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    • 专利摘要:
    cgrp antibodies. the present invention provides peptide antibodies related to the human planned calcitonin gene (cgrp) or its peptide binding fragments related to the human planned calcitonin gene (cgrp) or its antigen binding fragments for the treatment of osteoarthritis pain .
    公开号:BR112012031501B1
    申请号:R112012031501-3
    申请日:2011-06-07
    公开日:2020-02-11
    发明作者:Barrett Allan；Robert Jan BENSCHOP；Mark Geoffrey CHAMBERS；Ryan James DARLING
    申请人:Eli Lilly And Company；
    IPC主号:
    专利说明:

    Invention Patent Descriptive Report for HUMAN ENGINEERED ANTI-CGRP ANTIBODIES, FRAGMENT OF THE SAME, AND PHARMACEUTICAL COMPOSITIONS.
    The present invention is in the field of medicine, particularly in the field of antibodies to the peptide related to the calcitonin gene (CGRP). More specifically, the invention relates to CGRP antibodies and the use of CGRP antibodies for the therapy of osteoarthritis or migraine pain.
    The calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide secreted by the nerves of the central and peripheral nervous systems. CGRP is widely distributed in sensory nerves, in both the peripheral and central nervous systems and has a large number of different biological activities. The CGRP is reported to play a role. When released from the fibers of the trigeminal and other nerve, CGRP is supposed to mediate its biological responses by binding to specific cell surface receptors.
    CGRP has been reported to play a role in migraines when CGRP is released after sensory nerve stimulation and has potent vasodilatory activity. The release of CGRP increases vascular permeability and the subsequent leakage of plasma proteins (plasma protein leak) in the tissues innervated by trigeminal nerve fibers after stimulation of these fibers. In addition, studies have reported that the infusion of CGRP in patients suffering from migraines has resulted in symptoms similar to those of migraine.
    CGRP is also reported to play a role in osteoarthritis (OA). OA is a chronic condition that affects a large percentage of people worldwide. Arthritis pain is characterized by hyperalgesia (excessively sensitive to normally non-harmful stimuli) and spontaneous pain (pain at rest). Inflammation of the joint causes peripheral and central sensitization. In peripheral sensitization, nociceptors containing normally elevated threshold CGRP begin to respond to the slight pressure associated with normal joint movement. The CGRP acts as a local facilitator of inflammatory processes. This ongoing process
    Petition 870190077218, of 08/09/2019, p. 12/13
    2/27 eventually leads to central sensitization in the spinal cord in such a way that neurons become sensitive to the stimulation of inflamed as well as non-inflamed tissue. In preclinical models of OA pain in the rat, an increase in the number of positive CGRP nerve fibers was observed, 5 positively correlated with the amount of pain.
    The current standard of treatment for OA pain consists of non-steroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors. Over time, many patients will become insensitive to these treatments or are unable to tolerate the high doses required - 10 to relieve effective pain. Frequent treatments consist of intra-articular injections (hyaluronic acid) or opiates. The final treatment is total surgical replacement of the joint. Many of these treatments have negative side effects, ranging from gastrointestinal (NSAIDs) to cardiovascular (COX-2 inhibitors), are bothersome and / or not very effective 15 (intra-articular injections), carry the risk of abuse (opiates), or are restricted to just one joint (intra-articular injections, surgery).
    Although CGRP antibodies for the treatment of migraine and the treatment of pain from inflammation have been disclosed (migraines, WO 2007/076336 and WO 2007/054809; OA see, Antibody G1 of WO 20 2009 / 109908A1), there is still a need for alternatives. Preferably, the alternative treatment is safe and effective. Most preferably, the alternative treatment for OA will provide long-lasting pain relief across multiple joints that is tolerable for patients without the risk of addiction and / or abuse.
    The present invention provides a human-designed CGRP antibody comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein said LCVR comprises the amino acid sequences LCDR1, LCDR2 and LCDR3 and HCVR comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 that are taught from the group consisting of:
    a) LCDR1 is RASQDIDNYLN (SEQ ID NO: 3), LCDR2 is YTSEYHS (SEQ ID NO: 4), LCDR3 is QQGDALPPT (SEQ ID NO: 5), HCDR1 is
    3/27
    GYTFGNYWMQ (SEQ ID NO: 12), HCDR2 is AIYEGTGDTRYIQKFAG (SEQ ID NO: 13), and HCDR3 is LSDYVSGFSY (SEQ ID NO: 14);
    b) LCDR1 is RASQDIDNYLN (SEQ ID NO: 3), LCDR2 is YTSE-
    YHS (SEQ ID NO: 4), LCDR3 is QQGDALPPT (SEQ ID NO: 5), HCDR1 is
    GYTFGNYWMQ (SEQ ID NO: 12), HCDR2 is AIYEGTGKTVYIQKFAG (SEQ ID NO: 15), and HCDR3 is LSDYVSGFSY (SEQ ID NO: 14);
    c) LCDR1 is RASKDISKYLN (SEQ ID NO: 6), LCDR2 is YTSGYHS (SEQ ID NO: 7), LCDR3 is QQGDALPPT (SEQ ID NO: 5), HCDR1 is GYTFGNYWMQ (SEQ ID NO: 12), HCDR2 is AIYQTKK SEQ. 10 ID NO: 16), and HCDR3 is LSDYVSGFGY (SEQ ID NO: 39);
    d) LCDR1 is RASRPIDKYLN (SEQ ID NO: 8), LCDR2 is YTSE 'YHS (SEQ ID NO: 4), LCDR3 is QQGDALPPT (SEQ ID NO: 5), HCDR1 is
    GYTFGNYWMQ (SEQ ID NO: 12), HCDR2 is AIYEGTGKTVYIQKFAG (SEQ ID NO: 15), and HCDR3 is LSDYVSGFGY (SEQ ID NO: 39); and
    e) LCDR1 is RASQDIDKYLN (SEQ ID NO: 9), LCDR2 is YTSGYHS (SEQ ID NO: 7), LCDR3 is QQGDALPPT (SEQ ID NO: 5), HCDR1 is GYTFGNYWMQ (SEQ ID NO: 12), HCDR2 is AIYQTKKK SEQ ID NO: 15), and HCDR3 is LSDYVSGFGY (SEQ ID NO: 39), wherein the human planned CGRP antibody binds to humano human GRP.
    The present invention provides a human-designed CGRP antibody comprising an LCVR and an HCVR where LCDR1 is SEQ ID NO: 3, LCDR2 is SEQ ID NO: 4, LCDR3 is SEQ ID NO: 5, HCDR1 is SEQ ID NO: 12, HCDR2 is SEQ ID NO: 13, and HCDR3 is SEQ ID NO: 14, wherein the planned human CGRP antibody binds to human CGRP. The present invention provides a human planned CGRP antibody or antigen-binding fragment thereof comprising an LCVR and an HCVR where LCDR1 is SEQ ID NO: 3, LCDR2 is SEQ ID NO: 4, LCDR3 is SEQ ID NO: 5, HCDR1 is SEQ ID NO: 12, HCDR2 is SEQ ID NO: 15, and HCDR3 is 30 SEQ ID NO: 14, wherein the human planned CGRP antibody binds to human CGRP. The present invention provides a human-designed CGRP antibody comprising an LCVR and an HCVR where LCDR1 is
    4/27
    SEQ ID NO: 6, LCDR2 is SEQ ID NO: 7, LCDR3 is SEQ ID NO: 5, HCDR1 is
    SEQ ID NO: 12, HCDR2 is SEQ ID NO: 16, and HCDR3 is SEQ ID NO: 39, wherein the planned human CGRP antibody binds to human CGRP. The present invention provides a human-designed CGRP antibody with 5 comprising an LCVR and an HCVR where LCDR1 is SEQ ID NO: 8,
    LCDR2 is SEQ ID NO: 4, LCDR3 is SEQ ID NO: 5, HCDR1 is SEQ ID NO: 12,
    HCDR2 is SEQ ID NO: 15, and HCDR3 is SEQ ID NO: 39, wherein the human planned CGRP antibody binds to human CGRP. The present invention provides a human-designed CGRP antibody comprising a - 10 LCVR and an HCVR where LCDR1 is SEQ ID NO: 9, LCDR2 is SEQ ID
    NO: 7, LCDR3 is SEQ ID NO: 5, HCDR1 is SEQ ID NO: 12, HCDR2 is SEQ
    ID NO: 15, and HCDR3 is SEQ ID NO: 39, wherein the planned human CGRP antibody binds to human CGRP.
    The present invention provides a human-designed CGRP antibody comprising an LCVR and an HCVR where LCDR1 is
    RASXiX 2 IX 3 X4YLN (SEQ ID NO: 10), LCDR2 is YTSX 5 YHS (SEQ ID NO:
    11), LCDR3 is QQGDALPPT (SEQ ID NO: 5) and HCDR1 is
    GYTFGNYWMQ (SEQ ID NO: 12), HCDR2 is AIYEGTGX 6 TX 7 YIQKFAX 8 (SEQ ID NO: 37), and HCDR3 is LSDYVSGFX 9 Y (SEQ ID NO: 38), where
    Xi is Q, R, or K; X 2 is D or P, X 3 is D or S, X4 is N or K, X5 is G or E, X 8 is K or D, X 7 is V or R, X 8 is D or G, and X 9 is G or S, where the antibody binds to human CGRP.
    In another embodiment, the present invention provides a human-designed CGRP antibody comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises
    DIQMTQSPSSLSASVGDRVTITCRASXiX 2 IX 3 X 4 YLNWYQQKPGKAPKLLIYYTSX 5 YHSGVPSRFSGSGSGTD
    FTX 6 TISSLQPEDX 7 ATYYCQQGDALPPTFGX 8 GTKX 9 EIK where X1 is Q, K, or R; X 2 is D or P; X 3 is D or S; X4 is K or N; X 5 is E or G; X 6 is F or L; X 7 is I or F; X 8 is Q or G; and X 9 is L or V. (SEQ ID NO: 42) and HCVR comprises
    5/27
    QVQLVQSGAEVKKPGX1SVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTG X 2 TX 3 YIQKFAX4RVTX 5 TX 6 DX 7 STSTX 8 YMELSSLRSEDTAVYYCARLSDWSQFGY; X 2 = K or D; X 3 = V or R; X4 =
    G or D; X 5 = M or I; X 6 = R or A; X 7 = T or K; X 8 = V or A; Xg = G or S; and Xio = L or T. (SEQ ID NO: 43), where the antibody binds to human CGRP.
    In another embodiment, the present invention provides a human-designed CGRP antibody comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), where ΙΟΙ 0 VR and HCVR are selected amino acid sequences from the group consisting of:
    The. LCVR is SEQ ID NO: 17 and HCVR is SEQ ID NO: 22;
    B. LCVR is SEQ ID NO: 18 and HCVR is SEQ ID NO: 23;
    ç. LCVR is SEQ ID NO: 19 and HCVR is SEQ ID NO: 24;
    d. LCVR is SEQ ID NO: 20 and HCVR is SEQ ID NO: 25; and
    and. LCVR is SEQ ID NO: 21 and HCVR is SEQ ID NO: 26.
    The present invention provides a human-designed CGRP antibody comprising an LCVR of SEQ ID NO: 17 and an HCVR of SEQ ID NO: 22. In a preferred embodiment, the present invention provides a human-designed CGRP antibody comprising an LCVR of SEQ ID NO: 18 and an HCVR of SEQ ID NO: 23. The present invention provides a human planned CGRP antibody comprising an LCVR of SEQ ID NO: 19 and an HCVR of SEQ ID NO: 24. The present invention provides an antibody of Human planned CGRP comprising an LCVR of 25 SEQ ID NO: 20 and an HCVR of SEQ ID NO: 25. The present invention provides a human planned CGRP antibody comprising an LCVR of SEQ ID NO: 21 and an HCVR of SEQ ID NO: 26.
    The present invention also provides a human-designed CGRP antibody comprising a light chain (LC) and a heavy chain (HC), wherein the amino acid sequences of LC and HC are selected from the group consisting of:
    a) LC is SEQ ID NO: 27 and HC is SEQ ID NO: 32;
    6/27
    b) LC is SEQ ID NO: 28 and HC is SEQ ID NO: 33;
    c) LC is SEQ ID NO: 29 and HC is SEQ ID NO: 34;
    d) LC is SEQ ID NO: 30 and HC is SEQ ID NO: 35; and
    e) LC is SEQ OD NO: 31 and HC is SEQ ID NO: 36.
    In one embodiment, the human-designed CGRP antibody comprises an LC of SEQ ID NO: 27 and an HC of SEQ ID NO: 32. In another embodiment, the human-designed CGRP antibody comprises an LC of SEQ ID NO: 28 and an HC of SEQ ID NO: 33. In another embodiment, the human planned CGRP antibody comprises an LC of SEQ -10 ID NO: 29 and an HC of SEQ ID NO: 34. In another embodiment, the human planned CGRP antibody comprises an LC of SEQ ID NO: 30 and an HC of SEQ ID NO: 35. In another embodiment, the human planned CGRP antibody comprises an LC of SEQ ID NO: 31 and an HC of SEQ ID NO: 36. In one embodiment , the human planned CGRP antibody with 15 comprises two light chains and two heavy chains where each LC amino acid sequence is SEQ ID NO: 27 and each HC amino acid sequence is SEQ ID NO: 32. In one embodiment, the antibody of human planned CGRP comprises two light chains and two heavy chains where each LC amino acid sequence is SEQ ID NO: 28 and each 20 amino acid sequence of HC is SEQ ID NO: 33. In one embodiment, the human planned CGRP antibody comprises two light chains and two heavy chains where each LC amino acid sequence is SEQ ID NO: 29 and each sequence HC amino acid sequence is SEQ ID NO: 34. In one embodiment, the human planned CGRP antibody comprises two le25 chains and two heavy chains where each LC amino acid sequence is SEQ ID NO: 30 and each amino acid sequence of HC is SEQ ID NO: 35. In one embodiment, the human planned CGRP antibody comprises two light chains and two heavy chains where each LC amino acid sequence is SEQ ID NO: 31 and each HC amino acid sequence is SEQ ID NO: 36. The present invention also provides antigen-binding fragment of the antibodies described herein.
    The present invention also provides antibodies to CGRP pia
    7/27 human levels that have an IC 50 <1.0 nM in an assay essentially as described in Example 4. In one embodiment, the present invention provides a human-designed CGRP antibody comprising a
    LCVR and HCVR where LCDR1 is SEQ ID NO: 6, LCDR2 is SEQ ID NO: 7, LCDR3 is SEQ ID NO: 5, HCDR1 is SEQ ID NO: 12, HCDR2 is SEQ
    ID NO: 16, and HCDR3 is SEQ ID NO: 39, and where the planned human CGRP antibody binds to human CGRP and also has an IC 50 <1.0 nM in an assay essentially as described in Example 4. In one embodiment, the present invention provides a CGRP antibody designed
    - 10 human comprising an LCVR amino acid sequence of SEQ
    ID NO: 19 and an HCVR amino acid sequence of SEQ ID NO: 24, wherein the human planned CGRP antibody has an IC 50 <1.0 nM in an assay essentially as described in Example 4. In one embodiment, the present The invention provides a human planned CGRP antibody comprising an LC amino acid sequence of SEQ ID NO: 29 and an HC amino acid sequence of SEQ ID NO: 34, wherein the human planned CGRP antibody has an IC 50 <1.0 nM in an assay essentially as described in Example 4.
    The present invention also provides human-designed CGRP antibodies having an IC 50 <0.5 nM in an assay essentially as described in Example 4. In one embodiment, the present invention provides a human-designed CGRP antibody comprising an LCVR and a HCVR where LCDR1 is SEQ ID NO: 6, LCDR2 is
    SEQ ID NO: 7, LCDR3 is SEQ ID NO: 5, HCDR1 is SEQ ID NO: 12,
    HCDR2 is SEQ ID NO: 16, and HCDR3 is SEQ ID NO: 39, and where the human planned CGRP antibody binds to human CGRP and also has an IC 5 o <0.5 nM in an essentially conforming assay described in Example 4. In one embodiment, the present invention provides a human-designed CGRP antibody comprising an LCVR amino acid sequence of SEQ ID NO: 19 and an amino acid sequence of
    HCVR of SEQ ID NO: 24, wherein the human planned CGRP antibody has an IC 50 <0.5 nM in an assay essentially as described in
    8/27
    Example 4. In one embodiment, the present invention provides a human engineered CGRP antibody comprising an LC amino acid sequence of SEQ ID NO: 29 and an HC amino acid sequence of SEQ ID NO: 34, wherein the CGRP antibody human design has an IC 50 <5 0.5 nM in an assay essentially as described in Example 4.
    The present invention also provides a pharmaceutical composition comprising a human-designed CGRP antibody or its antigen-binding fragment of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. The present invention further provides a pharmaceutical composition which comprises a CGRP antibody of the present invention together with a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.
    In another aspect, the present invention provides a method of treating osteoarthritis pain comprising administering to a patient with his need for a planned human CGRP antibody or its antigen-binding fragment of the present invention. In another aspect, the present invention provides a method of treating migraine comprising administering to a patient with his need for a planned human CGRP antibody or antigen binding fragment thereof of the present invention.
    The present invention also provides a human-designed CGRP antibody or its antigen-binding fragment of the present invention for use in therapy. In another aspect, the present invention provides a human-designed CGRP antibody or its binding fragment 25 to the antigen of the present invention for the treatment of osteoarthritis pain.
    In a further aspect, the present invention provides a human-designed CGRP antibody or its antigen-binding fragment of the present invention for the treatment of migraines.
    The present invention also provides for the use of a planned human CGRP antibody or its antigen-binding fragment of the present invention for use in the treatment of osteoarthritis pain. In another aspect, the present invention also provides for the use of an antibody of
    9/27
    Human-designed CGRP or its antigen-binding fragment of the present invention for use in the treatment of migraines. The present invention also provides for the use of a planned human CGRP antibody or its antigen-binding fragment in the manufacture of a medicament for the treatment of osteoarthritis. The present invention also provides for the use of a human-designed CGRP antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment of migraines.
    A full-length antibody as it naturally exists is
    - 10 an immunoglobulin molecule comprising 2 heavy chains (H) and light chains (L) interconnected by disulfide bonds. The aminoterminal portion of each chain includes a variable region of about 100 to 110 amino acids, mainly responsible for the recognition of the antigen through the complementarity determining regions (CDRs) contained therein. The carboxy-terminal portion of each chain defines a constant region primarily responsible for the effector function.
    CDRs are interspersed with regions that are more conserved, called structure regions (FR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 20 of 3 CDRs and 4 FRs, arranged from the amino-terminal to the carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. At
    Light chain CDRs are referred to as LCDR1, LCDR2 and LCDR3 and the 3 heavy chain CDRs are referred to as HCDR1, HCDR2 and HCDR3. CDRs contain most of the residues that form specific interactions with the antigen. The numbering and positioning of the CDR amino acid residues within the LCVR and HCVR regions is in accordance with the well-known Kabat numbering convention.
    Light chains are classified as a cape or lambda, and are characterized by a particular constant region as known in the art. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, and define the isotype of an antibody as IgG, IgM, IgA, IgD or IgE, respectively. IgG antibodies can be further divided into subclasses
    10/27 months, for example, lgG1, lgG2, lgG3, lgG4. Each type of heavy chain is characterized by a particular constant region with a sequence well known in the art.
    As used herein, the term monoclonal antibody (Mab) refers to an antibody that is derived from a single copy or clone including, for example, any eukaryotic, prokaryotic or phage clone, and not the method by which it is produced. The Mabs of the present invention preferably exist in a homogeneous or substantially homogeneous population. Complete Mabs contain 2 heavy chains and 2 light chains. Antigen-binding fragments of such monoclonal antibodies include, for example, Fab fragments, Fab 'fragments, F (ab') 2 fragments, θ isolated chain Fv fragments. Monoclonal antibodies and their antigen-binding fragments of the present invention can be produced, for example, using recombinant technologies, phage display technologies, synthetic technologies, for example, CDR grafting, or combinations of such technologies, or other technologies known in the art. For example, mice can be immunized with human CGRP or its fragments, the resulting antibodies can be recovered and purified, and the determination of whether they have similar binding and functional properties or the same as the antibody compounds disclosed herein, can be evaluated. by the methods disclosed in the Examples below. Antigen-binding fragments can also be prepared by conventional methods. Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be seen, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapters 5-8 and 15, ISBN 0-87969-314-2.
    The term human-designed CGRP antibodies refers to monoclonal antibodies created and / or manipulated to have binding and functional properties according to the invention, that bind to human CGRP and that have structural regions that are substantially human or fully human surrounding the CDRs derived from an anti11 / 27 non-human body. Antigen-binding fragments of such human target antibodies include, for example, Fab fragments, Fab 'fragments, F (ab') 2 fragments, and isolated chain Fv fragments. The structural region or structure sequence refers to any of the structural regions 1 to 4. The human target antibodies and their antigen-binding fragments included by the present invention include molecules in which any one or more of the structural regions 1 to 4 4 is substantially or completely human, that is, in which any of the possible combinations of substantially or completely human structural regions from -10 1 to 4, is present. For example, this includes molecules in which backbone 1 and backbone 2, and backbone 1 and backbone 3, backbone 1, 2 and 3, etc., are substantially or completely human. Substantially human structures are those that have at least about 80% sequence identity to a known human germline structure sequence. Preferably, the substantially human structures have at least about 85%, about 90%, about 95%, or about 99% sequence identity to a known human germline structure sequence.
    The completely human ones are those that are identical to a known human germline structure sequence. Human structure germline sequences can be obtained from ImMunoGeneTics (IMGT) through their website http://imgt.cines.fr, or The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, ISBN 012441351. For example, germline light chain structures can be selected from the group consisting of: A11, A17, A18, A19, A20, A27, A30, L1, L11, L12, L2, L5, L15 , L6, L8, 012, 02, and 08, and the germline heavy chain structure regions can be selected from the group consisting of: VH25, VH2-26, VH2-70, VH3-20, VH3-72 , VHI-46, VH3-9, VH3-66, VH3-74, VH430 31, VHI-18, VHI-69, VI-13-7, VH3-11, VH3-15, VH3-21, VH3-23, VH3-30,
    VH3-48, VH4-39, VH4-59, and VH5-51.
    Humanly designed antibodies in addition to those disclosed herein12 / 27 of those having similar functional properties according to the present invention can be generated using several different methods. The specific antibody compounds described herein can be used as parent antibody templates or compounds for the preparation of additional antibody compounds. In one approach, the source antibody compound CDRs are grafted into a human structure that has a high sequence identity to the source antibody compound structure. The sequence identity of the new structure will generally be at least about 80%, at least about 85%, at least - 10 about 90%, at least about 95%, or at least about 99% identical to the sequence of corresponding structure in the parent antibody compound. This graft can result in a reduction in binding affinity compared to that of the parent antibody. If this is the case, the structure can be transformed back to the original structure in certain positions based on the specific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad. Know. USA 88: 2869. Additional references describing methods useful in humanizing mouse antibodies include US Patent Nos. 4,816,397; 5,225,539 and 5,693,761; ABMOD and ENCAD computer programs as described in Levitt (1983) J. Mol. Biol.
    Biol. 168: 595-620; and the method of Winter et al. (Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; and Verhoeyen etal. (1988) Science 239: 1534-1536.
    The identification of residues to consider the back mutation can be performed as follows:
    When an amino acid falls under the following category, the structural amino acid of the human germline sequence being used (the acceptor structure) is replaced by a structural amino acid from a structure of the original antibody compound (the donor structure):
    (a) the amino acid in the human structural region of the acceptor structure is unusual for human structures in that position, while the corresponding amino acid in the donor immunoglobulin is typical for
    13/27 the human structures in that position;
    (b) the position of the amino acid is immediately adjacent to one of the CDRs; or (c) any atom of the side chain of a structural amino acid is within about 5 to 6 angstrons (center to center) of any atom of an amino acid of the CDR in a three-dimensional immunoglobulin model.
    When each of the amino acids in the human structural region of the acceptor structure and a corresponding amino acid in the donor structure is generally uncommon for human structures in that position, that amino acid can be replaced by a typical amino acid for human structures in that position. This back mutation criterion allows to recover the activity of the original antibody compound.
    Another approach for generating human-designed antibodies that have similar functional properties to the antibody compounds disclosed herein involves randomly mutating amino acids within the grafted CDRs without altering the structure, and screening the resulting molecules for binding affinity and other functional properties that are as good as or better than those of the parent antibody compounds. Isolated mutations can also be introduced at each amino acid position in each CDR, followed by assessing the effects of such mutations on binding affinity and other functional properties. Isolated mutations that produce improved properties can be combined to assess their effects on combining with each other.
    In addition, a combination of both of the above approaches is possible. After CDR grafting, it is possible to return to specific structural regions in addition to the introduction of amino acid changes in the CDRs. This methodology is described in Wu et al. (1999) J. Mol. Biol. 294: 151-162.
    Applying the teachings of the present invention, a person skilled in the art can use common techniques, for example, site-directed mutagenesis, to replace the amino acids within the currently described CDR and the structural sequences, and thus generate other amino acid sequences from variable region from the present sequences
    14/27 cias. All alternative naturally occurring amino acids can be introduced at a specific substitution site. The methods disclosed herein can then be used to control these additional variable region amino acid sequences to identify the sequences having the 5 in vivo functions indicated. In this way, other sequences suitable for the preparation of human target antibodies and their antigen binding parts according to the present invention can be identified. Preferably, amino acid substitution within structures is restricted to one, two or three positions within any one or more of the 4 regions. 10 structural light chain and / or heavy chain disclosed herein. Preferably, amino acid substitution within the CDRs is restricted to one, two, or three positions within any one or more of the 3 light chain and / or heavy chain CDRs. Combinations of the various changes within these framework regions and CDRs described above are also possible.
    The term treatment (or treating) refers to processes that involve a delay, interruption, detention, control, stop, reduction or reversal of the progression or severity of a symptom, disorder, condition or disease, but does not necessarily involve a total elimination of all symptoms, conditions or disorders related to the disease associated with CGRP activity.
    The term migraine as used herein refers to migraines without aura (formerly common migraine) and migraines with aura (formerly classic migraines) according to the Headache Classification Committee of the International Headache Society (International 25 Headache Society, 2004). For example, migraines without aura, typically can be characterized as having a pulsating quality, moderate or severe intensity, are aggravated by routine physical activity, are unilaterally located and are associated with nausea and with photophobia and phonophobia. Migraines with aura can include disturbances in vision, disturbances in other senses, unilateral weakness, and, in some cases, difficulty with speech.
    The human target antibodies of the present invention can
    15/27 be used as medicines in human medicine, administered by a variety of routes. Most preferably, such compositions are for parenteral administration. Such pharmaceutical compositions can be prepared by methods well known in the art (See, e.g., Remington:.. The Science and Practice of Pharmacy, 19 th ed (1995), A. Gennaro et al, Mack Publishing Co.) and comprise a human target antibody as disclosed herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
    The results of the following tests demonstrate that the exemplified monoclonal antibodies and their antigen-binding fragments of the present invention can be used for the treatment of osteoarthritis pain.
    Example 1 - Production of Antibodies
    Antibodies from I to V can be produced and purified as follows. An appropriate host cell, such as HEK 293 EBNA or CHO, is transiently or stably transfected with an expression system to secrete antibodies using a predetermined HC: LC vector ratio or an isolated vector system that encodes both LC, such as such as SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31 for HC such as SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36. Clarified media in which the antibody was secreted are purified using any of the many commonly used techniques. For example, the medium can be conveniently applied to a Protein A or G Sepharose FF column that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4). The column is washed to remove non-specific binding components. The bound antibody is eluted, for example, by the pH gradient (such as 0.1 M sodium phosphate buffer pH 6.8 with 0.1 M sodium citrate buffer pH 3.0). Antibody fractions are detected, such as by SDS-PAGE, and are then pooled. Another purification is optional, depending on the intended use. The antibody can be concentrated and / or filtered sterile using common techniques. The soluble aggregate and multimers can be effective
    16/27 effectively removed using common techniques, including size exclusion, hydrophobic interaction, ion exchange or hydroxyapatite chromatography. The purity of the antibody after these chromatography steps is greater than 99%. The product can be immediately frozen at -70 ° C or can be lyophilized. The amino acid sequences for these exemplified antibodies are provided below.
    Table 1 - SEQ IP Antibody
    Antibody Heavy Chain Light Chain LCVR HCVR I 32 27 17 22 II 33 28 18 23 III 34 29 19 24 IV 35 30 20 25 V 36 31 21 26
    Antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 I 12 13 14 3 4 5 II 12 15 14 3 4 5 III 12 16 39 6 7 5 IV 12 15 39 8 4 5 V 12 15 39 9 7 5
    Example 2: Affinity measures (Kd) for human-designed CGRP antibodies
    The binding affinity of the exemplified antibodies to the CGRP is determined using a superficial plasmon resonance assay on a Biacore T100 instrument provided with HBS-EP + operating buffer (GE Healthcare, 10 mM Hepes pH 7.4 + 150 mM NaCI + 3 mM EDTA + 0.05% surfactant P20) and analysis temperature adjusted to 37 ° C. A CM5 chip containing immobilized protein A (generated using the standard NHS-EDC amine coupling) in all four flow cells (Fc) is used to employ a capture methodology. Antibody samples are prepared at 2 pg / ml by diluting in operating buffer. Human CGRP samples are prepared in final concentrations of 5.0, 2.5, 1.3, 0.63, 0.31 and 0 (white) nM by dilution in size
    17/27 operation bread. A new single-use rate of CGRP is used for each repeated experiment to avoid multiple freeze-thaw cycles. Each analysis cycle consists of (1) capture of antibody samples in separate flow cells (Fc2, Fc3 and Fc4), (2) injection of 350 pl (210 sec) of CGRP over all Fc at 100 μΙ / min , (3) return to the buffer flow for 10 minutes to monitor the dissociation phase, (4) regeneration of the chip surfaces with an injection of 5 μΙ (15 sec) of glycine, pH 1.5, (5) surface balance the chip with a 5 μΙ (15 sec) injection of HBS-EP +. Each concentration of CGRP is injected • 10 in duplicate. The data are processed using standard double reference and adjustment for a 1: 1 connection model using the Biacore T100 Evaluation software, version 2.0.1, to determine the association rate (k on units, M ' 1 s' 1 ), dissociation rate (units (kOff, s' 1 ), θ Rmax (units RU). The equilibrium dissociation constant (Kd) is calculated from the connection KD = k o n / kon. θ is in molar units. The values provided in the
    Table 2 are the averages of n numbers of experiments. Table 2 demonstrates that the exemplified antibodies of the present invention bind to CGRP with affinities <200 pM.
    Table 2 - Antibody Binding Affinities.
    Antibody K D (pM) Mean + SD n I 190 1 II 26 + 6 3 III 31 + 19 6 IV 24 + 10 4 V 24 + 6 2
    Example 3 - Pain reduction in an MIA model.
    The injection of monoiodoacetic acid (MIA) in the knee joint of rats produces an acute inflammatory insult that later develops into chronic degeneration of the joint tissues in the injected joint. The pain resulting from joint damage can be measured through the differential weight behavior of the hind legs using an inability checker.
    The MIA model has been well described in the literature and has been used to demonstrate effectiveness vs. a variety of mechanisms and compounds. The effectiveness
    18/27 is routinely measured by the ability of a compound to partially normalize weight distribution.
    To determine the ability of the exemplified antibodies of the present invention to reduce pain, male Lewis rats (Harlan, Indiana5 polis, IN) approximately eight weeks old at the time of MIA injection are used. The rats are housed in groups of two or three per cage and kept at a constant temperature and on a cycle of 12 hours of light / 12 hours in the dark. Animals have free access to food and water at all times, except during data collection. All experiments were carried out in accordance with the protocols approved by the Eli Lilly Institutional Animal Care and Use Committee.
    The right knees of each rat were injected with 0.3 mg of MIA in 50 pl of saline and the left knees with 50 μΙ of saline on day 0. On a given day after application of MIA, control of human IgG4 or 15 antibody of Test CGRP is administered subcutaneously in PBS (n = 6 per group). Three days after dosing with test CGRP antibody, pain is measured using the disability test which measures the difference in weight behavior of the hind paw between the knees treated with MIA and injected with saline. Each measurement is the average of three separate measurements of 20 each measurement for 1 second.
    The data are presented as percentage of pain inhibition calculated by dividing the mean of the group treated with CGRP antibody by the mean of the control antibody group, subtracting this value from 1 and multiplying that resulting value by 100. The groups treated with 25 CGRP they were also compared with vehicle groups by means of analysis of means and by Dunnett's test, using the statistical analysis program JMP (versions 5.1 and 6) (SAS Institute Inc., Cary, NC). Differences should be considered significant if the P value is less than 0.05. As shown in Table 3, the data demonstrates that the exemplified CGRP antibodies of the present invention significantly reduce pain.
    Table 3 - Percentage of MIA-induced pain inhibition
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    Antibody Dose and route % inhibition with MIA Significant by Dunnett's test (p <0.05) I 20 mg / kg sc 20% Yes II 4 mg / kg sc 30% Yes III 4 mg / kg sc 37% Yes IV 4 mg / kg sc 37% Yes V 4 mg / kg sc 19% Yes
    EXAMPLE 4 - Inhibition of CGRP-induced cAMP formation in SK-N-MC cells
    To compare the ability of an antibody of the present invention to Antibody G1 (LCVR - SEQ ID NO: 40 and HCVR - SEQ ID NO: 41), the inhibition of CGRP-induced cAMP formation in SK-N-MC is determined. The binding of CGRP to its receptor results in stimulating the production of cAMP. The CGRP receptor is a heterotrimeric complex consisting of the Receptor similar to the Calcitonin receptor (CLR, a receptor coupled to the G protein) and the Receptor Activity Modifying Protein (RAMP) -1, coupled in a cytoplasmic manner to the Component Protein of the Receiver (RCP). The human neuroepithelioma cell line SK-N-MC expresses these three molecules naturally and can therefore be used to evaluate the effect of CGRP on signal transduction events. Production of cAMP is a standard measure for activation of the G protein-coupled receptor.
    SK-N-MC cells are cultured in MEM, containing 10% FBS, 1X MEM non-essential amino acids, 1X 100 mM MEM Sodium Pyruvate, 1X Pen / Strep, and 2 mM L-glutamine. After harvesting, the cells are washed once and resuspended in assay buffer (stimulus buffer (HBSS with Mg and Ca, 5 mM HEPES, 0.1% BSA, 100 uM ascorbic acid) diluted 1: 2 with Dulbecco's PBS containing a final concentration of 0.5 mM IBMX) and plated in 96 well plates in 15000 cells per well. The test antibody or a control human IgG4 antibody is added (4-fold serial dilutions in assay buffer, 10 concentrations) to the cells, followed by a fixed amount of human α-CGRP (2 nM; Bachem H1470). The plates are incubated
    20/27 for 1 hour at room temperature. CAMP levels are measured by a homogeneous time-determined fluorescence assay system (HTRF) (Cisbio).
    The amount of cAMP induced by 2 nM human CGRP in the presence of varying concentrations of antibody is calculated as a percentage of inhibition compared to CGRP alone. The antibody concentration that produces a 50% inhibition of cAMP production (IC 5 o) is then calculated from a 4-parameter curve fitting model. After the procedures as described here, antibody III (IC50 of 0.41 nM) inhibited the amount of CGRP-induced cAMP to a much greater extent than antibody G1 (IC50 of 5.36 nM), possibly due to the K rate on Antibody III fastest (Kon rate measured by Biacore). EXAMPLE 5 - Model of Extravasation of Duras Plasma Protein in Rats (PPE)
    The rat EPP model is a well-established preclinical model that can be used to assess the effectiveness of anti-CGRP antibodies for the treatment of migraine.
    A 2 mg / ml solution of an anti-CGRP antibody (Ab) is prepared in saline. All subsequent dilutions are prepared with saline. A 2 mg / ml solution of monoclonal isotype control antibody (IgG) is also prepared in saline.
    Male Sprague-Dawley rats from Harlan Laboratories (250 to 350 g) were anesthetized with Nembutal (60 mg / kg, ip.) And placed in a stereotaxic structure (David Kopf Instruments) with the incisor bar fixed at -2.5 mm. Following an incision in the midline sagittal scalp, two pairs of bilateral holes were drilled through the skull (3.2 mm later, 1.8 and 3.8 mm laterally, all coordinates referred to the bregma). Pairs of stainless steel stimulation electrodes (Rhodes Medical Systems Inc), insulated except at the tips, are reduced through the holes in both hemispheres to a depth of 9.2 mm below the dura. The test antibodies or saline vehicle are administered intravenously via the femoral vein (1 ml / kg). Eight mi
    21/27 nutos later a solution of fluorescein isothiocyanate tincture (FITC) labeled bovine serum albumin (BSA) (FITC-BSA, Sigma A9771) (20 mg / kg, iv.) Is injected into the femoral vein to function as a marker for protein leakage. Ten minutes after dosing with the test or vehicle antibodies, the left trigeminal ganglion is stimulated for 5 minutes at a current intensity of 1.0 mA (5 Hz, 5 ms duration) with a Model S48 Grass Instrument Stimulator. Five minutes after stimulation, the mice are killed by exsanguination with 40 ml of saline. Exsanguination also rinses residual FITC / BSA out of - 10 blood vessels. The upper part of the skull is removed to collect the dural membranes. Membrane samples are removed from both hemispheres, rinsed with water, and dispensed horizontally on microscope slides. The slides are dried for 15 minutes in a slide heater and slid-covered with a 70% glycerol / water solution.
    A fluorescence microscope (Zeiss) equipped with a grid monochromator and a spectrophotometer are used to quantify the amount of FITC-BSA dye in each dura sample. The microscope is equipped with a motorized stage in interface with a personal computer. This facilitates the computer-controlled movement of the stage, with fluorescence measurements at 25 points (500 pm steps) on each dural sample. The extravasation induced by the electrical stimulus of the trigeminal ganglion is an ipsilateral effect (that is, it occurs only on the side of the dura mater on which the trigeminal ganglion is stimulated). This allows the other (unstimulated) half of the dura to be used as a control. The leakage ratio (that is, the ratio of the amount of leakage in the dura from the stimulated side compared to the unstimulated side) is calculated.
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    SEQUENCES
    ACDTATCVTHRLAGLLSRSGGWKNNFVPTNVGSKAF (SEQ ID NO: 1) ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF (SEQ ID NO: 2)
    Antibody LCDR1 LCDR2 LCDR3 I RASQDIDNYLN (SEQ ID NO: 3) YTSEYHS (SEQ ID NO: 4) QQGDALPPT (SEQ ID NO: 5) II RASQDIDNYLN (SEQ ID NO: 3) YTSEYHS (SEQ ID NO: 4) QQGDALPPT (SEQ ID NO: 5) III RASKDISKYLN (SEQ ID NO: 6) YTSGYHS (SEQ ID NO: 7) QQGDALPPT (SEQ ID NO: 5) IV RASRPIDKYLN (SEQ ID NO: 8) YTSEYHS (SEQ ID NO: 4) QQGDALPPT (SEQ ID NO: 5) V RASQDIDKYLN (SEQ ID NO: 9) YTSGYHS (SEQ ID NO: 7) QQGDALPPT (SEQ ID NO: 5) Consensus RASX1X2IX3X4YLN (SEQ ID NO: 10) YTSX5YHS (SEQ ID NO: 11) QQGDALPPT (SEQ ID NO: 5)
    X) is Q, R or K;
    X 2 is D or P;
    X 3 is D or S;
    X4 is N or K; and
    X 5 is G or E.
    Antibody HCDR1 HCDR2 HCDR3 I GYTFGNYWMQ (SEQ ID NO: 12) AIYEGTGDTRYIQKFAG (SEQ ID NO: 13) LSDYVSGFSY (SEQ ID NO: 14) II GYTFGNYWMQ (SEQ ID NO: 12) AIYEGTGKTVYIQKFAG (SEQ ID NO: 15) LSDYVSGFSY (SEQ ID NO: 14) III GYTFGNYWMQ (SEQ ID NO: 12) AIYEGTGKTVYIQKFAD (SEQ ID NO: 16) LSDYVSGFGY (SEQ ID NO: 39) IV GYTFGNYWMQ (SEQ ID NO: 12) AIYEGTGKTVYIQKFAG (SEQ ID NO: 15) LSDYVSGFGY (SEQ ID NO: 39) V GYTFGNYWMQ (SEQ ID NO: 12) AIYEGTGKTVYIQKFAG (SEQ ID NO: 15) LSDYVSGFGY (SEQ ID NO: 39)
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    Antibody HCDR1 HCDR2 HCDR3 Consensus GYTFGNYWMQ (SEQ ID NO: 12) Alyegtgx 6 tx 7 yiq kfax 8 (SEQ ID NO: 37) LSDYVSGFX9Y (SEQ ID NO: 38)
    X 6 is K or D;
    X 7 is V or R;
    X 8 is D or G; and
    X 9 is G or S.
    DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKA PKLLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALP PTFGQGTKLEIK (SEQ ID NO: 17)
    DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKA PKLLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALP PTFGQGTKLEIK (SEQ ID NO: 18)
    DIQMTQSPSSLSASVGDRVTITCRASKDISKYLNWYQQKPGKA PKLLIYYTSGYHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDALP PTFGGGTKVEIK (SEQ ID NO: 19)
    DIQMTQSPSSLSASVGDRVTITCRASRPIDKYLNWYQQKPGKA PKLLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALP PTFGQGTKLEIK (SEQ ID NO: 20)
    DIQMTQSPSSLSASVGDRVTITCRASQDIDKYLNWYQQKPGKA PKLLIYYTSGYHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDALP PTFGGGTKVEIK (SEQ ID NO: 21)
    QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQA
    PGQGLEWMGAIYEGTGDTRYIQKFAGRVTMTRDTSTSTVYMELSSLRSED TAVYYCARLSDYVSGFSYWGQGTLVTVSS (SEQ ID NO: 22)
    QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQA
    PGQGLEWMGAIYEGTGKTVYIQKFAGRVTMTRDTSTSTVYMELSSLRSED
    TAVYYCARLSDYVSGFSYWGQGTLVTVSS (SEQ ID NO: 23)
    QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQA
    PGQGLEWMGAIYEGTGKTVYIQKFADRVTITADKSTSTAYMELSSLRSEDT AVYYCARLSDYVSGFGYWGQGTTVTVSS (SEQ ID NO: 24)
    24/27
    QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQA
    PGQGLEWMGAIYEGTGKTVYIQKFAGRVTMTRDTSTSTVYMELSSLRSED TAVYYCARLSDYVSGFGYWGQGTLVTVSS (SEQ ID NO: 25)
    QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQA
    PGQGLEWMGAIYEGTGKTVYIQKFAGRVTITADKSTSTAYMELSSLRSEDT AVYYCARLSDYVSGFGYWGQGTTVTVSS (SEQ ID NO: 26)
    DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKA PKLLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALP PTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ - 10 WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC (SEQ ID NO: 27)
    DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKA PKLLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALP PTFGQGTKLEIKRTVAAPSVFIFTPSDEKG
    WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
    HQGLSSPVTKSFNRGEC (SEQ ID NO: 28)
    DIQMTQSPSSLSASVGDRVTITCRASKDISKYLNWYQQKPGKA
    PKLLIYYTSGYHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDALP PTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ
    WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
    HQGLSSPVTKSFNRGEC (SEQ ID NO: 29)
    DIQMTQSPSSLSASVGDRVTITCRASRPIDKYLNWYQQKPGKA PKLLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALP PTFGQGTKLEIKRTVAAPSVFIFTPSDEKG
    WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
    HQGLSSPVTKSFNRGEC (SEQ ID NO: 30)
    DIQMTQSPSSLSASVGDRVTITCRASQDIDKYLNWYQQKPGKA PKLLIYYTSGYHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDALP PTFGGGTKVEIKRTVAAPSVFIFTPSK
    WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
    HQGLSSPVTKSFNRGEC (SEQ ID NO: 31)
    QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQA
    25/27
    PGQGLEWMGAIYEGTGDTRYIQKFAGRVTMTRDTSTSTVYMELSSLRSED
    TAVYYCARLSDYVSGFSYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSES
    TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP
    SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFL
    FPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPR
    EEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
    TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL
    SLG (SEQ ID NO: 32). 10 QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQA
    PGQGLEWMGAIYEGTGKTVYIQKFAGRVTMTRDTSTSTVYMELSSLRSED
    TAVYYCARLSDYVSGFSYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSES
    TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFL
    FPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPR
    EEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
    TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL
    SLG (SEQ ID NO: 33)
    QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQA
    PGQGLEWMGAIYEGTGKTVYIQKFADRVTITADKSTSTAYMELSSLRSEDT
    AVYYCARLSDYVSGFGYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSEST AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS
    SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLF
    PPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPRE
    EQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
    PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS
    LG (SEQ ID NO: 34)
    QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQA
    PGQGLEWMGAIYEGTGKTVYIQKFAGRVTMTRDTSTSTVYMELSSLRSED
    TAVYYCARLSDYVSGFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSES
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    TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
    REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLG (SEQ ID NO: 35)
    QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQA PGQGLEWMGAIYEGTGKTVYIQKFAGRVTITADKSTSTAYMELSSLRSEDT. 10 AVYYCARLSDYVSGFGYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSEST AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR 15 EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LG (SEQ ID NO: 36)
    EIVLTQSPATLSLSPGERATLSCKASKRVTTYVSWYQQKPGQA PRLLIYGASNRYLGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCSQSYNYP 20 YTFGQGTKLEIK (SEQ ID NO: 40)
    EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWISWVRQAPG KGLEWVAEIRSESDASATHYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDT AVYYCLAYFDYGLAIQNYWGQGTLVTVSS (SEQ ID NO: 41)
    DIQMTQSPSSLSASVGDRVTIT25 CRASX1X2IX3X4YLNWYQQKPGKAPKLLIYYTSX5YHSGVPSRFSGSGSGTD FTXeTISSLQPEDXyATYYCQQGDALPPTFGXsGTKXgEIK
    Xt = Q, K, or R;
    X 2 = D or P;
    X 3 D or S;
    X4 = K or N;
    X 5 = EouG;
    X 6 = F or L;
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    X 7 = I or F; X 8 = Q or G; and X 9 = Lou V. (SEQ ID NO: 42) QVQLVQSGA- 5 EVKKPGXiSVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTG X 2 TX 3 YIQKFAX4RVTX 5 TX 6 DX 7 STSTX 8 YMELSSLRSEDTAVYYCARLSDYVS GFX9YWGQGTX1; X 2 = K or D; . 10 X 3 = V or R; X 4 = G or D; X 5 = Mou I; X 6 = R or A; X 7 = T or K; 15 X 8 = I go to A; X 9 = G or S; and Xio = L or T. (SEQ ID NO: 43)
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    权利要求:
    Claims (10)
    [1]
    1. Human engineered calcitonin gene (CGRP) antibody, characterized by the fact that it comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR) with complete human structure regions 1 -4, wherein said LCVR comprises the amino acid sequences of LCDR1 (light chain complementarity determining region 1), LCDR2 (light chain complementarity determining region 2), LCDR3 (light chain complementarity determining region 3) and said HCVR comprises the amino acid sequences HCDR1 (heavy chain complementarity determining region 1), HCDR2 (heavy chain complementarity determining region 2), HCDR3 (heavy chain complementarity determining region 3), selected from the group consisting of:
    (a) LCDR1 is RASQDIDNYLN (SEQ ID NO: 3), LCDR2 is YTSEYHS (SEQ ID NO: 4), LCDR3 is QQGDALPPT (SEQ ID NO: 5), HCDR1 is GYTFGNYWMQ (SEQ ID NO: 12), HCDRDTR is AIYEGTG (SEQ ID NO: 13), and HCDR3 is LSDYVSGFSY (SEQ ID NO: 14);
    (b) LCDR1 is RASQDIDNYLN (SEQ ID NO: 3), LCDR2 is YTSEYHS (SEQ ID NO: 4), LCDR3 is QQGDALPPT (SEQ ID NO: 5), HCDR1 is GYTFGNYWMQ (SEQ ID NO: 12), HCDRK is AIYQTG (SEQ ID NO: 15), and HCDR3 is LSDYVSGFSY (SEQ ID NO: 14);
    (c) LCDR1 is RASKDISKYLN (SEQ ID NO: 6), LCDR2 is YTSGYHS (SEQ ID NO: 7), LCDR3 is QQGDALPPT (SEQ ID NO: 5), HCDR1 is GYTFGNYWMQ (SEQ ID NO: 12), HCDRK is AIYTG (SEQ ID NO: 16), and HCDR3 is LSDYVSGFGY (SEQ ID NO: 39);
    (d) LCDR1 is RASRPIDKYLN (SEQ ID NO: 8), LCDR2 is YTSEYHS (SEQ ID NO: 4), LCDR3 is QQGDALPPT (SEQ ID NO: 5), HCDR1 is GYTFGNYWMQ (SEQ ID NO: 12), HCDRK is AIYQTG (SEQ ID NO: 15), and HCDR3 is LSDYVSGFGY (SEQ ID NO: 39); and (e) LCDR1 is RASQDIDKYLN (SEQ ID NO: 9), LCDR2 is
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    [2]
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    YTSGYHS (SEQ ID NO: 7), LCDR3 is QQGDALPPT (SEQ ID NO: 5), HCDR1 is GYTFGNYWMQ (SEQ ID NO: 12), HCDR2 is AIYEGTGKTVYIQKFAG (SEQ ID NO: 15), and HCDR3 is LSDYVSG : 39).
    2. CGRP antigen-binding fragment, characterized by the fact that it is of the engineered human anti-CGRP antibody as defined in claim 1.
    [3]
    3. Pharmaceutical composition, characterized by the fact that it comprises the engineered human anti-CGRP antibody as defined in claim 1 and a pharmaceutically acceptable carrier, diluent or excipient.
    [4]
    4. Engineered human anti-CGRP antibody, characterized by the fact that it comprises an LCVR and an HCVR, in which the said LCVR and HCVR are sequences of amino acids selected from the group consisting of:
    (a) LCVR of SEQ ID NO: 17 and HCVR of SEQ ID NO: 22;
    (b) LCVR of SEQ ID NO: 18 and HCVR of SEQ ID NO: 23;
    (c) LCVR of SEQ ID NO: 19 and HCVR of SEQ ID NO: 24;
    (d) LCVR of SEQ ID NO: 20 and HCVR of SEQ ID NO: 25; and (e) LCVR of SEQ ID NO: 21 and HCVR of SEQ ID NO: 26.
    [5]
    5. Pharmaceutical composition, characterized by the fact that it comprises the engineered human anti-CGRP antibody as defined in claim 4 and a pharmaceutically acceptable carrier, diluent or excipient.
    [6]
    6. Human engineered anti-CGRP antibody, characterized by the fact that it comprises a light chain and a heavy chain, in which the amino acid sequences of the light and heavy chains are selected from the group consisting of:
    (a) light chain of SEQ ID NO: 27 and heavy chain of SEQ ID NO: 32;
    (b) SEQ ID NO: 28 light chain and SEQ ID NO: 33 heavy chain;
    (c) SEQ ID NO: 29 light chain and SEQ ID heavy chain
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    NO: 34;
    (d) SEQ ID NO: 30 light chain and SEQ ID NO: 35 heavy chain; and (e) SEQ ID NO: 31 light chain and SEQ ID NO: 36 heavy chain.
    [7]
    7. Pharmaceutical composition, characterized by the fact that it comprises the engineered human anti-CGRP antibody as defined in claim 6 and a pharmaceutically acceptable carrier, diluent or excipient.
    [8]
    8. Engineered human anti-CGRP antibody, characterized by the fact that it comprises two light chains and two heavy chains, in which each amino acid sequence of the light and heavy chains is selected from the group consisting of:
    (a) light chain of SEQ ID NO: 27 and heavy chain of SEQ ID NO: 32;
    (b) SEQ ID NO: 28 light chain and SEQ ID NO: 33 heavy chain;
    (c) light chain is SEQ ID NO: 29 and heavy chain of SEQ ID NO: 34;
    (d) SEQ ID NO: 30 light chain and SEQ ID NO: 35 heavy chain; and (e) SEQ ID NO: 31 light chain and SEQ ID NO: 36 heavy chain.
    [9]
    9. Human engineered anti-CGRP antibody according to claim 8, characterized by the fact that each light chain amino acid sequence is SEQ ID NO: 29 and each heavy chain amino acid sequence is SEQ ID NO: 34.
    [10]
    10. Pharmaceutical composition, characterized by the fact that it comprises the engineered human anti-CGRP antibody as defined in claim 8 and a pharmaceutically acceptable carrier, diluent or excipient.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    GB8308235D0|1983-03-25|1983-05-05|Celltech Ltd|Polypeptides|
    US5225539A|1986-03-27|1993-07-06|Medical Research Council|Recombinant altered antibodies and methods of making altered antibodies|
    US5530101A|1988-12-28|1996-06-25|Protein Design Labs, Inc.|Humanized immunoglobulins|
    RU2139351C1|1991-04-25|1999-10-10|Чугаи Сейяку Кабусики Кайся|H- and l-chains of monoclonal antibody pm-1 to human il-6r receptor and their v-region, modified monat, its h- and l-chains and their v-regions, cdr-sequence, dna-sequence|
    DE69233254T2|1991-06-14|2004-09-16|Genentech, Inc., South San Francisco|Humanized Heregulin antibody|
    AU2003271174A1|2003-10-10|2005-04-27|Chugai Seiyaku Kabushiki Kaisha|Double specific antibodies substituting for functional protein|
    WO2006110214A2|2005-04-08|2006-10-19|Medimmune, Inc.|Antibodies against mammalian metapneumovirus|
    UA94244C2|2005-11-14|2011-04-26|Ринат Нейросаенз Корп.|Antagonist antibody directed against calcitonin gene-related peptide and pharmaceutical composition comprising the same|
    WO2007076336A1|2005-12-22|2007-07-05|Eli Lilly And Company|Treatment of migraine with anti-cgrp antibodies|
    KR101304150B1|2008-03-04|2013-09-05|라브리스 바이올로직스 인코포레이티드|Methods of treating chronic pain|
    JP5745861B2|2008-03-04|2015-07-08|ファイザー・リミテッドＰｆｉｚｅｒ Ｌｉｍｉｔｅｄ|Methods for treating inflammatory pain|
    JO3330B1|2010-06-10|2019-03-13|Lilly Co Eli|Cgrp antibodies|US4923637A|1987-06-24|1990-05-08|Yazaki Corporation|High conductivity carbon fiber|
    UA94244C2|2005-11-14|2011-04-26|Ринат Нейросаенз Корп.|Antagonist antibody directed against calcitonin gene-related peptide and pharmaceutical composition comprising the same|
    KR101304150B1|2008-03-04|2013-09-05|라브리스 바이올로직스 인코포레이티드|Methods of treating chronic pain|
    CN102740884A|2009-08-28|2012-10-17|瑞纳神经科学公司|Methods for treating visceral pain by administering antagonist antibodies directed against calcitonin gene-related peptide|
    JO3330B1|2010-06-10|2019-03-13|Lilly Co Eli|Cgrp antibodies|
    CN107827982B|2011-05-20|2021-07-06|H.伦德贝克公司|Use of anti-CGRP antibodies and antibody fragments to prevent or inhibit photophobia or aversion to light in a subject in need thereof|
    TWI685505B|2011-05-20|2020-02-21|美商艾爾德生物控股有限責任公司|Anti-cgrp compositions and use thereof|
    KR102128628B1|2011-05-20|2020-06-30|앨더바이오 홀딩스 엘엘씨|Use of anti-cgrp or anti-cgrp-r antibodies or antibody fragments to treat or prevent chronic and acute forms of diarrhea|
    KR102134088B1|2012-08-24|2020-07-14|더 리젠츠 오브 더 유니버시티 오브 캘리포니아|Antibodies and vaccines for use in treating ror1 cancers and inhibiting metastasis|
    PE20161439A1|2014-03-21|2017-01-26|Teva Pharmaceuticals Int Gmbh|ANTIBODIES ANTAGONISTS DIRECTED AGAINST THE PEPTIDE RELATED TO THE CALCITONIN GENE AND METHODS USING THE SAME|
    US10556945B2|2014-03-21|2020-02-11|Teva Pharmaceuticals International Gmbh|Antagonist antibodies directed against calcitonin gene-related peptide and methods using same|
    CN107108715A|2014-10-24|2017-08-29|默沙东公司|The co-agonists of hyperglycemic factor and the acceptors of GLP 1|
    TWI595006B|2014-12-09|2017-08-11|禮納特神經系統科學公司|Anti-pd-1 antibodies and methods of use thereof|
    JOP20200116A1|2015-04-24|2017-06-16|Amgen Inc|Methods for treating or preventing migraine headache|
    AR104847A1|2015-06-17|2017-08-16|Lilly Co Eli|FORMULATION OF ANTI-CGRP ANTIBODY|
    BR112018007214A2|2015-10-30|2018-10-16|Lilly Co Eli|bispecific anti-cgrp / anti-il-23 antibodies and uses of these|
    CN105483091A|2015-12-29|2016-04-13|天津三箭生物技术股份有限公司|Mouse anti-human Calcitonin monoclonal antibody and hybridoma cell line secreting same|
    CN108473567A|2016-01-28|2018-08-31|伊莱利利公司|CGRP antibody and application thereof|
    EP3474854A4|2016-06-27|2020-02-19|The Regents of The University of California|Cancer treatment combinations|
    CN109952314A|2016-09-23|2019-06-28|泰瓦制药国际有限公司|Treat intractable migraine|
    EP3515488A1|2016-09-23|2019-07-31|Teva Pharmaceuticals International GmbH|Treating cluster headache|
    RU2019130867A3|2017-03-02|2021-07-02|
    TW201902926A|2017-05-03|2019-01-16|美商美國禮來大藥廠|anti-CGRP/anti-IL-23 bispecific antibody and use thereof|
    US20190046956A1|2017-06-21|2019-02-14|Cephalon, Inc.|Cation exchange chromatography wash buffer|
    WO2019050759A1|2017-09-06|2019-03-14|Eli Lilly And Company|Combination therapy of lasmiditan and a cgrp antagonist for use in the treatment of migraine|
    RU2020126721A3|2018-01-12|2022-02-14|
    WO2019231800A1|2018-05-31|2019-12-05|Eli Lilly And Company|Anti-cgrp antibodies for treating menstrual-related migraines|
    EP3840836A1|2018-08-22|2021-06-30|Eli Lilly and Company|Anti-cgrp antibodies for treatment-resistant patients|
    WO2021076620A1|2019-10-15|2021-04-22|Eli Lilly And Company|Recombinantly engineered, lipase/esterase-deficient mammalian cell lines|
    法律状态:
    2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-01-29| 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 |
    2019-06-11| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|
    2019-12-24| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-02-11| 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 07/06/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US35332310P| true| 2010-06-10|2010-06-10|
    US61/353,323|2010-06-10|
    PCT/US2011/039381|WO2011156324A1|2010-06-10|2011-06-07|Cgrp antibodies|
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