ANTI-PDGFR-BETA ANTIBODIES, THEIR USES AND PHARMACEUTICAL COMPOSITION

专利摘要:
anti-pdgfr-beta antibodies and their uses. the present invention relates to antibodies that bind to platelet-derived growth factor beta receptor (pdgfr-beta) and processes for using them. according to certain embodiments of the invention, antibodies are fully human antibodies that bind to human pdgfr-beta with high affinity. the antibodies of the invention are useful for the treatment of diseases and disorders associated with pdgfr-beta signaling and / or pdgfr-beta cell expression, such as eye diseases, fibrotic diseases, vascular diseases and cancer.
公开号:BR112015016312B1
申请号:R112015016312-2
申请日:2014-01-07
公开日:2020-12-15
发明作者:Stanley J. Wiegand；Ivan B. Lobov
申请人:Regeneron Pharmaceuticals, Inc.；
IPC主号:

专利说明:

Field of the Invention
[001] The present invention relates to antibodies, and their antigen binding fragments, which are specific to human PDGFR-beta, and processes for their use. Background
[002] Platelet-derived growth factors (PDGFs) are potent mitogens that exist as five different dimeric configurations composed of four different isoform subunits: A, B, C and D. The five dimeric forms of PDGFs are AA, BB, AB , CC and DD, which are formed by disulfide bonding of the corresponding individual PDGF monomers. PDGF ligands exert their biological effects through their interactions with PDGF receptors (PDGFRs). PDGFRs are single-pass, transmembrane tyrosine kinase receptors, composed of heterodimeric or homodimeric associations of an alpha (α) receptor chain (PDGFR-alpha) and / or a beta (β) receptor chain (PDGFR-beta). Thus, active PDGFRs can consist of pairs of αα, ββ or αβ receptor chains. PDGFRs share a common domain structure, including five extracellular immunoglobulin loops, a transmembrane domain, and an intracellular tyrosine kinase (TK) cleavage domain. The interaction between dimeric PDGF ligands and PDGFRs leads to receptor chain dimerization, receptor auto phosphorylation and intracellular signal transduction. It has been demonstrated in vitro that ββ receptors are activated by PDGF-BB and - DD, while αβ receptors are activated by PDGF-BB, -CC, -DD and - AB, and αα receptors are activated by PDGF-AA, -BB, - CC and -AB (see Andrae et al. (2008) Genes Dev 22 (10); 1276-1312).
[003] PDGF signaling has been implicated in several human diseases including diseases associated with pathological neovascularization, vascular and fibrotic diseases, tumor growth and eye diseases. Likewise, PDGF signaling inhibitors have been suggested for use in a variety of therapeutic settings. For example, PDGFR-beta inhibitors have been proposed for use in the treatment of various diseases and disorders (Andrae et al. (2008) Genes Dev 22 (10): 1276-1312). PDGFR-beta inhibitors include non-specific small molecule tyrosine kinase inhibitors such as imatinib mesylate, sunitinib malate and CP-673451, as well as anti-PDGFR-beta antibodies (see, for example, US Patent Nos. 7,060,271; 5 882 644; 7 740 850; and US patent application No. 2011/0177074). Anti-ligand aptamers (eg, anti-PDGF-B) have also been proposed for therapeutic applications. Nevertheless, there is a need in the art for new, highly specific and potent PDGF signaling inhibitors. Summary of the Invention
[004] The present invention provides antibodies that bind to human platelet-derived growth factor beta receptor ("PDGFR-beta"). The antibodies of the invention are useful, inter alia, for inhibiting PDGFR-beta-mediated signaling and for treating diseases and disorders caused by or related to PDGFR-beta activity and / or signaling. The antibodies of the invention are also useful for inducing cell death in cells that express high levels of PDGFR-beta on their surfaces.
[005] The antibodies of the invention may be of full length (for example, an IgG1 or IgG4 antibody) or may comprise only an antigen binding portion (for example, a Fab, F (ab ') 2 or scFv fragment), and can be modified to affect functionality, for example, to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164: 19251933).
[006] The present invention provides antibodies, or their antigen binding fragments comprising a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, and 322, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
[007] The present invention also provides an antibody or antigen binding fragment of an antibody comprising a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58 , 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, and 330, or a substantially similar sequence thereof, at least 90% 95%, at least 98% or at least 99% sequence identity.
[008] The present invention also provides an antibody or its antigen binding fragment comprising a pair of HCVR and LCVR (HCVR / LCVR) sequences selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42 , 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242 / 250, 258/266, 274/282, 290/298, 306/314, and 322/330.
[009] The present invention also provides an antibody or antigen binding fragment of an antibody comprising a heavy chain CDR3 domain (HCDR3) having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56 , 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, and 328, or a substantially similar sequence thereof, at least 90% 95%, at least 98% or at least 99% sequence identity; and a light chain CDR3 domain (LCDR3) having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, and 336, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity,
[0010] In certain embodiments, the antibody or antigen-binding portion of an antibody comprises a pair of HCDR3 / LCDR3 amino acid sequences selected from the group consisting of SEQ ID NO: 8/16, 24/32, 40/48, 56/64, 72/80, 88/96, 104/112, 120/128, 136/144, 152/160, 168/176, 184/192, 200/208, 216/224, 232/240, 248 / 256, 264/272, 280/288, 296/304, 312/320, and 328/336.
[0011] The present invention also provides an antibody or fragment thereof further comprising a heavy chain CDR1 domain (HCDR1) having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, and 324, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2 domain (HCDR2) having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214 , 230, 246, 262, 278, 294, 310, and 326, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a light chain CDR1 domain (LCDR1) having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220 , 236, 252, 268, 284, 300, 316, and 332, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a light chain CDR2 domain (LCDR2) having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, and 334, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
[0012] Certain exemplary non-limiting antibodies and antigen binding fragments of the invention comprise HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, having the amino acid sequences selected from the group consisting of: SEQ ID NOs: 4-6 -8-12-14-16 (for example, H1M3299N); 20-22-24-2830-32 (for example, H1M3305N); 36-38-40-44-46-48 (for example, H1M3310N); 52-54-56-60-62-64 (for example, H1M3361N); 68-70-7276-78-80 (for example, H2M3363N); 84-86-88-92-94-96 (for example, H2M3365N); 100-102-104-108-110-112 (e.g., H2M3368N); 116-118-120-124-126-128 (e.g., H2M3373N); 132-134-136140-142-144 (e.g. H2M3374N); 148-150-152-156-158-160 (e.g., H4H3094P); 164-166-168-172-174-176 (e.g., H4H3095S); 180-182-184-188-190-192 (for example, H4H3096S); 196-198-200-204-206-208 (for example, H4H3097S); 212-214-216220-222-224 (for example, H4H3098S); 228-230-232-236-238-240 (for example, H4H3099S); 244-246-248-252-254-256 (e.g., H4H3102S); 260-262-264-268-270-272 (for example, H4H3103S); 276-278-280-284-286-288 (for example, H4H3104S); 292-294-296300-302-304 (for example, H4H3105S); 308-310-312-316-318-320 (e.g., H4H3106S); and 324-326-328-332-334-336 (for example, H4H3107S).
[0013] In a related embodiment, the invention includes an antibody or antigen binding fragment of an antibody that specifically binds PDGFR-beta, where the antibody or fragment comprises the heavy and light chain CDR domains contained in variable region sequences of heavy and light chain (HCVR / LCVR) selected from the group consisting of SEQ ID NO2 / 10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130 / 138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298, 306/314, and 322/330 . Processes and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used for identification of CDRs within the specified HCVR and / or LCVR amino acid sequences shown here. Exemplary conventions that can be used to identify the limits of CDRs include, for example, the Kabat definition, the Chothia definition and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86: 9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.
[0014] In another aspect, the invention provides nucleic acid molecules encoding PDGFR-beta antibodies or their antigen binding fragments. Recombinant expression vectors carrying the nucleic acids of the invention, and host cells into which such vectors have been introduced, are also encompassed by the invention, as are antibody production processes by culturing host cells under conditions allowing production of antibodies, and recovery of antibodies produced.
[0015] In one embodiment, the invention provides an antibody or fragment thereof comprising an HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, or 321, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least minus 99% homology.
[0016] The present invention also provides an antibody or fragment thereof comprising an LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, and 329, or a substantially identical sequence having at least 90%, at least 95%, at least 98% or at least 99% homology.
[0017] The present invention also provides an antibody or antigen binding fragment of an antibody comprising an HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 7, 23, 39, 55,71, 87, 103, 119, 135, 151, 167, 183, 199, 215, 231, 247, 263, 279, 295, 311, and 327, or a substantially identical sequence having at least 90%, at least 95%, at least 98 %, or at least 99% homology; and an LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207, 223, 239, 255 , 271, 287, 303, 319, and 335, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology.
[0018] The present invention also provides an antibody or fragment thereof which further comprises an HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307, and 323, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least less 99% homology; an HCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, 21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, 309, and 325, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology; an LCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 2219, 235, 251, 267, 283, 299, 315, and 331, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology; and an LCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237, 253 , 269, 285, 301, 317, and 333, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology.
[0019] According to certain modalities, the antibody or its fragment comprises the heavy and light chain CDR sequences encoded by the nucleic acid sequences of SEQ ID NOs: 1 and 9 (for example, H1M3299N), 17 and 25 (for example H1M3305N), 33 and 41 (for example, H1M3310N), 49 and 57 (for example, H1M3361N), 65 and 73 (for example, H2M3363N), 81 and 89 (for example, H2M3365N), 97 and 105 (for example, H2M3368N), 113 and 121 (for example, H2M3373N), 129 and 137 (for example, H2M3374N); 145 and 153 (for example, H4H3094P), 161 and 169 (for example, H4H3095S), 177 and 185 (for example, H4H3096S), 193 and 201 (for example, H4H3097S), 209 and 217 (for example, H4H3098S), 225 and 233 (for example, H4H3099S), 241 and 249 (for example, H4H3102S), 257 and 265 (for example, H4H3103S), 273 and 281 (for example, H4H3104S), 289 and 297 (for example, H4H3105S), 305 and 313 (for example, H4H3106S), or 321 and 329 (for example, H4H3107S).
[0020] The present invention includes anti-PDGFR-beta antibodies having a modified glycosylation pattern. In some applications, modification to remove unwanted glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody-dependent cell cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277: 26733). In other applications, modification of galactosylation can be done in order to modify complement-dependent cytotoxicity (CDC).
[0021] In another aspect, the invention provides a pharmaceutical composition comprising a recombinant human antibody or a fragment thereof that specifically binds PDGFR-beta and a pharmaceutically acceptable carrier. In a related aspect, the invention features a composition that is a combination of an anti-PDGFR-beta antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-PDGFR-beta antibody. Exemplary agents that can be advantageously combined with an anti-PDGFR-beta antibody include, without limitation, other agents that inhibit PDGFR-beta activity (including other antibodies or their antigen binding fragments, peptide inhibitors, small molecule antagonists, etc. .) and / or agents that do not directly bind PDGFR-beta but nevertheless interfere with, block or attenuate signaling mediated by PDGFR-beta. Additional combination therapies and co-formulations involving the anti-PDGFR-beta antibodies of the present invention are shown elsewhere herein.
[0022] In yet another aspect, the invention provides therapeutic processes for inhibiting PDGFR-beta activity using an anti-PDGFR-beta antibody or antigen binding portion of an antibody of the invention, where the therapeutic processes comprise administration of a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen binding fragment of an antibody of the invention. The treated disorder is any disease or condition that is improved, improved, inhibited or prevented by removing, inhibiting or reducing PDGFR-beta activity or signaling. Anti-PDGFR-beta antibodies or antibody fragments of the invention may function to block the interaction between PDGFR-beta and a PDGFR-beta binding partner (for example, a PDGFR ligand), or otherwise inhibit signaling activity of PDGFR-beta.
[0023] The present invention also includes the use of an anti-PDGFR-beta antibody or antigen binding portion of an antibody of the invention in the manufacture of a medicament for the treatment of a disease or disorder related to or caused by PDGFR activity -beta on a patient.
[0024] Other modalities will become apparent from a review of the following detailed description. Brief Description of the Figures
[0025] Figure 1 is a histogram showing the results of a PDGFR ligand blocking assay where PDGFR-beta was captured on a biosensor surface and PDGF ligand (BB, DD or AB) was applied to the surface following treatment with several anti-PDGFR-beta antibodies of the invention or control antibody. The results are shown as Rus.
[0026] Figure 2 is a matrix showing the results of an antibody cross-competition assay where a first anti-PDGFR-beta antibody (mAb # 1) was applied to a sensor tip coated with PDGFR-beta, followed by treatment with a second anti-PDGFR-beta antibody (mAb # 2). Binding responses (numerical values - 0.01 to 0.36) for each antibody combination tested are shown. Light gray boxes with black front represent a call response for self-competition. Antibodies competing in both directions, regardless of the order of antigen binding, are highlighted in black boxes with a white front. No competition, suggesting different connecting regions, is represented as white boxes with a black front. Detailed Description
[0027] Before the present invention is described, it is to be understood that this invention is not limited to the particular processes and experimental conditions described, insofar as such processes and conditions may vary. It is also to be understood that the terminology used here is only for the purpose of describing particular modalities, and is not intended to be limiting, since the scope of the present invention will only be limited by the stated claims.
[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those versed in the technique to which this invention belongs. As used herein, the term "fence", when used in reference to a particular recited numerical value, means that the numerical value can vary from the recited value by no more than 1%. For example, as used herein, the expression “about 100” includes 99 and 101 and all values in between (for example, 99.1, 99.2, 99.3, 99.4, etc.).
[0029] Although any processes and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred processes and materials are now described. Definitions
[0030] The terms "beta-platelet-derived growth factor receptor", "PDGFRβ", "PDGFR-beta", "PDGFRb" and the like, used herein, refer to the human PDGFR-beta protein having the amino acid sequence SEQ ID NO: 341 (see also UniProtaccession No. P09619). All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being of a non-human species (for example, “PDGFR-beta of mouse ”,“ monkey PDGFR-beta ”, etc.).
[0031] As used herein, "an antibody that binds PDGFR-beta" or an "anti-PDGFR-beta antibody" includes antibodies, and their antigen binding fragments, that bind a soluble fragment of a PDGFR-beta protein (for example example, all or a portion of the extracellular domain of PDGFR-beta) and / or PDGFR-beta expressed on cell surface. The term "PDGFR-beta expressed on cell surface" means a PDGFR-beta protein or a portion thereof that is expressed on the surface of a cell in vitro or in vivo, so that at least a portion of the PDGFR-beta protein ( for example, amino acids 33 to 532 of SEQ ID NO: 341) are exposed to the extracellular side of the cell membrane and are accessible to an antigen-binding portion of an antibody. "PDGFR-beta expressed on cell surface" includes PDGFR-beta molecules in the context of ββ receptor homodimers as well as PDGFR-beta molecules in the context of αβ heterodimers. Soluble PDGFR-beta molecules include, for example, monomeric and dimeric PDGFR-beta constructs as described herein in Example 3 (for example, “PDGFRb.mmh”, SEQ ID NO: 337 [monomeric], “PDGFRb.mFc”, SEQ ID NO: 338 [dimeric] and "PDGFRb.hFc", SEQ ID NO: 339 [dimeric], or constructions substantially similar thereto.
[0032] The term "antibody", as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (for example, PDGFR -beta). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, as well as their multimers (for example, IgM). Each heavy chain comprises a heavy chain variable region (hereinafter abbreviated HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (here abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region comprises a domain (CL1). The VH and VL regions can still be subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called structure 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. In different embodiments of the invention, the FRs of the anti-PDGFR-beta antibody (or its antigen binding portion) can be identical to human germ line sequences, or can be modified naturally or artificially. A consensus amino acid sequence can be defined based on a side-by-side analysis of two or more CDRs.
[0033] The term "antibody", as used herein, also includes antigen binding fragments of entire antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically, synthetically or genetically engineered polypeptide that is specifically bound to an antigen to form a complex. Antigen binding fragments of an antibody can be derived, for example, from whole antibody molecules using any appropriate standard techniques involving manipulation and expression of DNA encoding variable and optionally constant antibody domains. Such DNA is known and / or is readily available from, for example, commercial sources, DNA libraries (including, for example, phage - antibody libraries), or can be synthesized. DNA can be sequenced and chemically manipulated or through the use of molecular biology techniques, for example, to arrange one or more variable and / or constant domains in an appropriate configuration, or to introduce codons, create cysteine residues, modify , add or delete amino acids, etc.
[0034] Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) F (ab ') 2 fragments; (iii) fragments Fd; (v) single-stranded Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (for example, an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a restricted FR3-CDR3-FR4 peptide . Other engineered molecules, such as domain specific antibodies, single domain antibodies, suppressed domain antibodies, chimeric antibodies, antibodies grafted with CDR, diabodies, tribodies, tetribodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.). ), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also covered by the term "antigen binding fragment" as used herein.
[0035] An antigen binding fragment of an antibody will typically comprise at least one variable domain. The variable domain can be of any size or composition of amino acids and will generally comprise at least one CDR that is adjacent to or in structure with one or more structure sequences. In antigen binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be located relative to each other in any appropriate arrangement. For example, the variable region can be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen binding fragment of an antibody can contain a monomeric VH or VL domain.
[0036] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Exemplary, non-limiting configurations of variable and constant domains that can be found within an antigen binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL.
[0037] In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be linked either directly to each other or can be linked by a ligating or articulation region, total or partial. An articulation region can consist of at least 2 (for example, 5, 10, 15, 20, 40, 60 or more) amino acids that result in flexible or semi-flexible link between variable and / or adjacent domains in a molecule of simple polypeptide. In addition, an antigen binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and / or with one or plus monomeric VH or VL domain (for example, via disulphide bond (s)).
[0038] As with whole antibody molecules, antigen-binding fragments can be monospecific or multispecific (for example, bispecific). A multispecific antigen binding fragment of an antibody will typically comprise at least two different variable domains, where each variable domain is capable of specific binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats shown here, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using available routine techniques.
[0039] The antibodies of the present invention can function through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of cells expressing antigen by an antibody of the invention in the presence of complement. “Antibody dependent cell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells that express Fc receptors (FcRs) (eg, natural killer cells (NK), neutrophils, and macrophages) recognize antibody bound to a target cell and therefore leads to lysis of the target cell. CDC and ADCC can be measured using assays that are well known and available in the art. (See, for example, U.S. Patent 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95: 652656). The antibody constant region is important in an antibody's ability to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody can be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity.
[0040] In certain embodiments of the invention, the anti-PDGFR-beta antibodies of the invention are human antibodies. The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site specific mutagenesis in vitro or via somatic mutation in vivo), for example, in CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germ line of another mammalian species, such as a mouse, have been grafted onto human structure sequences.
[0041] The antibodies of the invention may, in some embodiments, be recombinant human antibodies. The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, raised or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected in a host cell (further described) antibodies isolated from a human combinatorial antibody library (further described below), antibodies isolated from an animal (for example, a mouse) that is transgenic to human immunoglobulin genes (see, for example, Taylor et al. (1992 ) Nucl. Acids Res. 20: 6287-6295) or antibodies prepared, expressed, raised or isolated by any other means that involve binding human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when a transgenic animal for human Ig sequences is used, in somatic mutagenesis in vivo) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from, and related to human germ line VH and VL sequences, cannot naturally exist within the human antibody germ line repertoire in vivo.
[0042] Human antibodies can exist in two forms that are associated with joint heterogeneity. In one form, an immunoglobulin molecule comprises a stable four-chain construction of approximately 150-160 kDa where the dimers are held together through an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via interchain disulfide bonds and a molecule of about 75-80 kDa is formed composed of covalently coupled light and heavy chains (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.
[0043] The frequency of appearance of the second form in several intact IgG isotypes is due to, but not limited to, structural differences associated with the antibody hinge region isotype. A simple amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30: 105) to levels typically seen using human IgG1 hinge. The present invention encompasses antibodies having one or more mutations in the joint, CH2 or CH3 region that may be desirable, for example, in production, to improve the yield of the desired form of antibody.
[0044] The antibodies of the invention can be isolated antibodies. An "isolated antibody", as used herein, means an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody" for the purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have undergone at least one purification or isolation step. According to certain embodiments, an isolated antibody can be substantially free of other cellular material and / or chemical compounds.
[0045] The present invention includes neutralization and / or blocking of anti-PDGFR-beta antibody. A "neutralizing" or "blocking" antibody, as used herein, is intended to refer to an antibody whose binding to PDGFR-beta: (i) interferes with the interaction between PDGFR-beta or a fragment of PDGFR-beta and a linker PDGF (for example, PDGF-BB, PDGF-CC, PDGF-DD, PDGF-AB, etc.); (ii) interferes with the formation of ββ and / or αβ receptor dimers; and / or (iii) results in inhibition of at least one biological function of PDGFR-beta. The inhibition caused by a blocking or neutralizing antibody to PDGFR-beta need not be complete as far as it is detectable using an appropriate assay. Exemplary assays for detecting PDGFR-beta inhibition are described herein in the Working Examples.
[0046] The anti-PDGFR-beta antibodies shown here may comprise one or more amino acid substitutions, insertions and / or deletions in the structure and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germ line sequences from which the antibodies were derived. Such mutations can be easily determined by comparing the amino acid sequences shown here to germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and their antigen-binding fragments, which are derived from any of the amino acid sequences shown here, where one or more amino acids within one or more structure and / or CDR regions are mutated to the corresponding residue (s) of the germ line sequence from which the antibody was derived, or for the corresponding residue (s) of another human germ line sequence, or for a conservative amino acid substitution of the corresponding germ line residue (s) (such sequence changes are collectively referred to here as “germ line mutations”). Those skilled in the art, starting from the heavy and light chain variable region sequences shown here, can easily produce numerous antibodies and antigen binding fragments that comprise one or more individual germ line mutations or combinations thereof. In certain embodiments, all CDR and / or structure residues within the VH and VL domains mutate back to the residues found in the original germ line sequence from which the antibody was derived. In other embodiments, only certain residues mutate back to the original germ line sequence, for example, only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the residues that mutated under CDR1, CDR2 or CDR3. In other embodiments, one or more of the structure residues and / or CDRs mutate to the corresponding residue (s) of a different germ line sequence (that is, a germ line sequence that is different from the germ line sequence from which the antibody was originally derived). In addition, the antibodies of the present invention can contain any combination of two or more germ line mutations within structure regions and / or CDRs, for example, where certain individual residues are mutated to the corresponding residue of a particular germ line sequence while certain other residues that differ from the original germ line sequence are maintained or mutated to the corresponding residue from a different germ line sequence. Once obtained, antibodies and antigen binding fragments that contain one or more germ line mutations can be easily tested for one or more desired properties such as improved binding specificity, increased binding affinity, improved or increased antagonistic biological properties. or agonists (as may be the case), reduced immunogenicity, etc. Antibodies and antigen binding fragments obtained in this generic manner are encompassed in the present invention.
The present invention also includes anti-PDGFR-beta antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences shown herein having one or more conservative substitutions. For example, the present invention includes anti-PDGFR-beta antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions with respect to any of the HCVR, LCVR, and / or CDR amino acid sequences shown herein.
[0048] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A simple antigen can have more than one epitope. Thus, different antibodies can bind to different areas on an antigen and can have different biological effects. Epitopes can also be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In some circumstances, an epitope may include saccharide moieties, phosphoryl groups, or sulfonyl groups on the antigen.
[0049] The term "substantial identity" or "substantially identical", when referring to a nucleic acid or its fragment, indicates that, when optimally aligned with appropriate insertions or deletions of nucleotides with another nucleic acid (or its complementary strand ), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by a well-known sequence, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain examples, encode a polypeptide having an identical or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.
[0050] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as through GAP or BESTFIT programs using default clearance weights, share at least 95% of sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, positions of residues that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (group R) with similar chemical properties (for example, charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from one another by conservative substitutions, the percentage of sequence identity or degree of similarity can be adjusted upward to correct the conservative nature of the substitution. Means for obtaining this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenyl alanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred groups of convergent amino acid substitution are: valine - leucine - isoleucine, phenyl alanine - tyrosine, lysine - arginine, alanine - valine, glutamate - aspartate, and asparagine - glutamine. Alternatively, a conservative substitution is any change having a positive value in the PAM250 probability-log matrix shown in Gonnet et al. (1992) Science 256: 1443-1445. A “moderately conservative” substitution is any change having a non-negative value in the probability matrix - log PAM250.
[0051] Sequence similarity to polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software adjusts similar sequences using similarity measures attributed to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit that can be used with default parameters for determining sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA using recommended or default parameters, a GCG Version 6.1 program. FASTA (for example, FASTA2 and FASTA3) provides alignments and sequence identity percentage of the regions of best overlap between the search and question sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-402. PH-dependent binding
[0052] The present invention includes anti-PDGFR-beta antibodies with pH dependent binding characteristics. For example, an anti-PDGFR-beta antibody of the present invention may exhibit reduced binding to PDGFR-beta at acidic pH as compared to neutral pH. Alternatively, the anti-PDGFR-beta antibody of the invention can exhibit improved binding to its antigen at acidic pH compared to neutral pH. The expression “acidic pH” includes pH values of less than about 6.2, for example, about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As used herein, the term "neutral pH" means a pH of about 7.0 to about 7.4. The term "neutral pH" includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.
[0053] In certain examples, "reduced binding to PDGFR-beta at acidic pH compared to neutral pH" is expressed in terms of a ratio of the KD value of the antibody binding to PDGFR-beta at acidic pH to the KD value of the antibody ligand PDGFR-beta at neutral pH (or vice versa). For example, an antibody or its antigen binding fragment can be seen to exhibit "reduced binding to PDGFR-beta at acidic pH compared to neutral pH" for purposes of the present invention if the antibody or its antigen binding fragment exhibits a reason Acid / neutral KD of about 3.0 or greater. In certain exemplary embodiments, the acidic / neutral KD ratio for an antibody or antigen binding fragment of the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 , 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0. 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or greater.
[0054] Antibodies with pH-dependent binding characteristics can be obtained, for example, by selecting a population of antibodies for reduced (or improved) binding to a particular antigen at acidic pH compared to neutral pH. In addition, modifications of the antigen binding domain at the amino acid level can yield antibodies with pH-dependent characteristics. For example, by replacing one or more amino acids in an antigen binding domain (for example, within a CDR) with a histidine residue, an antibody with reduced antigen binding at acidic pH relative to neutral pH can be obtained . Anti-PDGFR-beta antibodies comprising Fc variants
[0055] In accordance with certain embodiments of the present invention, anti-PDGFR-beta antibodies are provided comprising an Fc domain comprising one or more mutations that enhance or decrease antibody binding to the FcRn receptor, for example, at acidic pH as compared to pH neutral. For example, the present invention includes anti-PDGFR-beta antibodies comprising a mutation in the CH2 or CH3 region of the Fc domain, where the mutation (s) increases the affinity of the Fc domain for FcRn in an acidic environment (for example, in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations can result in an increase in serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, a change in position 250 (for example, E or Q); 250 and 428 (for example, L or F); 252 (for example, L / Y / F / W or T), 254 (for example, S or T), and 256 (for example, S / R / Q / E / D or T); or a change in position 428 and / or 433 (for example, H / L / R / S / P / Q or K) and / or 434 (for example, H / F or Y); or a change in position 250 and / or 428; or a change in position 307 and / or 308 (for example, 308F, V308F), and 434. In one embodiment, the change comprises a change of 428L (for example, M428L) and 434S (for example, N434S); a modification 428L, 259I (for example, V259I), and 308F (for example, V308F); a 433K modification (for example, H433K), and a 434 (for example, 434Y); a modification 252, 254, and 256 (for example, 252Y, 254T and 256E); a 250Q and 428L modification (for example, T250Q and M428L); and a modification 307 and / or 308 (e.g., 308F or 308P).
For example, the present invention includes anti-PDGFR-beta antibodies comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (for example, T250Q and M248L); 252Y, 254T and 256E (for example, M252Y, S254T and T256E); 428L and 434S (for example, M428L and N434S); and 433K and 434F (for example, H433K and N434F). All possible combinations of the previous Fc domain mutations, and other mutations within the antibody variable domains shown here, are contemplated within the scope of the present invention. Biological characteristics of antibodies
The present invention includes anti-PDGFR-beta antibodies and their antigen binding fragments that bind soluble monomeric or dimeric PDGFR-beta molecules with high affinity. For example, the present invention includes antibodies and antibody antigen binding fragments and bind monomeric PDGFR-beta (for example, at 25oC or 37oC) with a KD of less than about 30 nM as measured by surface plasmon resonance, for example example, using the assay format as defined herein in Example 3. In certain embodiments, the antibodies or antigen binding fragments of the present invention bind monomeric PDGFR-beta with a KD of less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, or less than about 1 nM, as measured by surface plasmon resonance, for example, using the test format as defined herein in Example 3, or a substantially similar test.
[0058] The present invention also includes antibodies and their antigen binding fragments that bind dimeric PDGFR-beta (for example, at 25oC or 37oC) with a KD of less than about 250 pM as measured by surface plasmon resonance, for example example, using the assay format as defined herein in Example 3. In certain embodiments, the antibodies or antigen binding fragments of the present invention bind dimeric PDGFR-beta with a KD of less than about 240 pM, less than about 230 pM, less than about 220 pM, less than about 210 pM, less than about 200 pM, less than about 190 pM, less than about 180 pM, less than about 170 pM, less than about 160 pM less than about 150 pM, less than about 140 pM, less than about 130 pM, less than about 120 pM, less than about 110 pM, or less than about 100 pM, as measured by plasma resonance of surface, for example, using the test format as defined here in Example 3, or a subs test substantially similar.
The present invention also includes anti-PDGFR-beta antibodies and their antigen binding fragments that block the binding of one or more PDGF ligands (for example, PDGF-BB, -AB, - CC, or -DD) to PDGFR-beta. For example, the present invention includes anti-PDGFR-beta antibodies that block the binding of PDGF-BB to monomeric PDGFR-beta in vitro, with an IC 50 value of less than about 300 pM, as measured by an ELISA-based assay. , for example, using the assay format as defined in Example 4 (A), or a real-time bioassay, for example, using the assay format as defined in Example 4 (B), or a substantially similar assay. In certain embodiments, the antibodies or antigen binding fragments of the present invention block binding of PDGF-BB to monomeric PDGFR-beta in vitro with an IC 50 value of less than about 280 pM, less than about 260 pM, less than about 240 pM, less than about 220 pM, less than about 200 pM, less than about 180 pM, less than about 160 pM, less than about 150 pM, less than about 140 pM, less than about 130 pM, less than about 120 pM, less than about 110 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, or less than about 75 pM, as measured by an ELISA immunobased assay, for example, using the assay format as defined herein in Example 4 (B), or a substantially similar assay.
[0060] The present invention also includes anti-PDGFR-beta antibodies and their antigen binding fragments that inhibit PDFG ligand-mediated activation of PDGFR-beta expressed on the cell surface. For example, the present invention includes anti-PDGFR-beta antibodies and their antigen binding fragments that inhibit PDGF-BB or PDGF-DD mediated activation of PDGFR-beta expressed on cell surface, with an IC50 value of less than 500 pM, as measured in a cell-based blocking bioassay, for example, using the assay format as defined herein in Example 6, or a substantially similar assay. In certain embodiments, the antibodies or antigen binding fragments of the present invention block PDGF-BB or PDGF-DD-mediated activation of PDGFR-beta expressed on cell surface with an IC 50 of less than about 400 pM, less than about 350 pM, less than about 300 pM, less than about 250 pM, less than about 200 pM, less than about 150 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, or less than about 30 pM, as measured by my cell-based blocking bioassay, for example example, using the assay format as defined herein in Example 6, or a substantially similar assay.
The present invention also includes anti-PDGFR-beta antibodies and their antigen binding fragments that are internalized in cells expressing PDGFR-beta. For example, the present invention includes anti-PDGFR-beta antibodies and their antigen-binding fragments that are effectively internalized in cells expressing PDGFR-beta as measured using a cell-based antibody internalization assay as defined herein in Example 7, or a substantially similar test.
[0062] The antibodies of the present invention may have one or more of the biological characteristics mentioned above, or any combination thereof. Other biological characteristics of the antibodies of the present invention will be evident to those skilled in the art from a review of the present disclosure including the present working examples. Epitope mapping and related technologies
The present invention includes anti-PDGFR-beta antibodies that interact with one or more amino acids found within the extracellular domain of human PDGFR-beta (for example, within Ig domains 1, 2, 3, 4 and / or 5 of extracellular domain of PDGFR-beta). Ig domains 1 to 3 (for example, amino acids 1 to 227 of SEQ ID NO: 337) are known to be involved in ligand binding. The present invention includes anti-PDGFR-beta antibodies that interact with one or more amino acids found within the Ig 1 domain (for example, amino acids 1 to 88 of SE ID NO: 337), Ig 2 domain (for example, amino acids 97 to 178 of SEQ ID NO: 337) and / or Ig 3 domain (for example, amino acids 182 to 277 of SEQ ID NO: 337), and therefore effectively block receptor / ligand interaction. In certain exemplary embodiments of the present invention, antibodies are provided that interact specifically with the Ig 2 domain (for example, within amino acids 97 through 178 of SEQ ID NO: 337; see, for example, Example 8). The epitope to which antibodies bind may consist of a simple contiguous sequence of 3 or more (for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20 or more) amino acids located within the extracellular domain of PDGFR-beta. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within the extracellular domain of PDGFR-beta.
[0064] Various techniques known to those skilled in the art can be used to determine whether an antibody "interacts common or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assay such as that described in Antibodies, Harlow and Lane (Cold spring Harbor Press, Cold Spring Harb., NY), mutation analysis of alanine scanning, peptide spot analysis ( Reineke, 2004, Methods Mol Biol 248: 443-463), and analysis of peptide cleavage. In addition, processes such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9: 487-496). Another process that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange process involves labeling the protein of interest with deuterium, followed by antibody binding to the deuterium-labeled protein. Then, the protein / antibody complex is transferred to water to allow hydrogen - deuterium exchange to occur in all residues except the residues protected by the antibody (which remains marked with deuterium). After dissociation of the antibody, the target protein is subjected to cleavage with protease and mass spectrometry analysis, thereby revealing deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267 (2): 252-259; Engene and Smith (2001) Anal. Chem. 73: 256A-265A.
The present invention further includes anti-PDGFR-beta antibodies that bind the same epitope as any of the specific exemplary antibodies described herein (for example, H1M3299N, H1M3305N, H2M3368N, H4H3096S, H1M3310N, H2M3373N, H4H3097S, H1M3361N, H2 H4H3098S, H2M3363N, H4H3094P, H4H3099S, H2M3365N H4H3095S H4H3102S H4H3103S, H4H3104S, H4H3105S, H4H3106S, H4H3107S, etc.).
[0066] Likewise, the present invention also includes anti-PDGFR-beta antibodies that compete for binding to PDGFR-beta with any of the exemplary specific antibodies described herein (for example, H1M3299N, H1M3305N, H1M3310N, H1M3361N, H2M3363N, H2M3365N, H2M3368N, H2M3373N, H2M3374N, H4H3094P, H4H3095S, H4H3096S, H4H3097S, H4H3098S, H4H3099S, H4H3102S, H4H3103S, H4H3104S, H4H3105S, H4H3105S, H4H3106, 7, 10 For example, the present invention includes anti-PDGFR-beta antibodies that cross-compete for binding to PDGFR-beta with one or more “Bin 1” antibodies as defined herein in Example 5 (for example, H4H3365N, H4H3374N, H4H3103S and H4H3094P). The present invention also includes anti-PDGFR-beta antibodies that cross-compete for binding to PDGFR-beta with one or more "Bin2" antibodies as defined herein in Example 5 (for example, H4H3099S, H4H3107S, H4H3305N and H4H3310N).
[0067] One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference anti-PDGFR-beta antibody using routine procedures known and exemplified herein. For example, to determine whether a test antibody binds to the same epitope as a reference anti-PDGFR-beta antibody of the invention, the reference antibody is allowed to bind to a PDGFR-beta protein (for example, a soluble portion of the extracellular domain of PDGFR -beta or PDGFR-beta expressed on cell surface). Next, the ability of a test antibody to bind to the PDGFR-beta molecule is assessed. If the test compound is able to bind to PDGFR-beta following saturation binding with the reference anti-PDGFR-beta antibody, it can be concluded that the test antibody binds to an epitope different from the reference anti-PDGFR-beta antibody . On the other hand, if the test antibody is unable to bind to the PDGFR-beta molecule following saturation binding with the reference anti-PDGFR-beta antibody, then the test antibody can bind to the same epitope as the antibody-bound epitope anti-PDGFR-beta reference of the invention. Additional routine experimentation (eg, peptide mutation and binding analyzes) can then be performed to confirm that the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if stereo blocking (or another phenomenon) is responsible for the observed lack of connection. Such experiments can be performed using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present invention, two antibodies bind (or overlap) the same epitope if, for example, a 1-, 5-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50%, but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, for example, Junghans et al., Cancert Res. 1990: 50: 1495-1502). Alternatively, two antibodies are thought to bind to the same epitope if essentially all of the amino acid mutations in the antigen that reduce or eliminate binding to one antibody reduce or eliminate binding to the other. Two antibodies are thought to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
[0068] To determine whether an antibody completes by binding (or competes - crossed by binding) with an anti-PDGFR-beta reference antibody, the binding methodology described above is carried out in two orientations: in a first orientation, the reference antibody is allowed to bind to a PDGFR-beta protein (for example, a soluble portion of the extracellular domain of PDGFR-beta or PDGFR-beta expressed on cell surface) under saturating conditions followed by evaluation of binding of the test antibody to the PDGFR- beta. In a second orientation, the test antibody is allowed to bind to a PDGFR-beta molecule under saturating conditions followed by evaluation of binding of the reference antibody to the PDGFR-beta molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the PDGFR-beta molecule, then it is concluded that the test antibody and the reference antibody compete for binding to PDGFR-beta (see, for example, the assay described here in Example 5, where soluble PDGFR-beta protein is captured on sensor tips and the PDGFR-beta coated sensor tips are treated with a reference antibody [mAb # 1] and a test anti-PDGFR-beta antibody [mAb # 2] sequentially and in both bsinding orders). As will be appreciated by those skilled in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody through binding of an overlapping or adjacent epitope . Preparation of human antibodies
[0069] Processes for generating monoclonal antibodies, including fully human monoclonal antibodies are known in the art. Any such known processes can be used in the context of the present invention to obtain human antibodies that specifically bind to human PDGFR-beta.
[0070] Using VELOCIMMUNE technology, for example, or any other known process for generating fully human monoclonal antibodies, high affinity chimeric antibodies to PDGFR-beta are initially isolated having a human variable region and a constant mouse region. As in the experimental section below, antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. If necessary, mouse constant regions are replaced with a desired human constant region, for example, wild-type or modified IgG1 or IgG4, to generate an entirely human anti-PDGFR-beta antibody. Although the selected constant region may vary according to specific use, high affinity antigen binding and target specificity characteristics reside in the variable region. In certain examples, fully human anti-PDGFR-beta antibodies are isolated directly from positive B cells - antigen. Bioequivalents
[0071] The anti-PDGFR-beta antibodies and antibody fragments of the present invention encompass proteins having amino acid sequences that vary from those of the described antibodies, but retain the ability to bind human PDGFR-beta. Such variant antibodies and antibody fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to the source sequence, which exhibits biological activity that is essentially equivalent to that of the described antibodies. Likewise, the DNA sequences encoding anti-PDGFR-beta antibody of the present invention encompass sequences that comprise one or more nucleotide additions, deletions, or substitutions when compared to the sequence shown, but which encode the anti-PDGFR-beta antibody or antibody fragment that is essentially bioequivalent to an anti-PDGFR-beta antibody or antibody fragment of the invention. Examples of such a variant amino acid and DNA sequences are discussed above.
[0072] Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered in the same molar dose under similar experimental conditions, both single and multiple doses. Some antibodies will be considered equivalent or pharmaceutical alternatives if they are equivalent in the extent of their absorption, but not in their absorption rate and can still be considered bioequivalent because such differences in the absorption rate are intentional and are reflected in the labeling, are not essential for obtaining effective concentrations of drugs in the body in, for example, chronic use, and are considered medically insignificant for the particular drug product studied.
[0073] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity and potency.
[0074] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be exchanged one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity , or decreased effectiveness, compared to continued therapy without such switching.
[0075] In one embodiment, two antigen-binding proteins are bioequivalent if both act through a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.
[0076] Bioequivalence can be demonstrated through in vivo and in vitro processes. Bioequivalence measurements include, for example, (a) an in vivo test on humans or other mammals, where the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of in vivo bioavailability data; (c) as an in vivo test on humans or other mammals where the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical experiment that establishes safety. Effectiveness, or bioavailability or bioequivalence of an antibody.
[0077] Bioequivalent variants of anti-PDGFR-beta antibodies of the invention can be constructed through, for example, modality of various substitutions of residues or sequences or suppression of residues or terminal or internal sequences not necessary for biological activity. For example, cysteine residues not essentially for biological activity can be suppressed or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges with denaturation. In other contexts, bioequivalent antibodies may include anti-PDGFR-beta antibody variants comprising changes in amino acids that modify the glycosylation characteristics of antibodies, for example, mutations that eliminate or remove glycosylation. Species selectivity and species cross-reactivity
[0078] The present invention, according to certain modalities, provides anti-PDGFR-beta antibodies that bind human PDGFR-beta, but not PDGF-beta from other species. The present invention also includes anti-PDGFR-beta antibodies that bind human PDGFR-beta and PDGFR-beta from one or more non-human species. For example, the anti-PDGFR-beta antibodies of the invention may bind to human PDGFR-beta and may or may not bind, as may be the case, to one or more of mouse, rat, guinea pig PDGFR-beta, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cinomolgo, marmoset, rhesus or chimpanzee. In accordance with certain exemplary embodiments of the present invention, anti-PDGFR-beta antibodies are provided that specifically bind human PDGFR-beta (e.g., monomeric and / or dimeric hPDGFR-beta constructs) and cynomolgus monkey PDGFR-beta (for example, Macaca fascicularis) (for example, monomeric and / or dimeric mfPDGFR-beta constructs). (See, for example, Example 3). Immunoconjugates
[0079] The invention encompasses anti-PDGFR-beta monoclonal antibodies conjugated to a therapeutic half ("immune conjugate"), such as a cytotoxin, chemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxic agents include any agent that is harmful to cells. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming immune conjugates are known in the art, (see, for example, WO 05/103081). Multispecific antibodies
[0080] The antibodies of the present invention can be monospecific, bispecific, or multispecific. Multispecific antibodies can be specific for different epitopes of a target polypeptide or can contain specific antigen binding domains for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147: 60-69; Kufer et al., 2004, Trends Biotechnol. 22: 238-244. The anti-PDGFR-beta antibodies of the present invention can be linked or coexpressed with another functional molecule, for example, another peptide or protein. For example, an antibody or fragment thereof may be functionally linked (for example, through chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment for produce a bispecific or multispecific antibody with a second binding specificity. For example, the present invention includes bispecific antibodies where one arm of an immunoglobulin is specific for human PDGFR-beta or a fragment thereof, and the other arm of the immunoglobulin is specific for the second therapeutic target or is conjugated to a therapeutic half.
An exemplary bispecific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, where the first and second Ig CH3 domains differ one on the other by at least one amino acid, and where at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bispecific antibody lacking an amino acid difference. In one embodiment, the first CH3 Ig domain binds Protein A and the second CH3 Ig domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 can still comprise a modification Y96F (by IMGT; Y436F by EU). Still modifications that can be found within second CH3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies. Variations on the bispecific antibody format described above are contemplated within the scope of the present invention.
[0082] Other exemplary bispecific formats that can be used in the context of the present invention include, without limitation, for example, bispecific scFv-based or bispecific formats, IgG-scFv fusions, dual variable domain (DVD) -Ig, Quadroma, bulges-in-holes, common light chain (e.g., common light chain with bulges-in-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF) -IgG, and bispecific Mab2 formats (see, for example, Klein et al. 2012, mAbs 4: 6, 1-11, and references cited there, for a review of previous formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, where unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody - oligonucleotide conjugates that then perform self-assembly in multimeric complexes with defined composition, valence and geometry . (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]). Therapeutic formulation and administration
[0083] The invention provides pharmaceutical compositions comprising the anti-PDGFR-beta antibodies or their antigen binding fragments of the present invention. The pharmaceutical compositions of the invention are formulated with appropriate vehicles, excipients, and other agents that provide improved transfer, release, tolerance, and the like. A variety of appropriate formulations can be found in the form known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, sprays, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (such as LIPOFECTIN, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in emulsions -water and water-in-oil, carbo wax emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbo wax. See also, Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.
[0084] The dose of antibody administered to a patient may vary depending on the age and size of the patient, target disease, conditions, route of administration, and the like. The preferred dose is typically calculated according to body weight or body surface area. When an antibody of the present invention is used to treat a condition or disease associated with PDGFR-beta activity in an adult patient, it may be advantageous to administer the antibody of the present invention intravenously normally in a single dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. Effective dosages and schedules for administration of anti-PDGFR-beta antibodies can be determined empirically; for example, patient progress can be monitored by periodic evaluation, and the dose adjusted in the same way. In addition, interspecies dosage scales can be performed using processes well known in the art (for example, Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).
[0085] Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing an antibody or other therapeutic protein of the invention, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). The antibodies and other therapeutically active components of the present invention can also be released using gene therapy techniques. Introduction processes include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition can be administered via any convenient route, for example, by infusion or injection of relatively large amounts, by absorption through epithelial or mucocutaneous linings (for example, oral mucosa, rectal and intestinal mucosa, etc.) and can be administered along with other biologically active agents. Administration can be systemic or local.
[0086] A pharmaceutical composition of the present invention can be released subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous release, a pen release device easily has applications in releasing a pharmaceutical composition of the present invention. Such a release pen device can be reusable or disposable. A reusable release pen device generally uses a refillable cartridge that contains a pharmaceutical composition. Once the entire pharmaceutical composition inside the cartridge has been administered and the cartridge is emptied, the emptied cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The release pen device can then be reused. In a disposable release pen device, there is no replaceable cartridge. Before, the disposable release pen device is previously filled with the pharmaceutical composition retained in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
[0087] Numerous autoinjector and reusable pen delivery devices have applications in the subcutaneous release of a pharmaceutical composition of the present invention. Examples include, but are not limited to AUTOPEN (Owen Mumford, Inc., Woodstock, UK), DISETRONIC pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25 pen, HUMALOG pen, HUMALIN 70/30 pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR (Novo Nordisk, Copenhagen, Denmark), BD pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN, OPTIPEN PRO, OPTIPEN STARLET, and OPTICLIK (Sanofi-Aventis, Frankfurt, Germany), to name just a few. Examples of disposable release pen devices having subcutaneous release applications of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTAR pen (Sanofi-aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), SURECLICK Autoinjector (Amgen, Thousand Oaks, CA), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA Pen (Abbott Labs, Abbott Park IL), to name just a few.
[0088] In certain situations, the pharmaceutical composition can be released in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in close proximity to the composition target, thus requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled-release systems are discussed in the review by Langer, 1990, Science 249: 1527-1533.
[0089] Injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared using publicly known processes. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium used conventionally for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with an appropriate solubilizing agent such as an alcohol (for example, ethanol ), a poly alcohol (for example, propylene glycol, polyethylene glycol), a non-ionic surfactant [for example, polysorbate 80, HCO-50 (polyoxyethylene (50 moles) hydrogenated castor oil adduct)], etc. As the oily medium, sesame oil, soy oil, etc. are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled into an appropriate ampoule.
[0090] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms in an appropriate unit dose to adapt a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforementioned antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the injection form, it is preferred that the antibody mentioned above is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms. Therapeutic uses of antibodies
[0091] The antibodies of the invention are useful, inter alia, for the treatment, prevention and / or amelioration of any disease or disorder associated with or mediated by PDGFR-beta expression, signaling, or activity, or treatable by blocking interaction between PDGFR-beta and a PDGFR-beta ligand (e.g., PDGF-BB, PDGF-CC, PDGF-DD, PDGF-AB, etc.) or otherwise inhibiting PDGFR-beta activity and / or signaling. For example, the present invention provides processes for treating eye diseases, fibrotic diseases (fibrosis), vascular diseases and / or cancer (inhibiting tumor growth) by administering an anti-PDGFR-beta antibody (or pharmaceutical composition comprising an anti-PDGFR-beta antibody) as described herein to a patient in need of such treatment. In the context of the treatment processes described herein, the anti-PDGFR-beta antibody can be administered as a monotherapy (i.e., as the sole therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described here in other parts).
[0092] Exemplary eye diseases that are treatable by administering anti-PDGFR-beta antibodies of the invention include age-related macular degeneration (e.g., "wet" AMD), exudative AMD, diabetic retinopathy (e.g., proliferative diabetic retinopathy) , retinal venous occlusive diseases such as central retinal vein occlusion (CRVO), iris neovascularization, neovascular glaucoma, post-surgical glaucoma fibrosis, proliferative vitreo-retinopathy (PVR), choroidal neovascularization, optic disc neovascularization, corneal neovascularization, neovascularization retinal, vitreal neovascularization, pannus, pterygium, macular edema, diabetic macular edema (DME), vascular retinopathy, retinal degeneration, uveitis, and inflammatory diseases of the eye.
Exemplary fibrotic diseases that are treatable by administration of anti-PDGFR-beta antibodies of the invention include pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis, bleomycin-induced pulmonary fibrosis, asbestos-induced pulmonary fibrosis, and bronchiolitis obliterans syndrome) , chronic asthma, fibrosis associated with acute lung damage and acute respiratory disease (for example, bacterial pneumonia-induced fibrosis, trauma-induced fibrosis, viral pneumonia-induced fibrosis, ventilator-induced fibrosis, non-pulmonary sepsis-induced fibrosis, and fibrosis aspiration-induced), silicosis, radiation-induced fibrosis, chronic obstructive pulmonary disease (COPD), ocular fibrosis (eg, fibrotic eye scarring), skin fibrosis (eg, scleroderma), liver fibrosis (eg, cirrhosis, fibrosis alcohol-induced liver disease, non-alcoholic hepatitis (NASH), bile duct damage, primary biliary cirrhosis, liver fibrosis infection or virus-induced [eg, chronic HCV infection], autoimmune hepatitis), kidney fibrosis (kidney), cardiac fibrosis, atherosclerosis, tube restenosis, and myelofibrosis.
Exemplary vascular diseases that are treatable by administration of anti-PDGFR-beta antibodies of the invention include vasoproliferative diseases, pulmonary arterial hypertension, restenosis, vascular healing, etc.
[0095] The present invention also includes processes for treating cancer, inhibiting tumor growth, promoting tumor regression, inhibiting metastasis, and / or inhibiting pathological angiogenesis (e.g., tumor growth-related angiogenesis) through administration of an anti-PDGFR-beta antibody as described herein to a patient in need of such treatment. For example, the antibodies and antigen binding fragments of the present invention can be used to treat, for example, primary and / or metastatic tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract female, muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood-forming cells and bone marrow, urinary tract and liver, and special sensory organs such as the eye. In certain embodiments, the antibodies and antigen-binding fragments of the invention are used to treat one or more of the following cancers: renal cell carcinoma, pancreatic carcinoma, breast cancer, head and neck cancer (e.g., brain cancer, oral cavity, oropharynx, nasopharynx, hypopharynx, nasal cavity, paranasal bells, larynx, lips, etc.), prostate cancer, urinary bladder cancer, malignant gliomas, osteosarcoma, osteoblastoma, osteochondroma, colorectal cancer, gastric cancer (for example , gastric cancer with MET amplification), malignant mesothelioma, astrocytoma, glioblastoma, medulloblastoma, retinoblastoma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, thyroid cancer connective tissue, Kaposi's sarcoma, basal cell carcinoma, squamous cell carcinoma, or melanoma. Combination therapies and formulations
[0096] The present invention includes compositions and therapeutic formulations comprising any of the anti-PDGFR-beta antibodies described herein in combination with one or more additional therapeutically active components, and treatment processes comprising administering such combinations to individuals in need.
[0097] The anti-PDGFR-beta antibodies of the present invention can be co-formulated with and / or administered in combination with, for example, a VEGF antagonist, for example, a "VEGF trap" such as aflibercept or other fusion protein. inhibition of VEGF as shown in US 7,087,411, and anti-VEGF antibody or its antigen binding fragment (e.g., bevacizumab, ranibizumab), a small molecule kinase inhibitor of the VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib) ), or an anti-VEGF receptor antibody. The anti-PDGFR-beta antibody can also be combined with a PDGF ligand antagonist (for example, an anti-PDGF-BB antibody, an anti-PDGF-DD antibody, an anti-PDGF-CC antibody, an anti- PDGF-ASB, or another PDGF ligand antagonist such as an aptamer [for example, an anti-PDGF-B aptamer such as Fovista, Ophthotech Corp., Princeton, NJ], an antisense molecule, a ribozyme, a siRNA, a peptibody , a nanobody or an antibody fragment directed against a PDGF linker). In other embodiments, the anti-PDGFR-beta antibodies of the present invention can be co-formulated with and / or administered in combination with an EGFR antagonist (for example, an anti-EGFR antibody [for example, cetuximab or panitumumab] or inhibitor small molecule EGFR [e.g. gefitinib or erlotinib]), an antagonist of another EGFR family member such as Her2 / ErbB2, ErbB3 or ErbB4 (for example, anti-ErbB2, anti-ErbB3 or anti-ErbB4 or small molecule inhibitor of ErbB2, ErbB3 or ErbB4 activity), a specific antagonist for EGFRvIII (for example, an antibody that specifically binds to EGFRvIII), a cMET antagonist (for example, an anti-cMET antibody), an antagonist IGF1R (for example, an anti-IGF1R antibody), or a B-raf inhibitor (for example, vemurafenib, sorafenib, GDC-0879, PLX-4720). In certain examples, the anti-PDGFR-beta antibodies of the present invention are combined, co-formulated and / or administered in combination with a PDGFR-alpha inhibitor (for example, an anti-PDGFR-alpha antibody), a DLL4 antagonist (for example , an anti-DLL4 antibody shown in US 2009/0142354 such as REGN421), an Ang2 antagonist (e.g., an anti-Ang2 antibody shown in US 2011/0027286 such as H1H685P), etc. Other agents that can be beneficially administered in combination with the anti-PDGFR-beta antibodies of the invention include cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-2 , IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or their respective receptors.
[0098] The anti-PDGFR-beta antibodies of the invention can also be administered and / or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, metal chelators, IFN-gamma, and / or NSAIDs. The anti-PDGFR-beta antibodies of the invention can also be administered as part of a treatment regimen that also includes treatment with conventional radiation and / or chemotherapy (for example, in the context of cancer treatment processes or inhibition of tumor growth) .
[0099] Any of the additional therapeutically active components can be administered in combination with any of the anti-PDGFR-beta antibodies of the present invention for the treatment of any disease or disorder where administration of an anti-PDGFR-beta antibody is beneficial, including , for example, any of the eye diseases, fibrotic diseases, vascular diseases, and / or cancers mentioned herein. For example, in the context of treating an eye disease (for example, wet AMD, diabetic retinopathy, CRVO, or any of the other eye diseases described herein), an anti-PDGFR-beta antibody of the present invention can be co-formulated with , and / or administered in combination with a VEGF antagonist, for example, a "VEGF trap" such as aflibercept or other VEGF inhibiting fusion protein as shown in US 7,087,411, or an anti-VEGF antibody or fragment thereof of antigen binding (for example, bevacizumab, or ranibizumab).
[00100] In exemplary embodiments in which an anti-PDGFR-beta antibody of the invention is administered in combination with a VEGF antagonist (for example, a VEGF trap such as aflibercept), including administration of co-formulations comprising an anti-PDGFR- antibody beta and a VEGF antagonist, the individual components can be administered to an individual and / or co-formulated using a variety of dosage combinations. For example, anti-PDGFR-beta antibody can be administered to an individual and / or contained in a co-formulation in an amount selected from the group consisting of 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, and 5.5 mg and the VEGF antagonist (e.g. a VEGF trap such as aflibercept) can be administered to the individual and / or contained in a co-formulation in an amount selected from the group consisting of 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg , 1.7 mg, 1.8 mg, 1.9 mg, 2.0 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg and 3.0 mg. Exemplary dosage combinations of anti-PDGFR-beta / aflibercept antibody of the present invention include, for example: (i) 0.2 mg of anti-PDGFR-beta antibodies + 2 mg of aflibercept; (ii) 0.5 mg of anti-PDGFR-beta antibodies + 2 mg of aflibercept; (iii) 1 mg of anti-PDGFR-beta antibody + 2 mg of aflibercept; (iv) 3 mg anti-PDGFR-beta antibody + 2 mg aflibercept; and (v) 4 mg anti-PDGFR-beta antibody + 2 mg aflibercept. The combinations / co-formulations can be administered to an individual according to any of the administration regimens shown here elsewhere, including, for example, once a week, once every 2 weeks, once every 3 weeks, once a week. once a month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, etc.
[00101] The additional therapeutically active component (s) can be administered to an individual prior to administration of an anti-PDGFR-beta antibody of the present invention. For example, a first component can be judged to be administered “before” a second component if the first component is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 hours before, 4 hours before, 3 hours before, 2 hours before, 1 hour before, 30 minutes before, 15 minutes before, 10 minutes before, 5 minutes before, or less than 1 minute before administration of the second component. In other embodiments, the additional therapeutically active component (s) can be administered to an individual after administration of an anti-PDGFR-beta antibody of the present invention. For example, a first component can be judged to be administered “after” a second component if the first component is administered 1 minute after, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hours after, 60 hours after, 72 hours after administration of the second component. In still other embodiments, the additional therapeutically active component (s) can be administered to an individual simultaneously with administration of an anti-PDGFR-beta antibody of the present invention. "Simultaneous" administration for purposes of the present invention includes, for example, administration of an anti-PDGFR-beta antibody and an additional therapeutically active component to an individual in a simple dosage form (for example, co-formulated), or in the form of a separate dosage administered to the individual within about 30 minutes or less of each other). If administered in a separate dosage form, each dosage form can be administered via the same route (for example, both the anti-PDGFR-beta antibody and the additional therapeutically active component can be administered intravitreally, subcutaneously, etc.); alternatively, each dosage form can be administered intravitreally, and the additional therapeutically active component can be administered systemically). In any case, administration of components in a single dose from, in separate dosage forms through the same route, or in separate dosage forms through different routes are all considered "concurrent administration", for the purposes of the present exhibition. For the purposes of the present exhibition, administration of an anti-PDGFR-beta antibody “before”, “simultaneously with”, or “after” (as those terms are defined herein above) administration of an additional therapeutically active component is considered administration of a anti-PDGFR-beta antibody “in combination with” an additional therapeutically active component.
[00102] The present invention includes pharmaceutical compositions in which an anti-PDGFR-beta antibody of the present invention is co-formulated with one or more additional therapeutically active components as described elsewhere herein.
[00103] The present invention also includes additional therapeutic compositions comprising a combination of a PDGF antagonist and a VEGF antagonist. PDGF antagonists according to this aspect of the invention include PDGF receptor antagonists as well as PDGF ligand antagonists. Likewise, VEGF antagonists according to this aspect of the invention include VEGF receptor antagonists as well as VEGF ligand antagonists. Administration regimes
[00104] In accordance with certain embodiments of the present invention, multiple doses of an anti-PDGFR-beta antibody (or a pharmaceutical composition comprising a combination of an anti-PDGFR-beta antibody and any of the additional therapeutically active agents mentioned herein) may be used. be administered to an individual over a defined course of time. The processes according to this aspect of the invention comprise sequential administration to an individual of multiple doses of an anti-PDGFR-beta antibody of the invention. As used herein, “sequential administration” means that each dose of anti-PDGFR-beta antibody is administered to the individual at a different time point, for example, on different days separated by a predetermined interval (for example, hours, days, weeks or months). The present invention includes processes that comprise sequential administration to the patient of a single initial dose of an anti-PDGFR-beta antibody, followed by one or more secondary doses of the anti-PDGFR-beta antibody, and optionally followed by one or more tertiary doses of anti-PDGFR-beta antibody.
[00105] The terms "starting dose", "secondary dose", and "tertiary doses", refer to the timing of administration of the anti-PDGFR-beta antibody of the invention. Thus, the “starting dose” is the dose that is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”): the “secondary doses” are the doses that are administered after the initial dose; and "tertiary doses" are the doses that are administered after secondary doses. The initial, secondary and tertiary doses can all contain the same amount of anti-PDGFR-beta antibody, but they can generally differ from each other in terms of frequency of administration. In certain embodiments, however, the amount of anti-PDGFR-beta antibody contained in the initial, secondary and / or tertiary doses varies from one to another (for example, adjusted up and down when appropriate) during the course of treatment. In certain embodiments, two or more (for example, 2, 3, 4, or 5) doses are administered at the start of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (for example , “Maintenance doses”).
[00106] In certain exemplary embodiments of the present invention, each secondary and / or tertiary dose is administered 1 to 26 (e.g. 1, 1%, 2, 2%, 3, 3%, 4, 4%, 5, 5% , 6, 6%, 7, 7%, 8, 8%, 9, 9%, 10, 10%, 11, 11%, 12, 12%, 13, 13%, 14, 14%, 15, 15% , 16, 16%, 17, 17%, 18, 18%, 19, 19%, 20, 20%, 21, 21%, 22, 22%, 23, 23%, 24, 24%, 25, 25% , 26, 26% or more) weeks after the immediately preceding dose. The phrase "the immediately preceding dose", as used herein, means, in a sequence of multiple administrations, the dose of anti-PDGFR-beta antibody that is administered to a patient prior to the administration of any subsequent dose in the sequence without intervening doses.
[00107] The processes according to this aspect of the invention may comprise administering to a patient any number of secondary and / or tertiary doses of an anti-PDGFR-beta antibody. For example, in certain modalities, only a simple secondary dose is administered to the patient. In other embodiments, two or more (for example, 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain modalities, only a single tertiary dose is administered to the patient. In other embodiments, two or more (for example, 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.
[00108] In modalities involving multiple secondary doses, each secondary dose can be administered at the same frequency as the other secondary doses. For example, each secondary dose can be administered to the patient 1 to 2 weeks or 1 to 2 months after the immediately preceding dose. Similarly, in modalities involving multiple tertiary doses, each tertiary dose can be administered at the same frequency as other tertiary doses. For example, each tertiary dose can be administered to the patient 2 to 12 weeks after the immediately preceding dose. In certain embodiments of the invention, the frequency at which secondary and / or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration can also be adjusted during the course of treatment by a doctor depending on the needs of the individual patient following clinical examination.
[00109] The present invention includes administration regimens in which 2 to 6 doses of loading are administered to a patient at a first frequency (for example, once a week, once every two weeks, once every three weeks, once per month, once every two months, etc.), followed by administration of two or more maintenance doses to the patient on a less frequent basis. For example, according to this aspect of the invention, if loading doses are administered at a frequency of, once a month (for example, two, three, four, or more loading doses administered once a month), then maintenance doses can be administered to the patient once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every ten weeks, once every twelve weeks, etc. .). Diagnostic uses of antibodies
[00110] The anti-PDGFR-beta antibodies of the present invention can also be used for detecting and / or measuring PDGFR-beta or cells expressing PDGFR-beta in a sample, for example, for diagnostic purposes. For example, an anti-PDGFR-beta antibody, or fragment thereof, can be used to diagnose a condition or disease characterized by aberrant expression (eg, overexpression, underexpression, lack of expression, etc.) of PDGFR-beta. Exemplary diagnostic tests for PDGFR-beta may comprise, for example, contacting a sample, obtained from a patient, with an anti-PDGFR-beta antibody of the invention, where the anti-PDGFR-beta antibody is marked with a reporter or marker molecule detectable. Alternatively, an unlabeled anti-PDGFR-beta antibody can be used in diagnostic applications in combination with a secondary antibody which is itself detectably labeled. The detectable marker or reporter molecule can be a radioisotope, such as 3H, 14C, 32P, 35S or 125I; a fluorescent or chemiluminescent half such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, beta galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure PDGFR-bea in a sample include enzyme-linked sorbent immunoassay (ELISA), radio immunoassay (RIA), and fluorescence activated cell classification (FACS).
[00111] Samples that can be used in PDGFR-beta diagnostic assays according to the present invention include any tissue or fluid sample obtainable from a patient that contains detectable amounts of PDGFR-beta protein, or fragments thereof, under normal or pathological. Generally, levels of PDGFR-beta in a particular sample obtained from a healthy patient (for example, a patient not afflicted with a disease or condition associated with abnormal levels or activity of PDGFR-beta) will be measured to initially establish a baseline level , or standard, of PDGFR-beta. This baseline level of PDGFR-beta can then be compared against the levels of PDGFR-beta measured in samples obtained from individuals suspected of having a PDGFR-beta-related disease or condition. Examples
[00112] The following examples are shown in order to provide those skilled in the art with a complete explanation and description of how to obtain and use the processes and compositions of the invention, and are not intended to limit the scope of what the inventors see as your invention. Efforts have been made to ensure accuracy with respect to numbers used (eg quantities, temperature, etc.), but some errors and experimental deviations should be considered. Unless otherwise indicated, parts are by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is, or close to, atmospheric. Example 1: Generation of human antibodies to PDGFR-beta
[00113] An immunogen comprising the ecto PDGFR-beta domain was directly administered, with an adjuvant to stimulate the immune response, to a VELOCIMMUNE mouse comprising DNA encoding variable regions of human immunoglobulin light and heavy chain. The antibody immune response was monitored using an PDGFR-beta immunospecific assay. When a desired immune response was obtained, splenocytes were harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. Hybridoma cell lines were separated and selected to identify cell lines that produce specific antibodies to PDGFR-beta. Using this technique, several chimeric anti-PDGFR-beta antibodies (i.e., antibodies having human variable domains and mouse constant domains) were obtained; exemplary antibodies generated in this manner were designated as follows: H1M3299N, H1M3305N, H1M3310N, H1M3361N, H2M3363N, H2M3365N, H2M3368N, H2M3373N and H2M3374N. The human variable domains of the chimeric antibodies were subsequently cloned into human constant domains to obtain fully human anti-PDGFR-beta antibodies as described herein.
[00114] Anti-PDGFR-beta antibodies were also isolated directly from antigen-positive B cells without myeloma cell fusion, as described in US 2007 / 0280945A1. Using this process, several fully human anti-PDGFR-beta antibodies (i.e., antibodies having human variable domains and human constant domains) were obtained; exemplary antibodies generated in this way have been designated as follows:
[00115] H4H3394P, H4H3095S, H4H3096S, H4H3097S, H4H3098S, H4H3099S, H4H3102S, H4H3103S, H4H3105S, H4H3105S, H4H3106S, H4H3107S.
[00116] Certain biological properties of the exemplary anti-PDGFR-beta antibodies generated according to the procedures of this example are described in detail in the Examples shown below. Example 2: Light and heavy chain variable region amino acid sequences
[00117] Table 1 shows the selected light and heavy chain variable region amino acid sequence pairs of selected anti-PDGFR-beta antibodies and their corresponding antibody identifiers. Table 1

[00118] Antibodies are typically referred to here according to the following nomenclature: prefix Fc (for example, "H1M", "H2M", "H4M"), followed by a numeric identifier (for example, "3299", "3363" , or “3094” as shown in Table 1), followed by a suffix “P”, “N”, or “S”. Thus, according to this nomenclature, an antibody can be referred to here as, for example, "H1M3299N", "H2M3363N", "H4H3094", etc. The prefixes H1M, H2M and H4H over the antibody designations used herein indicate the particular Fc region isotype of the antibody. For example, an "H1M" antibody has mouse Fc Igg1, while an "H4H" antibody has human Fc IgG4. As will be appreciated by those skilled in the art, an antibody having a particular Fc isotype can be converted to an antibody with a different Fc isotype (for example, an antibody with mouse IgG1 Fc can be converted to an antibody with a human IGG4, etc. .), but in any case, the variable domains (including CDRs) - which are indicated by the numeric identifiers shown in Table 1 - will remain the same, and the binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain. Construction control used in the examples that follow
[00119] An anti-PDGFR-beta control antibody has been included in the following examples for comparative purposes. The control antibody is referred to herein as Control I: a human anti-PDGFR-beta antibody with “2C5” heavy and light chain variable domain sequences as shown in US 7 740 850. Example 3. Antibody binding to human PDGFR-beta as determined by surface plasmon resonance
[00120] Binding affinities and kinetic constants for antigen binding to purified PDGFR-beta ati-human monoclonal antibodies were determined using a real-time plasma surface resonance biosensor assay (Biacore T100, GE Healthcare Life Sciences, Piscataway, NJ) at 25oC and 37 ° C antibodies, expressed as mouse Fc (prefix H1M; H2M) or human Fc (prefix H4H), were captured on their respective anti-Fc sensing surfaces (Mab capture format). Different concentrations of soluble monomeric PDGFR-beta constructs (hPDGFRb.mmh [SEQ ID NO: 337], PDGFRb.mmh from Macaca fascicularis [SEQ ID NO: 340] or dimeric PDGFR-beta constructions (human PDGFRb.mFc [SEQ ID NO: 338] or human PDGFRb.hFc [SEQ ID NO: 339]) were injected onto the captured surface of monoclonal anti-PDGFR-beta antibody at a flow rate of 50 microliters / minute. Dissociation rate constants (Kd) and association (Ka) were determined through data processing and adaptation for a 1: 1 link model using Scrubber 2.0 curve fitting software. Link dissociation equilibrium (KD) constants and dissociative half-lives (t1 / 2) were calculated from the kinetic rate constants as: KD (M) = kd / ka; and t1 / 2 (minutes) = (ln2 / 60 * kd). Kinetic binding parameters for different anti-PDGFR- monoclonal antibodies beta are shown in Tables 2 to 5. (NB = no connection observed under the conditions used; NT = no tested).
[00121] Table 2: Binding characteristics of anti-PDGFR-beta antibodies (mouse Fc format) for monomeric and dimeric PDGFR-beta constructs at 25oC


[00122] Table 3: Binding characteristics of anti-PDGFR-beta antibodies (human Fc format) for monomeric and dimeric PDGFR-beta constructs at 25oC



[00123] Table 4: Binding characteristics of anti-PDGFR-beta antibodies (mouse Fc format) for monomeric and dimeric PDGFR-beta constructs at 37oC


[00124] Table 5: Binding characteristics of anti-PDGFR-beta antibodies (human Fc format) for monomeric and dimeric PDGFR-beta constructs at 37oC


[00125] As shown in Tables 2-5, several anti-PDGFR-beta antibodies of the present invention showed subnanomolar affinity for the M. fascicularis and human PDGFR-beta constructs. In addition, several clones showed stronger binding (lower KD) to the PDGFR-beta constructs than the reference antibody (Control 1). Example 4. Anti-PDGFR-beta antibodies block binding of PDGF ligands to PDGFR-beta A. Receptor / ligand block assessed using an ELISA-based immunoassay
[00126] The ability of certain anti-human PDGFR-beta antibodies of the invention to block receptor binding to their PDGF-BB ligand was first evaluated with an ELISA-based immune assay. In summary, plates were coated with human PDGF-BB (2 micrograms / ml). Separately, 250 pM of biotinylated soluble hPDGFR-beta.mmh (“biot-hPDGFR-beta-mmh”, SEQ ID NO 337) was pre-mixed with serial diluted anti-PDGFR-beta antibodies (0-100 nM) for 1 hour at room temperature (25oC). The balanced antibody / PDGFR-beta solutions were added to ligand-coated plates, allowed to incubate for 1 hour, and washed. Levels of bound biot-hPDGFR-beta.mmh were detected using streptavidin conjugated to HRP. Data were analyzed using Prism software and IC50 values were calculated as the amount of antibody required to obtain 50% reduction of ligand-bound hPDGFR-beta-mmh. Maximum blocking values have also been calculated and reflect the ability of the antibody to block from the baseline. The absorbance measured in the constant 250 pM amount of biot-hPDGFR-beta-mmh on the dose curve is defined as 0% block and the absorbance without any added PDGFR-beta is defined as 100%. The absorbance of the wells containing the highest antibody concentration determined the maximum blocking percentage. Results are shown in Table 6. ("E" indicates that the antibody is an enhancer, that is, the signal was greater in the presence of some concentrations of the antibody than in the absence of the antibody).
[00127] Table 6: Blocking of anti-PDGFR-beta antibody binding PDGF-BB to PDGFR-beta
* Represents the average IC50 of three separate experiments.
[00128] As shown in Table 6, several antibodies of the invention potentially block the interaction of PDGFR-beta with its natural ligand PDGF-BB, with IC50 values ranging from about 7.6 nM (H1M3299N) to about 66 pM (H4H3107S ), and certain enhanced antibody ligand - receptor interactions (for example, H4H3097S, H4H3098S and H4H3102S). B. Receiver / ligand block evaluated using a real-time biosensor assay
[00129] The ability to select anti-human PDGFR-beta antibodies to block ligand binding (PDGF-BB, PDGF-DD and PDGF-AB) to human PDGFR-beta was also evaluated using a real-time SPR biosensor assay (Biacore 3000).
[00130] In summary, 400 Rus of soluble human PDGFR-beta.mFC (SEQ ID NO: 338) was captured on a Biacore derived surface (covalently coupled) with polyclonal rabbit mouse Fc antibody (GE Healthcare Life sciences, Piscataway, NJ). The captured surface was saturated with 300 nM of selected anti-PDGFR-beta antibodies for 4 minutes followed by an injection of 30 nM of ligand (PDGF-BB, PDGF-DD or PDGF-AB) for an additional 4 minutes at 25oC. Real-time binding response was monitored throughout the course of the assay and was compared to the measured binding response when PDGF ligand was applied to the captured control surface derived in the absence of captured antibody. Results are illustrated in Figure 1.
[00131] As seen in Figure 1, all antibodies showed the ability to block PDGF-BB and PDGF-AB ligands with fewer antibodies allowing efficient blocking of PDGF-DD when compared to the control without antibody. Of note were antibodies H4H3094P, H4H3374N, and Control I, which showed the least amount of RU response when ligand was applied on the Biacore sensing surface. Example 5. Cross-competition analysis of anti-PDGFR-beta antibodies
[00132] A cross-competition assay was conducted to assess the ability to select antibodies to compete with each other for binding to human PDGFR-beta. In summary, human soluble PDGFR-beta.mmh (SEQ ID NO: 337), was captured on anti-Penta-his Octet sensor tips (ForteBio Corp., Menlo Park, CA). Each sensor tip coated with PDGFR-beta.mmh was saturated for 5 minutes with a first anti-PDGFR-beta antibody (Mab # 1; 50 µg / mL). Next, each sensor tip was saturated with a solution of a second anti-PDGFR-beta antibody (Mab # 2). The real-time response of Mab # 2 binding to PDGFR-beta.mmh pre-complexed with Mab # 1 was then monitored. All tests were performed at 25oC with a flow rate of 1000 rpm ( ) on an Octet RED384 biosensor in Octet HBST buffer according to manufacturer's instructions (ForteBio Corp., Menlo Park, CA). The results are illustrated in Figure 2.
[00133] Binding responses of less than 0.1 nM are shown in Figure 2 in black or gray shading and indicate that the corresponding pairs of antibodies compete with each other for binding to PDGFR-beta. Binding responses greater than 0.2 nM (shown in white boxes in Figure 2) represent pairs of antibodies and do not compete with each other for binding to PDGFR-beta.
[00134] The results of this example indicate that the anti-PDGFR-beta antibodies of the invention can be grouped into two distinct "bins" on epitope binding characteristics: Bin 1 includes Control I, H4H3365N, H4H3374N, H4H3103S, and H4H3094P. Bin 2 includes H4H3099S, H4H3107S, H4H3305N and H4H3310N. The results of this example suggest that the Bin 1 antibodies bind different regions on PDGFR-beta than the Bin 2 antibodies. Example 6. Inhibition of ligand-mediated receptor activation and MAPK signaling with anti-PDGFR-beta antibodies
[00135] To further characterize anti-PDGFR-beta antibodies of the present invention, a bioassay was developed to detect the activation of PDGFR-beta through its known binding ligands, PDGF BB and DD. The interaction between PDGFR-beta receptors and their ligands is necessary for the induction of several cellular processes including proliferation, survival, migration and morphogenesis (Hoch and Soriano, 2003, Development 130: 5769-4784). PDGF receptors are tyrosine kinase receptors and are formed by homo- or heterodimerization of alpha and beta receptors with activation by PDGF BB and DD. With activation, autophosphorylation is induced and several cascades of signal transduction pathways are triggered, including the Ras-MAPK pathway (mitogen-activated protein kinase).
[00136] To detect activation of the MAPK signal transduction pathway via ligand binding to PDGFR beta, a stable HEK293 cell line was generated to express full-length human PDGFR-beta along with a luciferase reporter (Responsive Element - Serum [ SER-luciferase]). HEK293 / hPDGFR-beta cells were seeded in a 96-well plate and kept in low-serum media containing 0.1% FBS overnight. Following incubation, PDGF BB or DD, serially diluted 1: 3, was added to cells in concentrations ranging from 100 nM to 0.002 nM, to determine dose response. To examine ligand-activated MAPK signaling cascade inhibition, antibodies were serially diluted 1: 3 and added to cells in a concentration ranging from 100 nM to 0.002 nM. Concentrations of PDGF BB and DD remained constant at 250 pM and 400 pM respectively and luciferase activity was detected after 5.5 hours. PDGF BBe DD activated human PDGFRb with ECs50 of 0.04-1.11 nM and 0.34-1.82 nM respectively. The concentration of antibody required to inhibit 50% of PDGFR-beta mediated signaling (IC50) was determined for each antibody. The results are summarized in Table 7. (NB = no block; isotype 1 = mouse IgG negative control irrelevant antibody; isotype 2 = human IgG negative control irrelevant antibody).
[00137] Table 7: IC50 values for anti-PDGFR-beta antibodies blocking PDGF-BB and PDGF-DD ligand activation


[00138] As shown in Table 7, several of the anti-PDGFR-beta antibodies of the present invention potentially block ligand-dependent PDGFR-beta activation, with ICs50 in the subnanomolar range. In addition, both mouse IgG (Isotype 1) and human IgG (isotype 2) negative controls do not block receptor ligand activation. Example 7. Internalization of anti-PDGFR-beta antibodies on cells expressing PGDFR-beta
[00139] To study antibody-mediated receptor internalization, experiments were performed using engineering cells for expression of human PDGFR-beta (HEK293 / SER-luc / PDGFRb cells). In summary, 20,000 HEK293 / SRE Luc / PDGFRb / well cells were coated overnight in whole media (FBS 10%, Pen / Strep / Glut, NEAA, and G418 in DMEM) and stained with anti-PDGFR-beta antibodies in 10 ug / mL for 30 minutes at 4oC. Cells were washed twice and stained with Dylight 488 Fab conjugated secondary goat anti-human IgG antibody (10 µg / mL; Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 minutes at 4oC. Next, cells were incubated at 37oC for 2 hours to allow internalization of the receptor. Alexa-488 fluorescence was rapidly cooled by incubating cells washed with anti-Alexa fluoride 488 (Invitrogen Corp., Carlsbad, CA) for 45 minutes at 4oC to differentiate surface-bound antibodies from internalized antibodies. Images were taken with ImageXpress Micro XL (Molecular Devices LLC, Sunnyvale, CA) and point analysis was performed using Columbus software (Perkin Elmer, Waltham, MA). Relative internalization was calculated by comparing rapid cooling staining (i.e., internalized antibody) of each antibody to that of the Control 1 antibody. Results are summarized in Table 8.
[00140] Table 8: Internalization of selected anti-PDGFR-beta antibodies

[00141] As shown in Table 8, all anti-PDGFR-beta antibodies studied showed robust internalization in this assay format, reflecting the potential ability of the antibodies to effectively target cells expressing PDGFR-beta in various therapeutic contexts. Example 8. Anti-PDGFR-beta antibodies bind to different domains on PDGFR-beta
[00142] The extracellular portion of PDGFR-beta consists of 5 Ig-like C2-type domains, referred to as D1-D5. D1 through D3 are required for high binding binding affinity. In this example, experiments were conducted to determine which extracellular domain (s) of certain anti-PDGFR-beta antibodies of the invention interact.
[00143] For this experiment, four different extracellular domain constructs of PDGFR-beta were used: D1 (SEQ ID NO: 342), D1-D2 (SEQ ID NO: 343), D1-D3 (SEQ ID NO: 344) , and D1-D4 (SEQ ID NO: 345), as well as full length PDGFR-beta. Four different anti-PDGFR-beta antibodies were tested for binding to the various constructs using surface plasmon resonance (Biacore). In summary, 150-200 anti-PDGFR-beta antibody Rus were captured via an anti-human F5 CM5 chip. Then, the individual domain constructs, or full length PDGFR beta, were applied to the antibody-bound surface at a concentration of 50 nM. The ability of the various antibodies to bind to the various domain constructs was measured. The results are shown in Table 9. (-) = no binding observed; (+) = observed connection; ND = not determined.
[00144] Table 9. Observed binding of selected anti-PDGFR-beta antibodies to full length PDGFR-beta and PDGFR-beta domains

[00145] As summarized in Table 9, all antibodies bound to full length PDGFR-beta. Two antibodies, H4H3094P and H4H3374N, were determined to bind to domain 2. Interestingly, these two antibodies are also ligand blockers based on the ELISA immunoassay, confirming that domain 2 is important for ligand binding (PDGF-BB). The two other exemplary antibodies tested, H4H3099S and H4H3305N, do not bind to any of the domain constructs, suggesting that these antibodies may need the amino acids between domains 4 and 5 and / or domain 5 itself for high binding affinity. Example 9. Anti-PDGFR-beta antibodies deplete pericytes in a retinal model in vivo
[00146] Two exemplary anti-PDGFR antibodies, H4H3374N and H4H3094P, were tested in a model of retinal pericyte depletion in vivo. Pericytes are smooth muscle-like cells that express PDGFR-beta. PDGFR-B, expressed on endothelial cells, plays a role in recruiting parakeets for newly formed vessels, thus promoting angiogenesis and the establishment of vascular architecture. However, the interaction between pericytes and the endothelium, and PDGF-B / PDGFR-beta signaling, is interrupted during pathogenic angiogenesis, contributing to uncontrolled vessel formation. In diseases of the eye, this neovascularization can lead to visual morbidity and blindness.
[00147] In a first experiment, pupae from humanized PDGFR-beta mice were injected subcutaneously (sc) with 3 mg / kg of H4H3374N, H4h3094P, control I (2C5) or human Fc (hFc) to see the effect of signaling blocking PDGF-B / PDGFR-beta in vasculature being recently formed. In summary, humanized PDGFR-beta pupae (P2) on postnatal day 2 were injected subcutaneously with 3 mg / kg of hFc control or PDGFR-beta antibody. On postnatal day 5, pupae were sacrificed. Both eyes were collected and fixed in P.F.A. 4% for 1 hour. Eyes were washed 3x with PBS and retinas were dissected by removing hyaloid vessels. Retinas were stained O / N at room temperature with a primary anti-rabbit NG2 chondroitin sulfate antibody prepared in antibody dilution serum (ADS; 1% BSA in 0.05% Triton-X-100 in PBS). After incubation, all retinas were washed 3x for 15 minutes in PBS and then stained O / N at 4oC with fluorescein-labeled Griffonia Simplicifolia lectin and a secondary labeled with 594 goat anticoelho prepared in ADS. After incubation, all retinas were again washed 3x for 15 minutes in PBS. Retinas were mounted - flat on slides and covered - sliding using Fluoromount-G without DAPI.
[00148] Retinas were made images using a Nikon 80i fluorescent microscope. Images were analyzed using Adobe Photoshop and Fovea. The average positive NG2 area, normalized for hFc, was measured for each treatment group. Both, image formation and analysis were performed in a blind manner. Statistical analyzes were performed using one-way ANOVA in prism software. The results are summarized in Tables 10-11.
[00149] Table 10: Reduction in NG2 positive retinal area after treatment with 3 mg / kg of H4H3374N, Control I or hFc

[00150] Table 11: Reduction in NG2 positive retinal area after treatment with 3 mg / kg of H4H3094P, Control I or hFc

[00151] As shown in Tables 10-11, the positive average NG2 area of the retina was decreased in mice treated with anti-PDGFR-beta antibodies compared to hFc. The NG2 positive area was significantly decreased (p <0.001) for antibodies H4H3374N and H4H3094P in relation to hFc. In addition, H4H3374N showed the highest reduction in NG2 positive area when compared to both H4H3094P and the Control I antibody.
[00152] In a separate set of experiments, pupae of C57BI / 6 mice were injected subcutaneously (SC) in P2 with anti-mouse PDGFR-beta antibody “mAb39” (having the variable regions of the antibody referred to as APB5, see Uemura et al., J, Clin. Invest. 2002; 110 (11): 1619-1628) in doses of 50 mg / kg, 25 mg / kg, 12.5 mg / kg, or 6.25 mg / kg, or with Fc in 50 mg / kg as a control (Study 1). The effect on periscite coverage was assessed at P5 using a primary antibody 4 anti-rabbit NG2 proteoglycan chondroitin sulfate. In the development of retinal vessels, all doses of mAb39> 12.5 mg / kg inhibited blood vessel periscite coverage.
[00153] In another study (Study 2), P2 pupae were injected SC with 25 mg / kg of mAb39 or control. Retinas were collected in P5 and stained with Griffonia simplicifolia lectins (“GS Lectin I”, Vector Labs). At a dose of 25 mg / kg, mAb39 moderately decreased vascularized retinal areas and vessel density compared to controls.
[00154] In a set of separate experiments (Study 3), eyes left from pupae were injected intravitreally (IVT) with 5 micrograms (0.5 uL) of mAb39 or control in P4 and collected in P6. A single administration of intravitreal anti-PDGFR-beta antibody almost completely depleted mural cells and produced marked effects on retinal vascular differentiation and morphology, for example, irregular blood vessel caliber. Additional experiments were conducted to investigate the neutralizing effect of PDGFR-beta in the eyes and adult mice. In particular, left eyes of adult mice were injected IVT with mAb39 (5 micrograms or 10 micrograms) or control (5 micrograms or 10 micrograms). Eyes were collected 48 hours later and stained with anti-NG2 and GS Lectin I. In adult mice, mAb39 produced no evidence of any loss of pericyte or any vascular morphological changes.
[00155] These studies collectively demonstrate that selective pharmacological neutralization of PDGFR-beta is effective in promoting pericyte depletion and contributes to changes in morphology and vascular growth in the development of retinal neovessels. In contrast, this same inhibition does not appear to have any effect on periscites and mature vessels in the vasculature established in the adult mouse retina. Example 10. A Phase 1 clinical trial of a combination formulation comprising an anti-PDGFR-beta antibody and a VEGF antagonist in patients with age-related macular degeneration Study overview
[00156] A phase 1 clinical trial is conducted to test the safety of an anti-PDGFR-beta antibody of the invention released via intravitreal injection in patients with neovascular age-related macular degeneration (AMD) in conjunction with intravitreal aflibercept (IVT) . The amino acid sequence of aflibercept (also known as VEGFR1R2-FcΔC1 (a)), as well as the nucleic acid sequence encoding the same, are shown, for example, in WO2012 / 097019.
[00157] The primary objective of this study is to investigate the safety of intravitreal anti-PDGFR-beta (IVT) antibody in patients with neovascular AMD. The secondary objectives are to explore the anatomical effects of anti-PDGFR-beta IVT on corneal neovascularization (CNV) in patients with neovascular AMD, and to determine the kinetic drugs of anti-PDGFR-beta and aflibercept in humans. Another objective of this study is to determine the presence of antibodies against the anti-PDGFR-beta and / or aflibercept antibody in individuals treated with these agents. Target population
[00158] The target population for this study is men and women 50 years and older with neovascular AMD. Approximately 3-6 patients will be enrolled in four planned cohorts. A total of 15-24 patients are planned. Six patients will be enrolled at the maximum tolerated dose (BAT), if identified, or the highest dose level. Key Inclusion / Exclusion Criteria
[00159] The key inclusion criteria for this study are as follows: (1) men or women 50 years of age or older; and (2) secondary subfoveal CNV active for AMD, including juxtavoveal lesions that affect the fovea as evidenced by AF in the study eye.
[00160] The key exclusion criteria are as follows: (1) anti-VEGF IVT therapy in the study eye within 8 weeks of study initiation (Day 1); (2) any previous treatment with PDGF or PDGFR inhibitors; (3) intraocular pressure greater than or equal to 25 mmHg in the study eye; (4) evidence of infectious blepharitis, keratitis, scleritis, or conjunctivitis in any eye; (5) any intraocular inflammation / infection in any eye within 3 months of the screening visit; (6) current neovascularization of the iris, vitreous hemorrhage, or retinal detachment of visible traction in the selection evaluations in the study eye; (7) evidence of CNV due to any cause other than AMD in any eye; (8) evidence of diabetic retinopathy or diabetic macular edema in any eye; (9) inability to obtain photographs, FA or OCT to document CNV, for example, due to opaque media, allergy to fluorescein dye or lack of venous access; and (10) systemic anti-VEGF (IV) administration within 6 weeks of Day 1. Study Design
[00161] Patients will be evaluated for study choice at the selection visit, up to 2 weeks before Day 1 / baseline (Visit 2). On Day 1 / baseline (Visit 2), patients will undergo safety assessments before receiving the first study drug dose.
[00162] Eligible patients will be enrolled in the current cohort that is open for enrollment. The initial cohort will receive anti-PDGFR-beta / aflibercept (co-formulated in 0.2 mg: 2 mg). On Day 1 and Day 29 (+/- 3 days), patients will receive an injection of anti-PDGFR-beta / aflibercept.
[00163] The dose of anti-PDGFR-beta / aflibercept will be escalated based on safety and tolerability assessed during the previous cohort (starting from the first patient, first dose 2 weeks following the second dose of the last patient in that cohort, or approximately Week 6) . Also, the first patient enrolled in each cohort will be observed for at least 1 week after the first dose before additional patients are dosed. Climbing the next dose cohort will occur once the data has been reviewed. Intra-patient dose ladder will not be allowed.
[00164] Patients will be evaluated during study visits for eye and systemic safety (including ophthalmic examination, laboratory evaluations, etc.) and efficacy (OCT, FA / FP, CNV area, classic CNV size, total lesion size, macular volume , imaging, and BCVA using 4 meter0 ETDRS protocol and will be followed up for Week 24. Drug Treatment Study
[00165] Four different co-formulations of anti-PDGFR-beta / aflibercept will be administered to patients. Co-formulations are summarized in Table 12.
[00166] Table 12

[00167] Each formulation will consist of 10 mM sodium phosphate, pH 6.2, 0.03% (weight / volume) polysorbate 20, sucrose 5% (weight / volume), and 40 mM sodium chloride.
[00168] The various co-formulations of anti-PDGFR-beta / aflibercept will be released via IVT injection and the injection volume will be 50 microliters. As noted above, patients will receive two separate co-formulation administrations. The first administration will be on Day 1, and the second administration will be on Day 29. Primary and secondary end points
[00169] The primary end point of the study is study drug safety. Secondary end points are: (1) change in central retinal thickness from baseline (measured by OCT) at Week 8 and Week 12; (2) proportion of patients with complete retinal fluid resolution (measured by OCT) at Week 8 and Week 12; (3) change in CNV area from baseline (measured by OCT) in Week 8 and Week 12; (4) change in CNV size from baseline (measured by FA) at Week 8 and Week 12; (5) change in leakage area from baseline (measured by FA) at Week 8 and week 12; (6) change in BCVA from baseline; and (7) kinetic drugs and development of anti-drug antibodies.
[00170] The present invention is not limited in scope by the specific modalities described herein. Indeed, various modifications of the invention in addition to those described herein will become visible to those skilled in the art from the previous description and the accompanying figures. Such modifications are intended to fall within the scope of the stated claims.

权利要求:
Claims (6)
[0001]
1. Isolated antibody or antigen binding fragment thereof, characterized by the fact that it specifically binds to the dimeric human platelet-derived growth factor beta receptor (PDGFR-beta) with a binding dissociation equilibrium constant (KD) less than 200 pM measured in a 37 ° C surface plasmon resonance assay, in which the antibody or antigen binding fragment of the same blocks the binding of at least one PDGF ligand to the PDGFR-beta; the antibody or antigen binding fragment thereof blocks PDGF-BB ligand binding to soluble monomeric PDGFR-beta with an IC50 value of less than 150 pM measured in an in vitro receptor / ligand binding assay at 25 ° C; and the antibody or antigen binding fragment thereof inhibits PDGF-signaling PDGF-beta ligand-mediated activation in cells expressing PDGFR-beta; wherein the antibody or antigen binding fragment thereof comprises: three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) comprising SEQ ID NOs: 132, 134 and 136, respectively; and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) comprising SEQ ID NOs: 140, 142 and 144, respectively.
[0002]
2. Antigen or antigen binding fragment according to claim 1, characterized in that the antibody or antigen binding fragment thereof comprises a heavy chain variable region (HCVR) comprising a SEQ ID NO: 130, and a light chain variable region (LCVR) comprising a SEQ ID NO: 138.
[0003]
Antibody according to claim 1 or 2, characterized by the fact that it is an IgG1 or IgG4 antibody.
[0004]
Pharmaceutical composition, characterized by the fact that it comprises antibody as defined in any one of claims 1 to 3, or an antigen binding fragment, as defined in claim 1 or 2, and a pharmaceutically acceptable carrier or diluent.
[0005]
Pharmaceutical composition according to claim 4, characterized by the fact that it further comprises a VEGF antagonist selected from the group consisting of aflibercept, bevacizumab, or ranibizumab.
[0006]
6. Use of a pharmaceutical composition as defined in claim 4, characterized in that it is for preparing a medicament for the treatment of an eye disease selected from the group consisting of age-related macular degeneration (AMD), exudative AMD, diabetic retinopathy, central vein occlusion retina (CRVO), iris neovascularization, neovascular glaucoma, post-surgical glaucoma fibrosis, proliferative vitreous-retinopathy (PVR), choroidal neovascularization, optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus pterygium, macular edema, diabetic macular edema (DME), vascular retinopathy, retinal degeneration, uveitis, and inflammatory eye diseases.

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NZ709683A|2019-01-25|

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-06| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-13| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-20| B06I| Publication of requirement cancelled [chapter 6.9 patent gazette]|Free format text: ANULADA A PUBLICACAO CODIGO 6.6.1 NA RPI NO 2462 DE 13/03/2018 POR TER SIDO INDEVIDA. |

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2019-08-20| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-10-01| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-05-12| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-10-13| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-15| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/01/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361750437P| true| 2013-01-09|2013-01-09|

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US61/750,437|2013-01-09|

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US201361863452P| true| 2013-08-08|2013-08-08|

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US61/863,452|2013-08-08|

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US201361909421P| true| 2013-11-27|2013-11-27|

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US61/909,421|2013-11-27|

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PCT/US2014/010395|WO2014109999A1|2013-01-09|2014-01-07|ANTI-PDGFR-beta ANTIBODIES AND USES THEREOF|

---