the flow rate was set as 0.4 mL/min. 10 μg of each sample was injected. Detection wavelength was set at 280 nm and the run time was 8 minutes. Data was analyzed by Agilent CDS Software.
Results are shown in Table 12 and
C. Four ANT39 variants were produced at a 15 L scale after transfection at a 2:1:3 Knob chain: Hole chain: Light chain ratio. Protein titre was measured and is shown in Table 13. SEC HPLC purity was measured, and the results shown in Table 13 and
The Melting Point (Tm) of each molecule was determined using Differential Scanning calorimeter. Differential scanning calorimetry (DSC) is a thermos-analytical technique used to characterize the thermal stability of protein samples and assess conformational differences between them. Measurements were performed on MicroCal PEAQ DSC (Malvern) for thermal transition midpoint (Tm) and onset of unfolding (TOnset) testing. Samples were diluted to 1 mg/mL with the reference buffer. Experimental parameters were set such that the scan temperature ramped from 10 to 95° C. at a scan rate of 200° C./h. Data analysis was performed in MicroCal PEAQ-DSC automated data analysis software. The melting points for each of the molecules tested were higher that 50° C., showing a high stability for each molecule (Table 14)
D. The stability of four ANT39 variants was assessed by applying stress to the molecules in buffer solution and compared to a benchmark IgG. After the application of stress, characteristics of the molecules were determined. 5 mg/mL of each molecule was prepared in formulation buffer (20 mM Histidine, 8% sucrose, 0.04% PS80, pH6.0). The conditions for each test are described in Table 15, with measurements taken before the stress testing (TO) and at each time point described.
At each time point, the sample were clear, colorless and free of visible particles, with the exception of 40C-4W wherein the samples had a slight yellow coloring.
DLS was conducted on samples submitted to Oxidative, Agitation and Freeze-Thaw Stress to determine the hydrodynamic radiuses (Rh). The detection of Rh was performed on Wyatt DynaPro Plate Reader II. 20 μL of the sample was added into corresponding position, and the well plate was then centrifuged for 5 minutes at 5° C. with the speed of 4000 rpm. During the experiment, Rh was detected under 25° C., and the data was analyzed by the DYNAMICS 7.7.0.125 software. There was no obvious change under stress conditions compared to the starting formulations (TO). Results are shown in Table 16.
SEC analysis was performed on all samples. The purity of the monomer did not show obvious changes after Agitation and Freeze Thaw Stress compared to T0. Thermal and Oxidation Stress resulted in decreased percentage monomer compared to T0, as shown in Table 17. After Oxidation Stress, ANT39 LALAPs showed a higher percentage monomer (lower decrease compared to TO) than the other molecules tested.
iCIEF was conducted to determine the percentage of main charge isoform present in each sample (Table 17). For iCIEF, the protein sample was mixed with specific master mixture and then analyzed with iCE3 Capillary Isoelectric Focusing Analyzer equipped with a fluorocarbon (FC)-coated whole-column detection capillary. The pI value and relative abundance of the resolved peaks were quantitated using chromatographic software. ANT39 LALAPS showed the highest percentage of the main charge isoform compared to T0 after both Thermal and Oxidation Stress.
Caliper-SDS was performed to determine the purity of samples. Caliper-SDS was performed on a PerkinElmer Caliper automated electrophoresis using non-reduced samples. The sample denaturing solution was prepared by mixing sample buffer with 10% sodium dodecyl sulfate (SDS) and 100 mM N-Ethylmaleimide (NEM). Prepared samples were loaded, stained, separated and detected in the High-throughput Protein Express LabChip filled with destain-gel, gel-dye and maker. The raw data was analyzed with LabChip GX Reviewer software. The results are shown in Table 17. ANT39 LALAPS Merchant S-S showed the highest purity after Thermal Stress, whereas ANT39 LALAPS showed the highest purity after Oxidation Stress.
The potency of each molecule after Thermal Stress and Oxidative Stress was calculated according to Example 3. The results are shown in
Overall, the ANT39 LALAPS molecule showed increased stability and potency after stress compared to the other molecules.
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the inventions. Various substitutions, alterations and modifications may be made to the invention without departing from the spirit and scope of the invention. Other aspects, advantages, and modifications are within the scope of the invention.
The contents of all references, issued patents, and published patent applications cited through this application are hereby incorporated by reference. The appropriate component, process and methods of those patents, applications and other documents may be selected for the invention and embodiments thereof.
Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.
To clarify the use of and to hereby provide notice to the public, the phrases “at least one of <A>, <B>, . . . and <N>” or “at least one of <A>, <B>, . . . or <N>” or “at least one of <A>, <B>, . . . <N>, or combinations thereof” or “<A>, <B>, . . . and/or <N>” are defined by the Applicant in the broadest sense, superseding any other implied definitions hereinbefore or hereinafter unless expressly asserted by the Applicant to the contrary, to mean one or more elements selected from the group comprising A, B, . . . and N. In other words, the phrases mean any combination of one or more of the elements A, B, . . . or N including any one element alone or the one element in combination with one or more of the other elements which may also include, in combination, additional elements not listed. Unless otherwise indicated or the context suggests otherwise, as used herein, “a” or “an” means “at least one” or “one or more.”
WVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQ
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGK
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKTSDKT
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAED
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMN
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGKAPKL
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCP
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFA
TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS
HWV
YADSVKGRFTISADTSKNTAYLQMNSLRAE
DYWGQGTLVTVSSGGGGSDIQMT
VAWYQQKPGKAPKLLIY
GVP
TFGQGTKVEIKGGGGSG
WVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
HWV
YADSVKGRFTISADTSKNTAYLQMNSLRAE
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSA
VAWYQQKPGKAPKLLIY
GVPSRFSGSRS
TFGQGTKVEIKGGGGSGGGGSEPKS
HWVRQAPGKGLEWVA
YADSV
DYWGQGTL
VAWYQQ
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
HWV
YADSVKGRFTISADTSKNTAYLQMNSLRAED
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRV
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLT
TGQGTKVEIKGGGGSGGGGSEPKTSDKTHTC
HWVRQAPGKGLEWVA
YADSVKGRFTISADTS
DYWGQGTLVTVSSASTKGPS
HWV
YADSVKGRFTISADTSKNTAYLQMNSLRAED
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASV
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGT
TFGQGTKVEIKGGGGSGGGGSEPKSS
HWVRQAPGKGLEWVA
YADSVKGRFTI
DYWGQGTLVTVSSAS
VAWYQQ
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
HWVR
YADSVKGRFTISADTSKNTAYLQMNSLRAED
DYWGQGTLVTVSSGGGGSDIQMTQS
VAWYQQKPGKAPKLLIY
GVPSR
TFGQGTKVEIKGGGGSG
HEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPMVFDLPPSREEMTKNQVSLWCMVKGFYPS
HWVRQAPGKGLEWVA
YADS
DYWGQGT
HWVR
YADSVKGRFTISADTSKNTAYLQMNSLRAED
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTI
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTI
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCP
HWVRQAPGKGLEWVA
YADSVKGRFTISAD
DYWGQGTLVTVSSASTK
VAWYQQ
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
HW
YADSVKGRFTISADTSKNTAYLQMNSLRA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVG
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGT
TFGQGTKVEIKGGGGSGGGGSEPK
HWVRQAPGKGLEWVA
YA
DYWGQ
HWV
YADSVKGRFTISADTSKNTAYLQMNSLRAE
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRV
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTL
TFGQGTKVEIKGGGGSGGGGSEPKSSDKT
HWVRQAPGKGLEWVA
YADSVKG
DYWGQGTLVT
VAWYQ
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
HW
YADSVKGRFTISADTSKNTAYLQMNSLRA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGD
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDF
TFGQGTKVEIKGGGGSGGGGSEPKTSD
HWVRQAPGKGLEWVA
YADSVKG
DYWGQGTLVT
HWVR
YADSVKGRFTISADTSKNTAYLQMNSLRAEDT
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTIT
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTIS
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTC
HWVRQAPGKGLEWVA
YADSVKGRFTISA
DYWGQGTLVTVSSAST
WYQQ
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
V
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKGGGGSGGGGSEPKTSDKTHTCPPCPAPELLGGPSVF
HWVR
YADSVKGRFTISADTSKNTAYLQMNSLRAEDT
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFAT
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGP
HW
YADSVKGRFTISADTSKNTAYLQMNSLRAE
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQAHYFPITFGQGTKVEIKRTV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
GVPSRFSGSRSGTDFTLTISSLQPEDFATY
TFGQGTKVEIKGGGGSGGGGSEPKTSDKTHTCPPCPAPELLGGP
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQM
DYWGQGTLVTVSSASTKGPSVFPLAPSS
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPE
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAY
DYWGQGTLVTVSSASTKGPSVFPL
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGVyLFTFGQGTKVEIKRT
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDF
TFGQGTKVEIKLEDKTHTKVEPKTSDKTHTCPPCPAPELLG
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMN
DYWGQGTLVTVSSGGGGSD
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK
HWVRQAPGKGLEWV
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDF
TFGQGTKVEIKLEDKTHTKVEPKSSDKTHNCPPCPAPELLG
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNS
DYWGQGTLVTVSSGGGGSDIQMTQSPS
VAWYQQKPGKAPKLLIY
GVPSRFS
TFGQGTKVEIK (SEQ ID NO:
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMN
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRV
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSL
TFGQGTKVEIK* (SEQ ID NO: 921)
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMN
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRV
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSL
TFGQGTKVEIK* (SEQ ID NO: 922)
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQM
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDR
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISS
TFGQGTKVEIK* (SEQ ID NO: 923)
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQM
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDR
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISS
TFGQGTKVEIK* (SEQ ID NO: 924)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HWVRQAP
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
V
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK* (SEQ ID NO: 925)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
SGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
V
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK* (SEQ ID NO: 926)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HWVRQ
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HWVRQ
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK* (SEQ ID NO: 928)
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYL
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFT
TFGQGTKVEIK* (SEQ ID NO: 929)
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYL
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFT
TFGQGTKVEIK* (SEQ ID NO: 930)
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYL
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFT
TFGQGTKVEIK* (SEQ ID NO: 931)
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYL
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFT
TFGQGTKVEIK* (SEQ ID NO: 932)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HWV
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATY
TFGQGTKVEIK* (SEQ ID NO: 933)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GGSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HW
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFAT
TFGQGTKVEIK* (SEQ ID NO: 934)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GGSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HW
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFAT
TFGQGTKVEIK* (SEQ ID NO: 935)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GGSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HW
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFAT
TFGQGTKVEIK* (SEQ ID NO: 936)
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAW
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKD
HWVRQAPGKGLEWVA
YADSVK
DYWGQGTLVTVSSA
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAW
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKD
HWVRQAPGKGLEWVA
YADSVK
DYWGQGTLVTVSSA
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAW
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKD
HWVRQAPGKGLEWVA
YADSVK
DYWGQGTLVTVSSA
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAW
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKD
HWVRQAPGKGLEWVA
YADSVK
DYWGQGTLVTVSSA
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVT
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKG
GGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
AVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEY
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAY
DYWGQGTLVTVSSASTKGPSVFPLAPSSKS
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVT
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKG
GGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTA
DYWGQGTLVTVSSASTKGPSVFPLAPSS
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVT
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKG
GGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNT
DYWGQGTLVTVSSASTKGPSVFPLAPS
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVT
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKG
GGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNT
DYWGQGTLVTVSSASTKGPSVFPLAPS
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPP
HWVRQAPGKGLEWVA
Y
DYWGQGTLV
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPP
HWVRQAPGKGLEWVA
Y
DYWGQGTLV
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPP
HWVRQAPGKGLEWVA
Y
DYWGQGTLV
WVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPP
HWVRQAPGKGLEWVA
Y
DYWGQGTLV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGT
VAWYQQKPGKAPKL
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTS
DYWGQGTLVTVSSASTKGPSVFPL
WVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGT
VAWYQQKPGKAPKL
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTS
DYWGQGTLVTVSSASTKGPSVFPL
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGT
VAWYQQKPGKAPKL
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTS
DYWGQGTLVTVSSASTKGPSVFPL
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGT
VAWYQQKPGKAPKL
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTS
DYWGQGTLVTVSSASTKGPSVFPL
VAWYQQKPGK
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQG
VAWYQQKPGKAPKLLIY
SGV
TFGQGTKVEIKRTVAAPSVFI
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMN
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRV
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSL
TFGQGTKVEIK* (SEQ ID NO: 953)
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMN
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRV
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSL
TFGQGTKVEIK* (SEQ ID NO: 954)
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
WVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQM
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDR
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISS
TFGQGTKVEIK* (SEQ ID NO: 955)
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQM
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDR
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISS
TFGQGTKVEIK* (SEQ ID NO: 956)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
V
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK* (SEQ ID NO: 957)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
SGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
V
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK* (SEQ ID NO: 958)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HWVRQ
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK* (SEQ ID NO: 959)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HWVRQ
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK* (SEQ ID NO: 960)
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYL
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFT
TFGQGTKVEIK* (SEQ ID NO: 961)
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYL
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFT
TFGQGTKVEIK* (SEQ ID NO: 962)
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYL
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFT
TFGQGTKVEIK* (SEQ ID NO: 963)
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYL
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFT
TFGQGTKVEIK* (SEQ ID NO: 964)
WVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HWV
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATY
TFGQGTKVEIK* (SEQ ID NO: 965)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GGSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HW
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFAT
TFGQGTKVEIK* (SEQ ID NO: 966)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GGSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HW
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFAT
TFGQGTKVEIK* (SEQ ID NO: 967)
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
GGSGGGSGGGSGGGSGSTGEVQLVESGGGLVQPGGSLRLSCAASGF HW
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFAT
TFGQGTKVEIK* (SEQ ID NO: 968)
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAW
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKD
HWVRQAPGKGLEWVA
YADSVK
FDYWGQGTLVTVSSA
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAW
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKD
HWVRQAPGKGLEWVA
YADSVK
DYWGQGTLVTVSSA
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAW
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKD
HWVRQAPGKGLEWVA
YADSVK
DYWGQGTLVTVSSA
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAW
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKD
HWVRQAPGKGLEWVA
YADSVK
DYWGQGTLVTVSSA
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVT
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKG
GGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
AVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEY
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAY
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVT
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKG
GGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTA
DYWGQGTLVTVSSASTKGPSVFPLAPSS
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVT
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKG
GGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNT
DYWGQGTLVTVSSASTKGPSVFPLAPS
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVT
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKG
GGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNT
DYWGQGTLVTVSSASTKGPSVFPLAPS
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPP
HWVRQAPGKGLEWVA
Y
DYWGQGTL
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPP
HWVRQAPGKGLEWVA
Y
DYWGQGTL
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPP
HWVRQAPGKGLEWVA
Y
DYWGQGTL
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPP
HWVRQAPGKGLEWVA
Y
DYWGQGTL
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGT
VAWYQQKPGKAPKL
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTS
DYWGQGTLVTVSSASTKGPSVFPL
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGT
VAWYQQKPGKAPKL
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTS
DYWGQGTLVTVSSASTKGPSVFPL
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGT
VAWYQQKPGKAPKL
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTS
DYWGQGTLVTVSSASTKGPSVFPL
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGT
VAWYQQKPGKAPKL
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKV
HWVRQAPGKGLEWVA
YADSVKGRFTISADTS
DYWGQGTLVTVSSASTKGPSVFPL
VAWYQQKPGK
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGT
VAWYQQKPGKAPKLLIY
SGV
TFGQGTKVEIKRTVAAPSVFIF
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFAT
FGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFL
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVT
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKGGGGSGGGGSEPKTSDKTHTCPPCPAPELLGGPSVFLFP
HWVRQAPGKGLEWVA
YYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTV
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFAT
FGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFL
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVT
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKGGGGSGGGGSEPKTSDKTHTCPPCPAPELLGGPSVFLFP
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTV
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFAT
TFGQGTKVEIKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFL
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVT
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKGGGGSGGGGSEPKTSDKTHTCPPCPAPELLGGPSVFLFP
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYYDMDYWGQGTLVTV
VAWYQQKPGK
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGT
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKLEDKTHTKVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPK
HWVRQAPGKGLEWVA
YADSV
DYWGQGTLVT
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKLEDKTHTKVEPKSSDKTHNCPPCPAPELLGGPSVFLFPPK
HWVRQAPGKGLEWVA
YADSV
DYWGQG
VAWYQQKPGKAPK
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKV
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKLEDKTHTKVEPKTSDKTHTCPPCPAPELLGGPSVFLFPPK
HWVRQAPGKGLEWVA
YADS
MDYWGQGTLV
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYY
TFGQGTKVEIKLEDKTHTKVEPKSSDKTHNCPPCPAPELLGGPSVFLFPP
HWVRQAPGKGLEWVA
YADSV
DYWGQG
VAWYQQKPGKAPK
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
FTFGQGTKV
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYY
TFGQGTKVEIKLEDKTHTKVEPKTSDKTHTCPPCPAPELLGGPSVFLFPP
HWVRQAPGKGLEWVA
YADS
DYWGQGTLV
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKLEDKTHTKVEPKSSDKTHNCPPCPAPELLGGPSVFLFPPK
VAWYQQKPGKAPKLLIY
GVPSRFSGSR
TFGQGTKVEIKGTTAASGSSGGSSSGAE
HWVRQAPGKGLEWVA
DYWGQ
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKLEDKTHTKVEPKTSDKTHTCPPCPAPELLGGPSVFLFPPK
VAWYQQKPGKAPKLLIY
GVPSRFSGS
TFGQGTKVEIKGTTAASGSSGGSSSGA
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARAHYFPWAGAMDYWG
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKLEDKTHTKVEPKSSDKTHNCPPCPAPELLGGPSVFLFPPK
VAWYQQKPGKAPKLLIY
GVPSRFSGSR
TFGQGTKVEIKGTTAASGSSGGSSSGAEV
HWVRQAPGKGLEWVA
Y
DY
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKLEDKTHTKVEPKTSDKTHTCPPCPAPELLGGPSVFLFPPK
VAWYQQKPGKAPKLLIY
GVPSRFSGS
TFGQGTKVEIKGTTAASGSSGGSSSGAEV
HWVRQAPGKGLEWVA
Y
DY
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKLEDKTHTKVEPKSSDKTHNCPPCPAPELLGGPSVFLFPPK
VAWYQQKPGKAPKLLIY
GVPSRFSGSR
TFGQGTKVEIKGTTAASGSSGGSSSGAEV
HWVRQAPGKGLEWVASIYSSYGYTSY
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKLEDKTHTKVEPKTSDKTHTCPPCPAPELLGGPSVFLFPPK
VAWYQQKPGKAPKLLIY
GVPSRFSGS
TFGQGTKVEIKGTTAASGSSGGSSSGAEV
HWVRQAPGKGLEWVA
Y
DY
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKLEDKTHTKVEPKSSDKTHNCPPCPAPELLGGPSVFLFPPK
VAWYQQKPGKAPKLLIY
GVPSRFSGSR
TFGQGTKVEIKGTTAASGSSGGSSSGAEV
HWVRQAPGKGLEWVASIYSSYGYTSY
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKLEDKTHTKVEPKTSDKTHTCPPCPAPELLGGPSVFLFPPK
VAWYQQKPGKAPKLLIY
GVPSRFSGS
TFGQGTKVEIKGTTAASGSSGGSSSGA
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARAHYFPWAGAMDYWG
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTIT
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPE
TFGQGTKVEIKLEDKTHTKVEPKSSDKTHNCPPCPAPELLGG
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQ
VA
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK (SEQ ID NO: 1002)
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYY
TFGQGTKVEIKLEDKTHTKVEPKTSDKTHTCPPCPAPELLGGPSVFLFPP
HWVRQAPGKGLEWVA
AD
DYWGQGTLVT
VAWYQQK
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFG
VAWYQQKP
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQ
H
YADSVKGRFTISADTSKNTAYLQMNSLRAEDT
DYWGQGTLVTVSSLEDKTHTKVEPKSSDKTHNCPPCPAPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VA
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK (SEQ ID NO: 1004)
VAWYQQKP
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGO
H
YADSVKGRFTISADTSKNTAYLQMNSLRAEDT
DYWGQGTLVTVSSLEDKTHTKVEPKTSDKTHTCPPCPAPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
VAWYQQKP
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQ
H
YADSVKGRFTISADTSKNTAYLQMNSLRAEDT
DYWGQGTLVTVSSLEDKTHTKVEPKSSDKTHNCPPCPAPE
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGTTAAS
HWVRQAPGKGLEW
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSS (SEQ ID NO: 1006)
VAWYQQKP
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQ
H
YADSVKGRFTISADTSKNTAYLQMNSLRAEDT
DYWGQGTLVTVSSLEDKTHTKVEPKTSDKTHTCPPCPAPE
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIKGTT
HWVRQAPGKG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSS (SEQ ID NO: 1007)
VAWYQQKPGK
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGT
HWVRQAPG
ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMN
DYWGQGTLVTVSSGGGGSDIQMTQSP
VAWYQQKPGKAPKLLIY
GVPSRFSGSRS
TFGQGTKVEIK (SEQ ID NO: 1009)
HWVRQAPG
ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMN
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSA
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDF
TFGQGTKVEIK (SEQ ID NO: 1010)
HWVRQAPG
ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMN
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSA
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDF
FTFGQGTKVEIK (SEQ ID NO: 1011)
HWVRQAPG
ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYY
TFGQGTKVEIKGTTAASGSSGGSSSGAEVQLVESGGGLVQPGGSLRLS
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTA
DYWGQGTLVTVSS (SEQ ID NO:
HWVRQAPG
ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYY
TFGQGTKVEIKGTTAASGSSGGSSSGAEVQLVESGGGLVQPGGSLRLSC
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAY
DYWGQGTLVTVSS (SEQ ID NO:
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTIT
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPE
TFGQGTKVEIKLEDKTHTKVEPKSSDKTHNCPPCPAPELLGGPS
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQG
HWVRQAP
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYY
TFGQGTKVEIKLEDKTHTKVEPKTSDKTHTCPPCPAPELLGGPSVFLFPP
HWVRQAPGKGLEWVA
YAD
DYWGQGTLVT
HWVRQAPG
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
HWV
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITC
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPED
TFGQGTKVEIK (SEQ ID NO: 1016)
VAWYQQKPGK
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGT
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
T
HWVRQA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
TFGQGTKVEIKDKTHTKVEPKTSDKTHTCPPCPGQPREPQVYTLPPSREE
HWVRQAPGKGLEWVA
YA
DYWGQGTL
VAWYQQKPGKAPKLLI
TFGQGTKVEIK
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
VAWYQQ
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TF
HWVRQAPGKGLEWVA
YADSVKGRFTISADT
DYWGQGTLVTVSSGGG
VAWYQQKPGKAPKLLIY
TFGQGTKVEIK (SEQ ID
indicates data missing or illegible when filed
indicates data missing or illegible when filed
HW
YADSVKGRFTISADTSKNTAYLQMNSLRA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDR
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFT
TFGQGTKVEIKLEDKTHTKVEPKTSDKT
HWVRQAPGKGLEWVA
YADSVKGRFTISAD
DYWGQGTLVTVSSGG
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEI
HW
ADSVKGRFTISADTSKNTAYLQMNSLRA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDR
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFT
TFGQGTKVEIKLEDKTHTKVEPKSSDKT
HWVRQAPGKGLEWVA
YADSVKGRFTISAD
DYWGQGTLVT
VAWYQQKPGKAPK
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQ
HW
YADSVKGRFTISADTSKNTAYLQMNSLRAE
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTI
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTIS
TFGQGTKVEIKLEDKTHTKVEPKTSDKTHTCPP
HWVRQAPGKGLEWVA
YADSVKGRFTISADTS
DYWGQGTLVTVSSGGGG
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEIK
HW
YADSVKGRFTISADTSKNTAYLQMNSLRAE
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTI
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTIS
TFGQGTKVEIKLEDKTHTKVEPKSSDKTHNCPP
HWVRQAPGKGLEWVA
YADSVKGRFTISADTSKN
DYWGQGTLVTVSSGG
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTKVEI
HW
YADSVKGRFTISADTSKNTAYLQMNSLRA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRV
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLT
HWVRQAPGKGLEWVA
YADSVKGRFTIS
DYWGQGTLVTVSS
VAWYQQKPGKAPKLLI
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTK
HW
YADSVKGRFTISADTSKNTAYLQMNSLRA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRV
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTDFTLT
TFGQGTKVEIKLEDKTHTKVEPKSSDKTH
HWVRQAPGKGLEWVA
YADSVKGRFTISADT
DYWGQGTLVTVSS
VAWYQQKPGKAPKLLI
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGT
HW
YADSVKGRFTISADTSKNTAYLQMNSLRA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASV
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGT
TFGQGTKVEIKLEDKTHTKVEPKTSDK
YADSVKGRFTIS
DYWGQGTLVTVSS
VAWYQQKPGKAPKLLI
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTK
HW
YADSVKGRFTISADTSKNTAYLQMNSLRA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASV
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGT
TFGQGTKVEIKLEDKTHTKVEPKSSDKT
HWVRQAPGKGLEWVA
YADSVKGRFTISA
DYWGQGTLVT
VAWYQQKPGKAPK
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQ
HW
YADSVKGRFTISADTSKNTAYLQMNSLRA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVG
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTD
TFGQGTKVEIKLEDKTHTKVEPKSSDK
HWVRQAPGKGLEWVA
YADSVKGRFTISA
DYWGQGTLVT
VAWYQQKPGKAPK
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQ
YADSVKGRFTISADTSKNTAYLQMNSLRA
DYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVG
VAWYQQKPGKAPKLLIY
GVPSRFSGSRSGTD
TFGQGTKVEIKLEDKTHTKVEPKTSDK
HWVRQAPGKGLEWVA
YADSVKGRFTIS
DYWGQGTLVTVSS
VAWYQQKPGKAPKLLI
GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
TFGQGTK
AATAG (SEQ ID NO: 1030)
ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGC
AACTGCAACTGGAGTACATAGCGAAGTTCAGCTCG
AATAG (SEQ ID NO: 1031)
ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGC
AACTGCAACTGGAGTACATAGCGAAGTTCAGCTCG
TAATAG (SEQ ID NO: 1032)
ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGC
AACTGCAACTGGAGTACATAGCGAAGTTCAGCTCG
TAATAG (SEQ ID NO: 1033)
ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGC
AACTGCAACTGGAGTACATAGCGAAGTGCAGTTGG
ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGC
AACTGCAACTGGAGTACATAGCGAAGTGCAGTTGG
ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGC
AACTGCAACTGGAGTACATAGCGAAGTGCAGTTGG
ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGC
AACTGCAACTGGAGTACATAGCGAAGTGCAGTTGG
TAATAG (SEQ ID NO: 1048)
ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGC
AACTGCAACTGGAGTACATAGCGAGGTGCAGCTGG
TCGAGTCCGGTGGTGGCCTAGTACAACCCGGCGGC
TCTCTCCGGCTGTCCTGCGCTGCCTCCGGATTTGA
CTTCTCCTCAAGTTCCATTCACTGGGTCAGGCAGG
CTCCTGGCAAAGGACTGGAATGGGTTGCCAGTATC
TCCTCCTCTTACGGCTACACCTACTACGCGGACTC
AGTTAAGGGGAGATTCACCATCTCCGCGGATACCA
GCAAGAATACTGCTTACCTGCAAATGAATAGTTTG
CGGGCCGAGGATACTGCTGTGTATTATTGCGCCCG
GAGTTGGGCTATGGACTACTGGGGCCAGGGGACTC
TCGTGACCGTGTCTTCCGGCGGCGGTGGATCTGAC
ATCCAGATGACACAGTCTCCAAGCTCATTGTCTGC
CTCTGTTGGAGACCGAGTGACAATCACATGCCGGG
CCAGCCAGTCTGTGTCTTCTGCCGTGGCTTGGTAC
CAACAAAAACCTGGCAAGGCTCCCAAATTGCTCAT
CTATTCCGCATCCGACTTATACTCTGGCGTCCCTT
CTCGCTTCTCTGGGAGCAGATCCGGAACCGATTTT
ACATTGACCATCTCTAGTCTGCAACCTGAGGACTT
CGCCACCTACTATTGCCAACAATATGCCGGCGCTG
GACTCATTACTTTTGGACAAGGTACCAAAGTGGAG
ATCAAGGGTGGCGGTGGAAGTGGTGGAGGAGGTAG
TGAGCCCAAATCTTCCGACAAAACTCACACATGCC
CACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCG
TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACAC
CCTCATGATCTCCCGGACCCCTGAGGTCACATGCG
TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC
AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA
TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACA
ACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA
GTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTGCACCCTGCCCCCATCCCG
GGAGGAGATGACCAAGAACCAGGTCAGCCTGAGCT
GCGCCGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGAC
AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG
CTCCGTGATGCATGAGGCTCTGCACAACCACTACA
CGCAGAAGAGCCTCTCCCTGTCTCCGGGCAAAGGC
GGAGGAAGCGGCGGAGGCAGCGGAGGCGGTAGCGG
CGGCGGATCAGGAAGTACAGGGGAGGTACAGCTGG
TGGAGAGCGGAGGGGGGCTAGTGCAACCTGGGGGC
AGTCTGCGACTGTCATGTGCTGCAAGCGGGTTTAC
CCTGTCTTCATATAGCATGCACTGGGTCCGACAAG
CTCCCGGCAAGGGCTTGGAATGGGTCGCATACATT
TCAAGTTACGACTCAATCACTGACTATGCCGATTC
CGTGAAGGGCCGGTTCACCATTTCCGCGGACACCT
CCAAAAACACAGCATATCTTCAAATGAACAGTCTA
AGGGCGGAAGATACCGCTGTTTACTATTGTGCACG
ACCCGCGGTTGGTCATATGGCTTTTGACTACTGGG
GCCAGGGCACCCTAGTGACAGTCTCTTCCGCTTCC
ACTAAGGGGCCCTCTGTCTTTCCTCTGGCACCATC
CTCCAAATCAACGTCAGGTGGCACAGCCGCCCTCG
GCTGTCTGGTTAAGGACTACTTTCCCGAACCCGTT
ACCGTTTCTTGGAACTCTGGCGCCCTCACATCCGG
AGTCCACACATTTCCTGCTGTCCTACAATCTTCTG
GACTGTACTCCTTGAGCTCCGTAGTTACGGTGCCC
TCATCTAGCCTGGGTACTCAGACTTACATTTGTAA
CGTGAACCACAAGCCCAGCAACACAAAAGTGGACA
AGAAGGTTGAGCCCAA
GTCCTGTGACAAAACCCACACATAATAG
GAGGTGCAGCTGGTCGAGTCCGGTGGTGGCCTAGT
ACAACCCGGCGGCTCTCTCCGGCTGTCCTGCGCTG
CCTCCGGATTTGACTTCTCCTCAAGTTCCATTCAC
TGGGTCAGGCAGGCTCCTGGCAAAGGACTGGAATG
GGTTGCCAGTATCTCCTCCTCTTACGGCTACACCT
ACTACGCGGACTCAGTTAAGGGGAGATTCACCATC
TCCGCGGATACCAGCAAGAATACTGCTTACCTGCA
AATGAATAGTTTGCGGGCCGAGGATACTGCTGTGT
ATTATTGCGCCCGGAGTTGGGCTATGGACTACTGG
GGCCAGGGGACTCTCGTGACCGTGTCTTCCGGCGG
CGGTGGATCTGACATCCAGATGACACAGTCTCCAA
GCTCATTGTCTGCCTCTGTTGGAGACCGAGTGACA
ATCACATGCCGGGCCAGCCAGTCTGTGTCTTCTGC
CGTGGCTTGGTACCAACAAAAACCTGGCAAGGCTC
CCAAATTGCTCATCTATTCCGCATCCGACTTATAC
TCTGGCGTCCCTTCTCGCTTCTCTGGGAGCAGATC
CGGAACCGATTTTACATTGACCATCTCTAGTCTGC
AACCTGAGGACTTCGCCACCTACTATTGCCAACAA
TATGCCGGCGCTGGACTCATTACTTTTGGACAAGG
TACCAAAGTGGAGATCAAGGGTGGCGGTGGAAGTG
GTGGAGGAGGTAGTGAGCCCAAATCTTCCGACAAA
ACTCACACATGCCCACCGTGCCCAGCACCTGAAGC
CGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCCT
GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGA
AGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG
GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAG
CGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG
GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGC
CAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCC
TGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGAGCTGCGCCGTCAAAGGCTTCTATCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAA
GCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
CACAACCACTACACGCAGAAGAGCCTCTCCCTGTC
TCCGGGCAAAGGCGGAGGAAGCGGCGGAGGCAGCG
GAGGCGGTAGCGGCGGCGGATCAGGAAGTACAGGG
GAGGTACAGCTGGTGGAGAGCGGAGGGGGGCTAGT
GCAACCTGGGGGCAGTCTGCGACTGTCATGTGCTG
CAAGCGGGTTTACCCTGTCTTCATATAGCATGCAC
TGGGTCCGACAAGCTCCCGGCAAGGGCTTGGAATG
GGTCGCATACATTTCAAGTTACGACTCAATCACTG
ACTATGCCGATTCCGTGAAGGGCCGGTTCACCATT
TCCGCGGACACCTCCAAAAACACAGCATATCTTCA
AATGAACAGTCTAAGGGCGGAAGATACCGCTGTTT
ACTATTGTGCACGACCCGCGGTTGGTCATATGGCT
TTTGACTACTGGGGCCAGGGCACCCTAGTGACAGT
CTCTTCCGCTTCCACTAAGGGGCCCTCTGTCTTTC
CTCTGGCACCATCCTCCAAATCAACGTCAGGTGGC
ACAGCCGCCCTCGGCTGTCTGGTTAAGGACTACTT
TCCCGAACCCGTTACCGTTTCTTGGAACTCTGGCG
CCCTCACATCCGGAGTCCACACATTTCCTGCTGTC
CTACAATCTTCTGGACTGTACTCCTTGAGCTCCGT
AGTTACGGTGCCCTCATCTAGCCTGGGTACTCAGA
CTTACATTTGTAACGTGAACCACAAGCCCAGCAAC
ACAAAAGTGGACAAGAAGGTTGAGCCCAAGTCCTG
TGACAAAACCCACACATAATAG
ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGC
AACTGCAACTGGAGTACATAGCGAGGTCCAGCTCG
ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGC
AACTGCAACTGGAGTACATAGCGAGGTCCAGCTCG
ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGC
AACTGCAACTGGAGTACATAGCGAGGTCCAGCTCG
ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGC
AACTGCAACTGGAGTACATAGCGAGGTCCAGCTCG
ATGGAGACAGACACACTCCTGCTATGGGTACTGCT
GCTCTGGGTTCCAGGCTCCACCGGCGACATCCAGA
| Number | Date | Country | |
|---|---|---|---|
| 63127408 | Dec 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IB2021/061972 | Dec 2021 | US |
| Child | 17846846 | US |
