TW201041902A - Humanized antibodies specific for von willebrand factor - Google Patents

Abstract

The present disclosure relates to humanized antibodies or binding fragments thereof specific for human von Willebrand factor (vWF), methods for their preparation and use, including methods for treating vWF mediated diseases or disorders.

Description

201041902 VI. Description of the Invention [Technical Field to Which the Invention Is Applicable] [〇〇〇1] The present invention is more specific to anthropomorphic humans having a specificity of Wylie factor, or a binding fragment thereof, particularly regarding the inclusion of CDRs and right, Tian几 Anthropomorphic antibodies or their binding factor-specific NMCM tHs. ^The CDRS corresponds to the murine antibody. [Prior Art] ❹ [00〇2] The regulation of platelet attachment to the vascular wound site involves, if, the good father is on the side, and it plays both in the stop and thrombosis. important role. - Such a protein that promotes platelet adhesion is a large multi-audio protein of the Vonville-type factor/, Γ. The hypothesis is that VWF can (4) interact with the platelet receptor Gpib_a in its A1 domain (d〇main) to thereby promote platelet rolling and adhesion (MGake et al(10) Clm. I said st. 78:1456 state. Platelet rolling and adhesion Thereafter, a platelet/fibrin plug that causes bleeding to stop can be formed; however, excessive platelet and/or coagulation reactions may result in a pathological thrombus state. [0003] Inhibition of platelet activation (eg wGHIbIna, ADp 〇 receptor, cyclooxygenase or phosphodiesterase antagonist) or coagulation (eg thrombin and sputum factor inhibitor) treatment and bleeding In association with complications, there is a need to develop a dosage form capable of substantially inhibiting thrombus formation without severely impairing the hemostatic response. SUMMARY OF THE INVENTION [0004] The present invention generally relates to having human VW Willin factor (vWF) specificity. Anthropomorphic antibody or binding fragment thereof, preparation and use thereof, comprising a vWp vector disease or abnormal treatment method, having human VWF specificity The humanized antibody or the -σ S fragment thereof may comprise a complementarity determining region (CDRs) derived from a non-human antibody (eg, mouse CDRs) and a human framework region (biman 201041902 framework regions). 5| Providing a humanized antibody having VW specificity* or a binding fragment thereof k: a heavy chain variant region (Μ-*vanablereg·) sequence as shown in SEQ ID NO: 19 and a light chain variant as shown in SEQ ID NO: 28. The light chain variable region sequence. [0006] The present invention provides a humanized antibody having a t vWF property or a binding fragment thereof comprising the heavy chain sequence as set forth in SEQ ID NO: 237 and as shown in seqidn〇. Light chain sequence. 0007 ❾ [0007] The present invention provides a humanized antibody having a vWF specificity or a binding fragment thereof comprising (8) heavy and light chain complementarity determining regions (CDRs) corresponding to the presence of a murine antibody NMC -4 heavy and light chain variant regions (SEQ IDN: CDRs in sputum and sputum, (b) heavy chain framework regions and/or light chain architecture regions, the former corresponding to the presence of VH4-59 derived human antibodies (eg The domain of the antibody in the variant region of 181651 (seq^nom)) The latter corresponds to the framework region present in the variant region of human antibody ^94808 (1 018) (SEQ ID NO: 6). The present invention provides a humanized antibody having vWF specificity or a binding fragment thereof comprising one or more of the following heavy chain CDRs: HCDR1: gfsltdygvd (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8) And/or HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9). The invention also provides a humanized antibody or binding fragment thereof having vWF specificity, comprising HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9) And the heavy chain architecture region from the variant region of human antibody AAC18165.1 (SEQ ID NO: 4). The invention also provides a humanized antibody or binding fragment thereof having vWF specificity, comprising one or more of the following light chain CDRs: LCDR1: SASQDINKYLN (SEQ ID NO: 10) 'LCDR2: YTSSLHS (SEQ ID NO: 11) ), and / or LCDR3: QQYEKLPWT (SEQ ID NO: 12). [0011] The invention also provides a humanized antibody having a vWF specificity or a binding fragment thereof, comprising the following light chain CDRs: LCDR1: SASQDINKYLN (SEQ ID NO: 201041902 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO.. 12). In certain embodiments, the anthropomorphic antibody or binding fragment thereof can further comprise: a light chain architecture region from a variant region of human antibody AAK94808 (SEQ ID NO: 6).

The invention also provides a humanized antibody or binding fragment thereof having vWF specificity, comprising: heavy chain CDRs, HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR3 : DPADYGNYDYALDY (SEQ ID NO: 9); and light chain CDRs, LCDR1: SASQDINKYLN (SEQ ID NO: 10) > LCDR2: YTSSLHS (SEQ ID 0 NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12) . In certain embodiments, the anthropomorphic antibody or binding fragment thereof may further comprise a light chain framework region from a variant region of human antibody AAK94808 (SEQ ID NO: 6), and/or from human antibody AAC18165.1 (SEQ ID NO: 4) The heavy chain architecture area of the variation zone. The present invention provides a humanized antibody having VWF specificity or a binding fragment thereof, comprising one or more of the following heavy chain variant regions: H2 (SEQ ID NO: 13), H4 (SEQ ID NO: 14) > H5 (SEQ ID NO: 15) > H6 (SEQ ID NO: 16) > H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ Π) NO : 20), H13 (SEQ Π) NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146). The present invention provides a humanized antibody or a binding fragment thereof having vWF specificity, comprising one or more of the following light chain variant regions: L5 (SEqIDN〇: 23), L4 (SEQ ID NO: 24), L6 ( SEQ ID NO: 25), L7 (SEQ E) NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), L10 (SEQ ID NO: 29) or L11 (SEQ ID NO: 30). The present invention provides a humanized antibody having a vWF specificity or a binding fragment thereof, comprising (1) one or more of the following heavy chain variant regions: H2 (SEqIDN〇: 13), H4 (SEQ ID NO: 14) > H5 (SEQ ID NO: 15) > H6 (SEQ ID NO: 16) > H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20) > H13 (SEQ ID NO: 21) > H14 (SEQ ID NO: 22), HI5 (SEQ ID NO: 145), or HI6 (SEQ ID NO: 146); and (2) 201041902 One or more of the light chain variant regions: L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24); L6 (SEQ ID NO: 25) > L7 (SEQ ID NO: 26) > L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), L10 (SEQ ID NO: 29) or L11 (SEQ Π) NO: 30).

For example, an anthropomorphic antibody or binding fragment thereof can comprise: L5 (SEQ ID NO: 23) and H2 (SEQ ID Να 13); L5 (SEQ ID NO: 23) and H4 (SEQ ID NO: 14) L5 (SEQ ID NO: 23) and H5 (SEQ ID NO: 15); L5 (SEQ ID NO: 23) and H6 (SEQ ID NO: 16); L5 (SEQ ID NO: 23) and H7 (SEQ) ID NO: 17); L5 (SEQ ID NO: 23) and H8 (SEQ ID NO: 18); L4 (SEQ ID NO: 24) and H2 (SEQ ID NO: 13⁄4 L6 (SEQ ID NO: 25) and H2 (SEQ ϋ Π) NO: 13); LI 1 (SEQ ID NO: 30) and H2 (SEQ ID NO: 13); L7 (SEQ ID NO: 26) and H2 (SEQ ID NO: 13); L9 (SEQ ID NO: 28) and H9 (SEQ ID NO: 19); L8 (SEQ ID NO: 27) and H9 (SEQ ID NO: 19); L7 (SEQ ID NO: 26) and H9 (SEQ ID NO: 19) L6 (SEQ ID NO: 25) and H9 (SEQ ID NO: 19); L4 (SEQ E) NO: 24) and H9 (SEQ ID NO: 19); L5 (SEQ ID NO: 23) and H9 (SEQ) ID NO: 19); L10 (SEQ ID NO: 29) and H9 (SEQ ID NO. 19); L9 (SEQ Π) NO: 28) and H9 (SEQ ID NO: 19); L9 (SEQ ID NO: 28) and H12 (SEQ ID NO: 20); L9 (SEQ ID NO: 28) and H13 (SEQ ID NO: 21); L9 (SEQ ID NO: 28) and H14 (SEQ ID NO: 22); Lll O (SEQ ID NO: 30) and H9 (SEQ ID NO: 19); or LI 1 (SEQ Π) NO: 30) and H14 (SEQ ID NO: 22). The invention also provides a humanized antibody or binding fragment thereof having vWF specificity, comprising (1) one or more of the following heavy chain CDRs: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), and/or HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9); and (2) one or more of the following light chain CDRs: LCDRl· SASQDINKYLN (SEQ ID NO. 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and / or LCDR3: QQYEKLPWT (SEQ ID NO: 12). In certain embodiments, the anthropomorphic antibody or binding fragment thereof may further comprise a light chain construct 201041902 region from a variant region of the human antibody AAK94808 (SEQ ID NO: 6), and/or a variant region from a human antibody with a red snap. Heavy chain architecture area. [0018] The present invention provides a humanized antibody or binding fragment that binds to vWF as described herein, which has an affinity (Kd) of 1 () or less for VWF, preferably 5 or less, preferably less than or equal to nM, most preferably Preferably, it is at least about 2 nM to about 4 nM. The present invention also provides a humanized antibody or binding sheet k which is competitively bound to vWF as described herein, which has an affinity (magic) of 1 (10) nM or less, preferably 5 or less, preferably 1 〇 nM or less, most Preferably, it is at least about 02 nM to about 5 〇ώΜ.

[0019] The present invention also provides an anthropomorphic antibody or binding fragment that binds to the field of WF1 of VWF, which has an affinity (Kd) of less than 1 〇 nM for vWP, preferably less than 5 nM, and preferably less than InM. At least about 2 ships to about 4nM. The present invention also provides an anthropomorphic antibody or binding fragment thereof that competes for binding to the A1 domain of vWF, which has lOOnjy [Ki) for vWF, preferably at least nnM below 50 nMw, preferably at least about 〇 2nM to about 5.0nM. The invention also provides a humanized antibody or binding fragment of a Fab fragment having a thermostability temperature greater than 65 °C. The present invention provides a humanized antibody having a vWF specificity or a binding fragment thereof, comprising: HCDR1 (GFSLTDYGVD; SEQ ID NO: 7), HCDR2 ❹ (MIWGDGSTDYNSALKS; SEQ ID NO: 8), HCDR3 (DPADYGNYDYALDY; SEQ ID) NO·· 9); and light chain CDR1, light chain CDR2 and LCDR3 (QQYEKLPWT; SEQ ID NO.12), with the proviso that at least one of LCD1 and/or LCD2 is not SASQDINKYLN (SEQ ID NO: 10), respectively. Or YTSSLHS (SEQ ID NO: 11). The present invention also provides a humanized antibody having a vWF specificity or a binding fragment thereof, comprising: HCDR1 (GFSLTDYGVD; SEQ ID NO: 7), HCDR2 (MIWGDGSTDYNSALKS; SEQ ID NO: 8), HCDR3 (DPADYGNYDYALDY; SEQ ID) NO: 9), LCDR1 (SASQDINKYLN; SEQ ID NO: 10), LCDR2 (YTSSLHS; SEQ ID NO: 11) and LCDR3 (QQYEKLPWT; SEQEDNO: 12); corresponding to the human antibody germline family (germline family) VH4 The architectural regions 1, 2, and 3 of the architectural regions 1, 2, and 3; 201041902 and the structural regions 1, 2, and 3 corresponding to the structural regions of the human antibody germ cell family YJQ, 2, and 3. The invention also generally relates to an isolated nucleic acid, which is a humanized antibody having human vWp specificity as disclosed in the present invention. In a certain embodiment, a vector (veeto:) may comprise a nucleic acid as disclosed in the present invention; in another embodiment, a host cell may comprise the disclosed nucleic acid. [0024] The present invention generally also relates to a method for the preparation of a humanized antibody having VWF specificity. The method comprises: culturing a host cell of the present invention such that the nucleic acid is expressed and producing a shot, the method further comprising a self-hosting cell culture solution ( She ure)

Recycling the body, in some embodiments, the anti-system is recovered from the host cell medium; in some embodiments, prior to culturing, the vector comprising the nucleic acid encoding the heavy chain, and A vector comprising a nucleic acid encoding a light chain variant region co-infects a host cell. ~ The present invention generally relates to a composition comprising an anthropomorphic antibody having a specificity and a Ph_aceutically acceptable carrier. [Γ^6] η· The present invention also provides a second antibody comprising a first anthropomorphic antibody or a nucleic acid thereof as described herein, and an A1 domain that binds to vWF. In certain embodiments, the second antibody is AJW200. ^7], the present invention generally also relates to a method of treating a mediator, a disease or an abnormality in a subject (e.g., a patient) by administering a therapeutically effective amount of the v. Anthropomorphic antibodies or fragments thereof. In certain embodiments, the subject, in which the amount is effective, is sufficient to inhibit platelet aggregation but is insufficient to cause significant bleeding clinical symptoms. [0028] The present invention also provides the use of an anthropomorphic antibody or a binding agent thereof as described herein, and the invention also provides for the use of a humanized antibody or a mouth thereof as described herein for the preparation of a vWF vector disease or disorder. Handle the agent. In certain embodiments/examples = effective therapeutic amount is sufficient to inhibit platelet aggregation but not sufficient to cause significant bleeding. In certain embodiments, the vWF vector disease or abnormality is a thrombotic disease, in some embodiments, A thrombotic abnormality is a cardiovascular disease or a cerebrovascular disease such as local 201041902 ischemic stroke; in some embodiments, the cardiac management disease is atherosclerosis, restenosis , angina, acute myocardial infarction, acute coronary syndrome, or cardio-tube abnormalities associated with diabetes; in certain embodiments, thrombotic disease or Abnormal vascular inflammation, venous thrombosis, sickle cell disease, xenograft rejection, peripheral vascular disease, embolic thrombocytopenic purpura (blood (4) "price thr〇mb〇cytopenic purpura ), cystic fibrosis, vascular dementia (vascuiar 0 dementia), Raynaud's disease (Ray Naud's disease), rheumatoid arthritis or diabetes; in certain embodiments, the cerebrovascular disease is vascular dementia, ischemic stroke, or recurrence Sexual Stroke (recurren); stroke. [0030] In certain embodiments, a humanized antibody having vWF specificity or a binding fragment thereof lacks an effector function; in certain embodiments, anthropomorphic antibody Included in the Fc region derived from IgG4. [0031] In certain embodiments, a humanized antibody having a vWF specificity, or a binding fragment thereof, binds to the A1 domain of the Von Wylie factor. [0032] In certain embodiments, An antibody binding fragment having vWF specificity is ° Fab, Fab', Fab, _SH, Fv, scFv, F(ab,) or diabody. [0033] In certain embodiments, having vWF specificity The antibody binding fragment is not Faib. [0034] In certain embodiments, the anthropomorphic antibody having vWF specificity is a full length antibody (Ml length antibody). [0035] In certain embodiments, the anthropomorphic antibody can comprise One of HCDR1 Substituents such as F27Q L29I, T30S and/or V34W substituents; in certain embodiments, the anthropomorphic antibody may comprise more than one substituent of HCDR2, such as a S61P and/or A62S substituent; In embodiments, the anthropomorphic antibody may comprise more than one substituent in LCDR1, such as a S24Q, N30S and/or K31N substituent; in certain embodiments, the anthropomorphic antibody may comprise one of LCDR2 to 9 201041902 Substituents such as Y50D, Τ51Α, S53N, Η55Ε and/or S56T substituents. In certain embodiments, a 'humanized antibody' may comprise: one or more substituents, such as F27Q L29I, T30S and/or V34W substituents; (2) in HCDR2 More than one substituent, such as a S61p and/or a62S substituent; (3) one or more substituents in LCDR1, such as S24q, N3〇s and/or K31N substituents; and (4) in LCDR2 More than one substituent, such as Y5〇D, T5ia, S53N, H55E and/or S56T substituents. [Embodiment] Ο J [〇〇47J_ The present invention provides a virulence antibody having human Von Wylie factor (vWF) specificity or a conjugate thereof, comprising having more than one CDRs corresponding to those present in the murine anti-Mnmc_4 Or the CDR regions of some CDRs; νΜ[·4 antibody binding = GPH>a binding site in the field of WF1 (see, for example, j7ujimura et B1〇〇d 77:113-20, 1991, · Shimaetal JNara MedAssoc., 36: 662 1985). The anthropomorphic antibody of the present invention may further comprise a modified silk-modified human domain, for example, the human antibody AAC18165.1 (SEQid NO: 4) is characterized: ^ framework region and corresponding to the human antibody ΛΛΚ9 side (SEQ. 2-6 == The concept of bribes. #极录人顾减为观C-4 ^可^体》, containing the murine wrist c_4 heavy chain and light chain = (^family) showing a high degree of homology; the most interesting In the case of Budi II, the NMC·4 CDRs are transferred to the complex heavy chain transformation region. Among them, the light chain variant region is composed of: and the potential of the antibody to block the vWF vector to plate response. Anthropomorphic [0048] The term "chimeric antibody" encompasses the sequence of a variant region derived from a single antibody + from another antibody', such as a variant region, from the mutual region. Antibody. 曰趴家乳抗体和恒^049] The term "personalization of a few bodies" includes the CDR'm-, milking, and Antibody. In the human age construct sequence and the CDR sequence derived from the other n-structural cell line _ additional cultivation of the framework region can be performed 10 201041902 [0050] The term "antibody" includes an antibody having a variant region in which both the framework region and the CDR region are derived from a human germ cell strain immunoglobulin sequence; in addition, if the antibody comprises a constant region, the constant region is also derived from An immunogenic globulin sequence of a human lean cell line. The human antibody of the present invention may comprise an amino acid residue not encoded by an immunoglobulin sequence of a human germ cell line (eg, by in vitro random or specific site (site_specific) a mutation introduced by a mutagensis; however, the term "human antibody" as used herein does not encompass the transplantation of a CDR sequence of a germ cell strain derived from another mammalian species (eg, a mouse) into humans. An antibody on a sequence. [0051] As described herein, if the antibody is derived from a system that utilizes a human germ cell strain immunoglobulin gene, the human antibody comprises a sequence derived from a particular germ cell line. Heavy or light chain variants. This system involves the use of related antigens to transfer genes with human immunoglobulin genes (t Ransf〇rmati〇n) The mouse is immunized, or the relevant antigen is used to screen the human immunoglobulin gene library displayed on the phage. By comparing the amino acid sequence of the human antibody with the amino acid sequence of the human germ cell immunoglobulin And selecting the human germ cell strain immunoglobulin sequence closest to the (maximally identical %) human antibody sequence in the sequence, thereby recognizing the human antibody "derived from" the human germ cell strain immunoglobulin sequence. For example, Naturally occurring somatic mutations or intentional introduction of site-directed mutagenesis (site_directed ❹ mutati〇n ) 'Human antibodies derived from the specific human germ cell line immunoglobulin sequence may contain amino acids compared to the sequence of the germ cell line Difference; however, in the amino acid sequence, at least 8% of the selected human antibody or fragment thereof is generally identical to the one encoded by the human germ cell line immunoglobulin sequence, and is included in other species. Germ cell line immunoglobulin amino acid sequence (eg, murine genital wart line sequence) To the human amino acid residues. 'In some cases, the amino acid sequence of a human antibody may have at least, or even at least 95% 96%, 97%, 98%, or 99% with, for example, 80%, 81%, 82%, 83% , 84%, '85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, And 1%% of the germ cell strain immunoglobulin genes are encoded by the chiral acid sequence identity. 11 201041902 [0052] The present invention provides a humanized antibody having a vWF specificity or a binding fragment thereof (eg, Fab, Fab', Fab'-SH, Fv, scFv, F(ab,) 2, a diabody or a single chain antibody > comprises a heavy chain variant region sequence as set forth in SEQ ID NO: 19 and a light chain variant region sequence as set forth in SEQ ID NO: 28; the invention also provides a humanized antibody having vWF specificity or a combination thereof A fragment comprising the heavy chain sequence set forth in SEQ ID NO: 237 and the light chain sequence set forth in SEQ ID NO: 238. [0053] The invention also provides a humanized antibody having a vWF specificity or a binding fragment thereof 'comprising (1) one of the following heavy chain CDRs: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: 0 MTWGDGSTDYNSALKS (SEQ ID NO: 8), and/or HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9); (2) One or more of the following light chain CDRs: LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and/or LCDR3: QQYEKLPWT (SEQ ID NO: 12) ° [0054] The invention also provides a humanized antibody or binding fragment thereof having vWF specificity, comprising (1) one or more of the following heavy chain CDRs: HCDR1 : GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), and/or HCDR3: DPADYGNYDYALDY (SEQ ID NO.. 9); or (2) the following light chain CDRs, one or more of which: LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and/or LCDR3: QQYEKLPWT (SEQ ID NO: 12). [0055] The present invention also provides anthropomorphism with vWF specificity An antibody or binding fragment thereof, comprising (1) heavy chain CDRs, HCDR1: GFSLTDYGYD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO. 8), and HCDR3: DPADYGNYDYALDY (SEQ Π) NO: 9); Light chain CDRs, LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12). The invention also provides a humanized antibody or binding fragment thereof having vWF specificity, comprising (1) heavy chain CDRs, HCDR1: GFSLTDYGYD (SEQ ID NO: 7), 12 201041902 HCDR2: MIWGDGSTDYNSALKS (SEQ ED NO: 8), &HCDR3·· DPADYGNYDYALDY (SEQ ID NO: 9); or (2) Light chain CDRs, LCDRl:

SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12). [0057] In certain embodiments, a 'humanized antibody or binding fragment thereof can comprise a heavy chain variant region framework region, wherein the framework region comprises a heavy chain architecture sequence present in an antibody from a human VH4 family (eg, architecture 1 ( FW1), Architecture 2 (FW2), Architecture 3 (FW3), and/or Architecture 4 (FW4)). In some embodiments, the 'humanized antibody or binding fragment thereof can comprise a light chain ◎ ❹

A region of variation region architecture, wherein the framework region comprises sequences of light chain architecture regions present in antibodies from the human VK1 family (eg, architecture 1 (FW 3 (FW3), and/or architecture 4 (FW4)). In some embodiments, an anthropomorphic antibody or binding fragment thereof can comprise more than one (eg, one, two, three, and/or four) heavy chain framework regions present in antibodies from the human VH4 family. Paste (eg, silkworm (FW1), architecture 2 (FW2 conformation 3 (FW3), and/or architecture 4 (FW4)), and more than one (eg, one, two, =, and domain four) exist from the human νκι family Light chain architecture in the antibody sequence ==) 1) (10)), turn 2 (just) 'All 3', shape ί

:0] Members of the VH4 family and their individual heavy chain architectures 込2 and ==ί, then 147: 147, 148 and 149), 4-28 (respectively Q ID 4-30 W 1 and 52, 4_mi respectively (respectively SEQIDN〇: 153, 154 and !55), NO 1 if not for Qing ^ N〇: 156, 157 and 158), 4-30.4 (SEQ ID

Να 9,16〇^ 161), 4.31 〇: ^ ^EQID 3 = not SE(10) and 65,166 and 167), 4_39 (3 fE=, =8, (10) and 9 respectively (minutes _ ( =:=〇: Members of the 174, 175 and TM^ families and their individual light chain architecture zones 1, 2 and 3 contain: (knife is deleted, (8) and 182), (10) (g = ◦: 13 201041902 183, 184 and 185 respectively) , 018 (SEQ ID NO: 186, 187 and 188, respectively), 08 (SEQ ID NO: 189, 190 and 191, respectively), A20 (SEQ ID NO: 192, 193 and 194, respectively), A30 (respectively SEQ ID NOs: 195, 196 and 197), L14 (SEQ ID NO: 198, 199 and 200, respectively), LI (SEQ ID NO: 201, 202 and 203, respectively), LI5 (SEQ ID NO: respectively) 2〇4, 2〇5 and 2〇6), 14 (SEQ ID NO: 207, 208 and 209, respectively), L18 (SEQ ID NO: 210, 211 and 212, respectively), L5 (SEQ ID NO, respectively) : 213, 214 and 215), L19 (SEQ ED NO: 216, 217 and 218, respectively), L8 (SEQ ID NO: 219, 220 and 221, respectively), L23 (SEQ ID NO: 222, 223 and 224), L9 (SEQ ID NO: 225, 226 and

0 227), L24 (SEQ 冚NO·· 228, 229 and 230, respectively), LI 1 (SEQ ID NO: 231, 232 and 233, respectively) and LI2 (SEQ ID NO: 234, 235 and 236, respectively) .

The present invention provides a humanized antibody having vWF specificity or a binding fragment thereof comprising one or more of the following heavy chain CDRs: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8) And/or HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9).

The present invention provides a humanized anti-sputum or binding fragment thereof having vWF specificity, comprising one or more of the following heavy chain CDRs: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 〇 8), and/or HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9) and the heavy chain framework region from the variant region of human antibody AAC18165.1 (SEQ ID NO: 4). /,

The present invention provides a humanized antibody or a binding fragment thereof having vWF specificity, comprising one or more of the following heavy chain CDRs: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8) And/or HCDR3: DPADYGNYDYALDY (SEQH) NO: 9) and a heavy chain framework region from the variant region of human antibody AAC18165.1 (SEQ ID NO: 4), wherein the heavy chain framework region does not comprise more than one murine residue .

The present invention provides a humanized antibody having vWF specificity or a binding fragment thereof comprising one or more of the following heavy chain CDRs: HCDR1: GFSLTDYGVD 14 201041902 (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), &/4HCDR3.DPADYGNYDYALDY(SEQID1SiO:9)&&> heavy chain architecture region from the variant region of human antibody AAC18165.1 (SEQID]SK>.4), wherein the heavy chain architecture region further comprises a The above mouse residues.

The invention also provides a humanized antibody or binding fragment thereof having vWF specificity, comprising one or more of the following heavy chain CDRs: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8) ), and HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9).

The invention also provides a humanized antibody or binding fragment thereof having vWP specificity, comprising one or more of the following heavy chain CDRs: HCDR1: GFSLTDYGVD^ (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9) and the heavy chain framework region from the variant region of human antibody AAC18165.1 (SEQ ID NO: 4).

The invention also provides a humanized antibody or binding fragment thereof having vWF specificity, comprising one or more of the following heavy chain CDRs: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8 And HCDR3: DPADYGNYDYALDY (SEQ ED NO: 9) and the heavy chain framework region from the variant region of human antibody AAC18165.1 (SEQEDNOj) wherein the heavy bond framework region does not comprise more than one good class residue. 〇[〇°69] The present invention also provides a humanized antibody having a vWF specificity or a binding fragment thereof, comprising one or more of the following heavy chain CDRs: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9) and a heavy chain framework region from the variant region of human antibody AAC18165.1 (SEQ ID NO: 4), wherein the heavy chain framework region further comprises more than one mouse Residues. The invention also provides a humanized antibody or binding fragment thereof having vWF specificity, comprising one or more of the following light chain CDRs: LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11) ), and / or LCDR3: QQYEKLPWT (SEQ ID NO: 12). The invention also provides a humanized antibody having vWF specificity or a binding thereof 15 201041902 fragment comprising one or more of the following light chain CDRs: LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO) : 11), and/or LCDR3: QQYEKLPWT (SEQ ID NO: 12), and the light chain architecture region from human antibody AAK94808 (SEQ ID NO: 6). The invention also provides a humanized antibody or binding fragment thereof having vWF specificity, comprising one or more of the following light chain CDRs: LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID 11) And/or 1^汜: (3(3¥£10^1'(8£(510 excitation12)) and the light-key architecture region from human antibody AAK94808 (SEQ ID NO: 6), where the light-key architecture region Does not contain more than one murine residue. The present invention also provides a humanized antibody having a vWF specificity or a binding fragment thereof, comprising one or more of the following light chain CDRs: LCDR1: SASQDINKYLN (SEQ ID NO: 10) , LCDR2: YTSSLHS (SEQ ID 1^0:11), and/or 1^3: (3(3¥£10^1' beer (210:^0:12) and human antibody AAK94808 (SEQIDNO: 6) a light chain architecture region, wherein the light chain architecture region further comprises more than one murine residue.

The present invention provides a humanized antibody having a vWF specificity or a binding fragment thereof comprising a light chain CDRs LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT 〇 (SEQ ID NO: 12). The present invention provides a humanized antibody or binding fragment thereof having vWF specificity, comprising a light chain CDRs LCDR1: SASQDINKYLN (SEQ ED NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT ( SEQ ID NO: 12) and the light chain architecture region from human antibody AAK94808 (SEQ ID NO: 6). The present invention provides a humanized antibody or binding fragment thereof having vWF specificity, comprising a light chain CDRs LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT ( SEQ ID NO: 12) and a light chain framework region from human antibody AAK94808 (SEQ ID NO. 6), wherein the light chain framework region does not comprise more than one murine residue. 16 201041902 [0077] The present invention provides a humanized antibody or binding fragment thereof having vWF specificity, comprising a light chain CDRs LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLIiS (SEq ID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12) and the light chain architecture region from human antibody AAK94808 (SEQ ID NO: 6), wherein the light chain architecture region further comprises more than one murine residue.

The present invention provides a humanized antibody having vWF specificity or a binding fragment thereof comprising: heavy chain CDRs HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MTWGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9); light chain CDRs LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID Ό N0: u), and LCDR3: QQYEKLPWT (SEQ ID NO: 12); The light chain framework region comprising the variant region of human antibody AAK94808 (SEQ ID NO: 6) and/or the heavy bond framework region from the variant region of human antibody AAC18165.1 (SEQ ID NO: 4). The present invention also provides an amino acid sequence variant having a vWF-specific anthropomorphic antibody or a binding fragment thereof, and an amino acid sequence variant having a vWp-specific anthropomorphic antibody or a binding fragment thereof An amino acid sequence comprising a heavy chain and/or light chain framework region having at least 80% and a heavy or light chain, respectively, of the heavy and light chain variant region sequences of SEQ ID NO: 19 or SEQ ID NO: 28, respectively The original 〇 〇 humanized antibody has the same key and/or light key. Preferably, the amino acid sequence identity of the sequence of the heavy bond and/or light bond framework regions is at least 85%, more preferably at least 90%, most preferably at least 95%, especially 96%, more particularly 97%, still more particularly 98. %, most particularly 99%, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, and 1〇〇〇/0. Here, the identity and homology (h〇m〇l〇gy) of this sequence are defined as: after arranging the sequence and, if necessary, introducing a gap to complete the maximum sequence identity, the candidate sequence has The percentage of the amino acid residues of the VWF-specific anthropomorphic antibody or its binding fragment, such that sequence identity can be determined by standard methods generally used to compare the positional similarity of the amino acids of the two peptides. . Using a computer program such as BLAST or FASTA, for the best match of its individual amino acids (along a 17 201041902 skin size = long, or along a predetermined part of one or two sequences) Defining (defaUltGpenmgpenalty) and default, aPPenalty), and can be combined with computer programs such as Gu 250 ί 仔, matrix '' see DayhGff et a1., "Graphic Set of Protein Sequences and Structures" on Γ 〇 f Pr〇 Tm Sequence and Structure, νο15, SUPP.3 (1978)) The knife P can calculate the percent identity in the following way: by multiplying the total = the total number of the illusion by the 're-divided (four) with the span (talking) The length of the longer sequence in the ) plus the sum of the number of gaps introduced into the longer sequence for arranging the two sequences ° '^

因此 〇] Accordingly, the present invention provides a humanized antibody having vWF specificity or an ', 'α σ fragment thereof, wherein the humanized antibody or binding fragment thereof comprises a heavy chain variant region sequence: the heavy chain variant region sequence comprises at least 8 〇% is the same framework region as the SEqIDN〇: 19 architecture region, and/or a light chain variant region sequence having at least 8% of the architecture region identical to the architecture region of SEQ ID 23 . The invention also provides a humanized antibody or binding fragment thereof having the specificity, wherein the humanized antibody or binding fragment thereof comprises a heavy chain variant region sequence comprising at least 8% and SEqIDn〇: 237 A sequence of light chain variant regions having the same architectural region of the architectural region and/or an architectural region having the same architectural region of at least N: 238. An anthropomorphic antibody may optionally comprise more than one substituent, such as a F27Q L29I, T30S and/or V34W substituent in HCDR1; in certain embodiments, an anthropomorphic antibody may comprise more than one substituent. For example, in S61P, and/or A62S substituents; in certain embodiments, the anthropomorphic antibody may comprise more than one substituent, such as the S24Q, N30S and/or K31N substituents in LCDR1; In embodiments, the anthropomorphic antibody may comprise more than one substituent, such as the Y50D, T51A, S53N, H55E and/or S56T substituents in LCDR2. In certain embodiments, an anthropomorphic antibody can comprise: (1) more than one substituent, such as a F27Q L29I, T30S and/or V34W substituent in HCDR1; (2) more than one substituent is included a S61P and/or A62S substituent in HCDR2; (3) one or more substituents, including S24Q, N30S and/or K31N substituents in LCDR1; (4) more than one substituent, such as Y50D in LCDR2 , T51A, 18 201041902 S53N, H55E and/or S56T substituents. The present invention provides a humanized antibody having a vWF specificity or a binding fragment thereof

Segment ' contains one of the following heavy chain variant regions: H2 (SEQ ID NO: 13), H4 (SEQ)

ID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ)

ID NO: 17) 'H8 (SEQ ID KO: 18) > H9 (SEQ ID NO: 19) > H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146) (Polynucleotides encoding the above heavy chain variant regions are respectively SEQ ID NOs: 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138 and 139 are provided). The present invention provides a humanized antibody having a vWF specificity or a binding fragment thereof

Segment 'includes one of the following light chain variant regions: L5 (SEQ ID NO: 23), L4 (SEQ Π) NO: 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), L10 (SEQ ID NO: 29) or L11 (SEQ ID NO: 30) (Polynucleotides encoding the above-described light chain variant regions are respectively represented by SEQ ID NOs : 120, 121, 122, 123, 124, 125, 126, and 127 are provided). The present invention also provides a humanized antibody having a vWF specificity or a binding fragment thereof comprising: (1) one of the following heavy chain variant regions: H2 (SEQ ID NO: 13), H4 (SEQ ID NO: 14) &gt H5 (SEQ ID NO: 15) 'H6 (SEQ ID NO: 16) 'H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ ID NO: 19), Ο H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ Π) NO: 146) (encoding the above-described heavy chain variation region) The polynucleotides are provided by SEQ ID NOs: 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138 and 139, respectively; (2) one of the following light chain variant regions: L5 (SEQ ID NO) : 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25) > L7 (SEQ ID NO: 26) ' L8 (SEQ ID NO: 27) > L9 (SEQ ID NO: 28) L10 (SEQ ID NO: 29) or L11 (SEQ ID NO: 30).

The present invention provides a humanized antibody having a vWF specificity or a binding fragment thereof, comprising: L5 (SEQ ID NO: 23) and H2 (SEQ ID NO: 13); L5 (SEQ ID NO: 23) and H4 ( SEQ Π) NO: 14); L5 (SEQ ID NO: 23) and H5 (SEQ ID NO.15); L5 (SEQ ID NO: 23) and H6 (SEQ ID NO: 16); L5 (SEQ ID 19) 201041902 Ο 〇NO: 23) Α Η7 (SEQ ID NO: 17); L5 (SEQ ID NO: 23)^ H8 (SEQ ID NO: 18); L4 (SEQ ID NO: 24) and H2 (SEQ ID NO: 13); L6 (SEQ ID NO: 25) and H2 (SEQ ID NO: 13); Lll (SEQ ID NO: 30) and H2 (SEQ ID NO: 13); L7 (SEQ ID NO: 26) and H2 ( SEQ ID NO: 13); L9 (SEQ ID NO: 28) and H9 (SEQ ID NO: 19); L8 (SEQ ID NO: 27) and H9 (SEQ ID NO: 19); L7 (SEQ ID NO: 26) And H9 (SEQ ID NO: 19); L6 (SEQ ID NO: 25) and H9 (SEQ ID NO: 19); L4 (SEQ ID NO.. 24) and H9 (SEQ ID NO: 19); L5 ( SEQ ID NO: 23) and H9 (SEQ ID NO: 19); L10 (SEQ ID NO: 29) and H9 (SEQ ID NO: 19); L9 (SEQ Π) NO: 28) and H9 (SBQ ID NO: 19); L9 (SEQ ID NO: 28) and H12 (SEQ ID NO: 20); L9 (SEQ ID NO: 28) and H13 (SEQ ID NO: 21); L9 (SEQ ID NO: 28) and H14 ( SEQ ID NO: 22) LI 1 (SEQ ID NO: 30) and H9 (SEQ ID NO: 19); or LI 1 (SEQ ID NO: 30) and H14 (SEQ ID NO: 22). The present invention provides a humanized antibody or binding fragment that binds to VWF as described herein, which has an affinity (Kd) of 10 nM or less for vWF, preferably 5 nM or less, more preferably less than InM, most preferably At least about 2 nM to about 4 nM (e.g., from about 0.21, 0.28 or 0.34 to about 0.25, 0.32 or 0.38 ιι). The invention also provides a humanized antibody or binding fragment that competes for binding to vWF as described herein, which has an affinity (Ki) of less than 1 〇〇 nM for vWF, preferably less than 5 〇 祖, 1 〇 Preferably, it is at least about 0.2 to about 5 〇 (e.g., from about ,22, 〇28 or 0.34 to about 2.3, 3.5 or 4.7 nM). The invention also provides an anthropomorphic antibody that binds to the ai field of the er, or has an affinity (v) of ι 〇 or less for the ai field of the vw, and is preferably 1 or less, preferably at least about 0.2 to about 〇. 4 or G·34 to about G·25, (4)). The affinity of the anthropomorphic antibody in the A1 field of the present invention, the affinity of (=5), preferably 50 - [〇〇88] The present invention also provides the thermal stability temperature of the secret fragment is greater than the hunger 20 201041902 The humanized antibody or binding fragment is preferably greater than 7 ° C, more preferably greater than 75 ° C, and most preferably greater than 80 ° C. Analysis of the thermal stability of the Fab fragment uses the differential scanning calorimetry measurement to identify the midpoint melting temperature of the Fab fragment in the context of the full-length IgG. It is known to those skilled in the art and can be implemented according to, for example, Garber and Demarest (2〇〇7), BBRC 355:751-7. Surprisingly, we have found that the anthropomorphic antibody of the invention has a thermostability temperature greater than that of a parent non-humanized antibody, which is typically a murine antibody, especially a murine Antibody NMC-4. The present invention also provides a humanized antibody having a vWF specificity of a Fab fragment having a thermostability temperature greater than that of a parent non-humanized antibody or a U segment thereof. [=89] The present invention provides a humanized antibody having vWF specificity or a binding fragment thereof I's comprising the following hypervariable region amino acid sequence: HCDR1 (GFSLTDYGVD; SEQ ID NO: 7), HCDR2 (MIWGDGSTDYNSALKS) SEQ E) NO: 8), HCDR3 (DPADYGNYDYALDY; SEQ ID NO: 9); and LCDR3 (QQYEKLPWT; SEQ ID NO: 12), with the proviso that at least one of LCD1 and/or LCD2 is not SASQDINKYln (SEQ ID NO) : 10) or YTSSLHS (SEQ Π) N〇: n). Surprisingly, the anthropomorphic gastric heart 4 antibody lacking NMC-4 LCDR1 and/or LCDR2 retains the nanomolar binding affinity for vWP. [00=] In certain embodiments, anthropomorphic antibodies may further comprise a human antibody heavy chain, region 'in certain embodiments, the heavy chain architecture region corresponds to a heavy chain present in a 4-59-derived human antibody The framework region; in certain embodiments, the heavy bond framework region present in the 4_59-derived human antibody further comprises more than one murine residue; in certain embodiments, the weight present in the 4-59-derived human antibody The chain architecture region does not contain more than one murine residue. [ooyi] In certain embodiments, the anthropomorphic antibody may further comprise a human antibody light chain scaffolding region. In some embodiments, the light chain architecture region corresponds to a wheel chain present in the 018 derived human I population. The framework region; in certain embodiments, the light chain architecture region present in the 〇18-derived human anti-21, 201041902 body further comprises more than one murine residue; in certain embodiments, the 018-derived human antibody is present The light chain architecture region does not contain more than one murine residue. [0092] LCDR1 and/or LCDR2 can be obtained from a human source. In certain embodiments, LCDR1 and/or LCDR2 can be obtained from the same antibody (e.g., a human antibody); in other embodiments, 'LCDR1 and/or LCDR2 can be obtained from different antibodies (e.g., two human antibodies). If LCDR2 is derived from a human source, it is preferably DASNLET (SEQ ID NO: 118). [0093] The present invention also provides a humanized antibody having a vWF specificity or a binding fragment thereof comprising: HCDR1 (GFSLTDYGVD; SEQ ID NO: 7), HCDR2 〇 (MIWGDGSTDYNSALKS; SEQ ID NO: 8), HCDR3 ' (DPADYGNYDYALDY SEQ ID NO: 9), LCDR1 (SASQDINKYLN; SEQ ID NO: 10), LCDR2 (YTSSLHS; SEQ ID NO: 11) and LCDR3 (QQYEKLPWT; SEQ ID NO: 12); corresponding to human antibody germ cell line sequence ( In the germline sequence 4-59, the heavy chain framework regions 1, 2, and 3 of the framework regions 1, 2, and 3, wherein the heavy chain architecture region 1 is QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO: 171), and the heavy chain architecture region 2 is WIRQPPGKGLEWIG (SEQ ID NO: 172), and the heavy chain framework region 3 is RVTISTOTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: Q 173); and corresponds to the framework regions 1, 2 present in the human antibody light chain germ cell line sequence 〇i8, and 3 light chain architecture zones 1, 2, and 3, wherein light chain architecture zone 1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 186), light chain architecture zone 2 is WYQQKPGKAPKLLIY (SEQ ID NO: 187), and light chain architecture zone 3 is GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 188). The present invention provides a humanized antibody having a vWF specificity or a binding fragment thereof, comprising: HCDR1 (GFSLTDYGVD; SEQ ID NO: 7), HCDR2 (MIWGDGSTDYNSALKS; SEQ ID NO: 8), HCDR3 (DPADYGNYDYALDY; SEQ ED NO : 9), LCDR1 (SASQDINKYLN; SEQ ID NO: 10), LCDR2 (YTSSLHS; SEQ ID NO: 11) and LCDR3 22 201041902 (QQYEKLFWT; SEQ ID NO: 12); corresponding to human antibody germ cell line sequence 4-34 The heavy chain architecture zones 1, 2, and 3 of the architecture zones 1, 2, and 3, wherein the heavy chain architecture zone 1 is QVQLQQWGAGLLKPSETLSLTCAVY (SEQ ID NO: 165), and the heavy chain architecture zone 2 is WIRQPPGKGLEWIG (SEQ ID NO: 166) And the heavy chain architecture region 3 is RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 167); and the light chain architecture region 1, 2 corresponding to the framework regions 1, 2, and 3 present in the human antibody light chain germ cell line sequence 018 • And 3, wherein the light chain architecture area 1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 186), the light chain architecture area 2 is WYQQKPGKAPKLLIY (SEQ ID NO: 187), and the light chain architecture area ^ 3 is GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 188 188) 〇[0095] The present invention also provides a 领1 collar with vWF Domain-specific anthropomorphic antibody or binding fragment thereof, comprising: HCDR1 (GFSLTDYGVD; SEQ ID NO: 7), HCDR2 (MIWGDGSTDYNSALKS; SEQ ID NO: 8), HCDR3 (DPADYGNYDYALDY; SEQ ID NO: 9), LCDR1 (SASQDINKYLN SEQ ID NO: 10), LCDR2 (YTSSLHS; SEQ ID NO: 11) and LCDR3 (QQYEKLPWT; SEQ ID NO: 12); corresponding to antibodies present in the human antibody germline family VH4 The heavy chain architecture regions 1, 2, and 3 of framework regions 1, 2, and 3; and the light chain architecture corresponding to framework regions 1, 2, and 3 present in antibodies from human antibody germ cell family VK1 Zones 1, 2, and 3. The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in sequence 4-04 of the heavy chain mutant germ cell line (eg, FW1: QVQLQESGPGLVKPSGTLSLTCAVS) (SEQ ID NO: 147), FW2: WVRQPPGKGLEWIG (SEQ ID NO: 148) and FW3: RVTISVDKSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 149)). The anthropomorphic antibody or binding fragment thereof can comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in sequences 4-28 of the heavy chain mutant germ cell line (eg, FW1: QVQLQESGPGLVKPSDTLSLTCAVS) (SEQ ID NO: 150), FW2: WIRQPPGKGLEWIG (SEQ E) NO: 151) and FW3: 23 201041902 RVTMSVDTSKNQFSLKLSSVTAVDTAVYYCAR (SEQ ID NO: 152)). The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 f > ^FWl corresponding to framework regions 1, 2, and 3 present in sequence 4-30.1 of the heavy chain mutant germ cell line: QVQLQESGPGLVKPSQTLSLTCTVS (SEQ ID NO.. 153), FW2: WIRQHPGKGLEWIG (SEQ ID NO: 154) and FW3: RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 155)). The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in heavy chain variable 0 germ cell line sequence 4-30.2 (eg, FW1) : QLQLQESGSGLVKPSQTLSLTCAVS (SEQ ID NO: 156), FW2: WIRQPPGKGLEWIG (SEQ ID NO: 157) and FW3: RVTISVDRSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 158)). [00100] The anthropomorphic antibody or binding fragment thereof can comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in sequence 4-30.4 of the heavy chain mutant germ cell line (eg, FW1: QVQLQESGPGLVKPSQTLSLTCTVS) (SEQ ID NO: 159), FW2: WIRQPPGKGLEWIG (SEQ ID NO: 160) A FW3: RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: O 161) 〇 [00101] Anthropomorphic antibodies or binding fragments thereof may comprise a corresponding to a heavy chain variation The framework regions 1, 2, and 3 of the framework regions 1, 2, and 3 in the germ cell line sequence 4-31 (eg, FW1: QVQLQESGPGLVKPSQTLSLTCTVS (SEQ ID NO: 162), FW2: WIRQHPGKGLEWIG (SEQ ID NO: 163)^. FW3 : RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 164) ) 〇 [00102] The anthropomorphic antibody or binding fragment thereof may comprise an architectural region corresponding to framework regions 1, 2, and 3 present in sequence 4 - 34 of the heavy chain mutant germ cell line. 1, 2, and 3 (eg, FW1: QVQLQQWGAGLLKPSETLSLTCAVY (SEQ ID NO: 165), FW2: WIRQPPGKGLEWIG (SEQ ID NO: 166) and FW3: 24 201041902 RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ id no: 167)) [00103] Anthropomorphic antibody Or a binding fragment thereof may comprise a corresponding To the framework regions 1, 2, and 3 of the framework regions 1, 2, and 3 present in the sequence of the heavy-bond mutant cell line 4-39 (eg, FW1: QLQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO: 168), FW2: WIRQPPGKGLEWIG (SEQ) ID NO: 169) and FW3: RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 170). [00104] The anthropomorphic antibody or binding fragment thereof may comprise an architectural region corresponding to the sequence 4-9 in the heavy bond-changing 0 germline cell line. 1, 2, and 3 architectural regions 1, 2, and 3 (>^Ι^1··(^ν(3Ι^Ε30Ρ0ΕνΚΡ3ΕΤΙ^ΙΤσΓν8(3Ε0·· 171), FW2: WIRQPPGKGLEWIG (SEQ ID NO: 172) and FW3: RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 173) ) 〇 [00105] The anthropomorphic antibody or binding fragment thereof may comprise an architectural region 1, 2 corresponding to sequence 4 - 61 present in the heavy bond mutant germ cell line, and 3 architectural regions 1, 2, and 3 (^ί^ρ\νΐ: (5Ι^Ι^Ε30Ρ〇υνΚΡ8ΕΤΙ^υΓσΓν8(3Ε(3ΙΕ)ΝΟ: 174), FW2: WIRQPPGKGLEWIG (SEQ ID NO: 175) and FW3 : RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 〇176)). [00106] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in sequence 4b of the heavy chain mutant germ cell line (eg, FW1: QVQLQESGPGLVKPSETLSLTCAVS) (SEQ ID NO: 177), FW2·· WIRQPPGKGLEWIG (SEQ ID NO: 178) and FW3: RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 179)). [00107] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the kappachain mutant germ cell line sequence 12 (eg, (SEQ ID NO: 180), FW2: WYQQKPGKAPKLLIY (SEQ ID NO: 181) 25 201041902 and FW3: GVPSRPSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 182)). [00108] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the kappa chain variant germ cell line sequence (2 (eg, FW1: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 183), FW2: WYQQKPGKAPKLLIY (SEQ ID NO: 184) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 185)). [00109] The anthropomorphic antibody or binding fragment thereof can comprise framework regions 1, 2, and 3 对应 corresponding to framework regions 1, 2, and 3 present in sequence 018 of the Karpa chain mutant germ cell line (eg, FW1: DIQMTQSPSSLSASVGDRVTITC) (SEQ ID Μ): 186), FW2: WYQQKPGKAPKLLIY (SEQ ID NO: 187) and FW3: GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 188)). [00110] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the Kappa mutated germ cell line sequence 08 (eg, FW1: DIQMTQSPSSLSASVGDRVTITC ( SEQ Π) NO: 189), FW2: WYQQKPGKAPKLLIY (SEQ ID NO: 190) and FW3: GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 〇 191)). [00111] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the sequence of the Karpa chain mutant germ cell line A20 (eg, FW1: DIQMTQSPSSLSASVGDRVTITC ( SEQ ID NO: 192), FW2: WYQQKPGKVPKLLIY (SEQ ID NO: 193) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 194)). [00112] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the sequence of the Karpa chain mutant germ cell line A30 (eg, FW1: DIQMTQSPSSLSASVGDRVTITC ( SEQ ID NO: 195), FW2: WYQQKPGKAPKRLIY (SEQ ID NO: 196) and FW3: 26 201041902 GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 197)). [00113] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the sequence of Lappa germ cell line L14 (eg, FW1: N1QMTQSPSAMSASVGDRVTITC ( SEQ ID NO.. 198), FW2: WFQQKPGKVPKHLIY (SEQ ID NO: 199) and FW3: GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 200)) [00114] The anthropomorphic antibody or binding fragment thereof may comprise a corresponding to the Kappa strand The framework regions 1, 2, and 3 of the framework regions 1, 2, and 3 in the variant germ cell line sequence L1 (eg, FW1: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 201), FW2: WFQQKPGKAPKSLIY (SEQ ID NO: 202) and FW3 : GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 203)). [00115] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the sequence of Lappa germ cell line sequence L15 (eg, FW1: DIQMTQSPSSLSASVGDRVTITC ( SEQ ID NO: 204), FW2: WYQQKPEKAPKSLIY (SEQ ID NO: 205) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: O 206)). [00116] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the sequence of Lappa germ cell line L4 (eg, FWl. AIQLTQSPSSLSASVGDRVTITC ( SEQ ID NO: 207), FW2: WYQQKPGKAPKLLIY (SEQ E) NO: 208) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 209)). [00117] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the sequence of Lappa germ cell line L18 (eg, FWl·· AIQLTQSPSSLSASVGDRVTITC) (SEQ ID NO: 210), FW2: WYQQKPGKAPKLLIY (SEQ ID NO: 211) and FW3: 27 201041902 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 212)). [00118] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the Kappa mutated germ cell line sequence L5 (eg, FW1: DIQMTQSPSSVSASVGDRYTITC ( SEQ ID NO: 213), FW2: WYQQKPGKAPKLLIY (SEQ Π) NO: 214) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 215)). [00119] The anthropomorphic antibody or binding fragment thereof may comprise an architectural region 1, 2, and ◎ 3 corresponding to the framework regions 1, 2, and 3 present in the sequence of the Karpa chain mutant germ cell line L19 (eg, FW1: DIQMTQSPSSVSASVGDRVTITC) (SEQ ID NO: 216), FW2: WYQQKPGKAPKLLIY (SEQ Π) NO: 217) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 218)). [00120] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the Kappa mutated germ cell line sequence L8 (eg, FW1: DIQLTQSPSFLSASVGDRVTITC ( SEQ ID NO: 219), FW2·· WYQQKPGKAPKLLIY (SEQ ID NO: 220) and FW3: GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 〇 221)). [00121] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the sequence of the Karpa chain mutant germ cell line L23 (eg, FW1: AIRMTQSPFSLSASVGDRVTITC ( SEQ ID NO: 222), FW2: WYQQKPAKAPKLFIY (SEQ ED NO: 223) and FW3: GVPSRPSGSGSGTDYTLTISSLQPEDFATYYC (SEQ ID NO: 224)) 〇 [00122] The anthropomorphic antibody or binding fragment thereof may comprise a corresponding to the mutation present in the kappa chain The framework regions 1, 2, and 3 of the framework regions 1, 2, and 3 in the germ cell line sequence L9 (eg, FWl·· AIRMTQSPSSFSASTGDRVTITC (SEQ ID NO: 225), FW2: WYQQKPGKAPKLLIY (SEQ ID NO: 226) and FW3 : 28 201041902 GVPSRPSGSGSGTDFTLTISCLQSEDFATYYC (SEQ ID NO: 227)). [00123] The anthropomorphic antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the sequence of the Karpa chain mutant germ cell line L24 (eg, FW1: VIWMTQSPSLLSASTGDRVTISC ( SEQ ID NO: 228), FW2: WYQQKPGKAPELLIY (SEQ ID NO: 229) and FW3: GVPSRFSGSGSGTDFTLTISCLQSEDFATYYC (SEQ ID NO: 230)) 〇 [00124] The anthropomorphic antibody or binding fragment thereof may comprise a corresponding to the mutation present in the kappa chain The framework regions 1, 2, and 3 U of the framework regions 1, 2, and 3 in the germ cell line sequence L11 (eg, FW1: AIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 231), FW2: WYQQKPGKAPKLLIY (SEQ ID NO: 232) and FW3 : GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 233)). [00125] The suspected antibody or binding fragment thereof may comprise framework regions 1, 2, and 3 corresponding to framework regions 1, 2, and 3 present in the Kappa mutated germ cell line sequence L12 (eg, FW1: DIQMTQSPSTLSASVGDRVTITC) (SEQ ID NO: 234), FW2: WYQQKPGKAPKLLIY (SEQ ID NO: 235) and FW3: GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (SEQ ID NO: 〇 236)).

The present invention also provides a humanized antibody having a specificity in the A1 domain of vWF or a binding fragment thereof, comprising: HCDR1.. GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), ^ HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9), LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12); corresponding to the presence of the following human The heavy chain framework regions 1, 2 and 3 - 4-04 of the framework regions 1, 2 and 3 in the antibody (SEQ ID NOS: 147, 148 and 149, respectively), 4-28 (SEQ ID NO: 150, 151 and 152), 4-30.1 (SEQ ID NO: 153, 154 and 155, respectively), 4-30.2 (SEQ ID NO: 156, 157 and 158, respectively), 4-30.4 (SEq id NO: 159, 160, respectively) And 161), 4-31 (points 29 201041902 are SEQ ID NO: 162, 163 and ία), Μ 4 (SEq ro N〇: 16s, 166 and 167 respectively), 4-39 (SEq melon N〇, respectively) : 168, 169 and 17〇), 4_S9 (SEQ ID NO: 171, 172 and 173, respectively), 4-61 (SEQ ID NO: 174, 175 and 176, respectively) or 4-b (SEq Jiang, respectively) N〇: lr?, 178 and 179); and the structure corresponding to the presence of the following human antibodies丨, 2 and 3 light chain architecture regions 12*3 - 〇 12 (SEQ ID NOS: 180, 181 and 182, respectively), 02 (SEQ ED NO: 183, 184 and 185, respectively), 018 (SEQ ID NO, respectively) : 186, 187 and 188), 08 (SEQ ID NO: 189, 190 and 191, respectively), A2〇 (SEQ ID NO: 192, 193 and 194, respectively), A30 (SEQ ID NO: 195, 196 and 197, respectively) , L14 (SEQ ID NO: I98, 199 and 200, respectively), LI (SEQ ID NO: 20i, 202 and 203, respectively), L15 (SEQ ID NO: 204, 205 and 206, respectively), L4 (respectively SEQ ID NOs: 207, 208 and 209), LI8 (SEQ ID NOS: 210, 211 and 212, respectively), L5 (SEQ ID NOS: 213, 214 and 215, respectively), L19 (SEQ ID NO: 216, respectively) , 217 and 218), L8 (SEQ ID NO: 219, 220 and 221, respectively), L23 (SEQ ID NO: 222, 223 and 224, respectively), L9 (SEQ ID NO: 225, 226 and 227, respectively) L24 (SEQ ID NOS: 228, 229 and 230, respectively), LI 1 (SEQ ID NOS: 231, 232 and 233, respectively) and L12 (SEQ ID NOS: 234, 235 and 236, respectively). The invention also provides an anthropomorphic antibody or binding fragment thereof having the specificity of the A1 domain of VWF as described herein, which retains the parental non-humanized antibody or ❹ comprises a parental non-humanized antibody and The chimeric antibodies of the human Fc region share the same activity. The parental non-humanized antibody is typically a murine antibody, particularly a murine antibody; a chimeric antibody comprising a parental non-humanized antibody typically comprises a variant region derived from a murine antibody, particularly from the murine antibody NMC-4. And human antibodies, preferably in the human Fc region of the invention as the human Fc region. [00128] The activity of a parental non-humanized antibody and a chimeric antibody as described herein as an anthropomorphic antibody or binding fragment thereof can be determined by, for example, determining EC5〇 as described in Example 1 with ristocetin ( Ristocetin) induces platelet aggregation activity to be measured. When the activity of the anthropomorphic antibody or binding phase thereof as described herein is the same as the activity of the 匚5〇, or up to 50%, preferably up to 30%, 20% (eg higher or lower) is different from the pro When a non-humanized antibody or chimeric anti-sputum EQq activity is achieved, anthropomorphic 30 201041902 antibody or a binding fragment thereof as described herein can be considered to retain the same activity as the parent non-humanized antibody or chimeric antibody. [00129] In a preferred embodiment of the invention, the anthropomorphic antibody or binding fragment thereof as described herein further comprises a heavy chain framework region from a human antibody, wherein the human heavy chain architecture region does not comprise more than one mouse residue base. [2〇130] In still another preferred embodiment of the present invention, the anthropomorphic antibody or binding fragment thereof as described herein further comprises a light chain architecture region derived from a human antibody, wherein the human light-slit architecture region does not include- More than one gas residue. [00131] The human heavy-key architecture region does not contain more than one mouse residue or "human"

The steroid light chain architecture region does not contain a singularity of the human heart. The gratitude does not contain more than one human heavy or light chain architecture region that contains only the murine residues in the murine, for example, does not contain back mutations ( Backimtation) to residues that are only present in the gas and are not present in humans. Thus, the human heavy or light chain framework region containing human residues also present in the murine is not excluded; thus, it is not excluded that the residues have been mutated to the average human (eg, most human framework regions) Common residues), but also exist in human heavy or light chain architecture regions in rodents. [00132] In the heavy chain or light chain framework region of the present invention, more than one mouse silk embodiment is included in the heavy chain or light chain framework region, which usually comprises 1G or less of murine bases, preferably 9 or less, more preferably For 8 gamma, 7 gamma is better, best =! ^, 5 or less is better '4 or less is better, 3 is better and less, 2 is better than 1' is especially ideal. [00133] The present invention provides an isolated sputum encoding a direct binding fragment of a humanized antibody having vWF specificity, which comprises, for example, seqidnq. 丨; the heavy chain of the region, such as the Qing dynasty: 28 cap-like variation Region Sequence = 34] The invention provides an isolated nucleic acid encoding a humanized antibody or complex-fragment having vWF specificity, comprising the SEQ ID NO: Μ heavy bond bank and light as described in SEQ ID NO: 238 Chain sequence. Suppressing the sequel to the CDRs of the CDRs having the VWF#I heterologous, and the CDR regions corresponding to the CDRs present in the transgenic antibody 31 201041902; a heavy chain framework region of the framework region present in the variant region of human antibody AAC18165.1 (SEQ ID NO: 4); and light corresponding to the framework region present in the variant region of human antibody AAK94808 (SEQ ID NO: 6) Chain architecture area. [00136] The invention also provides an isolated nucleic acid encoding a humanized antibody having a vWF specificity or a binding fragment thereof, comprising: HCDR1: GFSLTDYGVD (SEQ ID NO: 7) 'HCDR2: MIWGDGSTDYNSALKS (SEQ m NO: 8 ) 'HCDR3 : DPADYGNYDYALDY (SEQ ID NO: 9), and the heavy chain framework region from the variant region of human antibody AAC18165.1 (SEQroNO: 4). A nucleotide sequence of an exemplary human heavy chain framework region is disclosed in SEQ ID NO: 116. 〇[(8)137] The present invention also provides an isolated nucleic acid encoding a humanized antibody having a vWF specificity or a binding fragment thereof, comprising the following light chain CDRs - LCDR1: SASQDINKYLN (SEQ E) NO: 10) ^ LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12), and the light chain architecture region from the variant region of human antibody AAK94808 (SEQ ID NO: 6). The nucleotide sequence of the exemplary human light chain architecture region is disclosed in 117. [00138] The invention also provides an isolated nucleic acid encoding a humanized antibody or binding fragment thereof having vWF specificity, which comprises one of the following heavy chain variant regions: H2 (SEQ ID NO: 13), H4 (SEQ ID NO) : 14), H5 (SEQ ID NO: 15), H6 ❹ (SEQ 10 NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ ID NO: 19) &gt H12 (SEQ ID NO: 20) ^ H13 (SEQ ID NO: 21) > H14 (SEQ ID NO: 22) 'H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146). [00139] The invention also provides an isolated nucleic acid having a vWF-specific anthropomorphic antibody or binding fragment thereof, comprising one of the following light chain variant regions: L5 (SEQ ID NO: 23) - L4 (SEQ ID NO: 24) > L6 (SEQ ID NO: 25) ' L7 (SEQ ID NO. 26) ' L8 (SEQ ID NO: 27) > L9 (SEQ ID NO: 28) > L10 (SEQ ID NO: 29) Or LI 1 (SEQ ID NO: 30). The present invention provides an isolated nucleic acid encoding an anthropomorphic antibody or a delta fragment having a specificity of 7 which comprises (1) one of the following heavy chain variant regions: H2 32 201041902 (SEQ ID NO: 13), H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146); and (2) One of the following light chain variant regions: L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24) > L6 (SEQ ID NO: 25) > L7 (SEQ ID NO: 26) ' L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), L10 (SEQ ID NO: 29) or LI 1 (SEQ ID NO: 30). The present invention also provides a light chain comprising an isolated nucleic acid sequence encoding a vector GS264 which has been filed with DSMZ on January 23, 2008, and deposited in a microorganism. The accession No. is DSM21059. The present invention also provides that the heavy chain's vector GS265 comprising the isolated nucleic acid sequence encoding the vector GS265 was deposited with the DSMZ on January 23, 2008, and the accession number (accession No.) is DSM21060. The present invention also provides a humanized antibody having a vWF specificity or a binding fragment thereof which is encoded by a heavy chain of the nucleic acid sequence encoding the nucleic acid sequence of the vector GS264 and the nucleic acid sequence of the gamma vector GS265. The present invention provides a vector comprising an isolated nucleic acid encoding a humanized antibody having a vWF specificity or a binding fragment thereof, the humanized antibody or a kiosk thereof comprising the same as described in SEQ ID NO: 19. The heavy chain variant region sequence and the light chain variant region sequence as set forth in SEQ ID NO: 28. The present invention provides a vector comprising an isolated nucleic acid encoding a humanized antibody having a vWF specificity or a binding fragment thereof, the humanized antibody or binding fragment thereof comprising the heavy chain as set forth in SEQ ID NO: 237 Sequence and light bond sequence as set forth in SEQ ID NO: 238. The present invention provides a vector comprising an isolated nucleic acid encoding a humanized antibody having a vWJF specificity or a binding fragment thereof, the humanized antibody or a knot thereof comprising: corresponding to the murine antibody NMC-4 The CDRs of the CDRs in the region of the heavy chain region of the framework region in the variant region of the human antibody AAC18165.1 (SEQ ID NO: 4); and the variation corresponding to the human antibody AAK94808 (SEQ ID NO: 33 201041902 6) The light chain architecture area of the architectural area in the district. [00147] The invention also provides a vector comprising a nucleic acid encoding a humanized antibody having VWF specificity or a binding fragment thereof, and the humanized antibody or binding fragment thereof comprises HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9), and the heavy chain framework region from the variant region of human antibody AAC18165.1 (SEQ ID NO: 4). The invention also provides a vector comprising a nucleic acid encoding a humanized antibody having VWF specificity or a binding fragment thereof, and the humanized antibody or binding fragment thereof comprises a light chain CDRs - LCDR1: SASQDINKYLN (SEQ. ID NO: 10;), 〇LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLFWT (SEQ ID NO: 12), and the light chain from the variant region of human antibody AAK94808 (SEQ E) NO: 6) Architecture area.

[00149] The invention also provides a vector comprising a nucleic acid encoding a humanized antibody having VWF specificity or a binding fragment thereof, and the humanized antibody or binding fragment thereof comprises one of the following heavy chain variant regions: H2 ( SEQIDNO: 13), H4 (; SEQ

ID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ Π) NO: 16), H7 (SEQ ID NO: 17) 'H8 (SEQ ID NO: 18) > H9 (SEQ ID NO: 19) ^ H12 (SEQ ID NO: 20) > H13 (SEQ ID NO: 21) > H14 (SEQ ID NO: 22) 'H15 〇 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146 ).

[00150] The invention also provides a vector comprising a nucleic acid encoding a humanized antibody having V\YF specificity or a binding fragment thereof, and the humanized antibody or binding fragment thereof comprises one of the following light chain variant regions: L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25) 'L7 (SEQ ID NO: 26), L8 (SEQ Π) NO: 27), L9 (SEQ ID NO) : 28), L10 (SEQ ID NO: 29) or L11 (SEQ ID NO: 30). The invention also provides a vector comprising a nucleic acid encoding a humanized antibody having VWF specificity or a binding fragment thereof, and the humanized antibody or binding fragment thereof comprises (1) one of the following heavy chain variant regions: H2 ( SEQ ID NO: 13), H4 (SEQ ID NO: 14) > H5 (SEQ ID NO: 15) Ή6 (SEQ ID NO: 16) > H7 201041902 (SEQ ID NO. 17), Η8 (SEQ ID NO: 18 ), Η9 (SEQ ID NO: 19), Η12 (SEQ ID NO: 20) 'HI3 (SEQ ID NO: 21) n H14 (SEQ ID NO: 22) ^ H15 (SEQ ID NO: 145), or H16 ( SEQ ID NO: 146); and (2) the following light chain t: hetero-region where - L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25) ' L7 (SEQ ID NO: 26) - L8 (SEQ ID NO: 27) ' L9 (SEQ ID NO: 28), L10 (SEQ ID NO: 29) or LI 1 (SEQ ID NO: 30). The invention also provides a vector comprising an isolated nucleic acid comprising a light chain encoding the isolated nucleic acid sequence of vector GS264, the vector (7) being applied at 2〇〇8 Ο

On January 23, 2013, DSMZ was applied to deposit in a microorganism, and its registered number (form No.) was DSM21059. [00153] The present invention also provides a vector comprising an isolated nucleic acid comprising the heavy chain of the isolated nucleic acid sequence of the gamma vector GS265, which has been deposited with the DSMZ for storage in the microorganism on January 23, 2008. Its registration number (accessi〇n No) is DSM21060. [00154] The present invention provides a host cell comprising an isolated nucleic acid, the isolated human nucleic acid or a binding fragment thereof, and the humanized antibody or binding version thereof comprises as described in SEQ ID NO: 19. The sequence of the heavy bond variant region and the sequence of the light chain variant region as described in SEQ ID NO.28. [00155] The present invention provides a humanized antibody or a binding fragment thereof comprising a PD-specific host cell code having a polymorphic sequence, and the human or a binding fragment thereof comprises the heavy chain sequence ID muscle as described in SEQ ID NO. Light chain sequence as described in 238. [00156] The present invention also provides a host cell comprising an isolated nucleic acid, the continuation antibody or its ligated segment, _ anthropomorphic or 5 fragment comprising ... corresponding to the CDR region present in the murine antibody gastric fistula Corresponding to the framework of the heavy chain that exists in the framework region of the human antibody from 〇181651 (the region of the variant region; and corresponding to the region of variation present in the variant region of human body 94808 (SEQ ID NO: 6) _ $ [00157] Also provided is a host cell comprising an isolated nucleic acid encoding a humanoidized reduction or a binding limb thereof having vWP prevalence, and the human or the binding fragment thereof comprises: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8) ^ HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9), and the heavy chain framework region from the variant region of human antibody AAC18165.1 (SEQ ID NO: 4). [00158] The invention also provides A host cell comprising an isolated nucleic acid encoding a humanized antibody having a vWF specificity or a binding fragment thereof, and the humanized antibody or binding fragment thereof comprises the following light chain CDRs - LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR 2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12), and the light chain architecture region from the variant region of human antibody AAK94808 (SEQ ID NO: 6). [00159] The present invention also provides A host cell comprising an isolated nucleic acid encoding a humanized antibody having a vWF specificity or a binding fragment thereof, and the humanized antibody or binding fragment thereof comprises one of the following heavy chain variant regions: H2 (SEQ ID NO: 13) ), H4 (SEQ Π) NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 ( SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146). The present invention also provides a host cell comprising an isolated nucleic acid encoding a humanized antibody having a vWF specificity or a binding fragment thereof, and the humanized anti-steroidal or binding fragment thereof comprises one of the following light chain variant regions L5 (SEQ ID NO: 23) > L4 (SEQ ID NO: 24) > L6 (SEQ ID NO: 25) > L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), L10 (SEQ ID NO: 29) or LI 1 (SEQ ID NO: 30). [00161] The invention also provides a host cell comprising an isolated nucleic acid encoding a humanized antibody having a vWF specificity or a binding fragment thereof, and the humanized antibody or binding fragment thereof comprises one of the following heavy chain variant regions: H2 (SEQ ID NO: 13), H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16) > H7 (SEQ ID NO: 17) ^ H8 (SEQ ID NO: 18) > H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145) , or H16 (SEQ ID NO: 146); and (2) the next 36 201041902 one of the light chain variant regions: L5 (SEQ ID NO: 23), L4 CSEQ ID NO: 24) ' L6 (SEQ ID NO: 25) 'L7 (SEQ ID NO: 26) > L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), li〇 (SEQ ID NO: 29) or LI 1 (SEQ ID NO: 30) . [00162] The present invention also provides a host cell comprising an isolated nucleic acid encoding the light chain of the nucleic acid sequence of the vector GS264, which has been deposited with the DSMZ for storage in the microorganism on January 23, 2008. The number (accessi〇nN〇.) is DSM21059.

The present invention also provides a host cell comprising an isolated nucleic acid encoding the heavy chain of the isolated nucleic acid sequence of the vector GS265, which has been applied to the microorganism in the DSM2 on January 23, 2008. , its registration number ( accession

No.) is DSM21060. _64] The method for producing a humanized antibody or a binding sheet thereof, comprising the method of cultivating the host cell of the present invention, expressing the nucleic acid and producing the antibody, and the method for producing the anthropomorphic vWF antibody or the binding fragment thereof of the present invention The antibody may be further recovered from the host cell culture solution. In certain embodiments, the antibody is recovered from the host cell culture medium; in some implementations, prior to culturing, the vector comprising the nucleic acid comprising the singularity of the singularity and the light chain of the light chain is used together. Infect the host cell. [00165] The invention provides a composition comprising a humanized antibody having a vWF specificity, or a binding fragment thereof, comprising the sequence of a heavy chain variant region as described in SEQ. NO. 19, and such as SEqidΝ The sequence of the light bond variant region described in 〇·μ. [= The present invention provides a composition comprising an anthropomorphic antibody having vWF specificity or a two-part cassette thereof, the humanized antibody or a binding fragment thereof comprising the heavy chain variant region sequence as described in SEQ ID NO: 28, the light chain is subjected to the sequence of the heterogeneous region, and the acceptable redness of the medical red (earrieT). The invention provides a humanized antibody or composition thereof comprising vwp specificity, the humanized antibody or binding fragment thereof comprising the heavy chain sequence described in * SEQ m ••π and as described in S£qidno:238 Light key sequence 201041902 column. [〇_] & The invention provides a composition comprising a humanized antibody having a vWF specificity or a binding fragment thereof, the humanized antibody or binding fragment thereof comprising the heavy chain sequence as described in SEQ n) NO: 237, eg SEQn) N〇: The light chain sequence described in 238, and the carrier on the medical treatment. The present invention provides a composition comprising a humanized antibody having human Von Wyper factor (VWF) specificity or a binding fragment thereof, the humanized antibody or a binding fragment thereof, corresponding to the presence of the murine antibody NMC_4 The CDR region of the CDRs; the heavy chain framework region corresponding to the framework region in the variant region of human antibody AAC18165.1 (SEQ ID NO: 4); the light chain corresponding to the framework region in the variant region of human antibody AAK94808 (SEQ ID NO: 6) An architectural zone; and a pharmaceutically acceptable carrier. [00170] The present invention also provides a composition comprising a humanized antibody having a vWF specificity or a binding fragment thereof. The humanized antibody or binding fragment thereof comprises 11〇]]1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 : MIWGDGSTDYNSALKS (SEQ ID NO: 8) > HCDR3: DPADYGNYDYALDY (SEQ Π) NO: 9), a heavy chain framework region from the variant region of human antibody AAC18165.1 (SEQ ID NO: 4), and a pharmaceutically acceptable carrier.本 [00171] The invention also provides a humanized antibody comprising VWF specificity or

The composition of the binding fragment 'The anthropomorphic antibody or binding fragment thereof comprises the following light chain CDRs-LCDR1: SASQDINKYLN (SEQ ID NO: 10) > LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: (^YEKLPWT (SEQ ID 1 0: 12), and a light chain architecture region from a variant region of human antibody from 04808 (3 £ (31 〇): 6), and a pharmaceutically acceptable carrier. [00172] Also provided is a composition comprising a humanized antibody having a vWF specificity or a binding fragment thereof comprising: heavy chain CDRs ^ HCDR1: GFSLTDYGVD (SEQ ID NO: 7) 'HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR3: DPADYGNYDYALDY (SEQ ID N〇: 9); and light chain CDRs, LCDR1: 38 201041902 SASQDINKYLN (SEQ ID NO: 10) > LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3 : QQYEKLPWT (SEQ ID NO: 12); (not required) Light chain architecture region from the variant region of human antibody AAK94808 (SEQ Π) NO: 6) and/or variation from human antibody AAC18165.1 (SEQ ID NO: 4) The heavy chain architecture zone of the zone; and a pharmaceutically acceptable carrier. The present invention also provides a composition comprising a humanized antibody having a vWF specificity, or a binding fragment thereof, comprising the following heavy chain variation region: 112 (3 5 (3101^>.13) ), 114 (3 £ (5©) ^ 0._14), 115 (8 £ (311 ^ 0 · 15), H6 (SEQ ID NO: 16), H7 (SEQ ED NO: 17), H8 (SEQ ID NO: 18) > H9 (SEQ ID NO: 19) Ή12 (SEQ ID NO: 20) 'H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ED NO: 145), Or H16 (SEQ ID NO.. 146) and a pharmaceutically acceptable carrier. [00174] The invention also provides a composition comprising a humanized antibody having a vWF specificity or a binding fragment thereof, the humanized antibody or binding thereof The fragment contains the following light bond variants: L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), L10 (SEQ ID NO. 29) or L11 (SEQ ID NO: 30) and a pharmaceutically acceptable carrier. [00175] The invention also provides for the inclusion of vWF specific A composition of a humanized antibody or a binding fragment thereof, the humanized antibody or binding fragment thereof comprising : one of the following heavy chain variant regions: H2 (SEQ ID NO: 13), H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15) > H6 (SEQ ID NO: 16) ^ H7 (SEQ ID NO: 17) H8 (SEQ ID M): 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO. 22), H15 (SEQ ID NO: 145), or HI6 (SEQ ID NO: 146); one of the following light chain variant regions - L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO) : 25), L7 (SEQ E) NO: 26) > L8 (SEQ ID NO: 27) > L9 (SEQ ID NO: 28) > Ll〇 (SEQ ID NO: Kiss or Lll (SEQ ID NO: 3)); and pharmaceutically acceptable cuts. [00176] Also provided is a composition comprising a first anthropomorphic antibody, or a binding fragment thereof, as described herein, in paragraph 39 201041902, and a second antibody that binds to the field of vWF. [〇〇πη本_ is also provided in the treatment of the subject's disease or t (such as to, plate disease or abnormality) = middle ^ method = therapeutic amount of anthropomorphic antibody with VWF4 temple or its knot fEC) 3φσ =EQ m N0: The heavy chain variant region sequence shown in 19 and the light chain variant region sequence as shown in SEQ ID NO: 28. _78] The present invention also provides a method of treating a medium iii, such as a platelet disease or abnormality, in a subject (e.g., a human), the method comprising a therapeutically effective amount of a humanized antibody having vw specificity or Two pieces of the fragment = two s EQ] [DN 〇: 237 1 heavy chain sequence and light chain sequence as shown in SEQ 110 NO: 238. [00179] The present invention is also a method of treating a sputum vector disease or abnormality (e.g., platelet disease or abnormality) in a subject (e.g., a human), the method comprising administering to the subject an effective treatment (4) having a vWF (tetra)-synthesis fragment comprising: a CDR region corresponding to the presence of a rectifying antibody; a construct n corresponding to the framework n in the variant region of the human antibody (SEQ IDN〇: 4); to human (10) AAK948() 8 (Qing Still NO: 6) The light chain architecture area of the architectural area in the variation zone. [00180] The present invention provides a method for treating a vwp vector disease or a heterologous (eg, platelet disease or abnormality) in a subject (eg, a human), the method comprising administering to the subject an effective therapeutic amount. An anthropomorphic antibody or binding fragment thereof having vWF specificity comprising: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8) 'HCDR3· DPADYGNYDYALDY (SEQ ID NO: 9), And the heavy bond framework region from the variant region of human antibody AAC18165.1 (SEQ ID NO: 4). [00181] A platelet disease or abnormality may be a cardiovascular disease or a cerebrovascular abnormality such as an ischemic stroke. In certain embodiments, the cardiovascular disease is atherosclerosis, restenosis, angina pectoris, acute myocardial infarction, acute coronary artery disease (acute coronary) Syndrome), or cardiovascular abnormalities associated with diabetes; 40 201041902 In certain embodiments, the thrombotic disease or abnormality is vascular inflammation, venous thrombosis, sickle cell disease, allograft rejection ( Xen〇graft rejection), peripheral vascular disease vascuiardisease) 'thrombotic thrombocytopenic purpura, cystic fibrosis, vascular dementia, Raynaud's dise^ e) rheumatoid arthritis, or diabetes; in certain embodiments, cerebral vascular abnormalities may include cerebral artery infarct and small gap infarction (_u lacumr ringing) Ischemic stroke and vascular dementia (vasculardemen) Tia). Anthropomorphic vWF antibodies can also be used to prevent recurrent towel winds or early stroke caused by inflammation of the cerebral blood vessels. [00182] In certain embodiments, a platelet disease or abnormality can comprise cancer. [00183] The invention also provides a method of treating a vector disease or, (eg, a platelet disease or disorder) in a subject (eg, a human), the method comprising administering to the subject a therapeutically effective amount of vWF specificity Anthropomorphic antibody or binding fragment thereof comprising the following light chain CDRs - LCDR1: SASqDINKYDSi (;^E(3 DD NO. 10), LCDR2: YTSSLHS (SEQ Π) NO: 11), and LCDR3: QQYEKLFWT (SEQ ID NO: 12), and the light chain architecture region from the variant region of human antibody AAK948〇8 (SEq ID NO: 6). GG[GG184] The present invention also provides a method of treating a vector disease or abnormality (e.g., platelet disease or abnormality) in a subject (e.g., a human), the method comprising administering to the subject a therapeutic amount of vWF Anthropomorphic antibody or binding fragment thereof comprising: one of the following heavy chain variant regions - m (SEQ ID NO: 13) > H4 (SEQ ID NO: 14) > H5 (SEQ ID NO: 15) ^ H6 (SEQ ID NO: 16) > H7 (SEQ ID NO: 17) > H8 (SEQ ID NO: 18) > H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 ( SEQ ID NO.. 21), H14 (SEQ ID NO: 23⁄4, HIS (SEQ ID NO.. I45), or H16 (SEq ID N〇: 146). [00185] The invention also provides in a subject ( For example, a method for treating a vWF vector disease or abnormality (eg, a disease, a plate disease, or an abnormality) in a human, the method comprising administering to the tester a therapeutic amount of a humanized antibody or material 41 201041902 fragment having a specific touch. Contains one of the following light chain variant regions - 1^5 (3 ugly (^10:^0: 23) ' L4 (SEQ ID NO: 24) > L6 (SEQ ID NO: 25) > L7 ( SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), L10 (SEQ ID NO: 29) or LI 1 (SEQ ID NO: 30). [00186] The invention also provides VWF vector disease or abnormality in a subject (eg, a human) A method of treatment (e.g., a platelet disorder or abnormality), the method comprising administering to the serotype a therapeutically effective amount of a humanized antibody having a vWF specificity or a binding fragment thereof comprising: one of the following heavy chain variant regions (3 £(31〇]^0: 13) > H4 (SEQ ID NO: 14) > H5 (SEQ ID NO: 15) ^ H6 (SEQ ID NO: 16) 'H7 (SEQ ID NO: 17) 'H8 (SEQ ID NO: 18) 'H9 (SEQ ID NO: 19) Ή12 (SEQ ID NO: 20)^H13 (SEQ ID NO: 21) Ή14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146); one of the following light chain variant regions: L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO 25), L7 ( SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), L10 (SEQ ID NO: 29) or Lll (SEQ Π) NO: 30). [00187] In certain embodiments, a humanized antibody or a binding fragment thereof having vWF specificity lacks an effector function; in certain embodiments, the anthropomorphic 'antibody comprises an Fc region derived from IgG4. The present invention provides a human scorpion antibody having a specificity of Von Wagner (vWp) or a binding fragment thereof, which can be provided from about i to about 250 times the effective therapeutic amount of HD1Q0 without causing significant bleeding clinically. symptom. The human antibody or its ligated 3 fragment is preferably of the A1 domain specificity of human VWF, and the human antibody or binding fragment thereof having vWF specificity is more preferably a vWF-specific anthropomorphic & Fragment. [00189] The present invention also provides a method of treating a disease or disorder of a vWF vector, which is a humanized form having VWF specificity as described herein by administering to a subject (preferably a human) a therapeutically effective amount. An antibody or a binding fragment thereof. The therapeutic amount is from about 0.001 to about 100 mg/kg, preferably from about 〇〇〇2 to about 2 〇m_g, more preferably from about 002·002 to about 1 〇mg/kg, and more preferably from From about U to about (Umg/kg, more preferably from about _5 to about 〇. 2mg/kg, most preferably from about _201041902 to about 0.1 mg/kg. Π - Γ Γ also provides a vwp medium A treatment for a disease or disorder * t 2 ΐ T 2 requires a therapeutically effective amount of an anthropomorphic antibody as described herein to be achieved. The effective therapeutic amount is from about 1 to about ED1 of ED1 (X). About 1 to about 200 times, from ED1. About 1 'Π-ί hair, also provides a treatment for vWF vector disease or abnormality - or multiple small doses (sub-dGse) effective treatment amount Achieving a second anthropomorphic antibody or a binding fragment thereof for administration to a subject in need of such treatment. The invention also provides a therapeutic agent for a vWF vector disease or disorder, (iv) a therapeutically effective amount of anthropomorphic antibody or a combination thereof as described herein. Yuefeng is also subcutaneously administered to a subject in need of such treatment. The invention also provides a treatment for vWF-mediated diseases or abnormalities. An anthropomorphic antibody or a binding fragment thereof as described herein is also administered intravenously to a subject in need of such treatment. Ο Π-ίί! also provides a therapeutic agent for vWF vector disease or abnormality i It ΐττϋ, or in combination with radiotherapy (eg, irradiation or introduction of radiation π 〇, KUniSChe 0nkol ° ^ Springer-Verlag by intravenous administration of a therapeutic amount of anthropomorphic antibody or its binding fragment k as described herein requires such treatment Subjects to achieve. In the implementation or testing of this issue, _ on or equivalent to the methods and materials described herein can be made 'but the following are still better methods and materials. Anthropomorphic Von Wylie factor antibody manufacturing Anthropomorphic Marvel's factor (vwf) antibody (eg, murine body or combination method) is provided. Anthropomorphic anti-hunting with vWF specificity is derived from non-human axis (eg, mouse) and/or lir·f on CDS Or a portion thereof to the human New Zealand and Region I, the above wood region. Optionally, such a person present in the VH and/or VL region, the frame 43 201041902 31 ft maintains the binding affinity required or desired Non-complex ^Substitute. Non-essentially, the non-human amine-based residues present in the CDRs can be replaced by human residues. The classification of the framework and CDRs (except for the deletion of friends ϋ 卜 于 奸 奸 ( ( Kabatnumbeiings_ CDR. The CDS of the heavy chain contains the residues 31_35 (HCDR1), 5 secret (HC gift), 3 95-102^HCDR3); the light chain (10) is defined by the residue 24_34 (lcdri), force m And 89_97 (LCDR3). The structure area in Xie (such as heavy chain Ο wood (R1), heavy chain architecture zone 2 (ΗΡί〇, heavy chain architecture zone 3 (hpr3) and/or heavy chain architecture zone 4 (title 4)) The residues are composed of brain ^), 36-49 (HFR2), 66-94 (HFR3), and 1〇3_113 (HFR4), while the framework region in VL contains residues (2) (10) phantom), 35_49 (LFR2), 57_88 (lfr3) and 98-107 (LFR4) (W and Kabat, 1970 J and .ship 132:211). However, based on the structure of CDRs, Cho1ilia defines CDR1_H to contain residues 26_32 jChothia et al., 1992 J·1/227:799), and AbM (antibody modelling) CDR1-H contains residues 26-35. Oxford Molecular, a compromise between the two used by the AbM antibody modeling software, is the definition used herein for the anthropomorphic approach using NMC-4. [00198] Anthropomorphic antibody or a U-binding fragment thereof having Von Wylie factor (VWF) specificity can be produced by transferring heavy chain complementarity determining regions (CDRs) from NMC-4 to corresponding human antibodies The heavy chain framework region of the framework region in the variant region of (SEQ ID Να 4); and the transfer of the light chain CDRs from NMcy to the variant region corresponding to the human antibody 〇48〇8 (SEq ID Ν〇·6) The light chain architecture area of the architectural area in the middle. [00199] Anthropomorphic antibodies or binding fragments thereof having vWF specificity can also be produced by the heavy chain CDRs from murine NMC-4 (eg, 曰HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 : MIWGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR3: DPADYGNYDYALDY (SEQ ID NO.. 9)) One of the above is transferred to the human framework region (eg, the variant region from AAC18165.1 (SE, QIDNO: 4)). 44 201041902 [00200] Anthropomorphic antibodies or binding fragments thereof having vWF specificity can also be produced by: CDRs of light chains (eg, LCDR1: ^ SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ. ID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12)) wherein - the above is transferred to the human framework region (eg, from the variant region of AAK94S08 (SEQ ID NO: 6)). [00201] Anthropomorphic antibodies or binding fragments thereof having vWF specificity can be produced by the following methods: heavy chain CDRs from murine NMC-4 (eg, HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: 〇MIWGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9)) one of which is transferred to the human framework region (eg, the variant region from AAC18165.1 (SEQ ID NO: 4)); Chain CDRs (eg LCDR1: SASQDINKYLN (SEQ ID NO: 1〇),

One of the LCDR2·· YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKXPWT (SEQ ID NO: 12)) is transferred to the human framework region (eg, from the variant region of AAK94808 (SEQ ID NOJ)). [00202] Anthropomorphic antibodies or binding fragments thereof having vWF specificity can be made by: including inclusion in NMC-4 and human framework regions.

Modified heavy chain variant regions of CDRs (eg, η2 (SEQ ID NO: I3), H4 (SEQ ID)

D NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID

NO: 17) 'H8 (SEQ ID NO: 18) > H9 (SEQ ID NO: 19) ^ H12 (SEQ ID

NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146)) are transferred to the human constant region.

[00203] Anthropomorphic antibodies or binding fragments thereof having vWF specificity can also be produced by the inclusion of a cultivar light chain variant region (eg, L5 (eg, L5) that is present in CDRs and CDRs in the human framework regions. SEq is still NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27) > L9 (SEQ ID NO: 28) 'L10 (SEQ ID NO: 29)^, Lll (SEQ ID NO: 30)) is transferred to the human constant region. [00204] Anthropomorphic antibodies or binding fragments thereof having vWF specificity can be made by 45 201041902 in the following manner: a modified heavy chain variant region comprising CDRs present in the NMC-4 and human framework regions (eg, H2 (SEq) Still no: 13), H4 (SEQ Π)

NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID

NO: 17) > H8 (SEQ ID NO: 18) > H9 (SEQ ID NO: 19) > H12 (SEQ ID

NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146)) transferred to the human constant region; Modified light chain variant regions comprising CDRs present in NMC-4 and human framework regions (eg, L5 (SEQ ID NO: 23), L4 (SEQ ID NO..24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ Π) NO: 28), [10 (8 is called 1〇: 29) or B 11_(^1〇: ^〇: 30 )) Transfer to the human constant O zone. [00205] In an attempt to further reduce the antigenicity of anthropomorphic antibodies, residues in the CDRs of the human amino acid residues (e.g., murine residues) can be altered. For example, an anthropomorphic antibody can comprise one or more of the F27G, L29I, T30S and/or V34W substituents in HCDR1. In certain embodiments, the 'humanized antibody can comprise one or more of the S61P and/or A62S substituents in HCDR2; in certain embodiments, the anthropomorphic antibody can comprise S24Q, N30S and/or K31N in LCDR1. In one embodiment, the anthropomorphic antibody may comprise more than one substituent, such as the Y50D, T51A, S53N, H55E and/or S56T substituents in LCDR2. In certain embodiments, a 'humanized antibody' may comprise: one or more of the F27QL29I, T30S and/or V34W substituents in HCDR1; one or more of the S61P and/or A62S substituents in HCDR2; S24q in LCDR1 And one or more of N3〇s and/or K31N substituents; and one or more of y5〇d, T51A, S53N, H55E and/or S56T substituents in LCDR2. [00206] A variety of different forms of anthropomorphic antibodies are contemplated. For example, an anthropomorphic antibody can be, for example, an antibody fragment of a Fab that is arbitrarily conjugated to more than one cytotoxic agnet to produce an immunoconjugate (). Alternatively, the 'humanized antibody or affinity matured antibody can be an intact antibody, such as a full IgGl antibody. [00207] We have developed a variety of different antibody sequences for anthropomorphic antibodies 46 201041902. Conventionally, these fragments are obtained by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., o/5z-〇c/zemz'caZ and Methods, 24: 107-117 (1992); A Brennan et Al., Science, 229-81 (1985)); however, these fragments can now be directly produced by recombinant host cells, for example, antibody fragments can be isolated from the antibody phage library (antib〇dy phage library) discussed above; Alternatively, the FaV-SH fragment can be directly recovered from five c〇/z· and chemically coupled to form an F(ab,) 2 fragment (Carter et aL, ❸ 偷 偷/73⁄4如命1〇: 163-167 ( 1992)). According to another approach, the F(10),2 fragment can be isolated directly from recombinant host cell culture, and other methods of making antibody fragments will be apparent to those skilled in the art. In other embodiments, the selected antibody is a single chain Fv fragment (scFv), see WO 〇 1993/16185; U.S. Patent No. 1, No.; and U.S. Patent No. 5,5 δ 7,458. The antibodies may also be "linear antibodies" as described, for example, in U.S. Patent No. 5,641,87. * In the same method, it is possible to have at least a part of the desired binding specificity (antibody - silk fibroin mutual domain, nucleus), CH2 region and CH3 region, preferably (10) - The age-old mosquito area (CH1) is present in at least -3 chain mouth (if the dna of the prion protein/bond fusion and the immunoglobulin twitch (9); the ratio of each other to the (four) application ratio is two;; =;; The high yield or the coding sequence is as shown in the table. The insertion of the two or more peptides of the cytotoxic serotonin is shown in the 脰, and the antibody is chimeric to [deletion] and is considered to be formed in the anti-financial solution. Job leolytic) 201041902 Money to practice the nucleus of Wei enzyme [00211] There are a variety of radioisotopes that can be used to make radioconjugated anthropomorphic VWF antibodies, examples include At2i^ m(2) 9〇 to 186, such as 188,81111533 strict 2, ?32 and 1^ radioisotope., '5: [00212] The antibody and cytotoxin conjugate can be produced by using a variety of bifunctional protein surface agents, such as N-butyl succinimide-3_(2_pyridine N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDp)), succinimide-4-(N) -maleimide fluorenyl) cyclohexane small carboxylic acid ester

s (succinimidyl-4-(N-maleimidomethyl) cydohexane-1-caitoxylate), imin〇thi〇lane (IT), difunctional derivatives of imidoesters (eg Dimethyl imidate (HC1), active esters (such as disuccinimidyl suberate), genus (such as glutaraldehyde), bis-azido a compound (for example, bis(p-azidobenzoyl)hexanediamine), a bis-biszonium derivative (for example, bis-(p-diazonium benzoquinone) Bis) (bis-(p-diazonium benzoyl)-ethylenediamine), diisocyanates (eg toluene 2,6-diisocyanate), and di-active fluorine ( Bis-active fluorine compound (for example, 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. Sd (9) ce, 238: 1098 (1987). 1 -is〇thi〇cyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) labeled with carbon 14 as an exemplary radioactive nucleotide (radionucleotide) chelating agent to the antibody, see W0 1994/11026. The linking agent may be a "cleavable linker" that assists in the release of a cytotoxic agent in a cell, for example, an acid linker, a peptide-sensitive linker, a dimethyl linker, or a disulfide may be used. Attachment agent (〇^]^扣^7/.(^72£^7·· 52: 127-131 (1992)).

4S 201041902 [00213] Alternatively, a fusion protein comprising an anthropomorphic vWF antibody and a cytotoxin can be made, for example, by recombinant techniques or peptide synthesis. [00214] In yet another embodiment, an anthropomorphic VWF antibody can be ligated to a "receptor" (eg, streptividin) for use in tumor pretargeting, wherein The antibody-receptor conjugate is administered to the patient, followed by a clearing agent to remove unbound conjugate from the circulation, and then provides a "coordinator" that is conjugated to a cytotoxin (eg, a radionucleotide) (eg avidin). [00215] An active anticancer drug can also be obtained by conjugated a humanized vWF antibody to a pre-conversion drug (eg, a peptidyl chemotherapeutic agent, see WO 1981/01145) (see, eg, w〇1988〇7378 and U.S. Patent No. 4,975,278, prior to the use of a pr〇dmg-activating enzyme, the antibody of the present invention is used in ADEPT. [00216] The enzyme component of the immunoconjugate which can be used in ADEPT comprises any enzyme which acts on the prodrug in such a way as to convert it into a more active and cytotoxic manner. =0217] The enzymes that can be used include, but are not limited to, a test enzyme that facilitates the conversion of phosphate-containing precursors to free drugs; it helps to convert sulfate-containing precursors into free The aryl sulfatase enzyme of the drug (the town is like the blood); helps to convert the non-toxic 5-fluorocytosine into anti-cancer 5-fluoroadenine (5-fluorourad〇 cytosine) The cyt〇sine deaminase is a protease that facilitates the conversion of a peptide-containing prodrug into a free drug, such as a serratia protease, a thermolysin, or a bacillus protease. (subtilisin), carboxypeptidase and cathepsin (eg, cell autolysing enzymes B and L); can be used to convert 〇-propylamine carboxy carboxy peptides that are pre-drug containing amino acid substituents D-alanylcarboxypeptidase; a carbohydrate-cleaving enzyme that facilitates the conversion of giyc〇sylated uranium drive to free drug, such as β-galactosidase (p_gaiact〇sidase) and neuroamine Nitase (neuraminidase); help to be within β_ Amine (p_lactam) to derive the drug 49 201041902, into the drug β' glutamate (β-lactamase); and help to convert the odorous acetylene nucleus to the eclipse Pharmacological plate ί = other antibodies to penicillin V glutaminase and penicillin G amide activating enzymes (also referred to in the art as "catalyst! abzyme"), the present invention Previous drug conversion into free activity

Massey, chest, like: beginning rhyme (jump?)). The antibody-catalyzed antibody-transferred e-antibody can be delivered to the Lai cell population as described herein. [00218] The enzyme can be covalently bound to the imitation of the vWp Ο 抗体 antibody by techniques well known in the art, such as the use of a heterobiifimmental crosslinker as described above. Alternatively, recombinant DNA techniques well known in the art can be utilized to construct a fusion protein comprising at least an antigen binding region of an antibody of the invention, the antigen binding region linked to at least a functionally active portion of an appropriate enzyme (see, for example, - Yin et al., iVaiwre, 312: 604-608 (1984)). P0219] Other variants of antibodies are considered here. For example, the antibody can be linked to one of various non-proteinaceous polymers, such as polyethylene glycol (p〇lyethyiene glycol), polypropylene glycol (p〇lypr〇pyleneglyc〇1), polyoxymethylene oxide jpolyoxyalkylene) Or a copolymer of polyethylene glycol and polypropylene glycol; for example, 5' may also cause the antibody to be trapped by, for example, a clot technique or interface (for example, hydroxymethylcellubse, respectively) ) or gelatin-microcapsule and polymethyl methacrylate (Cpoly^nethylmethacylate)) microcapsules in colloidal drug delivery systems (eg liposomes, egg white microspheres, microemulsions, nanoparticles and nai Micules) or microcapsules prepared in a macroemulsion. Such techniques are disclosed in the book A. Ororwewtoz ZSd (16th edition, edited by Oslo, A. (1980)). [00220] The anthropomorphic VWF antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, for example: Epstein et al, Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et Proc. *Scz.. /75乂77:4030 (1980); U.S. Patent Nos. 4,485,045 and 4,544,545; and the disclosure of WO. Liposomes with enhanced cycle times are disclosed in U.S. Patent No. 5,013,556. [00221] Particularly useful liposomes can be produced by a reverse phase burst method using a lipid component comprising phosphatidylcholine, cholesterol, and pEG-derived phospholipid olamine ethanol (PEG-PE). . The liposomes are extruded through a filter defining the pore size to produce liposomes having the desired diameter. The Fab fragment of the antibody of the present invention can be conjugated to a liposome as described in Martin et al 7 〇/C/zem 257: 286-288 (1982) by a disulfide exchange reaction. The chemotherapeutic agent is contained in a liposome. See Gabizon(R)/, j.#如面81(19). ^ 1484 (1989). Vector, host cell, and recombinant method [00222] The present invention provides a humanoid encoding a vWF specificity An isolated nucleic acid 'vector of an antibody and a binding fragment thereof, and a host cell comprising the nucleic acid, and a recombinant technique for producing the antibody or a binding fragment thereof.

[00223] In the case of recombinant production of an antibody, the nucleic acid encoding the antibody can be isolated or inserted into a replicable vector to further perform basal-thinning (enlargement of DNA) or performance. A method (e.g., an oligonucleotide probe capable of visibly binding to a gene encoding a heavy chain and a light chain of an antibody) can be used to isolate and sequence the DNA encoding the monoclonal antibody. You can paste the township, the carrier domain contains f but not P. in the following: the message sequence, the origin of the replication (.rephcation), more than one marker gene (markergenes), enhanced element (enhanced element) Promoter (promote〇, and transcription-temiination sequence. (i) message sequence composition [=4] minus anthropomorphic vWF can be used to reorganize the 'f can be heterogeneous (heteiOlGgQUS) The peptide fusion polypeptide form ^ j has a special breaking position on the N-terminal (f) erminus of the mature protein f or the multi-peptide. (10) shouting shreds can be identified by the charm cells and 51 51 201041902

(ie, the message is broken by peptide fermentation). For prokaryotic host cells that fail to recognize and process the native anthropomorphic vWF antibody message sequence, they can be selected from, for example, an experimental phosphatase, a penicillin enzyme, an Ipp, or a heat-resistant enterotoxin leader (enterotoxin II leader). The prokaryotic message sequence is substituted for the message sequence. For yeast secretion, for example, the yeast transformase leader, the α_ factor leader (including the yeast () and the Kluyveromyces α- The factor leader), the acid-phosphatase leader, C. albicans (C. must/(3)) glucoamylase leader, or the message described in WO 1990/13646 is substituted. In mammalian cell expression, a nipple cell message sequence can be used as well as a viral secretory leader' such as a heipes simplex gD message. [00225] The DNA used in such precursor regions can be iigated in a reading frame to DNA encoding the anthropomorphic vWp antibody. (ϋ) copy the origin of the composition

[00226] Both expression and ci〇ning vectors comprise a nucleic acid sequence that enables the vector to replicate in more than one selected host cell. In general, when the vector is selected, this sequence allows the vector to replicate independently of the host chromosome and contains the origin of replication or the spontaneous replication sequence (aut〇n〇m〇usty replicatingsequences); Many bacterial, yeast, and viral sequences. The origin of replication from plastid PBR322 is suitable for most Gram-negative 2m plastid origins for yeast, and various viral origins (sv4〇, dioma, adenosis, VSV or BPV) help Colonization in mammalian cells In general, mammalian expression vectors (generally using SV4〇 origin, simply because, containing an early promoter) do not require replication of the origin of the composition. (ui) Screening for gene composition [=7] The expression and selection vector may comprise a screening gene, also known as a Uelectable marker. Representative screening gene coding -p column «(thorax than sigma mp), j^ne== ^ pjl Gth〇treXate) ' ^ , ω a lack (auxotroplncdefunency), or (6) supply cannot be compounded by the medium 201041902 (complexmedium A must-have or desired nutrient, such as the gene encoding the D-alanine racemase of Bacillus. [=228] One example of a screening protocol utilizes drugs to prevent the growth of host cells. These cells, which were successfully transformed with transgenic cells by the Xingyuan gene, were made to provide anti-muscle protein, and thus survived in the selection law (selecti〇nregimen). Examples of such advantages are the use of drugs such as neomycin, myc〇phen〇lic acid, and hygromycin. [00229] Another example of a suitable screening marker for a mammal is one that recognizes a cell capable of occupying a nucleic acid encoding a humanized vWF antibody, such as DHFR 'thymidine kinase (metallothionein-Ι & II) (preferably Long heavy metal protein gene), aden〇sine deaminase, omithinedecarb〇xylase, and the like. [00230] For example, by culturing a transformant in a medium containing methotrexate, cells that are transformed with a DHFR screening gene are first identified. The appropriate host cell when wild SDHFR is used is a Chinese hamster ovary cell strain lacking DHFR activity (CH〇(10)(f)). [^0023^1 Alternatively, by using a selection agent (such as an aminoglycoside antibody) such as an aminoglycoside antibody, such as kanamycin, neomycin, or guanidine 418, see U.S. Patent No. 4,965,199 Cell growth in the medium, selected to encode anthropomorphic = WF antibody, wild-type DHFR protein, and another screening marker (eg, aminoglycoside 3 -acid transferase (an^ogiycoside 3 Lph〇Spi!〇transferasepH) )) A DNA sequence host cell (especially a wild-type host comprising endogenous dhfr) that has been transformed or co-transformed. [00232] The appropriate f-selected gene for use in yeast may be the trpl gene present in the yeast plastid YRp7 (stinchcomb ei a/., Mziwre, 282:39 (1979)), and the trpl gene is in tryptamine Yeast mutants lacking growth ability in tryptophen (eg, ATCC44, 〇76 or ΡΕΡΦΙ. Jones, 85: IS (I" 7)) provide screening. The presence of the Kun 1 lesion (lesi〇n) in the yeast host cell genome (gen〇me) then provides an effective environment for detecting transformation by growth under leucine-free acid; A yeast strain lacking Leu2 (Leu2-deficeient) with a known plastid with Leu2 group 53 201041902 (ATCC 20, 622 or 38, 626). [00233] In addition, the transformation of Kluyveromyces cerevisiae can be carried out using a vector derived from the 1·6 μιη circular plastid pKDl; or, as described in Nicholas Vanden Berg, 8: 135 (1990), large-scale manufacturing recombination The performance system of calf chymosin. We have revealed a stable multi-c〇py expression vector for the secretion of mature recombinant human serum proteins by industrial strains of the genus Kluyveromyces, see Fleer e/ and 9: 968-975 (1991). (iv) Promoter Composition [00234] The expression and selection vectors typically comprise a promoter that can be recognized by the host organism and can be manipulated to bind to the vWF antibody-encoding nucleic acid. Promoters suitable for use in prokaryotic hosts include the ph〇A promoter, the β-endase and lactose promoter systems, the prodrug %lie enzyme, the tryptophan (trp) promoter system, and the tac promoter, for example. (hybrid) promoter, however, other known bacterial promoters are also suitable. The promoter for the bacterial system may also comprise a Shine-Dalgamo (S.D.) sequence operably linked to the DNA encoding the anthropomorphic vWF antibody. [00235] Promoter sequences of eukaryotes are known. The eukaryotic gene has an AT-rich region of about 25 to 30 bases upstream of the site of transcription initiation, found 70 to 8 bases upstream of the transcription initiation point of many genes. The other sequence is the CNCAAT (SEQ ID NO: 31) region, where N can be any nucleotide. At the 3' end of most eukaryotes is the AATAAA (SEQ ID NO: 32) sequence, which may be a signal that adds a poly A tail to the 3' end of the coding sequence. These sequences can be suitably inserted into expression vectors for eukaryotes, and suitable promoter sequences for yeast include: for 3-phosphiosphoglycerate kinase or other glycolytic enzymes (eg, sterolase) (enolase), glycerol by GIYCeraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofoxctokinase, glucose_6- Isotonic acid: (gIucose-6-phosphate isomerase), 3-phosphoglyceratemutase, pyruvate kinase (ργπιν &1; ε1ά]_ε), 201041902 (triosephosphatei), phosphoglucose (igosphoglucose iS0merase), and glucokinase (ghic〇ki_)) [00236] Other yeast promoters with a promoter that has additional transcriptional advantages controlled by growth conditions Can be the promoter region of the following proteins: ethanol dehydrogenase 2, isocytochrome c, acid pot acid, and nitrogen f: sweet 3, money enzyme, and negative Heteroaryl and m are as wheat _ European No. 73,657. It is also advantageous to use the yeast enhancer together with the 囷 yeast 囷 promoter. [0023] The humanized vWF antibody is transcribed from a vector in a mammalian host cell, such as a polymorphic (P〇ly_) virus, a fowlpQX virus: an adenovirus, a diseased bone 2), a bovine papilloma virus, a poultry Promoter, heterologous mammalian promoter (eg actin promoter ^ immunoglobulin) obtained from the sarcoma virus, cellular giant virus, retrovirus, hepatitis 3 virus and the genome of simian virus 40 (SV40) Promoter), heat-sister fresh control, provided that such a promoter is compatible with the host cell system. [00238] We can conveniently obtain the early and late promoters of the SV40 virus, q as a SV40 restriction fragment containing the origin of the replicated sv4 prion; we can conveniently obtain the immediate early initiation of the human cell giant virus. 2 is a Η--limit fragment. The use of bovine papilloma virus as a carrier has been disclosed as a carrier (10). See, for example, U.S. Patent No. 4,419,446, the disclosure of which is incorporated herein by reference. No. Reyes βα/., Gamma, 297: 598_6〇1 (1982) describes the expression of human & interferon cDNA in murine cells under control of the thymidine kinase promoter from a single ton of herpesvirus. ' (v) Enhancer element composition The DNA encoding the anthropomorphic VWF body of the present invention is transcribed by a eukaryotic organism such as a car bud, and the county can be inserted into a ray towel to enhance it. It is known that useful enhancer sequences from the porcine animal genes (blood globulin, elastase, protein, sputum type fetus 55 201041902) can be used; however, on the side of the f-familiar L bracket: the late child at the origin of the copy. f indicates that the promoter of the secret scorpion scorpion ^ Γ Γ Γ 豊 豊 豊 豊 豊 豊 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , Polyoma enhancer on the late side, and adenovirus enhancement

In ^ & Si]_ι, ... _ η —~ can be calculated from the anthropomorphic vWF promoter i 5, / recorded enhancer cut into vectors, but preferably in the (vi) transcription termination of the plant her ff core Biological host cells (eg yeast, fungi, insects,

Or the nucleated cells from other multicellular organisms. § The sequence necessary to terminate transcription or stabilization. Such sequences are untranslated from eukaryotes or viral DNAs *cDNAs. Region 5, Μ (%• is 3k) 'These untranslated regions are included in the encoding of the anthropomorphic antibody, and the untranslated part of the sputum is touched into a poly-glandularized (Saki-Cai (4)) piece, the nucleus Segments. - Useful to transfer the polyadenylation region of the oblique growth polymorphism (see, for example, W〇 1994/11〇26 and the expression vector of the bean disclosure). , (vii) Screening and Transformation of Host Cells [00241] Suitable host cells for the selection or expression of DNA in a vector comprise various prokaryotes, yeast g, or higher prokaryotic cells for use in For this purpose, prokaryotes comprise eubacteria (such as Gram-negative or Gram-positive organisms such as Enter〇bacteriaceae such as Eucheyces (such as R c) 〇li)), mer〇bacter, Erwinia, Crescens (Imperial Essie! Iay Proteus)

(Proteus), Salmonella (such as Salmonella typhimurium), Serratia (such as Serratiamarcescans), stalk meter M (ShigeUa), anti-stalk (Bacilli) such as B. subtilis · licheniformis, Pseudomonas' (Pseudomonas') such as P. aeruginosa anti-iron bird ^ (your job (10) fire with y!. five · co / z · breeding host contains ruler co / z · 294 (ATCC 31,446) , co/zB, pent. co/z_X1776 (ATCC 31, 537) and pent. co/fW311 〇 56 201041902 (ATCC 27, 325). [00242] In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are suitable The colonization or expression of the anthropomorphic vWF antibody encoding vector can be used to express (10) (7) plus or common baker's yeast. In addition, several other genera, species, and strains are also generally available and user-friendly. For example; Schizosaccharomycespombe' Kluyver〇myces host, eg K. lactis, K. fmgilis (ΑΎ(:(: Λ2Α24), K. bidgaricus (xrcc 16,045), K. wickeramii (ATCC 24,178) , K. waltii (ATCC 56,500), K. drosophilarum (NIC C 2>6,9Q6\ K. thermotolerans, anti-K. marxianus. Less catching Jane. (EP 402,226); Nai W Waga (EP 183,070); Candida 〇 (Cfl secret also); 知咖办r(10)b (EP 244,234); Neuvspom crassa., Schwanniomyces such as Schwanniomyces; and filamentous fungi such as Rhodobacter (valence-cut (four)), Penicillium CPenicmium, Tolyp〇cladium, genus (Aspergillus), such as A. nidulans, anti-A. niger. [00^43] Suitable host cells for expressing glycosylated anthropomorphic VWF antibodies can be obtained from multicellular organisms, eukaryotic cells. Contains plant, insect and vertebrate cells. We have identified many bac^loviral strains and variants, as well as from, for example, tillers (Ranunculus), Aedes aegyptK mosquitoes +), Aedes aWopictus (M+), Drosophila ( Fruit rope), and 5 (10) buckle ^ _rz • host corresponding to the allowable tailworm 佰 main cells. There are a number of viral strains for transfection that can be used by the public, such as Autographa califomica OTV and Bm-5 strains of five (10) morz. NPV, and such viruses can be utilized as the virus according to the invention herein, especially for Spodopterafrugiperda (four) version of the transfected soil. [00j44] Cotton, corn, horse yam, soybean, morning glory, tomato, and plant cell culture of grass can also be used as a host. [00245] However, the greatest interest has been in vertebrate cells, and the proliferation of spinal cells in culture (group, culture) has become a routine procedure. Examples of mammalian primary cell lines that can be used include: ChK2 cells (Chxomos Molecular Systems 57 201041902)

Inc_); a monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCCCRL 1651); a human embryonic kidney cell line (293 or a 293 cell for subcloned growth in suspension medium, Graham ei aZ., J. G(9) %〇/·, 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); human embryonic kidney (HEK) 293 cells (Simmons, 1990 Exp corpse 75:309 SP2 spleen-myeloma fusion cells (HaasandWabl, 1984, /WAS81:7185); Chinese hamster ovary cells/-DHFR (CHO, Urlaub a/., Prac. Mzi/. Scz.. 1/3⁄4 77:4216 ( 1980), including DG44 (Urlaub ei < Ce// face/M?/. G, 12: 555466 (1986)) and DP12 cell line); mouse sertoli cells (TM4, Mather, and 〇) /_ and India (7) especially 23: 243-251 (1980)); monkey kidney cells (CV1 ATCCCCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCCCCL2); canine kidney cells (MDCK, ATCCCCL70); buffalo liver cells (BRL3A, ATCC CRL1442); human lung cells (W138, ATCCCCL75); human liver cells (Hep G2, HB 8065); mouse breast tumors (MMT 060562, ATCC CCL 51); TRI cells (Mather ei a / ·, qe Zang 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2). Host cells are transformed using the above-described expression or selection vector for the production of anthropomorphic vWF antibodies, and cultured in a conventional medium adjusted as needed to induce promoters, sputum screening transformants, or amplification A gene encoding a desired sequence. [00246] Stable cell lines exhibiting high levels of antibodies can also be used to produce anthropomorphic antibodies of the invention. For example, a mutant lambda-integrase (eg, ACE integrase) can be used in conjunction with a shuttle vector to load a high-yield mammalian protein based on a murine artificial chromosome representation (ACE) platform with a heterologous gene sequence. In the system, the ACE platform has been designed to contain multiple site-specific recombination acceptor sites (Linenbaum ei ^ (A^c/32 (21): el72 (2004); US patent application) No. 3/0119104 A1 and 2006/0246586A1. This system can be used to produce stable cell-derived (viii) cultured host cells for the anthropomorphic variants and NMC-4 chimeras screened. 58 201041902 [00247] Host cells useful for the production of anthropomorphic vWF antibodies can be cultured in a variety of media. Commercial media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), RPMi_i64(Sigma) 'A Dulbecco s Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing host cells; in addition, 'for example: 58.44 (1979), Bames β such as β/·, i〇 2: 255 (1980); States Patent No. 4,767,704 'No. 4,657,866, No. 4,927,762, No. 4,560,655, brother or No. 5,122,469' WO 1990/03430; WO 1987/00195; or US

Any medium described in Japanese Patent Laid-Open No. 30,985 can be used as a medium for the host. Hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium, calcium, magnesium, and sulphate), buffers (such as HEPES) may be used as needed. , nucleotides (eg, adenine nucleotides, thymidine nucleotides), antibiotics (eg, trace elements (defined as inorganic compounds usually present in the final concentration of the micromolar range), and glucose or equivalent energy Supplement to any of these media; any other necessary supplements known to those skilled in the art to be appropriate concentrations may be included. Various culture conditions, such as temperature, pH, may be employed for the host cells for the performance of the screening. (ix) Anthropomorphic vWF Purification of antibodies [,] 248] § When using recombinant techniques, antibodies can be produced in the intercellular membrane region (penpla^nicspace) or directly secreted into the culture medium. If the antibody production step is carried out, the host cell or the cell debris tablets can be removed by, for example, centrifugation or ultrafiltration. Carter ek/., 〆1〇: 163_167 (1992) describes the separation of antibodies secreted into the co/z•membrane region, sodium acetate (pH 3.5), EDTA, and phenylsulfonyl fluoride &quot ;, (phenylmethylsulfonylfluoride (PMSF)) (cell minutes, can be borrowed (four) remove cell debris #. In the antibody secretion == 2^1, 11 often first concentrated from such a system above the liquid, including ρ3ρτ τ ΤΓ ^ ΤΜ 贝 贝 concentrating filter, such as AMIC0NTM4 PELUC〇NTM ultrafiltration unit. Protein such as PhenylmethylsulfQ_UQnde (PMSF) can be included in any of the steps of the previous 59 201041902 to inhibit protein. Decomposition, and may include antibiotics to prevent sporadic contamination from expanding. [00249] For example, hydroxylapatite chromatograpliy, gel electrophoresis, dialysis, and affinity layers may be utilized. The antibody component prepared by the cell is purified by an analytical method, wherein affinity chromatography is a preferred chromatographic technique. The suitability of diprotein A as an affinity ligand depends on any immunity present in the antibody. Globulin Fc domain And subtype (is〇切^). Protein A can be used to purify antibodies based on human γ1, γ2, or γ heavy chains ((五)船也 f α/., ////_咖/. 3)); protein ^ can be used as the old mouse antibody subtype and human (Guss ^.© to 5:15671575 (1986)). Affinity with the axis of the wire (matrix can be mixed (agaK) se), but Other matrices can also be used. A mechanically stable matrix, such as controlkd-pore glass or p〇ly (styrene dmnyl-benzene), can be tolerated compared to the use of acacia The faster processing time is shorter. In the case where the antibody comprises the CH3 domain, BAKERBONDABXTM^^ (JT Baker, Phillipsburg, NJ.) can be used; other proteins can be used depending on the antibody to be granulated, such as ion exchange column fractionation, ethanol Precipitation, reverse phase jjpLC, chromatographic on silica > (heparin) SEPHAROSETM® ^. cation cation f anion exchange resin chromatography (eg, polyaspartic acid (P〇lyaspartic acid) column), Chromatic focus, set (chr〇matof〇cusing), and sulphuric acid. Pharmaceutical Formulations [00250] g provides a drug-containing composition comprising a wWF-specific anthropomorphic antibody, and a drug-acceptable compound that is mixed with a drug, and a drug-acceptable product, {'m> mm > {Remingtons Pharmaceutical Sciences 16th Ed_n, Osol, A. Ed (1·)) 'Prepare a VW antibody with a cold-rolling formulation or an aqueous solution, and use the carrier, _agent, and virgin V] received in Formulation 1 at the dose used and It is not toxic to the recipient at the concentration, and it contains 60 201041902: buffers such as disc salts, citrates, and other organic acids; antioxidants, including anti-oxidation, blood acid, methionine (methionine) Preservatives such as octadecyldimetliyrbeiizyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzalkonium chloride Benzethonium chloride, phenol, butyl alcohol or benzyl ο alcohol, such as methyl p-benzoyl phthalate (me or yi paraben) or propyl benzoate Alkyl (propyl paraben ) Alkaline parabens, catechol, resorcin〇i, cyclohexanol (1), 3-pentanol, and m_cres〇Q; low component (less than about 10 residues) polypeptide, protein, such as serum protein, gelatin or immunoglobulin, hydrophilic polymer, such as polyethylene tetrahydropyridinium Polypyrorolidone; amino acids such as gyiCine, glutamine, asparagine, histidine (arginine), arginine or Lysine; monosaccharides, disaccharides, and other carbohydrates containing glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol (mannit〇) 1) trehalose or sorbit〇i; salts form relative ions (cminter-ion) 'eg sodium; metal complexes (eg, word-protein complexes); ο and/or Ionic surfactants such as TWEENTM, pLUR〇NICSTM, or polyethylene glycol 〇i, PEG). A preferred freeze-dried anthropomorphic vWp formulation is described in WO 1 "7/〇 sideband, which is incorporated herein by reference. [00251] The formulation herein may also comprise more than one or more active compounds necessary for the treatment of the symptoms, preferably having no J to each other; or, alternatively, the composition may further comprise a chemotherapeutic agent, a cell; (cytokine), growth inhibitors, antihormonal agents, anthropomorphic drugs, anti-blood

T f t#i ( antl'angiogenic a§ent)' A/m, ^ * ( cardioprotectant) J This is suitable as a combination of amounts that can be effective for a desired purpose. [Γ=2] Eight products will be coated with active ingredients by, for example, clot (__ti〇n) technology or; I face bearing (for example, thiomethyl cellulose 61 201041902 (hydroxymethylcellulose) or gelatin-microcapsule () Methyl methacrylate (poly^methyl^^acyhe)) in colloidal drug delivery systems (eg liposomes, egg protein microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions The microcapsules prepared are prepared. Such techniques are disclosed in Remington's Pharmaceutical Sciences (16th Edition, edited by Oslo, A. (1980)). [00253] A sustained-release preparation is prepared. Examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained release of mubes include polyesters, hydrogels (e.g., poly(2-ethyl methacrylate) or poly(vinylidene)), polylactide (U.S. Patent No. 3,773,919), L. - a copolymer of glutamic acid and γ-ethyl-L- face acid, a non-degradable ethylene-vinyl acetate copolymer, a degradable lactate-glyc〇lic acid copolymer (eg LUPRONDEPO) TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolideacetate) and poly-D_(mono)_3_hydroxybutyric acid). [00254] The formulation to be used for in vivo (wvz'w) administration must be sterile, which is accomplished by filtration through a sterile filtration membrane.治疗 Treatment with anthropomorphic vWF antibodies [00255] Anthropomorphic vWF antibodies or binding fragments thereof can be used to treat a variety of different vWF-associated diseases or abnormalities, including thrombotic diseases or abnormalities. A thrombotic disease or abnormality may include a cardiovascular disease or a cerebrovascular disease such as an ischemic stroke. In certain embodiments, the cardiovascular disease can be atherosclerosis, restenosis, angina (acne &), acute myocardial infarction, acute coronary artery disease ( Acme coronary syndrome ), or cardiovascular abnormalities associated with diabetes. In some embodiments, thrombotic or abnormal vascular inflammation, venous blood test, sputum opening, blood cell disease, xenograft rejection ( Xenograftrejection), peripheral vascular disease, embolic thrombocytopenic purpura, 62 201041902 (thrombotic thrombocytopenic purpura), cystic fibrosis, vascular dementia, Raynaud's disease (anti-noisy (10)^ disease), wind, house arthritis (also eumat〇id sister), or diabetes. In some examples, 'cerebral vascular disease consists of cerebral arte' infarcts and small The sinus infarction (arcs of smau iacunar) is caused by ischemic stroke and vascular dementia (vasc [ulardementia). Humanized vWF antibodies can also be used to prevent recurrent stroke or initiation of stroke caused by inflammation of the cerebrovascular. In the case of acute coronary artery disease, anthropomorphic antibodies or binding fragments thereof It is especially suitable for the treatment of 3D (ST_segmem) diagnostic subjects. ^ The anthropomorphic vWF antibody or its binding fragment can also be used for postoperative treatment to prevent clot formation. '[00256] Diagnostic assays to assess the overexpression of vWF, amplification, for example, by administering a molecule (eg, an antibody) that binds to a molecule and is measurable to the target, and externally scans the patient to locate the [00257] In certain embodiments, an immunoconjugate comprising an anthropomorphic vWF antibody conjugated to a cytotoxin can be provided to a patient; preferably an immunoconjugate and/or anthropomorphic combination thereof The vWF antibody is trapped inside the cell, thus enhancing the therapeutic utility of the immunoconjugate in killing the cancer cells to which it binds. In a preferred embodiment, the cytotoxin (including, for example, maytansine, 〇 (maytansmold) , calicheamicin, nbonuclease, and endonuclease to lock or interfere with nucleic acids in cancer cells; in another embodiment, cytotoxins (eg, taxane or angstrom) Epothilone can lock or interfere with microtubules in cancer cells and rely on microtubule-dependentmitosis. ' [00258] According to known methods, for example, in a large amount of bolus or by continuous infusion of _ _ _ _ _ _ sinfhsiQn, intravenous administration, intramuscular, intraperitoneal, cerebrospinal The anthropomorphic vWF antibody or neoadhesive acid is administered to the patient internally, subcutaneously, intra-articularly, intrasynovially, intrarenally, orally, topically, or inhaled. Intravenous, intraperitoneal, or subcutaneous administration of the antibody is preferred; The preferred dosage meter 63 201041902 draws γ for the acute abnormal single dose, and for chronic one, depends on the acceptance of the household 〜 / / /, hanging sputum, scoop mother two to four weeks to know other factors 疋 lactose animal, antibody type And the practitioner is familiar with r0025gi~, and other dosages can be used here. 々社人 can combine other treatment guidelines with anthropomorphic vWF antibody plexus;;;-;* (co-ad^on):;1;^ two-two-order continuous administration, of which the preferred one is There is a time when the new year is in its birth. In the U-example, the treatment may include anthropomorphic anti-vw antibody plate

Ο == 3fflbrinolyticagent) and / or combination of anti-platelet agents to give examples of ^ = protein agents such as altplase, batithrinase: = ase or microplasmin, Anti-blood Γ Ξϊ 疗 疗 由 由 心肌 心肌 心肌 心肌 心肌 = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = Administration of VWF antibodies and administration of antibodies directed against another associated antigen [00262] > In one embodiment, the treatment of the invention relates to anthropomorphic vWp antibodies, immunomodulatory-like regulators in mammals ( For example, cell kinase), and a combination of a chemotherapeutic agent or a growth inhibitor, including a combination of different chemotherapeutic cocktails; preferably a chemotherapeutic agent comprising a taxane (eg, paclitaxel)

(paclitaxel) and European paclitaxel (d〇cetaxel) and / or cranberry janthracycline) antibiotics. It can be determined according to the manufacturer's instructions or by the familiar practitioners, and the preparation and adjustment of such chemotherapeutic agents; the chemotherapeutic agents and preparations for such chemotherapeutics are also described in Chemotherapy Service. Ed., M. C

Perry, Williams & Wilkins, Baltimore, Md. (1992). A dose of an anti-hormone compound can be known to bind an anthropomorphic VWF antibody to such a molecule. Examples of anti-hormone compounds include, for example, an anti-estrogen compound of tamoxifen or an aromatase inhibitor such as anastrozazole; for example, an anti-lutein of onapristone (anti-progesterone) (see EP 616 812); or for example, fiutamide 64 201041902 f $ (anti_androgen). If the cancer to be treated is a hormone-independent person, it can have been treated with anti-drug therapy, and the cancer becomes hormone J J . Alternatively, the patient may be given a humanized vWF antibody (and other non-must diseases as described herein, and the appropriate amount of the antibody will depend on: the surface of the disease, the redness and the strict record Silk (whether given ο ϊΐ Γ 或 或 或 或 或 、 、 、 先前 先前 先前 先前 先前 先前 先前 先前 先前 先前 先前 先前 先前 先前 先前 先前 先前 先前 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体 抗体Disease _ and severity and purification of antibodies?! 2.7 disease red initial candidate dose is about 1 (four) ^ ~ 15 fine (for example, even the disease of the antibody 'regardless of, for example, by more than one time, independently administered or borrowed The daily dose range from t can be from about 1 to g to 100 beer, such as 荜, ΐ ΐ ΐ 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 期望The inhibitory effect is as follows. 〇] The preferred dose of the anthropomorphic vWF antibody can be from about 0.05 mg/kg to about Hg, and therefore, the patient can be about 0.3 mg/kg, 0.5 mg%, 0 mg/kg, 4.0 mg/kg, or 10 mg/k (or 1) 〇 antibody i. can be spliced from about 2 to about 2 〇 (eg 6) doses of anthropomorphic More than vWF times = ^1%% is given a higher initial dose (loadingd〇se), and then given a community of tablets two or two. The quantitative method of inclusion includes the administration of anthropomorphic vWF antibody or its method and method. This type of therapy can be performed by the conventional technique = pre-existing bed symptoms, except for the increase in bleeding from small wounds (Example 65 201041902, such as the template bleeding time measured in the incision bleeding test. ), and even more shocking, the clinical symptoms of facial bleeding have not been observed from about i to ED or 00, although there have been observed bleeding in small wounds. Therefore, The anthropomorphic vWF antibody or binding fragment thereof described herein is intended to treat a vWF vector disease or abnormality in humans. [00267] Thus, an effective therapeutic amount ranging from about _1 to about 100 mg/kg can be used, The anthropomorphic vWF antibody or binding fragment thereof is administered as a human (preferably human); preferably, the effective therapeutic amount ranges from about 〇〇〇2 to about f mg/kg, from about 0.002 to about 10 Mg/kg is especially preferred, especially from about 〇〇〇2 to about 〇* mg/kg, from about _5 to about 0.2. Preferably, the subject is administered a therapeutically effective amount from about 〇〇ι· to about 0.1 mg/kg, preferably a human, at a therapeutically effective dose, or a humanized vWF antibody or Combining the fragments into the tester's well-behaved effective treatment (4), the material is sufficient to invent the mad clinical form, but it is enough to inhibit the plate agglutination; in other words, it does not cause the duck to come out of bed symptoms (for example, does not produce Bleeding ^ = except for increased bleeding from small wounds.) (6) Normal [〇〇268] Milk nl ranges from about 〇·002 to about 〇·4 mg/kg or especially from about _5 to about 0.2 mg/ Kg, more particularly from about 〇〇1 to about 〇] 知 之 有效 知 知 知 知 知 知 知 知 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟Produce effects (eg, reduce formation). [00269] may range from ED (10) to about 2

About 1 f〇〇 times, from ED to about 1 to K) 〇倍尤佳的办如如人人化. Anti-Finance knots, Ke Zhi g Ming J column if there is no bleeding Clinical symptoms, in addition to increased bleeding from the small X mouth. A single or multiple anthropomorphic VWP antibody or a binding fragment thereof can be administered to a subject === rj-r : Li έι ® ^, , therapy ^I. The amount of bleeding through the subcutaneous/pathway is 1 to 3 times higher than that in the vein. 201041902 [=270] The clinical symptoms of bleeding can be referred to by __ a t17 Xiao, Pages 288 side and applied To the animal model Sc if j will be ds_ specifically developed into the bleeding ίί t td=e resistance to the board, the wire. Bleeds_ = Severity to assign points to clinical findings (fmd (4)), dreams are added: the scores produced. The symptoms of bleeding are gradually increased. ο Blood or a combination of the above (6 points per event), this is in the judgment of fish: blood two anti-thrombotic therapy associated with moderate to moderate bleeding "Yf: easy;: r The most serious bleeding complications. Superficial bleeding ^3 ;,}; tT/PeteChia) ^ ^ (eCCh^s) ^ m (hp〇ma).(epistaxis)^^^ ^ ❹ , blood, black I (=_), eye bleeding, hematuria (hema Hematemesis); warning bleeding contains the following criteria: need transfusion (transfusion) ^H ^ ^ A (intracranial hemorrhage [OOfl] in the 纟 纟 纟 经 经 经 经 经The arterial towel is a quantitative method that determines the amount of circulating flow reduction in the arterial trauma model (cyciic ^, and, therefore, inhibition of platelet aggregation can indicate effective administration) It is sufficient to reduce the number of CFRs in animals. Quantitative measurable can refer to a dose that can effectively improve the survival time of _ anti-symptoms, or long-term sputum, and the inner valley. Effective treatment as described here. The amount includes a vwf vector disease «normal vWF antibody amount which can effectively treat a subject; vWF antibody #效治疗量包括治疗67 201041902=Difficult to make a plate (for example, in the process of blood formation of aorta, peripheral arteries, arterioles). [=] 彳 练 或 or effective forging can be Effectively improve or prevent the disease, itching ΐΐ ΐΐ 剂量 ΐ ΐ ΐ ΐ 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 ΐ ΐ ΐ ΐ ΐ ΐ ΐ ΐ ΐ ΐ ΐ ΐ ΐ ΐ ΐ ΐ ΐ ΐ ΐ The vWF thief's sister has a heterozygous or _ ^ such as the online pulse, the end of the vein, the small artery, the vein thrombosis process) Hi dose ° effective treatment dose contains edigg, which is enough in 3q minutes The spider formation by the amount of domain reduction in the blood container reduces the effective dose; as described herein, (10) contains blood sufficient to measure the blood flow reduction in the blood trough state within 30 minutes. The therapeutic dose includes a dose that can reduce the thrombus formation as measured by a suitable in vitro (πνζ·νο) measurement, such as at least a blood sputum sputum, or a decrease in platelet aggregation. About 15% better i reduce at least 3G% 'more good machinery At least, it is optimal to reduce at least 80%, in particular to reduce at least 1% of thrombosis. Qiao 0274] Alternatively, the anthropomorphic vWF antibody can be treated continuously or in combination with radioactive treatment, such as irradiation or introduction of a grazing substance, like [00275] Thus, the present invention further provides a virulence factor (vwp); a t-type antibody or a binding fragment thereof, which may range from ED to 1 to about 250 ^ effective therapeutic dose to the needle, without the clinical symptoms of bleeding from the visceral. Preferably, the human antibody or binding fragment thereof has a specific soil in the A1 domain of human vWp. The human antibody or binding fragment thereof having vWF specificity is more preferably a specific humanized antibody or a binding fragment thereof. Article of Manufacture [00J^] In another embodiment of the invention, an article of manufacture (e.g., anthropomorphic vWF antibody) comprising materials useful for treating the above is provided. The article of manufacture may comprise a container and a label or copy associated with or associated with the container

6S 201041902 (package insert); suitable containers include, for example, bottles: containers can be formed from a variety of materials such as glass or plastic; accommodating (four) products, and can have a sterile access port (such as a small pinch of a needle-punching tooth) Glass bottle). In the composition, a ^ ^ ^ ί, the standard _ single can refer to cancer. In an embodiment, the label or the singularity may indicate that 匕3 anthropomorphic V is the composition of the sufficiency to treat vWF-related abnormalities. ο Cry, the manufactured article may comprise: (8) a second component containing the composition, wherein the group n contains the anthropomorphic antibody; and W contains the composition if, wherein the composition comprises a treatment other than the anthropomorphic antibody Agent. Ϊΐ: = The article of manufacture in the embodiment may further comprise a combination of the first treatment - __ or abnormality - the therapeutic agent may be in the preceding paragraph = any combination therapy (such as thrombolytic agents, antiplatelet agents, antibiotics) Blood (4) raw agents, anti-hormone compounds, heart-protecting drugs, and / or feeding ίί can adjust the package ί ', interleukin). Alternatively, or in addition, the article of manufacture may be ii ii with pharmaceutically acceptable buffers such as sulphate water for primary injection (BWFI), phosphate buffered saline,

G User's % appears to be a suitable material, including other probation, artistic benefits, injection needles, and syringes. "Non-therapeutic use of anthropomorphic vWF antibodies [00278] Anthropomorphic vWF antibodies and binding fragments thereof have further therapeutic applications. For example, antibodies can be used as affinity purifying agents; in this procedure, this can be utilized A method known in the art, which is anti-tweaked on a solid phase, such as SEPHADEXTM resin or filter paper. The immobilized antibody can be contacted with the vWF protein f (or a fragment thereof) to be purified, and then substantially In the Shifter, in addition to the anthropomorphic tvWF egg self-quality (shot binding to @定化抗体), the solvent is used to wash off the support (SUpp0rt). Finally, another coil 69 201041902 solvent (such as pH) 5.0 glycine buffer) to wash away the support, releasing the anthropomorphic tvWF protein in the lysed antibody. [00279] The anthropomorphic vWF antibody can also be used for the determination of human vWF protein, such as detecting specific cells, tissues, Or the performance in serum. Seek the application! 'T is labeled with a detectable moiety. There are labels that can be generally classified into the following categories: [00280] (8) Radioisotopes, such as 35s, 1 4C, 125I, 3h, and 131. For example, VQlumes i & a Coligen et al, Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991)

The technique described in 〇 is used to label antibodies, and radioactivity can be measured by sdntiUti〇n counting. [00281] A fluorescent label such as a rare earth chelating agent (cerium chelating agent) or fluorescent fluorescein and its derivatives, rose red (rll (iamine) and its derivatives, dansyl) can be used. ), (Lissamine), phycoerythrin (phycoerthrin), and Texas Red. Fluorescent labels can be conjugated to antibodies using techniques such as Cwrr gamma P & 沅/面圆/卿, supra; fluorescence can be utilized Quantitative fluorescence labeling. [〇〇f82] (c) Various enzyme-matrix labels can be used (see, for example, US Patent No. 4,275,149). These enzymes are usually catalyzed and can be measured using various techniques. The chemical change of the chromogenic substrate. For example, the enzyme can catalyze the color change of the substrate towel, which can be measured spectrophotometrically; or the enzyme can change the fluorescence or chemihiminescence, quantitative luminescence The technique of variation is as described above. The chemiluminescent substrate can be electronically excited by a chemical reaction, and it can then emit light that can be measured or supply energy to the fluorescent acceptor. The enzyme labeled j column contains luciferin. Enzyme (hx Dferases, such as firefly luciferase and bacterial luciferase, US Patent No. 4,737,456, luciferin, 2,3-dihydroquinone, 2,3-dihydrophthalazinediones, Malate dehydrogenase, urea urease, peroxidase (such as horseradish peroxidase (HRPO)), alkaline phosphatase (alkalinephosphatase), β-galactose Enzyme 〇 galact〇sldase), glucoamylase, lysozyme, saccharide 70 201041902 oxidases (eg glucose oxidase, galactose oxidase, and glucose _6_ sulphate) Wind _), heterocyclic oxidase (such as uric acid enzyme (ur|case) and xanthine. oxidase (xanthine oxidase), lactoperoxidase (lact), and microperoxidase (microperoxidase). Techniques for ligating enzymes to antibodies are described in 〇'Sullivan et al, "Methods for the Preparation of Enzyme-Antibody Conjugates for use m Enzyme Immunoassay, Methods in Enzym. (Ed., J.

Langone & H. Van Vunakis), Academic Press, New York 73-147-166 (1981). 'Examples of enzyme-matrix combinations include: ❹ Ο [00283] (Hydrazine catalase as a substrate for horseradish peroxidase (HRPO), in which catalase oxidizes dye precursors (eg o-phenylenediamine (〇rth) 〇phenylene diamine) or 3,3',5,5'-tetrahydrobenzidine hydrochloride (TMB)); [00284] (ii) p-nitrobenzene Phosphate (para_nitr〇phenylph〇sphate) is a chromogenic substrate of alkaline phosphate (AP); and [00285] (out) with a chromogenic substrate (eg, p-nitrobenzene_β_〇-galactosidase) or fluorescent Matrix 4-mercapto-formula _β1_galactosidase C^methylumbelliferyl-iS-D-galactosidase ) (β-D-Gal) ββ-D-galactosidase 2, 286], know the skill A variety of other enzyme-matrix combinations can be used (see, for example, Wu Guo Patent Nos. 4,275,149 and 4,318,98). The indirect bonding of the label to the antibody is familiar with the various techniques that the skilled artisan should be aware of. For example, the antibody can be combined with biotin (10), and the two major components can be labeled with avidin. The substance is selectively bound to avidin, so it can be The human 4 is ligated to the body; or 'to achieve a cleavage between the label and the antibody and with a small hapten (eg, Digoxin (d_)) anti-Glutinic antibody). Thus, the Jr antigen antibody (eg [caffe] in the hair = another === indirect junction of the target reading antibody. In another case of X yoke, the anthropomorphic vWF antibody does not need to be labeled with 71 201041902, and The presence thereof can be detected using a labeled antibody that binds to a humanized vWF antibody. [00289] The antibodies of the invention can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, And immunoprecipitation, see

Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press Inc 1987). [00290] In terms of immunohistochemistry, tumor samples may be fresh or frozen or embedded in a stone and fixed with a preservative such as fumarin.

Ο [00291] Antibodies can also be used in diagnostic assays in vivo. In general, radionuclides can be labeled with radionuclides (eg, (1) and 14匸131] i25: [3幵32p or S), so that tumors can be localized, for example, by immunoscintigraphy. . [00292] For convenience, the antibody of the present invention may be in the form of a kit (10). The kit for use in the assay of the assay may contain the fine or fluorophore required for the enzyme. ^, r" Bar 3 other additives, such as stabilizer, buffer solution i, ckbuffer) or cell lysis buffer (lysis buffe〇) are provided, including in the reading of Weng provided "appropriate concentration where the marker can be placed on the magnetic resonance ^ ^ this skill Ϋΐί Plus grading and treatment. The antibody can be used to force the family + Shi L, Yi Bu Ri., Taiwan Yi -_, _, any part detectable in the main body, including iodine (such as 3 and 131l), -.~:TM- no- "Two winds are marked 72 201041902 CU), wrong (such as 93⁄4), and 铢 (such as 186Re and 188). It can be attached to the egg by any radioisotope, including, for example, a metal chelate compound = ritual peroxidase, or tetrachlorodiphenylglycolide for iodidine (i〇dini). 〇gen) Technology. [00294] Material storage: The following materials have been deposited at the National Strain Conservation Center of Braunschweig, Germany (Deutsche Sammhmg von Mikroorganismen und Zellkulturen

GmbH (DSMZ)): (1) Microorganisms deposited on DSMZ on January 23, 2008, accession number DSM 21〇59 (five _' containing the vector GS264, and GS264 containing the coded anthropomorphic NMC 4: xenobiotic An isolated nucleic acid of a light bond of a nucleic acid sequence of H9L9 IgG4; (2) a microorganism (WC/z·) deposited on DSMZ, Access No. DSM 21〇6〇 on January 23, 2008, which comprises the vector GS265, and GS265 comprises an isolated nucleic acid having a heavy chain encoding the nucleic acid sequence of the anthropomorphic NMC-4 variant H9L9 IgG4. These deposits are based on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent. Procedures and their provisions under the (Budapest Agreement) [00295] Without further elaboration, it is believed that those skilled in the art will be able to make and utilize the invention. The method of the present invention is provided and the following working examples are provided to assist in the implementation of the invention, and it should not be construed as being critical. Example 1: Construction of chimeric antibody, [00296] Generation of NMC-4-human Fc drum: A chimeric antibody system comprising a variant region of the murine antibody NMC-4 and a human Fc region was constructed in the following manner. In an exemplary method, a synthetic gene sequence for the VH and VL regions of NMC-4 is produced using an anti-vWF antibody NMC_4 (eg, IgG1 λ: having the disclosed amino acid sequence of the variant region) as a template. (Celikel deduction, 1997

Blood Cells, Molecules and Diseases 23:123-134). For example, the synthetic gene sequences used for VH and VL of nmc" 73 201041902 are produced by using the amino acid sequence described in Celekel over β/· and using the software to generate the corresponding nucleotide sequence. ~~,.To find the primers of NMC-4 Xinqin body. H::,; ί- ·Ί,· w ·-ton f. _ Pr!^^ B __ — — — — , i Heavy chain NMC-VH-Eco S'-GACGCGAATTCGCAGGTGCAG NMC-VH-丨gG1-R 5,-CGGATGGGCCCTTGGTGGAAG Rt-F CTGAAGGAGAGC -3' CGCTGCTCACGGTCACGCTGGT -3' (SEQ ID NO: 34) (SEQ ID NO: 35) hlgG-F 5 '-GCTTCCACCAAGGGCC hlgG-R 5,-CCAGAGACAGGGAGAGGC CATCCG -3' TCTTCTG -3 (SEQ ID NO: 36) (SEQ ID NO: 37) lgG1-BamHI-R 5-ATTAGGATCCTTATCATTTACC CAGAGACAGGGAGAGGCT -3, (SEQ ID NO: 38 hFc-L235E-F 5,-CTCGAGGGGGGACCGTCAGTCT hFc-L235E-R 5,-AAGAGGAAGACTGACGGTCCCC TCCTCTT -3' C (SHQIDNO: 39) CTCGAG -3, (SEQ ID NO: 40) CH2-C1q(+F 5'-GGCGTACGCGTGCGCGGTCT CH2-C1q(-)-R 5-CCGCGCACGCGTACGCCTTGC CCAACAAAGC -3' CATTCAGCCA-3· (SEQIDN〇:41) (SEQ ID NO 42) 4-59 5'_ATTAAGCTTGCCGCCACCATGAAA lgG1-BamHi-R 5'- TAAGGATCCTTATCATTTAC leader-Hindiil CATCTGTGGnCTTCCTTCTCCTGGTG CCGGAGACAGGGAGAG-3, • NMC-4 GCAGC Ding CCCAGGTGGGTCCTGTCCCA GGTGCAGCTG AAGGAGAGC -3, (SEQ ID NO: 44) (SEQ ID NO: 43) Light chain NMC-VL-Eco 5-GACGCGAATTCGGACATCCA NMC-VL-Kappa-R 5,-GAAGACAGATGGTGCAGCCACA Rl-F GATGACCCAGAGCC -3f GT (SEQ ID NO: 45) TCGCTTCACCTCCAGCTTGGTGCC -3' (SEQ ID NO: 46) Kappa-F 5'-CGAACTGTGGCTGCACCAT Kappa-BamHI-R 5,-AATTCGGATCCTTACTAACACT CTGTCTT -3' CTCCCCTGTTGAAGCTCTT -3' (SEQ ID NO: 47) (SEQ ED NO: 48) 201041902

[00297] In the exemplary method, the pCR reaction is carried out by using Accuprime PFXDNA POLYMERASE KIT (Invitrogen); for example, a combination of the following 5^μ1 reaction mixture (^) is combined: lxpFX buffer, 〇2^M mixture, 1 unit PFX polymerase, 1 μΜ pre-introduction, 1 μΜ inverted primer and 100 ng of DNA template. The standard PCR procedure consists of starting denaturation at /94 for 1 minute, followed by 94 〇c for 3 sec at each cycle, 55 + C for 30 sec, and 68 ° C for 1 min. Loops, as well as at 68. 〇After stretching for 10 minutes, extend the extensi〇n step. The PCR product was permanently purified by acacia gum on 〇.8% Tae gel, and the expected size of one band was removed and purified by Qiagen GEL EXTRACTION KIT. Ligation of DNA fragments at room temperature in a volume of 10 μΐ containing Ό lx T4 DNA ligase (%ase) buffer (NEB), 0.5 μl Τ4 DNA ligase (Ν^Β) and 100 ng of each DNA 30 minutes; then 'use the μ μ1 ligation solution to transform the 侃 1〇9 five c〇/z• cells, and verify the PC.R by sequencing with a Beckman CEQ 8000 DNA analyzer. The resulting insertions (inserts). [00298] These chimeric antibodies comprising VH and/or VL from the murine antibody NMC-4 and human Fc are synthesized by PCR according to standard procedures (pr〇t〇c〇1) known in the art. In an exemplary method, the VH and/or VL from the fusion is fused to the human Fc by a primer specific for nmc-4 and a primer specific for human Fc. [00299] Optionally introducing an amino acid substituent into the IgGl Fc region to disrupt Fc and complement binding sites to eliminate cytotoxicity that is hypothesized to be caused by the wild-type gamma 1 Fc constant region (see, eg, SEQ ID) NO·· 143). For example, derived from IMAGE. cDNA clone #4764579 (ATCC) (SEQ ID NO: 33) Human IgG1 constant region (eg, Fc) utilizes the primers MgG-F (SEQ ID NO: 36) and hIgG-R (SEQ ID NO: 37) ) (Table 1) to enlarge. Amino acid substituents (eg, L235E (FcR binding region) and E318A, K320A) by, for example, site directed mutagenesis (Duncan &Winter; Nature. 332(6166): 738-40 (1988)) , K332A (at the Clq complement binding site)) was introduced into the IgG1 Fc region. For example, using the primer pair hFc-L235E-F (SEQ ID NO: 39) and 75 201041902

hFc-L235E-R (SEQ ID NO: 40) introduces the L235E mutation into the constant region; using the primer pair CH2-Clq(-)-F (SEQ Π) NO: 41) and CH2-Clq(-)-R) (SEQ ID NO) :42) (Table U introduces complement site mutations. The two external primers, hlgG-F (SEQ ID NO: 36) and IgG1-BamHI-R (SEQ ID NO. 38), were used to link the synthesis containing the four mutations. PCR product to generate a PCR product encoding a modified IgGIFc region (referred to as IgGl (dm)). [00300] In an exemplary method, the heavy chain variation of NMC-4 is mutated by recombinant techniques known in the art. The region is fused to the modified igG1 Fc region (eg, igG1(dm)). For example, the primer NMC-VH-EcoRI-F (SEQ ID NO: 34) and NMC introduced into the five-coRI selection site upstream of the N-terminus of NMC-VH are used. -VH-IgGl-R (SEQ ID Ο NO: 35), amplifying the nucleotide sequence encoding the heavy chain variation region of NMC-4 (SEQ ID NO: 1); in other words, by two-step recombinant PCR, The PCR product is ligated to the heavy chain constant region (eg, IgG1 (dm)), first using the primer pair NMC-VH-EcoRI-F (SEQ ID NO.34) and hlgG-R (SEQ ID NO: 37), followed by the primer For NMC-VH-EcoRI-F (SEQ ID NO: 34) and IgGl-B amHI-R (SEQ ID NO: 38) was subjected to a reverse reaction, and 〇111 and 5 legs 111 were decomposed ((^65〇 final PCR product, and the PCR product was cloned into pIRES2-EGFP-IgK vector (Clontecli) , in the EboRI and 5·ΗΙ sites of PaloAlto, CA), the vector is modified to include an IgK leader sequence that is selected for the guano 1 and 5 coRI sites.

MET (METDTLLLWVLLLWVPGSTGD) (SEQ ID NO.. 107)) (encoded by the polynucleotide of SEQ ID NO: 140). [00301] In an exemplary method, the light chain variant region of NMC-4 is fused to a modified igGi pc region (eg, igGi(dm)) by recombinant techniques as described below. For example, 'use the 3' end of the IgK light chain constant region to further guide the restriction

- meal introducer Kappa-F (SEQ ID NO: 47) and Kappa-BamHI-R (SEQ ID NO: 48), amplified from IMAGE clone #4704496 (ATCC) (SEQ Π) NO: 108) The IgK light chain constant region in the DNA (for example, KC1 (SEQ ID NO: 141)); the same as the 'introduction at the 5' end; the 0-inverse [introduction of the NMC-VL-EcoRI-F (SEQ ID NO: 45) NMC-VL-Kappa-R (SEQ ID NO: 46) (Table 1) 'Enlarge the light chain variant region from the synthetic VL gene; next, Li 76 201041902 with the primer NMC-VL-EcoRI-F (SEQ ID NO) : 45) and Kappa-BamHI-R (SEQ ID NO: 48) 'The nmc-4 variant region and the kCI fragment were joined by recombinant PCR. The final pCR product was decomposed with five coRI and, for example, mHI and cloned into the pIRES2-DsRed2-IgK vector at the same position. [00302] Compared to the piRES-DsRed plastids expressing the light chain mouse-human chimera, the expression level of the heavy chain in the PIRES2-EGFP vector may be insufficient, except for the use of both the strand and the heavy chain. The IgK leader sequence. Therefore, in order to improve the performance level of the heavy chain, the primer pair 4-59 leader-HindIII-NMC_4 (; SEQ ID NO: 43) and IgGl-BamHI-R (SEQ ID NO: 38) and pIRES2-EGFP-NMC4- The IgG (mut) vector was used as a template (Table i) to replace the IgK leader sequence with the leader sequence from the human 〇 germ cell line '59 VH (MKHLWFFLLLVAAPRWYLS;) ID Μ): 109); the fragment was decomposed by the ugly ship and PweI 'Partially selected to the pcDNA6-cMyc-A vector (Invitrogen, Carlsbad, CA). [00303] Construction of AJW200 reference antibody: AJW200 is another antibody directed against the A1 domain of vWF, which has the ability to interact with Gpib_a. The AJW200 reference anti-system is produced by designing the VH and VL sequences' described in U.S. Patent No. 6,228,36, to include, for example, additional functional Kozak sequences for improved performance. For example, the synthetic AJW2〇〇VH gene line is amplified by the Q sub-Hind m_Ko_AJW-F (SEQ ID NO: 49) and HuFab-HR (SEQ ID NO: 50) (Table 2) and selected It is thinned to the π and but αΐ sites of the pcDNA6-cMyc-A vector containing the human igGi(dm) heavy bond definite region (by replacing the VH of NMC-4); and the AJW200 VL system is used.

XhoI-Ko-AJW-F (SEQ ID NO: 51) and Kappa-BamHI-R (SEQ ID NO: 4S) were amplified and sub-selected into the melon with nmc-4 light chain chimera I and the price of the mHI part (by replacing the VL of nmc-4). 77 201041902 Table 2 Introduction to generate AJW200 performance plastids

Hind ΠΙ-Κο- 5' -GTTAAGCTTGCCGCCACCATGGATTT HuFab-HR 5 '-GAATGGGCCCTTGGTGGAAGCG AJW-F TGGGCTGATTTTTTTTATTGTT -3' GAGGAAACGGTCACGAGGGTA -3' (SEQ ID NO: 49) (SEQ ID NO: 50) XhoI-Ko-AJW- 5'- AATCTCGAGGCCGCCACCATGAGTG Kappa- 55 - AATTCGGATCCTTACTAAC ACTCT F TGCCCACTCAGGTCCTGG -3^ BamHI-R CCCCTGTTGAAGCTCTT -3' (SEQ ID NO: 51) (SEQ ID NO: 48) [00304] Antibody Production: Funeral antibodies can be known from the art Any method is produced. In an exemplary method, HEK239F cells were cultured in a Freestyle 293 expression matrix (Invitrogen) in a 120 rpm, 37 ° C, 8% C02 shake flask. The cells were granulated at 100 xg and suspended in 30 ml of Freestyle 293. The matrix was vortexed for 20 seconds to complete a single cell suspension. The number of cells was counted and seeded in 3.3 ll 〇8 cells in 2 L shake flasks containing a total volume of 330 ml of Freestyle 293 performance matrix. The transfection mixture is composed of an equal amount of DNA/OptiMEM (eg 165 pg of HC expressing plastids, 165 μ§ of LC expressing plastids, and room temperature OptiMEM (Invitrogen) to a total volume of 11 ml) and 293feCtin/0PtiMEM (eg 433) ΐ 293 fectin (Invitrogen) and room temperature OptiMEM (Imdtrogen) to a total volume of n ml). The DNA mixture was added to the 293fectin mixture, which was then mixed and incubated at room temperature for 2 Torr, and added to the existing matrix containing 293F cells; 〇, 8% C02, 120 rpm of the rate of culture of cells. At the hour after transfection, the suspension was centrifuged for 5 minutes to pellet the cells; the Mab (monoclonal antibody) supernatant was filtered through a 〇2μιη filter, and the eggs were purified from the f_A affinity column. [00305] A small amount of conditions from transient transfection of HEK-293F cells were cultured (CM? was applied to a sputum 3 ml protein that had been equilibrated with PBS). mlpBS cleans the column and washes out the protein with glycine acid. The blood of the blood is collected into the sputum of the sputum. The S antibody will be washed out in the first two washes; use 'ivaspin The ml centrifuge is used to collect and concentrate the two fractions to the final body 78 201041902 (for example, 0.2-0.3 ml). During this concentration step, the buffer is used to convert the buffer from Tris-glycine to pBS. The final concentrate is sterilized by filtration through, for example, a taro filter, and the anti-white matter concentration is determined by the labor method (BlGRadDC^_ method). [00306] For large-scale purification, ultrafiltration is used. Hollow fiber

Amersham Biosciences 30,000 NMWC/290 cm2 hollow fiber 耔Ο F F UFMO-C-mMA) Concentrate 2L conditioned medium from transiently transfected epiphyte cells (eg HEK-293T) until the volume drops to ~2 〇〇 This concentrated material or purely de-slurried for smaller transfections is equilibrated with Tris-HCl using 〇1, such as Protein A on the SEpHAR〇SE column, ί PH 8,0 TriS'HC1 purge column Until the reading of (10) 280 has been established. Wash the antibody with G] Μ, ΡΗ2·7 glycine and collect the final concentration of 121 Μ, ΡΗ 8·_ TriS_HC1 to αΐ 3 to adjust the final concentration of the peak protein of the peak protein. Value; the collection peaks are condensed to a volume of less than 7 by more than 遽 (eg on a centrifuge device) and then utilized on a 管 0 column (Amersham

Two separate rounds of Bi_ences/GE-Healthcare are desalted. [00=] Quantitatively and analytically derived from the culture of liquid solution = white shell by means of the method known from the art towel (for example, the Laoli protein egg (four) assay). In an exemplary method, the strain was analyzed by SDS_pAGE, and the protein was converted to a nitrocellulose membrane at room temperature at 5% milk.

Lit 1 hour' and cultured with a mouse-resistant mouse anti-human city (such as the linger chain L main cham specific), 1:1 〇, _) and mouse anti-human Kappa (such as κ-, special, sexual) 'Detect the signal with the ECL kit. In the assay of platelet aggregation induced by rist〇cetin, the activity was determined: in an exemplary method, the test was used to detect the binding specificity of 人类4 human 抗 ϊϊϊΐ: including, for example. For example, platelet Ί 1 Ό疋 is a quasi-platelet aggregometer (Bio^Data, m〇e from 4), human platelets dried using cold springs (Bio/Data, Horsham 79 201041902 T% i \ . .One

The purified vWF of μΐ was added to the tube to start the agglutination reaction 1 sec., and the baseline reading was recorded at 1.5 ′. Estimate the EQo value of the test antibody as a potent inhibitor of 〇5血小板% platelet aggregation. Compared with the parental single antibody, the aging body exhibits the efficacy of ___murine blue ^'^ body. [00309] Furthermore, for more accurate % values, chimeric antibodies are assayed using, for example, a plate reader method adapted from the mocroplate method. In an exemplary method, a 96-well COSTAR 3603 plate is added to each cell (Perwe11) 15 〇ml TBS (pH 7.5) containing 4.5 x 107 platelets fixed with parafimaldehyde, and purified human vW is added. (Calbiochem, SanDiego, CA) to a final concentration of 1.5 pg/mL per pool. After adding ristocetin to a final concentration of 1.5 mg/mL per cell, a series of test antibodies were added to initiate the agglutination reaction and set at 37 ° C for 6 minutes, while taking the 彳 环 ring There is a 20 second on-board oscillation (on_b〇ardshaking) of SPECTRAMAX PLUS PLATE READER (Molecular Devices)^ turbidity (for example, absorbance at 405 nm). An inhibitor compound (e.g., 2 〇 pl/weii) or a reference MAb (e.g., AVW-3) is added, and the mixture is cultured and monitored for 2 Torr. Yuan Hou's cultured with the ribasterin or Botrocetin (for example, 20 μΐ/well) and monitored the mixture for 40 minutes. The agglutination signal is monitored for the degree of decrease in absorbance (e.g., - Δ absorbance). [00310] The NMC-4-human Fc chimera was compared to the original: NMC-4 monoclonal antibody, and the selected AJW200 antibody. As shown in Figure 1, the NMC-4 chimeric antibody was similar in activity to the original NMC-4MAb in the inhibition of platelet aggregation assay, and slightly better than AJW200, with EC5G values of 〇.1, respectively. .17, and 0.27 nM. 5 Example 2: Construction of anthropomorphic antibodies [00311] Screening of human receptor architecture: available databases (eg human skinny 80 201041902 cell line database, human V region database (Vbase), or Kabat) Databases or published publications (eg Kabat et al., Sequences of Proteins of Immunological lnterest, 1992) to identify the subfamily of the murine heavy and light chain v regions and to determine the optimal human germ cell line Architecture to use as a receptor molecule. The use of VH and v1 sequences within these subfamilies as a screening for receptor sequences can be based on sequence homology and/or matching of typical structures of CDR1 and CDR2 regions to help preserve six CDRs after transplantation. Appropriate relative performance / [00312] For example, 'assuming that there is good homology between the NMC-4 VL architecture and the members of the ^^科i (VK1), use the human v-region database to indicate _心4 κ The light chain belongs to Kapa Iko. We observed that germ cell line sequence = 18 (SEQ 10 Ν 0: 5) has the highest homology and optimal conservation of the typical structure of the CDR loop, which is 78% up to the entire sequence of CDR3. The sequence identity is; in terms of the architectural region, it has 84% sequence identity. NMC-4 light chain, human heavy chain 〇18 (SEQ ID NO: 5), and AAK94808 (mature antibody from 018, used to provide LCDR3 and architecture 4 sequences to compare with NMC-4 in this region (SEQ ID NO: 6) Alignment (Zhan Qingcan) is shown in Table 3. The difference between NMC-4 and human antibodies is indicated in bold (numbering is based on Kabbah count (Kabat, 1978). )). [00313] Similarly, the human V-region database is used to indicate that the νπ 序列 sequence up to architecture 3 falls in subfamily IV. Within human VH subfamily IV, NMC-4 VH, 4-59 germ cell line sequence (SEQ ID NO: 3) has the highest sequence homology, up to the entire VH of CDR3, exhibiting murine vh 56 The sequence identity of %, in terms of the architecture area, shows 67% identity (Table 4). 'AAC18165.1 (SEQ ID NO: 4) is selected to provide HCDR3 and architecture 4 comparator sequences without being limited by the theory of the invention, as it has human germ cells in architectures 1 to 3 and HCDR1 and HCDR2 The same amino acid sequence of strain 4-59 VH. HCDR3 is known to be highly divergent, and FW4 is derived from a different domain of the independent gene product (J), so HCDR3 and architecture 4 are not included in the VH thin cell line sequence of the human V region database. In Table 4, the amino acid difference between the sequence of nmc-4 VH and AAC18165.1 (SEQ ID NO: 4) is highlighted in bold, 81 201041902, and its position is highlighted by an asterisk. Table 3 NMC-4 VL and human antibody AAK94808 Alignment, the latter has the same amino acid architecture, CDR1 and CDR2 sequences as human germ cell line VL, 018

Name FW1 CDR1 ------ A 2 — *— *-- — — — 3* — — — 12345678901234567890123 45678901234

Kabat No: NMC-4 VL (SEQ ID NO: 2) 018 (AAZ94808) (SEQ ID NO: 6) DIQMTQSPSSIiSASVGDRVTITC Name FW3 ---6---------7 Kabat No: 78901234567890123456789012345678 NMC-4 VL · GVPSRFSGSGSGTDYSLTISNLEPEDIATYYC 018 (AAK94808) GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC FW2 CDR2 ---------*· 5 *_*-** 567890123456789 0123456 SASQDINKYLN WYQQKPDGAVKLLIF YTSSLHS QAS QDISNYLN WYQQKPGKAP KLLIY DASNLET CDR3 FW4

901234567 QQYEKLPWT QQYDNLPLT

8901234567 PGGGTKLEVK FGGGTKVEIK Table 4 Comparison of NMC-4VH with human antibody AAC18165.1, which has the same architecture, CDR1 and CDR2 amino acid sequence as human germ cell line 4-59

Name FW1 CDR1 FW2 CDR2 ----*----1 — * ——**- -*------*-*3*-*** -*- -4------ -*_ 5-***·

Kabat No: 1234567 890123456789012345 67 89012345 67890123456789 0123456789012 345

NMC-4 VH

(SEQ ID NO: 1) QVQLKESGPGLVAPSQSLSITCTVS GFSLTDYGVDWVRQPPGKGLEWLGMIWGDGSTDYNSALKS 4-59 (ACC18165.1)

(SEQ E) NO; 4) QVQLQESGPGLVKPSETLSLTCTVS GGSXSSYYWS WXRQPPGKGLEWXG YIYYSGSTNYNPSLKS

Name FW3 CDR3 FW4 f10^ f---* 5

Kahat No: 67890123456789012abc345678901234 567890abcdef12 34567890123 NMC-4 VH RLS Worker TKDNSKSQVFIjKMNStiQTDDTARyycvR DPADYGNYDYALDY WGQGTSVTVSS

4-5 9 (AAC18165.1) RVTISVDTSKNQFSXjKLSSVTAADTAVYYCAR GYRPGVAAHSPFDY WGQGTLVTVSS

[00314] Since we hypothesized that direct grafting of CDRs from mouse antibodies to the human antibody architecture results in loss of antigen binding affinity (Foote and Winter J. MoZ. Biol. 224: 487-499 (1992); Xiang et al JMol Biol 253: 385-90 (1995); Homes 167:296-301 (2001)), so it may be desirable to make some of the residual money in the framework of 2010 2010902 in the position of the old base, this cake is called a mutation (bf Μ leg For example, Table 5 shows residues that may affect cd f as a potential candidate for back mutation to murine residues (eg, to emphasize the amino group at these positions between NMC-4 and the human architecture) Acid difference). ^Architecture residues that affect the configuration of CDRs; compare a and human receptors

[00315] We have identified amino acid residues that may be involved in the pairing of heavy and light chains to coordinate the expression of CDRs (H〇lmes Peri-J Immunol. 167:296-301 (2001)) and may be back mutations Candidates. Residues 44, 96 and 98 in the 'VL region are considered to be of importance without being limited by the theory of the invention, with additional contributions from residues 34, 36, 38, 46, 87, 89 and 91. Among these residues, the only significant difference between NMC-4 and acceptor 018 in the VL region is residue 44, which is vaune in NMC-4 VL, but in human 〇ι § The structure is proline. Without being bound by the theory of the present invention, residues 45 and 103 are of importance in terms of yl, and residues 35, 37, 39, 47, 91, 93 and 95 also interface with VH and VL (interface Packing) has contributed. Among the merging residues in the NMC-4 and Receptor 4-59 frameworks, the only difference is only in residue 93, where there is a conservative difference in lysine in nmc-4 compared to alanine in '59 (Table) 5). 83 201041902 [00316] Comparing the framework sequences of the 4-59 VH sequences of murine and human germ cell lines, it is known that among the residues important for CDR expression and interface stacking, many differences are clustered in architecture 3 (residue 67 , 71, 73, 78, 93), the additional difference is residues 37 and 48 in architecture 2; these residues are putative candidates for back mutation in the prototype (pr〇t〇type) anthropomorphic sequence. However, the difference between the two architecture 2 residues (37V vs 37I and 48L vs 481) is conservative, so the first prototype VH sequence can focus on the difference in the framework 3 region' and transport the following human-to-mouse back mutation: V67L, V71K, T73N, F78V and A93V (Table 6). The synthetic gene line was synthesized by Retrogen (San Diego, Calif.) and was designated as H2 and was selected for inclusion in the bacterial vector pRSFDuet (Novagen, Madison, Wisconsin) and was used as the site. The template of the embedded segment was amplified by 4-59- huNMC-F (SEQ Π) NO: 54) and hu-VH-R (SEQ ID NO: 55) primers (Tables 7 and 9). The fragment synthesized by αΐ is decomposed to decompose the plastid pcDNA6-NMC-HC contained in the chimeric heavy chain produced in Example 1 by τιΛΙΙ and αΐ, and in the presence of NTPs Kleno

The Klenow fragment blunt-ends the DNA fragment containing the pcDNA-IgGldm fragment. The VH-containing PCR product was ligated into a blunt-ended pcDNA-IgGldm fragment, followed by plastid DNA to transform the pure host strain 109. The Beckman CEQ 8000 DNA Sequencer was used to sequence individual clones (clones) to identify colonies with the correct sequence in the correct orientation (selection strain pcDNA-huVH- IgGldm). This vector was used as a template for H4, H5, H6, H7 and H8 variants, which were used to evaluate the effect of individually returning each residue to a human residue (summarized in Table 6). These variants were constructed using the primer pairs described in Table 7 (corresponding sequences are shown in Table 9). 018 was incorporated into the germ cell line 4_59 of different VL and VH variants and Table 6

"vl-: (human to mouse) 1 H2 V67L, V71K, T73N, F78V, A93V L5 P44V, Y49F, F71Y H4 V67L. V71K. T73R F78V L4 Y49F.F71Y 1 IT- ! H) V71K. T73N. F78V. A93V L6 | P44V. F71Y

F73L, G100Q F73L. GT〇n〇F73L, G1Q0 (S4 201041902 Table 7 Templates and primers for constructing anthropomorphic heavy chain variants Ο Ο VH variant template fragment-1 PCR primer fragment-2 PCR primer final VH PCR Primer vector selection site H2 from Retrogen synthesis dna 4-59-huNMC-F and hu-VH-R pcDNA6- igG1(dm) Hindll! Apal H4 H2in pcDNA6-lgG1(dm) pcDNA6-F and VH-V93A-Rev VH-V93A-For and hFc-L235E-R pcDNA6-F and hFc-L235E-R pcDNA6- igG1(dm) Hindlll Apal H5 H2 in pcDNA6-lgG1(dm) pcDNA6-F and HC-L67V-R HC-L67V- F and hFc-L235E-R pcDNA6-F and hFc-L235E-R pcDNA6- igG1(dm) Hindlll Apal H6 H2 in pcDNA6-igG1(dm) pcDNAS-F and HC-K71V-R HC-K71V-F and hFc- L235E-R pcDNA6-F and hFc-L235E-R pcDNA6- igG1(dm) Hindlll Apal H7 H2 in pcDNA6-lgG1( dm) PCDNA6-F and HC-N73T-R HC-N73T-F and hFc-L235E-R PCDNA6 -F and hFc 7235E-R pcDNA6- igG1(dm) Hindlll Apal H8 H2 in pcDNA6-lgG1( dm) PCDNA6-F and HC-V78F-R HC-V78F-F and hFc-L235E-R PCDNA6-F and hFc -L235E-R pcDNA6- lgG1(dm) Hindlll Apal H9 from Retrogen DNA 4-59-huNMC-F and hu-VH-R pcDNA6- lgG1(dm) Hindlil Apal H6 V67L, T73N, F78V, A93V L7 P44V. Y49F F73L, G100O H7 V67L, V71K, F78V, A93V L8 No back mutation F73L'G100〇H8 V67L, V71K, T73N, A93V L9 No-reversion mutation F73L, I83F, G100Q H9 No-reversion mutation [00317] Comparison of residues that can affect standard structure and interface stacking: NMC-4 VL and 018 human There are two differences between the vl receptor architecture (eg, residues 44, 49, and 71), so a prototype anthropomorphism with three to murine residues, Detroit, and rain can be designed. This is a kind of residue 73 that has been changed from amphetamine to leucine (Euche) because leucine is the more common residue in this position in the human antibody list. 201041902 In order to produce this variant, a variant strain (Regr〇gen) called VL4 with structural changes (eg Y49F, F71Y, and F73V) was synthesized and cloned using the primers listed in Table § Into the pETDuet vector. Using the obtained L4-pETDuet vector as a template 'using the primer pair shown in Table 8 to introduce the P44V mutation of L5; then substituting the L5 variant into the EcoRI and BamHI sites of the pIRES DsRed2 vector to replace the murine NMC-4 VL The L6 & L7 vector was then generated using the L5-pIRES DsRed vector as a template using the primer pairs listed in Table 8. ^8 Templates and primers used to construct anthropomorphic scorpion mutants from NMC4-VL-Ec〇RI-F L4 Retrogen and piRbS- EcoRI -~. _ Synthetic DNA Kappa-BamHI-R DsRed2-lg/c BamHI L4 in Fab-L-For LC-P44V-F Fab-bFor and Ndel L5 pETDuet-1 and Fab-L-Rev pETDuet-1 Xho! -- LC-P44V-R Fab-L-Rev L5in NMC4-VL- EcoRI-F L5 pETDuet-1 and pIRES- EcoRI Kappa-BamHl-R DsRed2-lgk BamHI L5 in NMC4-VL-EcoR|-F LC-F49Y-F NMC4-VL-Ec〇R!-F L6 pIRES- And pIRES- EcoRI DsRed2-lg/c LC-F49Y-R Kappa-BamHi-R Kappa-BamHi-R DsRed2-ig/c BamHI L5in NMC4-VL-EcoRI-F LC-Y71F-F NMC4-VL-Ec〇Ri -F L7 pIRES- . and piRES- EcoRI DsRed2-lgk LC-Y71F-R Kappa-BamHI-R Kappa-BamHI-R DsRed2-ig/c BamHi L7 in 5,IRES HuLC-V44PF49Y-F 5'IRES L8 pIRES- and and, and p 丨RES-DsRe EcoRI _ DsRed2-lg/( HuLC-V44P-F49Y-R 3'丨RES 31RES 62-\gk BamHi L9 from NMC4-VL-EcoR!-F Retrogen and pIRES- DsRe EcoRI Synthetic DNA Kappa-BamHI-R d2-lgk BamHi 86 201041902 9 Primer sequence for constructing various variant regions 4-59-huNMC-5'- GTTAAGCTTGCCGCCACCATGAA Hu-VH-R 5'-GGATGGGCCCTTGG Ding OGAAGC F ACATC Ding GTGG DTC TTC CCTTCTCCTGG Ding GGCAGCTCCCAGGTGGGTCCTGTCCC GGAGGAAACGGTCACGAGGGTA-3, AGGTGCAGCTGC AGGAATCCGG- 35 (SEQ ID NO: 55) (SEQ ID NO: 54) pcDNA6-F 5'- CACTGCTTACTGGCTTATCG hFc-L235E-R 5 '-AAGAGGAAGACTGACGGTCCCCC AAATTA-3, CTCGAG -35 (SEQ ED NO: 56) (SEQ ID NO: 40) VH-93A-For 5,-GACACCGCTGTTTACTACT VH-V93A-Rev 5'-AGTCAGCCGGGTCACGACCGCA GCGCTCGTGACCCGGCTGACT -3 ' GTAGTAAACAGCGGTGTC -3, (SEQ ID NO :57) (SEQ ID NO: 58) HC-L67V-F 5' -CTGAAATCCCGTGTTACCATC HC -L67V-R 5'-GTCTTTGGAGATGGTAACACGGG TCCAAAGAC -3, ATTTCAG -3, (SEQ ID NO: 59) (SEQ ID NO: 60) HC-N73T-F 5'-ACCATCTCCAAAGACACCTCC HC-N73T-R 5>_GTTTTTGGAGGTGTCTTTGGA AAAAAC-3 , GATGGT-3' (SEQ ID NO: 61) (SEQ ID NO: 62) HC-V78F-F 5 ' - AACTCC AAAAACC AGTTCT HC-V78F-R 5^_gtttcagggagaactggttttt CCCTGAAAC -3' GGAGTT-3, (SEQ ID NO: 63) (SEQ ID NO: 64) HC-K71V-F 5'-CTTACCATCTCCGTAGACAA HC-K71V-R 5,-GTTTTTGGAGTTGTCTACGGA CTCCAAAAAC -3' GATGGTAAG -3' (SEQ ID NO: 65) (SEQ ID NO: 66) hu-VH-K71V- 5'-CGTOTTACCATCTCCGTAGA hu-VH-K71V- 5'_丁丁TCGAGGTG丁CTACGGAGA丁 F(H9) CACCTCCAAA-3, R(H9) GGTAACACG-3, (SEQ ID NO: 67 (SEQ ID NO: 68) Fab-L-For 5,- ATACATATGGACATCCAGATG Fab-L-Rev 5!- AGACTCGAGTTATCAACACTCTCC ACCCAGAGC -3, CCTGTTGAAGCT (SEQ ID NO: 69) (SEQ ID NO; 70) NMC4-VL- Ec 55-GACGCGAATTCGGACATCCA Fab-L-Rev 5' - AGACTCGAGTTATCAACACTCTCC oRI-F GATGACCCAGAGCC -35 CCTGTTGAAGCT -3, (SEQ ID NO: 71) (SEQ ID NO: 70) 5,-IRES 5,-AGCTGGTTTAGTGA -3, SPIRES 5 '-CAAGCGGCTTCGGCCAG -3' (SEQ ID NO: 72) (SEQ ID NO: 73) LC-Y49F-F 5, -CCAAGCTGCTGATCTTCTAC LC-Y49F-R 5, TGGTGTAGAAGATCAGCAG ACCA-r CTTGG -3' (SEQ ID NO: 74 (SEQ ID NO: 75) LC-F83I-F 5'-CAGCCCGAGGACATCGCCAC LC-F83I-R 5, GCAGTAGTAGGTGGCGATGTCCT CTACTACTGC-3' CGGGCTG-3' (S EQ ID NO: 76) (SEQ ID NO: 77) 87 201041902 LC-P44V-F 5'-AAGCCCGGCAAGGCCGTC AAGCTGCTGATC -3s (SEQ IDN〇: 78) LC-P44V-R S'-GATCAGCAGCTTGACGGCCTTGC CGGGCTT-3, (SEQtDNO: 79 LC-F49Y-F 5,-GCCGTCAAGCTGCTGATCTA CTACACCAG -3 s (SEQ ID NO: 80) LC-F49Y-R 5'-CTGGTGTAGTAGATCAGCAGCT TGACGGC-3, (SEQ ID NO: 81) LC-Y71F-F 5,-GGCAGCGGCACCGACTTCA CCCTGACCATC -31 ( SEQ ID NO: 82) LC-Y71F-R 5,-GATGGTCAGGGTGAAGTCGGTG CCGCTGCC -3* (SEQ ID NO: 83) HuLC-V44P, F49Y-F 5*-GGCAAGGCCCCCAAGCTGCT GATCTACTACACCAG ·3, (SEQIDN〇:S4) HuLC-V44P, F49Y-R 5'-CTGGTGTAGTAGATCAGCAGC TTGGGGGCCTTGCC -3* (SEQ ID NO: 85) 〇[00318], parallel to the selection of the first set of variants, identified as having up to HCDR3 in the 31^3 3D search A 1D 〇pdb structure (eg, 2 3 angstrom resolution) of the highest sequence identity (88%) of the sequence and 89% sequence identity of the framework region was established to create a three-dimensional model of the computer-generated sequence of the 4-59 receptor germ cell line. The structure lA〇K.pdb was chosen as a template for the anthropomorphic vh sequence with 81% sequence identity. Two crystal structures of murine NMC-4 Fv can be utilized. 1 AOK.pdb (eg 2.2 human resolution) 'has bound antigen (Celikel ei a/, Nat. Struct. Biol. 5:189-194 (1998) ) and was also selected as the best fit in the ugly field of prototype anthropomorphic variants; and IFNS.pdb (2·0λ resolution), which has been incorporated into mutant antigens. ❹ 1 A0K 'Pdb and 1FNS-Pdb both have substantially the same VH/VL interface angle' so lAOK, pdb was used to model the overlapping human receptor model and the anthropomorphic VH and VL sequences. [00319] When overlapping the backbone structures of NMC-4 VH and 4-59, a three-zone difference was observed. First, the difference exists in the residue of 7 to 3, which contains a part of HCDR1. Without being bound by the theory of the invention, these residues contact HCDR2 and HCDR3 which together form part of the antigen binding site. We predicted that residue 34 (Val in murine NMC-4 VH and Trp in 4-59 sequence) is most likely responsible for the conformational change of the H27-33 loop and is therefore a candidate for back mutation. Second, the difference exists in residues H52 to H55, which form part of the CDR2 loop. We believe that the structural residue 71 (Lys in mice, Val in 4-59) is most likely related to this difference, 88 201041902. Three additional residues in the 4-59 structure were identified as moon-blocking antigen binding: Η37, 1 is π compared to Val in mice; Η73, which represents 4-59 compared to Asp in mice For the ==jH7S compared to the w in the mouse, which represents the version of 2_59 in the _2 〇Γ / -- ' difference exists in the CDR3 towel, can be expected to be the dissimilarity of the towel. έ±·························································································· , and L92. Ο Ο Ϊ rm.P W is a template for the anthropomorphic model because it matches 97/107 residues (eg 91% sequence identity). # =°4的凡 backbone and 〇18 where the structure of the good match meets the 2 phase = 'only - obvious structural difference in the loop composed of silk L39_L45 ^,, contact VH and thus affect the accumulation and then affect the binding capsule The shape of the bag (not limited to the theory of the present invention, the residue is private (for example, the mouse makes the Pro in each product U) may be the cause of this difference' and thus may be a candidate for back mutation based on computer modeling. In vitro (-fine) 在 in the test of platelet agglutination induced by rustatin. To test whether the computer model can accurately predict the effect on activity, 'VH2 prototype mutant and VL mutant (such as L4) , 5, 6 and 7) pairing, soil, = L5 and VH variants (eg H2, H4, taking H7 and H8) are temporarily transfected with 2 3 fertilizer cells to produce antibody variants, and Acupuncture ί ^c·4 chimerism recorded in Wei, will be touched and listened to Yangshen liquid [Removal 3] ruthenium-induced platelet aggregation assay is performed using cold 4 dry platelets as in Example i. Antibody-series dilution = measurement using the Spectromax reaction disc reader (M〇lecularDevices) The Pmm software was used to analyze the data to determine the EC5G values of various purified antibody variants (see, eg, Table 9). The first type of anthropomorphic antibody (consisting of m and L5) was presented with the parental sputum (eg The EC50 is 0.18 er) the same activity (for example, % is 〇13) 89 201041902 H〇), and then the test has a return to human f-deletion: the mutant strain exhibits a far-off strain (Table 9). Interestingly, 'this is a light-strained cockroach'. This suggests that the stability of the anthropomorphic heavy chain and the antibody is negative, because this unexpectedly suggests that the chemical does not affect the activity at all.日# . These results show that: the difference between the expected two ί i ί ^ _ and the ^ 4 variability _, including the 观察 匕 皮 = = 2 Shi Jia ^ Xi Tong ^ Fan Wang has no influence. Based on these results, construct transplants = (for example, just represent fresh coffee - to complete humans): (10) Transplantation activity (5) 5〇 is _ this table 10> Ming: directly transplanted from the murine antibody to the publicity of the "work", even if C, & Mo / Dz, .23: 123; CeHkeUtal ^ (Ct al; ^ π t along,, gamma wci5io /5:189), therefore,

The two expected the relative performance of the six CDRSs in the J-guided platelet aggregation assay

90 201041902 L5, H7 0.16 H9, L9 0.12 (n=2) L5, H8 0.28 L4, H2 0.14 L6, H2 0.14 L7, H2 0.15 [00325] Anthropomorphic CDR regions · Molecular Molding Suggestions - at] v^yfC -4 LCDR1 (eg residues 24, 30 and 31) and NMC-4 HCDR1 (eg residues 27, 29, 30 and 34) can tolerate additional changes in CDR1 designed to anthropomorphic light and heavy chains; Furthermore, 'we also believe that two residues (eg, 61 and 62) in the HCDR2 sequence represent tolerable changes (eg, Ser to Pro of residue 61 and Ala to Ser of residue 62). Therefore, a series of so-called "super anthropomorphic" variants (Tane^, 2002 J/ww Chi 9/. 169:1119-1125) are composed of templates and primer pairs as listed in Table 12 (for example, representing a fully human LCDR1). The mutant strain L10; part of the anthropomorphic HCDR1 region of H12 and H13 (Table 11)) was constructed. Table 11 shows mutations in murine CDR residues to their corresponding counterparts in 4-59

Country^»1 H12 F27G, L29I, T30S L10 -----=- S24Q, N30S, K31N H13 F27G, L29I, T30S, V34W Lll S24Q, N30S, K31N, Y50D, T51A, S53N, H55E, S56T H14 F27G, L29I, T30S, V34W, S61P, A62S H15 F27G, L29I, T30S, D31S, G33Y, V34W, G35S, S61P, A62S H16 • F27G, L29I, T30S, V34W, M50Y, W52Y, G53Y, D54S; D58N, S61P, A62S

[00326] In an exemplary method, computer modeling predictions are tested by performing more than one assay to determine the activity of anthropomorphic antibodies. For example, L11 variants were co-transfected with H9 and tested for antibodies in the restromycin-induced platelet aggregation assay. 91 201041902 Days can be tolerated by three variations to make LCDR1 _ completely (10) ^LCDRl ' without causing a loss of effectiveness on the crane. #Him &

When the heterologous strain binds to L9, the antibody of H12_L9 mutant strain is obviously I Ο, and the additional V34W mutation retains the complete efficacy, and the effect of adding s6ipnn has no effect on the effect, and the silk shows that the silk can not affect the condition of New Zealand. The next is converted to a 4-_59 sequence. Secondly, the importance of LCDR2 in maintaining the _blocking activity is evaluated by constructing a variant of the L10 chain. In the variant of the L1 〇 chain, the entire LCDR2 (YTSSLHS) (SEQ ID NO: 11) is encoded. The plastids of VL^ variant L10 and the primer pairs listed in Tables η and 12 were replaced by human germ cell line LCD18LCDR2 (DASNLET) (SEQ IDNa.118). This newly constructed mutant L11 was then paired with H14 to generate an antibody variant Ui_H14. Although this mutant still exhibits nM-class efficacy, its activity is reduced by a factor of 1% compared to the chimera (EC% of 1.63 nM) in the platelet agglutination test, which implies that LCDR2 may be the most active for anthropomorphic antibodies. Indispensable.

92 201041902 Table 12 summarizes how to construct a "super anthropomorphic" variant

H12 H9 in pcDNA6-IgG1 (dm) pcDNA6-F and huH12-R huH12-F and hFc-L235E-R pcDNA6-F and hFc-L235E-R pcDNA6- IgGl(dm) Hindlll Apal H13 H9 m pcDNA6-F huH13- F pcDNA6-F pcDNA6- Hindlll pcDNA6-IgG 1 (dm) and IgG1(dm) Apal huH13-R hFc-L235E-R hFc-L235E-R H14 H13 in pcDNA6-F huHH-F pcDNA6-F pcDNA6- HindTII pcDNA6-IgG1 (dm) and IgG 1 (dm) Apal huH14-R hFc-L235E-R hFc-L235E-R H15 H14m pcDNA6-F huH!5-F pcDNA6-F pcDNA6- Hindlll pcDN A 6 -IgG 1 (dm) and and IgG 1 (dm) Apal huH15-R hFc-L235E-R KFc-L235E-R H16 HI 5 m pcDNA6-F huH16-F pcDNA6-F pcDNA6- Hindlll pcDNA6-IgGl (dm) and IgG 1 (dm) Apal huH16-R hFc-L235E-R hPc-L235E-R L9 LI in pETDuet-l NMC4-VL-EcoRI-F LC-Y71F-F NMC4-VL-EcoRI-F pIRES-DsR EcoRI and And ed2-IgK BamHI LC-Y71F-R Kappa-BamHI-R Kappa-BamHI-R L10 L9in 5'IRES huLlO-F 5'IRES pIRES-DsR EcoRI pIRES-DsRed2-Ig^ and and ed2-IgK BaxnHI huLlO- R 3, IRES 3'IRES L11 LlOin 5,-IR£S huLl 1-F 5,-IRES pIR ES-DsR EcoRI pIRES-DsRed24g^) and ed2-IgK BamH huLH-R 3, IRES 3, TRES 201041902 Table 13 is used to construct the primer for humanized CDR variant 5, -IRES 5, _AGCTGGTTTAGTGA -3' (SEQ IDNO :72) 3,-IRES 5;-CAAGCGGCTTCGGCCAG o' (SHQIDNO:73) huL10-F 5'-ACCATCACCTGCCAAGCCAGCCAG GAC ATCAGC AACTACCTGAACTGG-3J (SEQ ID NO: 86) huLlO-R 5'-CCAGTTCAGGTAGTTGCTGATGT CCTGGCTOGCTTCGCAGGTCATOG D-3: (SEQ ID NO: S7) huLll-F 55 -CCC AAGCTGCTGATCTACGACGCC A GCAACCTGGAAACCGGCGTGCCC -3' (SEQ ID NO: 88) huLll-R 5?- GGGCACGCCGGTTTCCAGGTTGCTGG CGTCGTAG ATCAGCAGCTTGGG-35 (SEQ ID NO: 89) pcDNA6-F 5^- CACTGCTTACTGGCTTATCG AAATTAo5 (SEQ ID NO: 56) hFc-L235E-R 5,-AAGAGGAAGACTGACGG D CCCCC CTCGAG -r (SEQ ID NO: 40) huH12-F 5' -GTTTCCGGTGGCTCCATCTC CGACTACGGTGTTGACTGGA -3, (SEQIDKO:90) huHl2-R 5'-TCCAGTCAACACCGTAGTCGGAG ATGGAGCCACCGGAAAC -35 ( SEQ ID NO: 91) huHB-F 5, GTTTCCGGTGGCTCC ATCTCCGAT ACGGTTGGGACTGGATCCGTCAG ^ (SEQ ID NO: 92) huHl3-R 5!-CTGCAGGATCCAGTCCCAACCGT AGTCGGAGATGGAGCCACCGGAAAC -35 (SEQ ID NO: 93) huH14-F 5'-GTTCCACCGACTACAACCCC TCTCTGAAATCCCGT-3' (SEQ ID NO: 94) huH14-R 5 '-ACGGGA butyl TTCAGAGAGGGGTT GTAGTCGGTGGAAC -3' (SEQ ID NO: 95) huH15-F 5, GTTTCCGGTGGCTCCATCTCCTCCTACTATT GGTCCTGGATCCGTCAG ～3, (SEQ ID NO: 96} huH15-R 5'-CTGACGGA butyl CCAGGACCAATAGTA GGAGGAG human TGGAGCCACCGGAAAC '3, (SEQ ID NO: 97) huH16-F 5 '-GAATGGATCGGTTATATCTATTATTC CGGTTCCACCAACTACAACCCCTCT - (SEQ ID MO: 98) huH16- R 5^AGAGGGGTTGTAGTTGGTGGAACCG GAATAATAGATATAACCGATCCATTC -3* (SEQ ID NO: 99) [00327] Next, the importance of remaining murine residues (eg, H31, H33, and H35) in the anthropomorphic HCDR1 of variant H14 is These residues were altered to their human counterparts in the VH germ cell 4-59 sequence (eg, D31S, G33Y, and D35S). As a result of the construction, m5 has the assigned sequence GGSISSYYWS (SEQ 0: U0) compared to the part of the anthropomorphic sequence GGSISDYGWD (SEqIDN(): m) in m4; finally, the entire gift of m5 is converted into VH4- The human counterpart in 59 (from miwgdgstdynsalks 94 201041902 (SEQ ID NO: 8) to YIYYSGSTNYNPSLKS (SEQ Π) NO: 119), a total of 7 residues differ) 'to create another complete human HCDR1 and HCDR2 Mutant strain H16. Mutant strains H15 and H16 were each paired with a light chain mutant L1〇 to produce antibody variants H15-L10 and H16-L10, respectively. [00328] These mutant strains were evaluated by platelet aggregation assay to determine their activity. The data shown in Table 14 suggests that replacing the entire HCDR1 with a human sequence completely disrupts the anti-vWF activity. This implies that the remaining three residues in HCDR1 (eg, D at position H31, G at position H33, and D at position H35) are important for retention activity, even though these residues may not Direct contact with the antigen 'as indicated by the crystal structure proposed by Celikkel, et al (Ato. 5: 189). Table 14 EC50 values of the super anthropomorphic variants

* *;.·- H9, L9 (CDR-transplantation) NMC-4 chimera H12, L9 H13, L9 H14, L9 H13, L10 H14, L10 H14, L11 0.18+0.03 (π=9) 0.12 (η=2 0.29 0.16 (η=2) 0.13 (η=2) 0.20 0.22±0.05 (η=5) 1.63

H15, L10 | H16, L1Q

ND

ND Example 3: Subtype of Recombinant Antibody (IS0type) [00329] In an exemplary method, since IgG1 is an active subtype of complement (the activation and the effector response, we expect to record the mutation) The IgGl form is reconstituted into the IgG4 form, although IgG4 is relatively incompatible. For example, a candidate VH variant is converted from a mutated IgGl form to a formula (see, eg, SEQ ID NO: 144) to generate a candidate for development; In addition, ^ 201041902 new δ is also a light chain and heavy chain open reading frame (〇 卿 卿 哗 , , , , , , , , , , , , , , , 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含The restriction endonuclease site on the site was used to assist in the subsequent selection of multiple cloning sites for the expression vector PST0518 for downstream cell line development and large-scale antibody production (Table 15). In other words, by replacing the igG1 constant region with the IgG4 constant region, and introducing the ligament at the 5th end of the heavy chain, and introducing the red part at the 3' end, Two anthropomorphic mutants H9 and H14 were converted into igG4 form. IgGl and I Both gG4 contain a naturally occurring flI site at the junction near the variant and the site, and this site is used for colonization in IgG4 (substitution in IgGl). Place the 5 preposition 1 and the TVoil restriction On the 3' end of the sequence, to assist in the subsequent selection into the vector of pST〇518. Using Blue Henm Biotechnology (Bothell, WA), the custom synthesis has the addition of 如cr卩, The intron-deleted IgG4 constant region sequence to the stop codon. Furthermore, by designing the 5th end of the heavy chain variation region, the end includes the sequence and inserts the IgK signal sequence. The primers were subjected to overlapping PCR to change the signal sequence in the pST0518 vector to the IgK leader sequence. [00331] The H9 and H14 heavy chain sequences have the same primer region, so both use the same primer and selection. The strategy of generating two independent PCR products, each of which contains one of the necessary changes in the heavy chain variation region of huNMC4-H9 (and huNMC4-H14). Using pIRESdsRed-HUL10 as a template and primer igKLF (8 £ (3!0:^0:1〇〇) and 1§1〇11111^ ( 8 £ (5 still: ^0:1〇1) (see Tables 15 & and 15b) 'Generate the PCR reaction containing the guano 1 and the gn restriction site on the pre-priming primer and amplify the IgK signal peptide, and then use pCDNA6_H9 (or pCDNA6-H14) as a template and a second reaction step of overlapping 14 VHF (SEQ ID NO: 102) and 14 VHR (SEQ ID NO: 103) and overlapping with the first PCR reaction by 30 nucleotides: PCR product The five amino acids were included in the H9 (or H14) variant region and the IgG1 constant region before the αΐ site; the five amino acids in the constant region did not differ between IgG1 and IgG4. The reaction was added to the valence portion 96 201041902 position after the alpha Ι site to aid in the selection of the variant region prior to insertion into the Ig 〇 4 constant region. The Kappa front derivation is added upstream by the third PCR step (using the reaction products of the first two steps as a template, the first reaction pre-initiator (IgKLF), and the second reaction anti-introduction (MVHR)); The product from this reaction is decomposed and inserted into the plastid backbone pCIneo which is decomposed in the same way. This linkage (iigati〇n) produces the selection intermediate pCI-NMC4-VH9var (or pCI-NMC4-VH14var). It contains an Ig leader and a variant region of NMC4-H9 (or H14). Agar and Notl were used to decompose plastids from the Blue Heron Biotechnology containing the regulated IgG4 constant region, and gel-purified (gei_purified) 1 kb IgG4 constant region fragment and ligated into (10) Ι/Μ? pCI-]SiMC4-VH9var (or pCI-NMC4-VH14var)) ' This resulted in pCI-NMC4-VH9 and pCI-NMC4-VH14. After transformation into DH5a cells, the plastid-embedded segments from individual colonies were sequenced to confirm that they were correct. Table 15a for the name of the primer in the heavy chain PCR reaction sequence IgKLF 5'CCTATCTCGAGAAGCTTCCACCATGGAGACAGACACACTCCT (SEQ Π) job 1〇〇) — IgKHnmcR 5'ACCCGGACCGGATTCCTGCAGCTGCACCTGTCCAGTGGAACCTGGAACCCAGAGC (SEQ ID NO: 101) 14VHF 5'CAGGTGCAGCTGCAGGAATCCGGTCCG (SEQ ID NO: 102) 14VHR S' CCTATGCGGCCGCGGGCCCTTGGTGGAAGCGGAGGAAACGGT (SEQ ED NO: 103) Table 15b PCR reaction for heavy chain construction PCR pre-initiator reverse primer template product 1st IgKLF IgKHnmcR pIRESdsRed-huLl 0 Xhol, HindTII, IgK signal peptide 2nd 14VHF 14VHR pCDNA6-huH9, ( orpCDNA6-lmH14) hu-H9 variant region, Apal, Notl (or hu-H14 variant region, Apal, Notl) 3rd IgKLF 14VHR The product of the above two PCR reactions Hu-VH9 (or VH14) variant region [00332] L9 and L10 light The changes in the chain are done by PCR. Since the L9 and L10 light chains have the same primer region, both use the same primer and colonization 97 201041902 strategy. Each light chain produces two separate PCR templates. The first PCR step comprises a Ζ/ζαΙ and a wc/III restriction at the 5th end; the second reaction step overlaps the first reaction step by 30 nucleotides, and comprises at the 3' end of the fragment and Λ/〇 The two separate overlapping PCR products are used as templates in the third PCR reaction to generate an inclusion by amplifying the primers from the first PCR step and the reverse primer from the second step. These changes eventually overlap the PCR product; the product from the third PCR reaction is decomposed with phobia 1/Yao and inserted into a similarly decomposed plastid pCI-neo (Invitrogen) to produce plastid pci-NMC4 -VL9 and pCI-NMC4-VL10 (exemplary primers and strategies for light chain construction are shown in Tables 16a and 16b) 0 Table 16a for primers in light chain PCR reactions mm: culvert: 'Rui 6 II - IgKLF 5'CCTATCTCGAGAAGCTTCCACCATGGAGACAGACACACTCCT (SEQ ID NO: 1〇〇) IgKLNMCR 5'GCTGCTGGGGCTCTGGGTCATCTCGATGTCTCCAGTGGAACCTGGAACCCAGAGC (SEQ ID NO: 104) 10VLF 5'GACATCCAGATGACCCAGAGCC (SEQ ID NO: 105) hKcR _____ S'CCTATGCGGCCGCGGATCCTATCAACACTCTCCCCTGTTGAAGCTCT (SEQ ID NO: 106) , i-ΐit X* - ...·ί ΐ * ' . j -·一炎16b PCR reaction for light chain construction Sassen 1st 2 pIRESdsRED-huL10 Xhol, Hindlll, Signal.peptide _ — 2nd 3 I 4 pIRESdsRED-huL9 or pIRESdsRED-huLlO huL9 or huLlO variable region and hlgK constant region, BamHl, Νοΐΐ ~~* 3rd _— 1 4 1st and 2nd PCR product NMC4-VL9 or NMC4-VL10 IgG4 sequence cassettes [00333] H9 The IgG4 subtypes of -L9 and H14-L10 were generated from the HEK293T cells described above in Example 1, and purified by protein a affinity chromatography; followed by testing the purified antibodies by the vWF-mediated platelet aggregation assay. To determine the relative effectiveness. As shown in Table 17, conversion to the IgG4 subtype had no effect on potency. 98 201041902 Comparison of platelet aggregation induced by Swissomycin against vWFIgG1 and IgG4 variants

H9-L9 H9-L9 IgGl IgG4

1.25 nM (1.3 nM, 1.2 nM) 13〇nM (1.3nM, 1.3nM) 1·40ηΜ (1.3ηΜ, 1.5nM)

IgGl H14-L10 H14-L10

IgG4 2.15 nM (2.3 nM, 2.0 nM) Example 4: Binding of antibodies to the VWF or A1 domain [00334] The His-tagged A1 domain antigen: not limited to the theory of the present invention NMC-4 binds to the A1 domain of v\VF, which is generally only achievable when vWF is activated (eg, under high shear conditions). Another method 'we may wish to demonstrate the separation of vWF (is〇iated) in the A1 field, which is not exposed to GPIb-a (Celikel et al, 1997) in the case where its efficacy is equal to the full activation of vWF. The A1 field serves as a matrix for the binding studies of microwej. The plastid lineage containing full-length human VWFC DNA was purchased from ATCC (catalog No. 67122), and the vWF A1 field was based on the primer VWF-Al-For (5'-CCCAGGAATTCCTCGGAACCGCGTTGCAC-3,) (SEQ ID NO.. 112) &;vWF-Al-Rev

(5'-CCGATGCGGCCGCTCACCTCTTGGGCCC CAG-3,) (SEQ ID NO: 113) was amplified from this selection. The PCR product was gel purified, recombined with oRI and 7VM, and cloned into the pETDuet-Ι vector. The ligated product is transformed into a DH5a competent cell to produce an oxidized form of the A1 domain. [00335] To construct a plastid that expresses the field of mouse A1' follows the rules of the product manufacturer (Molecular Research Center, Inc., Cat# DN127, Cincinnati,

Ohio ) ' Mouse chromosomal DNA was isolated from mouse liver using DNAzol reagent; then, using primer Rat-vWP-Al-F 99 201041902

(5 '-AGCGAATTCCCCCGAACCCCCCCTGCAC AACTTC-3,) (SEQ ID NO: 114) and Rat-vWF-Al-R

(5,-AGTGCGGCCGCTTATCACCTTTTGGGTCCTGGTGATGAAA CC-3 ' () (SEQ ID NO: 115) ' The chromosomal DNA was used for the PCR reaction. The PCR product was decomposed with EcoRI and Notl and cloned into the same portion of the pETDuct-1 vector; the ligated product was transformed into DH5oc competent cells. [00336] 25 ml of overnight bacterial culture (eg, 质 with plastid p35 [pET-Duet-Rat-Al] or p36 [pET-Duet-humaii-Al]

Origami B strain), culture 1 liter of bacterial culture medium (LB or 2 χΥΤ) containing antibiotics Carbencillin, Kangmycin, tetracycline, and grow the culture solution to 〇D600 in a shake flask at 37 °C. It is 0.6-0.8. The expression of the recombinant protein was induced by the addition of IPTG to the end of m mM of 1 mM; then, the culture broth was continued at 37 Torr for an additional 4-5 hours; then centrifuged at 6000 rpm in a JA-10 rotor (Beckman) 'Collect bacteria. The cell pellet (ceUpellet) was frozen or immediately treated by resuspending the pellet in 20 ml PBS containing two ingots dissolved in complete protease inhibitor (Roche). The resulting cell suspension was on ice. Two 2 minute cell disruption cycles were performed (eg, Branson sonifier 250 equipped with a micropipette tip (nucro-tip) using a fixed load cycle setting and output control setting of 1-2). Centrifuge these cell lysates in a JA〇〇〇20 rotor (Beckman) for 16 minutes at 4 °c; filter the supernatant with a 0.45 μηη needle filter, then use the knot a buffer (5 mM imidazole, 〇·3 M Naa, 5 〇 Tris_Hcl, ρΗ 8 平衡 balanced and filled with His-SelectHF nickel affinity sigma column to adjust the flow rate to The i ml/min injection pump is used for the chromatography. The tube is washed with 2 〇 blood combined with the buffer, and the protein is washed with 25 〇, a3 M Naa, 5 〇 f 8·〇, and Collect 1 ml of the liquid; the majority of the protein is washed out. The protein is monitored on the polyaciylamidegd (C〇_Ssie) blue dyed (4), (28kD) ) and elementality. The collection tip ♦ is divided into crane liquid, if it is reduced to 2.5 仏 then the profit column (A job 100 201041902 =, using Lloyd (LQwry) protein method (BioRadDC protein method) to determine protein concentration [00337] Combining kinetics · Performing sensitivity measurements to evaluate & scale and scale numerical analysis Purification of the hydrazine (N1 chelating agent) antibody conjugate. Decomposition-enhanced 镧Green-free fine-graining method (DELFIA) is used to make the conjugated body and the scorpion-resolved A1 anti-like combination; [00338] for example and δ' Controlled antibodies (eg subtype control group MOPC-21' from human bone IgG IgG1/K; Sigma_Ald touch, stLc^isM〇) ❹ ❹ ^NMC-4 chimera; in other words, add antibodies to sterile filtration Treatment of sodium phosphate buffer (96 mM, pH 7.4) and extensive dialysis into the buffer solution (pBS, Phosphate Buffered Saline; 1.47 mM KH2P04, 8.1 mM Na2HP04, pH 7:4, l38 mM NaCl and Z^mMKCl) 'To remove the low molecular weight primary amine. Concentrate the dialyzed antibody in a rinse-off MicroSeP concentrator at 9500 rpm (7000 x g) for 2 min to contain a final concentration of 1 mM NaHC〇3, pH 9.3 The PBS was adjusted to 4.0 mg/ml. The bicarbonate mixture (0.250 ml) was mixed to contain Eu3+ (Eu-Nl-ITC; Perkin) by gently pipetting up and down with a pipet. Elmer Life Sciences, Waltham MA) chelated tv1-(p-isothiocyanatobenzyl) _Diethylenetriamine W, iv3-tetraacetic acid (^-(p-isoMocyanatobenzyQ-diethylenetriamine-^^^^-tetraac eticacid) 0.2mg, a mixture of antibody and amine reactive chelating agent at 4 ° C, no The reaction was carried out overnight with stirring. [00339] The calibrated antibody mixture was applied to a separate NAP-10 column pre-equilibrated with PBS (Amersham Biosciences, Piscataway, NJ), and the fractions were collected using PBS as a column buffer; the reaction was quantified by using victor2 Perkin-Elmer time-resolved fluorescence (TRP), diluted 1:10,000 in DELFIA Enhancement Solution (Perkin-Elmer), sampled using a SpectraMax384 absorbance reaction disc reader Total protein (eg, Bradford reagent; Bio-Rad Laboratories, Inc., Hercules, CA) and sputum. Collect the pair of proteins and sputum 101 201041902 and apply it to the new NAP~10 Ai, pre-equilibrated with running buffer (Running PH 7·4 (4) 138 and NaC1), I column set for protein And supplemental calibration, both of which are positive reactions, applied to the solitary 10 column pre-equilibrated with the electrophoresis buffer, collected, and Γ 铕 calibrated both of the riding reactions, and by The total protein and sputum contained in the sputum were determined by carefully calibrating the TRF. The receiver calculates the F··protein ratio. ^0=] Α is isolated from humans (for example, 25 ng in PBS, 3 mM TriS, pH 7.4 ❹ i iLiLiC1 or PBS without divalent cations) or 4 〇C) The small-rolled (for example, 5〇ng/well) vWp twisted gossip field coated ^unulon 4 reaction plate, pool. Wash buffer (WashBuffer, such as 5 〇福聊, blocked with _^ blocking buffer (for example, IgG-free sputum containing 3.0 _ηΙ and 〇1% sodium azide) at room temperature Road pH% 150 mM Naa, a5% Tween_20) The reaction plate was washed three times for a small 'B' and was washed 5 times with wash buffer before use. P0341] An equilibrium binding assay was performed as follows. The 铕_antibody was pre-diluted into a combination buffer such as 3.0 mg/ml of BSA without igG and 0.1% copper azide in a wash buffer and applied to each of the 96 cells, and the reaction disk was sealed with a SEALPLATE membrane. The plate is oscillated (e.g., if the plate is at least 6 seconds or at room temperature for 6 seconds), the PlateShak sink rate is set at 4 for Ti, and placed in a Nalgene box containing a wet tissue at 37. (: Incubate for 2 hours in a closed box. For the measurement without calibration, transfer the supernatant sample (4.0 μΐ) from each pool containing the binding mixture to the DELFIA-enhancing solution (l〇〇pi/weii) In one of the parallel pool groups. To evaluate the bound antibody, wash the A1 coating bath with the remaining binding mixture five times with wash buffer, pat dry on a paper towel, and add DELFIA Enhancement Solution (1〇〇_wdl) Add the empty pool to measure the bound antibody. For the calibration, add DELFIA enhanced Lok (100 μΐ/well) to the unused pool and add the pin standard solution (1〇μΐ/wd丨). Disk (for example, setting the Titer Plate Shaker rate to 5 for 1 minute at room temperature), using the Victor^ multi-calibration disk reader (Perkin_EImerWaUac Boston, ΜΑ) to read the time-resolved fluorescence (TRF) intensity, by individual The fluorine of the antibody: '102 201041902 = =ρ) and the chelating content of the ruthenium is normalized in the case of binding. Calculate the specific knot ==R of one of the high-affinity = the average of the secrets by subtracting the non-specific binding (n fic b human instance ^ the average combination of the subtype control groups calibrated) by ^ knots such as Eu_NMC_4 and The free Eu-NMC-4 nucleus is calculated from the TRP readings measured in the bath liquid phase. Ο Ο Λ analysis reveals the ugly binding site: ^ is Q_37 high affinity and 2 r if affinity part; similarly, it is combined with the anti-his phase regular mosquito binding kinetics, except at different time points to refer to the i box calibration The antibody is used in place of the washing buffer. Li (for example, set the TiterP1 Shato rate at room temperature, the liquid wash contains five times of the combined mixture, and the money towel is patted dry: the second month-reaction tray cleaning time. As stated, DELFIA The enhancement solution (°L, μ=11) was added to the empty cell used for the measurement and binding, by using ^ (GraphPad Software Inc., San Dieg0, CA) into the fine position. (ι-β· fitted to linear Equation: k.....= + ^

...•,", corpse Wi ‘ J OJJ

[00345] wherein the binding rate constant is the fitted slope, [the concentration of the secret En-NMC-4, and the dissociation rate & is the fitted intercept. The dissociation half-life calculated by ',,, _46] to the NMC-4 chimera from human or Xiaona Na is calculated by the following equation: 103 201041902 M2) 1/2, tiwsoc to human or mouse vWF The combination of His_Ai can be formulated as follows: The formula for the concentration of each labeled antibody can be obtained as shown in the figure: the binding speeds of the two antigens are all in the same order. 'And reveals that the ^C-4 scorpion fits the human heart, has a 〇·32 〇. 〇7 sac heart value, and for the mouse ai field has t2 soil r Jf1, the & value 1 in two cases These results are determined by the combination of balance: These results also suggest that the antigen-antibody complexes of the two A1 species exist, and the in vitro dissociation half-lives of human and mouse antigens are 44 and 69 minutes, respectively.

Table 18: Eu-NMC complexes to the power and equilibrium of His_M from human and mouse vWF (at 37 ° C), dynamic constant ϊ | ί^Γι:ζ^,Τ·~Κλϊ^Ϊ)-7 Balanced combination His-Al homologous competition (JQ His-Al kon 0VI'1 min') b (My dissociation half-length (min) k〇n 丨k0ff {Kd) 4.1xl〇7 1.6xl0'2 44 0.39 nM 0.316 ±0.06811 }^ (n = l〇) 0.275 ±〇.〇64 nM :4)

1.4xl07 l.OxlO.2 69 0-70 nM 0.276 ±0.011 nM^ (n = 5) 0.297 ±0.128 nM

[00348] A competitive binding study can be performed to determine & values (e. g., measuring antigen versus affinity). In addition to the case in this case, a binding buffer of 80 μΐ/wdl (for example, a wash buffer containing 0.1% sodium azide and 1% sodium azide) having a binding force of 80 μΐ/wdl is applied as described above. Then apply the uncalibrated competitive antibody of 1〇Eu_N]y[C_4 or I## a (4) and 1G μΐ/weu of the repeated serial dilution series ranging from 1〇]§ to 10/Μ; ; = concentration is 1 GGPM. In the presence of the chelating agent DTPA (1 μΜ), the level of =, winter binding is greatly reduced, so it is also included in the competitive binding assay; this scene 104 201041902 by the use of acetaminophen (K> The d0 acetyl) gel was purified and purified by Hi coating to remove the equation from the protein preparation liquid by the method of "heart 7/2 < PA^7w. 22.3099). Im uncalibrated should be the homologous competition of c_4 chimera to obtain the death value of 2, and the binding of Eu Wei_4 to the antigen is measured by the standardization of the binding test. Compilation of human Hls_A1 competing with ^_ end 5 body = Eu, c-4 combined strong inhibitor, job / table 19), consistent with the observed & value 〇 32 ± 〇〇 7nM; ,on

The job-collecting body has the same value of Q.29mi28nM #^value^12', and 〇.276±〇.011 nM. In comparison, benefit

Cfi? (from the human bone marrow slurry) has no inhibitory effect on the Eu__C-4 δ to A1 antigen. The binding activity of the anthropomorphic KMc variant strain selected in Table 19 (the competition determination of the magic ratio than the car father 1

0.60 ± 0.13 ηΜ(η=3) 4 ιΊ _ sand 1 dispersion 0.28 ± 0.06 ηΜ (η=4) cannot be measured

IgG/κ control group Ig(}1

0.96 ± 0·27 ηΜ (n=3) 3.51 ± 1.21 ηΜ (η=4) 3_53ηΜ(η=1) [00350] The efficacy of the anthropomorphic nmc_4 mutant strains ^9L9 IgG1 and IgG4 was then tested using a competition binding assay. The CDR_transplanted H9L9 variant of this two subtypes exhibited the same ηΜ activity, although less potent than the homologous NMcy chimera (Table 19). 105 201041902 [00351] 卩 competitive binding assay to test ΑΓν_antibody ί祖1 competition of anthropomorphic _4 (four) strain Ha

Binding to H1S-A1 is enhanced by channel A 200, and its EC5〇 is 21〇 pM

The Hill diagram of Eu pendulum C-4 bound to His-Al shows close to 0.909 and 0.957), which is consistent with the binding to the single site under C00perativity. In contrast, in the 丨.8 _ or 2〇_ AJW2〇〇, = Ο not shown in Figures 2C and 2D), the Eu-NMC-4 combined with the His-Al Hill map shows a large & er slope ( Ww. Call this to indicate positive (pQS=e)U (coopemtivity). The positive synergy by AJW200 as a medium was observed in two independent experiments conducted in two different experiments, and these results not only confirmed the coupling to The independent binding site of AJW200 in the A1 field of vWF also indicates that ~ AJW200 enables NMC-4 to bind to the Jpib_a binding site in the case of the isolated A1 fragment. Example 5: Anti-Zhao blocking platelet adhesion ability [ 00352] The ability of the antibody to block the GPIb-a receptor binding site on vWF is demonstrated to its ability to counter vWF-GPIb interaction under primitive flow conditions. It has been developed by Moake and colleagues (1986, /C/k/iwe) A method developed by 78:1456-61) found the fact that when histamine is used to activate endothelial cells, it secretes extra large vWp 〇 (ULvWF) 'where the A1 domain is in an open (eg active) configuration Due to the division of the ADAMS-13 media of ULvWF, endothelial cells rapidly disintegrate under the introduction of public liquid (Dong et al. 2002 00:4033-9) [00353] In one example of the method, the first migration (Pi) HUVECs were separated and inoculated onto a 35 mm dish at a density of 5 cells/dish. Cultured for 7 days and used on day 7 (2 to 3 days after its 100% clustering). Human platelets labeled with CFSE were easily attached to HUVECs at a flow rate of 1.2 mL/min (Fig. 3A); When the cells were pretreated with 25 μL of histamine for 10 minutes at room temperature, more platelets adhered to HUVECs (Fig. 3B); platelets were perfused in a buffer containing NMC-4 at a concentration of 10 pg/ml. In the case of a single layer, the adhesion of such platelets is completely inhibited (Fig. 3C); and when the platelets are perfused in the presence of an anti-GPIb-α antibody (for example, a dish) at a concentration of ig 106 201041902, it is also a small plate to a partial resistance. In contrast, the rodent control IgG of IS _nl did not prevent platelets from attaching to the vWF polymer (Fig. 3E), suggesting that platelet attachment f HUVECs is actually derived from epithelial-derived vWp and platelet Gpib_a. The inter-female interaction is used as a medium. When I am covered by 2 blood secrets taken from each round _And_ CGmpix has a negligible effect caused by group antibodies when sputum is added, _〇_4 reduces platelet adhesion to 卜. 0 Example 6: Anti-caries ability to prevent vascular occlusion [t〇0354], use A ferric chloride model of arterial thrombosis to assess the antithrombotic activity of chimeric and anthropomorphic derivatives (eg, H14, L10) compared to AJW200. The contralateral carotid artery is re-positioned through the salivary gland and accompanying adipose tissue to the cranial side of the incision. The carotid artery is exposed and placed into a stent that can be used as a carotid artery and provides ferric chloride (7.5%). A piece of filter paper on the surface of the solution (eg 4 mmx5 imn). Four minutes after the application of the FeCls solution, the flow probe was placed near the carotid artery and the flow was measured using the iransonicSystemSInc flow system (ithaca, NY) until the time of occlusion (typically 1 minute in the control group) Or 45 minutes. At a volume of 1 μΐ/g mouse body weight, each group of 4 mice (n=6 of physiological saline) was administered a NMC-4 chimera ranging from 5 to 〇·〇ΐ mg/kg, for example. .4, V14, L10, AJW200 or control group IgG. Sterile filter antibody preparation, 'and tested with LMULUS AMOEBOCYTE ASSAY (BioWMttaker) according to the manufacturer's rules to ensure low endotoxin content; and estimated monodispersity by HPLC analysis prior to use as animal studies . =0355] As shown in Fig. 4, both NMC-4 and AJW200 significantly suppressed blood clot obstruction. NMC-4 showed an ED% similar to AJW200 at doses of 0·03 and 〇! mg/kg, and the anthropomorphic derivative m4, L1〇 also appeared at doses between 〇〇3 and 〇1 mg/kg. Similar to Ed5〇. Example 7. Effect of antibody on bleeding time and blood loss 107 201041902 The harmful side effects, I (for anti-vWF antibody) are related to the syndrome of the disease. In the saline solution, t) and put the tail into the calculation of the blood time process towel, and the time required by the & blood (10); To this end, the red blood cells were pelleted by low-speed centrifugation, and the final volume of mr^7f was adjusted in the physiological saline solution of 5 mi (10) to measure the heme concentration of the solution by the absorbance of t疋 at 420 nm. The MC-4 conjugate showed the same milk dose 50 mg _mg/kg as the anthropomorphic derivative; the dose associated with the efficacy of the two antibodies in the FeCl3 model. There is no extension or loss of blood to increase the number of mosquitoes. The NMC_4|synthesis and its anthropomorphic derivative, strain, were slightly higher than AJW200 (which was closer to its anti-i-plug activity when the bleeding time was increased in 2ED5〇 in mice). Dose response (five) e response ) This suggests that NMC_4 provides a more improved therapeutic window than AJW2 (8). ' Table 20 Effect of NMC-4 chimera and its anthropomorphic derivative on bleeding time and blood loss in mice compared with AJW2〇〇

Physiological saline 3.1 ± 0.3 (16) 0.287 ± 0.088 (9) NMC-4 (chimera) = 0-09 mg/kg 0.01 mg/kg 2.7 ± 0.3 (2) 0.059 ± 0.009 (2) 0.03 mg/kg 4.1 ± 0.2(4) 0.07610.014 (4) 0.10 mg/kg 19.7 ±0.9 (2) 1.503 ±0.485 (2) 0.30 mg/kg 32.7 ±4.4 (3) 1.501 ±0.213 (3) 3.00 mg/kg 32.3 ± 2.3 (2) 1.106 ± 0.243 (4) H14, L10 (personalization) ED50 - 0,09 mg/kg 0.03 mg/kg 2.03 ±0.35(3) 0.094 ±0_〇35 (3) 0.10 rag/kg 15.30 ± 1.70 (4) 0-630 ± 0.294 (4) 0.30 mg/kg 26.45 ±3.09 (4) 1.883 ±0,312 (4) 108 201041902 AJW200 0.01 mg/kg 0.03 mg/kg 0.10 mg/kg 0-30 mg/kg 3.00mg /kg EDso = 0.05 mg/kg 2.8 ± 0.25 (2) 8.2 ± 1.56 (7) 25.1 ± 0.4 (5) 29.2 ± 0.72 (5) 30.9 ± 0.62 (3) 0.177 ± 0.059 (2) 0.250 ± 0.059 (4) 1.943 ± 0.420 (7) 2.074 ±0.521 (3) 2.912 ±0.243 (4) Ϊ I Harmful side effects on hemostasis - the parameter is blood loss. The second line is used to measure the blood collected during the bleeding time. In addition, these antibodies induced significant blood loss when they were at Gl mg/kg. However, at 0.3 and 3 mg/kg in d, the AJW2 〇〇 initiation was higher than the chimera at the same dose. The amount of blood loss, although these differences were only treated in the 3 G mg/kg group (where ^4 instead of n=3) were statistically significant (Table 2〇). The H14, L1〇 antibody variant did not show significant differences in the parental NMC-4 chimera. Example 8: Effect of antibodies on the number of platelets and white blood cell cycles [00359] It is hypothesized that certain antiplatelet agents may inhibit thrombosis, while at the same time may cause hypoxemia (thrQmb (Kytopenia) (H-face y β

Pharmacol Exp Ther. 298:165-71 (2001)) 〇NMC-4 ❹ The effect of white blood cells (WBC) and the number of platelet cycles in animals is injected with j tons (4) of NMC-4 (eg by intravenous means) ) to a control group of 3 mice weighing 23 〇 "26 〇 8 of 5 mice" and drug controlled (vehiclec〇ntr〇1) (eg, surgical grade) to 3 mice. For example, the number of blood cells in the baseline is measured by a hemorrhagic tail using HEMATOIOCT A>iALYZKlTM (Dl:ew Sdentific). The antibody or physiological saline is injected from the unanesthetized mouse by extraction from the eye of the fine straw. After water, blood samples are collected at predetermined time points up to 48 hours (eg, 3 minutes, 2, 4, 24, and 48 hours), and then about 4 〇rL of blood is converted to contain 5 acidified citrate glucose In the test tube of blood clot (d citrate dextrose anti-coagulant solution, ACD), a sample was taken from the ''Like/mMAVET cell counter to determine the number of platelets and white blood cells J. P. For each blood draw' The eyes are alternately used for sampling. 109 201041902 Very slight. At any time point of the main analysis, the effect of 4 on the number of platelets was shot at a minute. 'A brief decrease in white blood cells was observed by 37.5%. A similar reduction was observed when the human PBS drug was injected. In both the drug-treated group, the white blood cell levels were 2_4 after injection. Example 9: Establishment of a cell line for antibody expression developed a high-yield mammalian protein expression system based on the murine artificial chromosome expression (ace) platform. The ACE platform has been designed to contain site-specific λ•integrase (eg, ACE integrase) binding to a target shuttle vector and to be loaded with a heterologous gene sequence. Recombinant receptor sites (rec〇mbinati〇n accept〇r sites) (β竑(施32(21):el72 (2004); US Patent Application Nos. 2003/0119104 A1 and 2006/0246586 A1). Using this system, a stable cell line for expressing the anthropomorphic variants and chimeras screened is generated. [00362] Decomposition of plastid pCI by addition of deterrent (light chain vector) or melon and 5amHI (heavy chain vector) -NMC4- The VL10 and pCI-NMC4-VH14 were succumbed into the MCS 1 (light chain) and MCS2 (heavy chain) of the PST0518 vector. The pST0518 vector with the heavy and light chains in tandem is used as a shuttle vector for transport to ACE target vectors (ATVs) with different resistance genes derived from Lindenbaum et al. The stated target vector (Cudi/ and .32 (21): el72 (2004); US Patent Application Nos: 2003/0119104A1 & 2006/0246586 A1). In order to transport cassettes containing both heavy and light chain antibodies into ATVs, pST0518-VH14 was decomposed with I-Ceul and homing endonuclease (New England Biolabs MA). VL10 vector; gel purified VH14 plus VL1 (/ fragment, and selected into the same part of pZeo and pHygro-ATV pre-decomposed by the same I-Ceul and H-Scel endonuclease. This produces plastid pNHT605-H14L10-IgG4 (hygR gene) and pNHT607-m4L10JgG4 (p zeoR gene). 110 201041902 [00363] Similarly, 'constructing PST0518 target vector with NMC-4 IgG4 chimera' and inserting it into the sub-column Into the pZeo and pHygro-ATV vectors, 'the plastid PNHT623 (human IgG4 chimera plus hygR gene) and PNHT624 (human IgG4 chimera plus ze〇R gene) were generated. Ο [00364] Targeting integration to platform ACE, to 6 The pool was cultured at a density of χΐ〇4χΐ〇5 cells per cell and inoculated with the host ChK2 ACE platform cells and cultured overnight. The medium was changed in serum-free medium 3 hours before transfection; after 3 hours, the carrier was 1 μ§ 1 μ§ ^ according to the manufacturer's instructions The integrase expression vector complexed with the reagent (Invitrogen) was transfected. After 24 hours, the cells were expanded to a 15 cm dish; the next day, 3.0 pg/mi of zeomycin or hygromycin (hygr〇mydn) After adding to the culture medium, after 4 days of screening, the resistant colonies were isolated by the selection loop, and the individual selection strains were amplified for analysis of antibody production. Example 10: In vivo efficacy and safety of NMC-4 anti-caries [00365] The in vivo efficacy and safety of the anthropomorphic NMC-4 antibody is tested in an in vivo animal (eg, sputum) model. [00366] In the exemplified method, ketamine h^kodikmde ) (purchased from Premier Pharmaceuticals Co., Ltd., such as Caze Qiao Anesthesia), and heated, and, from & all in the stock secret and femoral in the femoral artery and femoral vein Cut a small incision and insert the blood vessel: ίΐίΓ, attached to the tip of the blood vessel to turn the arterial blood to ^ Γ Π t αΠ Γ Will be a tube-type super-wave flow probe (T_nic

Systems Inc, Maastricht Htwu,. The eight shovel of the money. 敕 fine L Netherlands)) attached to the sputum tube, allowing blood flow to stabilize for about 20 knives, continuous volume throughout the test, ^ ^ ^ liquid flow, the shunt for drug delivery and Take f for blood. Thousands of average and position (10) fiber) [00367] Its - person, using Martin needle sweet (five) brand also coffee 14cm, product code 20.634.14), + 旖 by the large depression to repress the endothelium continued 111 201041902 cutting edge fine The inner layer of the vein. Manufactured a two-flow two-week plastic constrictor placed above the wound site to gradually reduce the blood flow; we observed that the blood was taken out and the blood-carrying field was reduced to the claws. $511117111111, open the constrictor to form a plate t-test; secondly, once again narrow the outside and restart the blood Λ彳 ° this remnant of the new _ blood (four) 1 amount of slave-repeating bear CFRs holding volume reduction; _ New food and post-monitoring, ''' only another twenty minutes. The & 眶 变异-4 mutant H9L9 _ as in Example 3 was used. Anthropomorphism of the State, 3 A. The two methods were used to evaluate the bleeding status at the time of administration. The epidermal template bleeding time is determined at the point where the first method force is tapped. Apply pressure to the wound nearby. The epidermal bleeding time is defined as the induction and visual phase of the wound. Carefully wipe the blood with filter and paper every 15 seconds, and stop the measurement when the bleeding time exceeds _ seconds (for example, 15 minutes), and treat the bleeding time as 9 seconds. Li-Fang Caiyun was created by Wei Annexinv and rabbit carotid artery to estimate the blood loss of the incision (see for example RThiagarajanetal. (l997) CTO^〇7i %(7): 2339-47). Cut 2 峨 (10) cm cut in the groin and weigh the gauze to clean the cotton block. At the end of the 30-minute dose injection cycle, it is filled with sputum and replace the gauze sterilized cotton block; at the end of the study, all the gauze Scale ^ get Ϊ] blood loss. The ratio of gauze in the physiological saline control stage is expressed as Γΐ = Ϊ. The heartbeat was continuously monitored at intervals of 1Q minutes throughout the study period: pressure. [00370] At the end of each dose period, 1 ml of EDTA blood and 10 ml of citric acid-containing blood were drawn and the FBC platelet count, prothrombin time, and activated coagulating time were determined (4) Thromboplastin time), eighth factor and vWF. If necessary, a small dose of 3 〇Ομ1 under _8 冷 , , , , , 运送 运送 运送 运送 运送 运送 运送 额外 112 112 112 112 112 112 112 112 112 , , , , , , , , , , , , , , , , , , , , , , i, 2, 8, 24 and 48 hours = test _ Lai Shu, to 浦 浦 浦 浦 浦 血小板 血小板 血小板 371 371 371 371 371 371 371 371 371 371 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在 在Intramed) lasts 20 points ', but = adrenaline does not trigger platelet clotting in the dilute sputum W plate agglutination factor and restores the destroyed circulating stream 26:1^ G Anf〇SS1" (1996) E- J Clln ^ Ο Ο [00372] Efficacy and safety of GBR 600 · Perform 4 to determine the efficacy and safety of GBR 6GG. 1 ^ : Conduct a pilot study with (4) animals to establish an increasing amount of c-mass response (d 〇Seresp〇nse) curve, and identify the maximum 4 objects observed to take out the blood sample to establish the highest dose of anti-m interval 30 minutes to inject the following incremental doses, and in the study: Jt, 〇, 3mg/kg; #]. 2, 〇^ ^ every 1^5 shot = 41 (; ; and dose 5, (10) mg / kg. After the main shot, ι〇 is a bell test. [This phenomenon is caused by the arterial damage again == ftIir\6 (L〇〇〇mg/kg) ^ ^2·2^η4ίΐ; on the kidney It 3 reached the end, the plate agglutination is strong or weak. Because of the effect of epinephrine on blood pressure, the injection into the kidney is increased by j, but it does not reverse the inhibition of CFRs. 201041902 Table 21 GBR 600_^ Effect on CFRS [〇π _ Ν _SU (mg/kg) Cumulative dose (mg/kg) CFRs Baseline----"· 0 -- enchanting --- 8 saline 0 ~ ~~ ----—, 8 0.03 ---- 0.03 ' -----a 5 0.1 ------ 0.13 ' —-----_ 0 0.3 — 0.43 " _ --- 0 1 ------ 10 ^-------- 1.43 -------- 11.43 ----— 0 __ ~0 ^~~ Ο 〇 In research (10) to check the right:: := mu In Study 2 (see eg Figure 6 and Table 22), the effect of 0·01 m§/kg BR 600 on CFRs was observed (7 CFRs/3〇 minutes compared to 9 CFRs/30 of Physiology 7) Minutes; however, 'injecting extra 〇〇, = ΓΓ into the end of CFRs _, GBR _ ^ = (10) pulse re-injury does not reverse (3) inhibition, indicating that this is straight two suppression . The inhibitory effect was maintained at a higher dose, up to the maximum dose of lGm: , the effect of adrenaline on blood pressure, and the injection of erythropoietin resulted in a short increase in the flow of money, but did not reverse the inhibition of CFRs. Table 22 Effect on CFRs (0.01-10 mg/kg) Dose (mg/ks) dose (mg/^ Baseline saline _ 0.01 0.03 0.1 0.3 Cumulative dose (mg/kg) 0_ 0_ 0.01 0.04 Number of 0.14 0.44 CFRs 9_ 9_ _7_ 0 114 201041902

L〇〇378] Research

给予 Give the starting dose of _ _ knowing the hand, study 1 way, except (accumulated dose = 0.01 mg / kg), day #接# with another dose of 0.005 mg / kg 6 times. Gg) and the county called 4_mg/kg increase [00379] In study 3 (see example _ ^ 600 where the effect U ϊΐϊ (four) _ let the head 3 ==== tsr. 81 miscellaneous by the next table , the number of CFRs in the equation is good / dose cycle = _1 〇 9 χ GBR6 剂量 dose + 7 [00380] compared to the complete system caused by cumulative dose 〇 4 mg / kg in Study 2, study The ED (10) in 3 is 〇.〇7mg/kg. In Study 3, the time between the incremental doses was 30 minutes, and the observed difference in ED exposure may be caused by the blood removal due to the initial elimination of the drug. Initiation of GBR 600 decreased. Infusion of adrenaline reversed the inhibition of CFRs, which may be related to the shape of the CFR curve at the cumulative dose of 〇_〇7 mg/kg. At the cumulative dose of 0.07ιη§/ι^, inhibition The shape was slowly reversed, indicating that the thrombus is developing. Under these special conditions, adrenaline appears to be able to reverse CFR. Table 23 Effect of cumulative dose of GBR 600 on CFRs (〇〇〇5_〇〇7 mg/ Kg) _ Dose (mg/kg) Refining dose (mg/kg) Number of CFRs Baseline 0 8 Physiological saline 0 8 0.005 0.005 7 0.005 |0.01 115 201041902 0.01 0.02 0.01 0.03 ~ 0.01 0.04 0.01 0.05 0.01 0.06 0.01 0.07 — 5 2 Study 4 was carried out in a manner similar to Study 1, except that in a sputum, clopidogrel was used. The purpose is to compare GBR600 in i! 5 anti-J „) f and anti-protective effect and blood culvert, 2, 5, 5 and 1 amount [00382] in study 4, Baoshuotong to Buddha Buddha I II = 3: in *5_cumulative _ = =====24 in the peach 13 鄕 fresh. Inject xianxian 0 Ο ❹

Effect J is dragged 1 county ▲, one I dose (mg/kg) baseline physiological saline 1 1.5 2.5 5 10 cumulative dose (mg/kg) 0 0 2.5 10 20 number of CFRs 狒狒 1 13 13 Number of CFRs狒狒2 8 7 8 5 8 2 Number of CFRs 狒狒3 6 8 2 0 7 2 0 0 0 0 0 0 [00^83] "Template bleeding time: in studies i and 2, all above 〇〇4 mg/ The template bleeding time at kg I was longer than 15 minutes; in the positive control group study containing Bristol Myers Squibb/Sanofi Pharmaceuticals, the template bleeding time was extended to greater than 2.5 mg/kg cumulative dose 116 201041902 The same degree; in Study 3, the template from the template template bleeding time showed a high base linear two, longer than 15 minutes. The baseline value), so its scale is extremely fresh two (hat 狒 2, 3 _ 嶋 嶋 = = time itself (see, for example, Lmd❿/. He is, for example, in the adjustment of the surgical procedure, 疋1 has a blood loss through the incision and is more variability, because the blood test is performed in addition to the blood test, in addition to the template in the table 25 and 26

Table 26: The blood loss time of the template of the stagnation of the sputum [minutes] ο

Mm. (mg^cg) έ^μ^ΤΤ~7~7]~~^ 〇 117 201041902

Ο

2^84] Tables 27 and 28 show the self-limiting nature when the sputum and the GBR 600 are cut. In all studies, the highest blood loss observed in the four doses of the observed condition was reduced by the amount of blood absorbed by the gauze, and it appeared that the healing of the mouth was the same. In studies 1 and 2, the maximum amount of bleeding was similar to that of the slinger. Although the control system was 2-4 times ED (10) and the GBR 600 system was measured at up to 250 times, in Study 3, it was observed that the bleeding at all injection doses was negligible. Table 27 Incision Blood Loss Test [Multiple units of physiological saline value] Dose (mg/kg) Cumulative dose (mg/kel Study 1 Study 2 Study 3 Baseline 0 nanana Physiological saline 0 1 1 1 0.005 0.005 nana 0.13 0.005 z 0,01 nana 0.08 0.01 0.02 nana 0.05 0.01 0.03 nana· 0.05 0.01 0.04 nana 0.02 0.01 0.05 nana 0.03 0.01 0.06 nanand 0.01 0.07 nanand Π8 201041902 Ο

Dosage (mg/kg) cumulative dose (mg/kd 狒狒1 狒狒2 狒狒3 baseline 0 --------------- n. an,ana physiological saline 0 — 1 1 — 1 1 1 1.59 1.21 1.28 1.5 2.5 1.06 1 1.1 2.5 5 1.41 6.64 3.32 5 10 5.82 13.64 0.95 10 20 9.12 2.64 0.92 0.01 _〇.〇1 na 2.5 na 0.03 , 0.03/0.04 0.125 0.5 na 0.1 0.13/0.14 0.625 4.75 η. a. 0.3 ^.43/0.44 3.125 7.75 na 1 0.143/1.44 7.625 5.75 na 10 _11.43/11.44 4 1.75 11-3,.

[00385] _ treatment window (the therapeutic window) and bleeding points (bleedscore) · In Figure ι〇, the results of the incision test from the study i and 2 and the three shackles study on GBR_ and silk pass The dose is expressed as a multiple of its ED100s and plotted on a logarithmic scale. [=0386] Even at doses greater than 1 〇〇 of its ED (10), GBR 6 〇〇 still has a degree of bleeding that is visible in the claims to only 4 times its ED100. In the field, GBR600 _ bleeding risk is a treatment with unprecedented safety ^ D ° The only clinically relevant increase observed in this study is the increase in self-limiting bleeding from sputum, as by Template bleeding and incision bleeding ί, ΐ 密切 After close observation of the animal for 48 hours after surgery, no frontal or cutaneous bleeding signals were detected, such as bleeding, bleeding, ecchymoses, etc. Detection of bleeding in the coffee department, such as hematoma, nosebleed 119 201041902

Cepistaxis), blood loss from the mouth or vagina, melena, hema'toa, or hematemesis. These surgical wounds do not bleed and heal normally. ^88] As shown in Table 29, both Bosson and GBR 600 scored 1 in the BleedSc〇re 7-knife program. In addition to the increase in bleeding on the epidermal wound or the observation period of the study, 48 hours of the observation period were detected. I persimmon _ on the table [^__g pass and GBR 600^ BleedScore judge BleedScore aged if $ Ο Ο jk serious epidermal bleeding internal bleeding symptom scores check GBR 600 easy to eat blood from small wound bleeding bleeding point 1 0 0 0 Hematoma nosebleeds from the mouth or vagina, blood loss, black stool, eye bleeding, hematuria, vomiting blood 0 0 need blood transfusion, internal bleeding, life risk 0 0 0 warning bleeding BleedScore

Findings of efficacy and safety studies: Table purchases show the effect of GBR 600 in study i_3 [00389] 120 201041902 on vWp levels, eighth factor levels, white blood cell count, heme concentration (Hb), and platelet count (Plt), prothrombin time (PT), activated procoagulant time (aPTT). [00390] Regarding the Von Weir range obtained in Studies 1-3, no pattern was observed in the study}; however, a decrease in the code Weiss level was clearly observed in Studies 2 & 3. In Study 2 using higher doses, the effect was more pronounced than the use of relatively low doses of Study IX 3; in the study of anthropomorphic IgG4 antibodies using a control group not bound to vWF, no observations were made for the Von Weir range. Effects, such effects and implications should be carefully monitored in future studies. In all studies, GBR shame had no significant effect on the eighth factor.

〇[00391] f observed an increase in WBC, but this is a known effect of the invasive procedure, and up to now, this result is related to the anthropomorphic individual IgG4 antibody against the control group that does not bind vWF, and in this model The results observed for all other drugs tested were well correlated; no significant effect on the concentration of erythropoietin due to injection of GBR6〇〇 was observed; however, injection of GBR6〇〇 was effective for platelet count. Since platelet deposition is the cause of CFR inter-arterial obstruction, platelets are consumed in this sequence, gj, and effective inhibition of CFRs will reduce the consumption of platelets. This explains the large platelet consumption observed in the anthropomorphic individual IgG4 antibody that was not bound to the control group, and no inhibition of CFRg was observed. [〇°392^ GBR showed no significant effect on PT and aFrr indicating the integrity of the coagulation protein, and similar results were observed in the anthropotactic single lgG4 antibody in the control group not bound to vWF. 121 201041902 Table 30 Study 1 镰 Physiological woven water 0.03mg/kg 0.1mg/kg 0.3mg/kg 1.0mg/kg 10mg/kg WBCx 10S/1 7,48 6,92 7,89 9,32 11,57 12, 29 11,52 RBCx 1012/1 5,7 5,88 5,75 5,86 5,B3 5,79 5,8 Heme g/di 13,4 14,3 14,1 14,1 14,4 14 14 blood volume ratio ι/ι 40,5 43,5 42,5 43,5 43,4 0,4 41,4 MCVfl 71,1 74,0 73,9 74,2 74,4 74,6 71,4 MCHpg 23,5 24,3 24,5 24,1 24,7 24,2 24,1 MCMCg/dl 33,1 32,9 33,2 32,4 33,2 32,4 33,8 pit x 109/ 1 306 249 234 236 245 257 2S2 neutx 109/1 3,46 3,32 4,35 5,92 8,16 8,89 8,44 lymph x 109/1 3,62 3,21 3,04 2,80 2,75 2,88 2,32 Mononuclear leukocytes X 1〇e/i 0,36 0,36 0,46 0,58 0,61 0,50 0,73 Acidophilus X 109/丨0,03 0 02 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10,00 10 10 10 aPTT Set ^2. 44 43 夂A2. 45 FVIII 107 89 S3 88 03 78 78 vWF concentration 25 46 3Θ 15 26 48 17 chick % ^11 ί*- I ^nf. tear 1 m jhrr I Λτττ m ι m ^llf I ffll Γ. m 1 m no ❹ Table 31 Study 2 woven physiological hungry water 0.01mg/kg 0.03mg/kg 0.1mg/kQ 0.3mg/Kg 1.0mg/kg 1〇mg/kg WBCx 109/1 11,98 12,-G 12,49 11,89 11,14 10,91 11,D1 13,92 RBC x 1012/1 5,24 5,35 5 ,34 5,34 5,42 5,57 5,57 5,54 ]ME prime g«i 12,2 1^7 1^8 12,8 1^9 13,2 13,4 13.2 Blood volume ratio Ifl 34 ,1 34,9 34,8 34,7 35,1 36,1 36,1 36,1 MCVfl 65,1 65,1 65,2 65,0 54,8 64,8 64,8 65,2 MCHpg 23 ,3 23,7 24,0 24,0 23,8 23,7 24,1 23,8 MCMC g/d) 35,8 3^4 36β 36,9 36^8 36,6 37,1 36,6 Pit x 109/1 313 281 276 2B1 257 283 2B3 into yet x 109/1 9,37 9,44 9,12 8,60 8,03 7,96 8,15 11,33 lymph x 109/1 2,20 2,55 2,84 2,82 2,68 2,51 2,46 2,10 White blood cells X 103/1 0,38 0,40 0,47 0,42. 0,37 0,^ 0,32 0 ,38 13⁄43⁄43⁄4球 X 109/1 0,01 0,02 0,05 0,04 0,04 0,05 0,06 0,10 basophilic ball X 109/1 0,01 0,01 0,01 0 02 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 10 mm% No no no no no no no

UZ 201041902

Ο

=0393] GBR 600 seems to be a potent platelet deposition inhibitor during arterial thrombosis. This inhibition is also the case. Use · Harmful lx is now a serious harmful bleeding, even when the drug appears to be infused as it currently appears. At this (four) amount, the amount of bleeding by the incision r is generated and infused with an effective dose of 4-8 times.

Coagulation protein is not influential, this is PT-shaped, but there is no clear effect on the level of i-reducing factor in i. Because of the killing of the rat spirit, the peak of the K-phase money and the blood of the egg are from the circulation level. The effect of the blood count on the king's blood has not observed an unexpected effect. The thermal stability of the humanized nmc_4 mutant == the special heart =) = the body of the dazzle A \ Fab H 5 卩 in the context of the full length 1gG, can still easily extract the secret temperature Easily identified = monitored ^ 彻 彻 (4) 中 融 融 融 融 2010 2010 2010 2010 (differs scanning 123 201041902 microcalorimeter, MicroCal, Northampton, UK) on the heat: The cell volume was 0.128 ml; the heating rate was rc/min; and the excess pressure was maintained at 64 psi; all protein fragments were at a concentration of 1-0.5 mg/mL (74 μΜ) in the twist 7.4) use. Each part of the protein was estimated to have a molar capacity by comparison with a replicate sample containing the same solution (protein removed therefrom); part of the molar heat capacity and melting curve were analyzed using standard procedures. Prior to further analysis of the thermograms using the non-two state model (N〇n_Tw〇State model) in the software 〇riginv7.0, the baseline was corrected and the concentration normalized: for H14L10-IgG4 as mentioned in Example 3. An example of the obtained data 1 is shown in FIG. The Fab fragment of the murine NMC-4 showed a single transformation at 74.7X:, and the fragmentation of the fragment of the 10C was found at 81·Γ(:, which corresponds to a significant difference in stability). C). In order to detect the shadow of the human Fab stable area ^ prepared by transplantation to human IgGl (the most stable human subtype; Garberand

Demarest (2007), BBRC 355:751-7) The chimera composed of the mouse-busy-4 variant field. In the case of H14-L10Fab, the apparent Fab Tm values of the H14L10-IgG4 and NMC-4-IgGl chimeras still showed a significant increase in stability (ΔΤηι>1〇〇'' 曰 Example 12: The gene encoding the gene encoding GBR600 heavy chain (VH9) and light chain (VL9) [00396] The materials and methods for quarantining the gene encoding GBR 600 are listed below:

PfuUltra (Stratagene, Cat.-No.: 600380)

Spel (NEB, Cat-No.: R0133)

Hindm (NEB, Cat.-No.: R0104) CIP (NEB, Cat.-N〇.:M0290) pCR-blunt end (Invitrogen, Cat.-No.: 44-0302) Introduction ··Operon, Cologne, Germany

GLNPR107: TAACTAGTCGTGAGGCTCCGGTGCCCGTC GLNPR108: 124 201041902 AAGCTTACGGCTAGCTCACGACACCTGAAATGGAAG GLNPR139: CCTCAGACAGTGGTTCAAAG GLNPR176: GCTAGCGCCACCATGGAGACAGACACAC GLNPR177: TAAGCTTCTATCATTTACCCAGAGACAGGG GLNPR178: TAAGCTTCTATCAACACTCTCCCCTGTTG BGHREV: provided TMC vector pCI-NMC4-VL9 (pl56) of Fasteris and pCI-NMC4-VH9 (pl58) (supplied by Chromos )

Qiaquick Gel extraction kit (Qiagen, Cat-No.: 28706) lkb+ ladder (Fermentas, Cat.-N〇.:R0491) O pcDNA3.1 (I) (Invitrogen, Cat.-No: V795-20) pEF-Dest51[ CD19] (RZPD, Cat.-No.: RZPD〇839GO 167-pEF-DEST51) Sequencing · Fasteris SA (Geneva, Switzerland)

Gigaprep kit (Macherey-Nagel, Cat.-No.: Nucleobond PC10000) [00397] The expression vector pEFcDNA of the expression vector pEFcDNA3.1 was replaced by pcDNA3.1 by EF1-α promoter from pEF-DEST51. -) (Invitrogen) CMV promoter was constructed. For this purpose, the EFl-α promoter was amplified using the primers GLNPR107,108 and PMJltra (Stratagene, 30 cycles of decantering temperature 55 °C '; the primers amplified all of the EFl-α promoter and amplified the fragment The 5' end is attached to the el side and the 3' end is attached to the m/ΙΠ side. The PCR amplicon was cloned into pCR-pure end (Invitrogen) and the clones were analyzed by net decomposition. The Spel/calendar fragment from the selected strain #4 was excised and cloned into the PCDNA3.1(-) skeleton which was decomposed using the same enzyme combination and CIPed. Using the eI and imitation, ship analysis to select the lean strain, the selected strain #2 seems to be positive; the second decomposition of the skeleton and the strict entry section confirmed the correct size of the promoter fragment. One

[00398] GBR 600 was cloned into pEF cDNA using a PMJltra (standard conditions, annealing temperature 55. 〇, 3 循环 cycles) and primers GLNPR 176 and 177 to amplify GBR 600 VH9, template is TMC vector pl56; 125 201041902 The chain described above used the primers GLNPR 176 and 178 to amplify the gbr 600 VL9. The template used was the vector pl58. The primers were attached to the restriction portion 5 and the restriction portion 3' to the individual enhancers, and the obtained PCR fragment was cloned into the pCR-blunt end, and analyzed by applying d and by limiting decomposition. The light chain of the selected strain #1 and the heavy chain of the selected strain #3 were excised and cloned into the pEF cDNA using the enzyme Mel and the ηί/ϋΐ and ClPed. This restriction decomposition revealed that the light chain selection strain #6 and the heavy chain selection strain #1 contained fragments of the correct size, and the two selection strains were sent to the sequencing control group iFasteris as samples GS256 and GS257. ° Align the sequenced columns with the reference sequence. Due to the poor quality of a small amount of prepared DNA, the heavy chain sequence GS257 could not be confirmed to about 1%. G G GBR 600 heavy chain VH9 ( GS257 ) and GBR 600 light chain VL9 ( GS256 ) to prepare a multi-quantity # (Giga two) S). It is difficult to re-stay the pool to complete the sequence, 'this sample name is GS265 (GBR 600 heavy chain VH9) and GSS264 (GBR600 heavy chain VL9). Due to its better DNA quality, the sequence identity of the heavy and light chains for the reference sequence can be cut. The present invention has been described herein with reference to various specific materials, procedures, and embodiments. , additions, changes, and their equivalents. Therefore, the present invention should be considered as illustrative and not restrictive, and the spirit of the invention is represented by the following patent claims. The materials, patents, and patent applications referred to in this application are hereby incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS [0036] Figure 1 shows the inhibitory activity of NMC-4 & antibody in a REST〇cetin-induced vWF-mediated platelet agglutination assay, Compared with the original one monoclonal antibody and different anti-vWF antibody AJW200. 2A-B show the NMC-4 chimeric antibody to the sputum of the NMC-4 chimeric antibody calibrated with Eu alone and in combination with the uncalibrated NMC_4 chimeric antibody (homologous competition) (Fig. 2A). Figure 2A shows the anthropomorphic derivative of the A4W-derived AZW200 with the uncalibrated NMC-4 chimeric antibody, subtype control IgG, 126 201041902 strain on the mutated region of H9, L9. Competition for antibody binding; Figure 2B is a map of the competition in the presence of 20 _ "〇_存^ or non-existent (HiUPl〇t). Inside U iA-E shows that Li'4 is blocked in the case of shear flow Platelets are attached to η to: force 'includes adhesion to the visceral cells. Microplates of cells are h wp (Fig. 或) or 25 μΜ histamine (Fig. Ε Ε), plus 1 〇 Pg/ml VV 々, several bodies, Lan. 4 (Fig. C), 18 μ§/πύ anti-GPIb_a antibody, AK2 (Figure Λ, ί Hg/ml rodent 1gG (panel E) treated cell monolayer. Immediately before filling the early layers, Different antibodies are included in the corresponding platelet suspension. ^

[0039] Figures 4A-C show NMC-4 chimeras in a mouse model of iron oxide in arterial thrombosis compared to AJW200 (Fig. 4C) and a heterogeneous region with a table of H14, u〇 The activity of the anthropomorphic antibody (Fig. 4B). This tri-antibody was compared in a dose response study. '"ι [0040] Figure 5 shows the effect of increasing dose (0.03-10 mg/kg) of GBR 600 on circulatory flow reduction (CFRs) in sputum. Figure 6 shows the effect of increasing dose (0.01-K) mg/kg of GBR 600 on CFRs in sputum. Figure 7 shows the effect of increasing doses (0.005-0.07 mg/kg) of GBR 600 on CFRs in sputum. Figure 8 shows the dose response curves for cumulative doses of GBR 600 on CFRs in sputum. θ [0044] Figure 9 shows the effect of the escalating dose (1-1 〇 mg/kg) of the sputum in the sputum. [0045] FIG. 1A shows a comparison of the infusion effects of incremental doses of GBR600 and stagnation on the incision hemorrhage test. The dose has been expressed as a multiple of the effective dose (e.g., the cumulative dose at time zero). [0046] Figure 11 shows the thermal stability of the anthropomorphic NMC-4 variant as measured by differential scanning calorimetry. 127 201041902 [Key component symbol description]

Claims (1)

201041902 VII. Patent application scope: 1. A humanized antibody or a binding fragment thereof having a specificity of Von Whale's factor (vWF), comprising: (8) a sequence of a heavy chain variation region as described in SEQ ID NO: 19; b) a light chain variant region sequence as set forth in SEQ ID NO: 28. 2. A humanized antibody or binding fragment thereof having vWF specificity, the humanized antibody comprising: (8) a heavy chain variant region sequence as set forth in SEQ ID NO: 237; and (b) as set forth in SEQ ID NO: 238 The light chain variant region sequence. 3. A humanized antibody or binding fragment thereof having vWF specificity, the humanized antibody comprising: (a) heavy and light chain complementarity determining regions (CDRs) corresponding to the presence of the murine antibody NMC-4 Heavy and light chain variants (respectively guilty): 丨 and 2) CDRs; and (b) heavy chain framework regions and/or light chain architecture regions, the former corresponding to the human antibody AAC18165.1 The framework region in the variant region of (SEQ ID NO: 4), which corresponds to the framework region present in the variant region of human antibody AAK94808 (SEQ ID NO: 6). 4. A humanized antibody having vWF specificity or a binding fragment thereof, the humanized antibody comprising: 〇 selected from one or more of the heavy chain CDRs of the group consisting of: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR3. DPADYGNYDYALDY (SEQ ID NO: 9); and/or one or more of light chain CDRs selected from the group consisting of: LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12). 5. An anthropomorphic antibody or binding fragment thereof having vWF specificity, the humanized antibody comprising: heavy chain CDRs: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR3: 129 201041902 DPADYGNYDYALDY (SEQ ID NO: 9); and/or light chain CDRs: LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12) ). 6. The anthropomorphic antibody or binding fragment thereof having vWF specificity according to claim 4 or 5, further comprising: a heavy chain framework region derived from human antibody AAC18165.1 (SEQ ID NO: 4); and/or Light chain architecture region from human antibody AAK94808 (SEQ ID NO: 6). 7. The anthropomorphic antibody or binding fragment thereof having vWF specificity as claimed in claim 6 wherein the heavy chain framework region further comprises more than one murine residue. 8. A humanized antibody or binding fragment thereof having vWF specificity according to claim 6 wherein the heavy chain framework region does not comprise more than one murine residue. 9. The anthropomorphic antibody or binding fragment thereof having vWF specificity according to claim 6 wherein the light chain framework region further comprises more than one murine residue. 10. An anthropomorphic antibody having vWF specificity or a binding fragment thereof as claimed in claim 6 wherein the light bond framework region does not contain more than one murine residue. 11. The anthropomorphic antibody or binding fragment thereof having vWF specificity according to claim 4 or 5 wherein the anthropomorphic antibody comprises a heavy chain variation region selected from the group consisting of: H2 ( SEQ ID NO: 13), H4 (SEQ ID NO: 14), ❹ H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ H) NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: ? 145), Or H16 (SEQ ID NO: 146). 12. A humanized antibody having a vWF specificity, or a binding fragment thereof, according to claim 4 or 5, wherein the humanized antibody comprises a light chain variant region selected from the group consisting of L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ED NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28) 5 L10 (SEQ DD NO: 29), or Lll (SEQ ID NO: 3'0). 13. The vWF-specific anthropo-antibody or binding fragment thereof according to claim 4 or 5, wherein the anthropomorphic antibody comprises: 130 201041902 a heavy chain variation region selected from the group consisting of: Melon (3)^1〇^〇: 13), H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146); and a light chain variant region selected from the group consisting of L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), L10 (SEQ ID NO: 29), or Lll (SEQ ID NO) : 30). ' 14. The anthropomorphic antibody having vWF specificity or a binding fragment thereof according to claim 5, wherein the anthropomorphic antibody comprises: a heavy chain variant region sequence comprising at least 80% of the structure of SEQ ID NO: 19. The same framework region; and/or the light chain variant region sequence, which contains at least 80% of the framework region identical to the framework region of SEQ ID NO: 28. 15. The vWF-specific anthropomorphic antibody or binding fragment thereof according to claim 5, further comprising: a heavy chain framework region corresponding to the framework region of the human antibody family VH4; and corresponding to the human antibody family VK1 The light key architecture area of the architecture area. 16. The vWF-specific anthropomorphic antibody guanidine or binding fragment thereof according to claim 5, further comprising: a heavy chain structure corresponding to the framework region of the human antibody germline sequence 4-59 Regions 1, 2, and 3, wherein heavy chain architecture region 1 is QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO. 171), heavy chain architecture region 2 is WIRQPPGKGLEWIG (SEQ ED NO: 172), and heavy chain architecture region 3 is RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ) ID NO: Π3); and light chain architecture regions 1, 2, and 3 corresponding to the framework regions present in human antibody light chain germ cell line sequence 018, wherein light chain architecture region 1 is DIQMTQSPSSLSASVGDRVTITC (SEQIDNa 186), light The chain architecture region 2 is WYQQKPGKAPKLLIY (SEQ ID NO: 187), and the light chain architecture region 3 131 201041902 is GVPSRPSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 188). 17. The VWP-specific anthropomorphic antibody or binding fragment thereof according to claim 5, further comprising: a heavy chain framework region corresponding to the framework region of human antibody germ cell line sequence 4-34, 1, 2/ And 3, wherein the heavy chain architecture region 1 is QVQLQQWGAGLLKPSETLSLTCAVY (SEQ ID NO: 165), the heavy chain architecture region 2 is WIRQPPGKGLEWIG (SEQ ID NO: 166), and the heavy chain architecture region 3 is RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 167); and Ο corresponds to Light chain architecture regions 1, 2, and 3 present in the framework region of human antibody light chain germ cell line sequence 018, wherein light chain architecture region 1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 186), and light chain architecture region 2 is WYQQKPGKAPKLLIY (SEQIDNO) : 187), and the light chain architecture area 3 is GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 188). 18. The vWF-specific anthropomorphic antibody or binding fragment thereof according to any one of claims 1-5, 740, 14-17, wherein the anthropomorphic antibody system binds with an affinity (Kd) of 10 nM or less To vWF. 〇19. A vWP-specific anthropomorphic antibody or a binding fragment thereof according to any one of claims 1-5, 7-10, 14-17, wherein the anthropomorphic antibody system has an affinity below ΙΟΟηΜ ( Ki) Competition is combined with VWF. 20_ A humanized antibody having a vWF specificity or a binding fragment thereof according to any one of claims 1 to 5, 7-10, 14-17, wherein the anthropomorphic resistance system has an affinity of 10 nM or less (Kd) Bind to the A1 domain of vWF. 21. The vWF-specific anthropomorphic antibody or binding fragment thereof according to any one of claims 1 to 5, 740, 14-17, wherein the anthropomorphic antibody system binds to VWF with an affinity (Ki) of 1 (8) nM or less The A1 domain. 22. The vWP-specific anthropomorphic antibody or binding fragment thereof according to any one of the preceding claims, wherein the anthropomorphic antibody has a FAB fragment thermostability temperature of 132 201041902 greater than 65 °C. 23. A vWF-specific anthropomorphic antibody or binding fragment thereof according to any one of claims 1-5, 7-10, 14-17, wherein the anthropomorphic antibody has a higher anthropomorphism than the parent The FAB fragment of the antibody is thermostable temperature. 24. An anthropomorphic antibody or binding fragment thereof having vWF specificity, comprising: heavy chain CDR1, GFSLTDYGVD (SEQ ID NO: 7); heavy chain CDR2, MIWGDGSTDYNSALKS (SEQ ID NO: 8); heavy chain CDR3 , is DPADYGNYDYALDY (SEQ ID NO: 9); light chain CDR1; light chain CDR2; and light chain CDR3, is QQYEKLPWT (SEQ ID NO: 12), conditionally light chain CDR1 non-SASQDINKYLN (SEQ ID NO: 10) and/or The light chain CDR2 is not " O YTSSLHS (SEQ ID NO: 11) 〇, 25. The wWF-specific anthropomorphic antibody or /, ''σ σ fragment of the patent application scope 24 further contains the human antibody heavy chain architecture region And/or human antibody light-key architecture regions. 26. The anthropomorphic antibody having vWF specificity, or a binding fragment thereof, according to claim 25, wherein the heavy chain framework region corresponds to a heavy chain framework region present in a 4_59 derived human. The invention relates to a VWF-specific anthropomorphic antibody 〇 fragment of claim 26, wherein the towel is present in the heavy chain framework region of the 4_59-derived human antibody towel and further comprises more than one mouse residue. The anthropomorphic antibody having vwp specificity of the 26th item of the second paragraph is a segment of the human antibody t which is present in the 4_59 and does not contain more than one mouse residue. Or pot: Shi 9 j , the 25th item of the anthropomorphic antibody with vw specificity, the light chain structure area corresponds to the anthropomorphism of the human being The antibody further comprises more than one or more of the mouse (10) surnamed human antibody in the light chain framework region η. The anthropomorphic antibody having vwf specificity according to the fourth item of claim 4, 201041902 == upper == (10) derived human antibiotic Zhao Zhongzhi has a vWF-specific anthropomorphic antibody 5 H σ fragment according to the 26th item of the light chain architecture region, wherein the 4-59-derived human antibody is AAC18165.:1. Formula I The gentleman has a vWF-specific anthropomorphic antibody such as 专利,,,,,, in the patent scope of claim 29. 5 fragment, wherein the (10) derived human antibody is AAK94808. # 拟 申请 如 如 如 如 如 如 如 如 如 如 如 如 如 如 LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD LCD Ο 35. A humanized antibody or binding fragment having vWp specificity as disclosed in claim 34, wherein LCDR2 is DASNLET (SEQ ID NO: 118). 36. A VWF-specific anthropomorphic antibody or binding fragment thereof, according to any one of claims 1-5, 7-10, 14-17 and 24, wherein HCDR1 comprises more than one selected from F27Q An amino acid substituent of the group consisting of L29I, T30S and V34W. 37. A vWF-specific anthropomorphic antibody or binding fragment thereof, according to any one of claims 1-5, 7-10, 14-17 and 24, wherein the HCDR2 comprises more than one selected from the group consisting of S61P and Amino acid substituent of the group consisting of A62S. 38. A vWF-specific anthropomorphic antibody or a binding fragment thereof according to any one of claims 5, 7-1, 14_17 & 24, wherein the LCDR1 comprises more than one selected from the group consisting of S24Q, N30S and K31N Group of amino acid substituents. 39. A vWF-specific anthropomorphic antibody or binding fragment thereof according to any one of claims 1 to 5, 7-10, 14-17 and 24, wherein the HCDR1 comprises more than one selected from the group consisting of F27Q L29I, T30S And an amino acid substituent of the group consisting of V34W, the HCDR2 comprises one or more amino acid substituents selected from the group consisting of S61P and A62S, and the LCDR1 comprises more than one selected from the group consisting of S24Q, N30S and An amino acid substituent of the group consisting of K31N. 40. The vWF-specific anthropomorphic antibody or binding fragment thereof according to claim 4 or 5, further comprising a heavy chain framework region derived from a human antibody, wherein the human heavy chain architecture region does not comprise more than one mouse Residues. 134 201041902 41. The vWF-specific anthropomorphic antibody or binding fragment thereof according to claim 4 or 5, further comprising a light chain architecture region from a human antibody, wherein the human light chain architecture region does not comprise more than one Murine residue. 8. The anthropomorphic antibody or binding fragment thereof having the specificity of any one of claims 15-17, wherein the heavy chain framework region does not comprise more than one murine residue. 43. The anthropomorphic antibody or binding fragment thereof having vWp specificity according to any one of claims 15-17, wherein the light chain framework region does not comprise a murine residue. 44', as in any one of claims 1-5, 7-10, 14-17 and 24-35, having a vWF-specific anthropomorphic antibody or a binding fragment thereof, wherein the anthropomorphic antibody retains The non-humanized antibody, or the same activity as the chimera from the variant region of the parental non-humanized antibody and the human Fc region. ', 45. The anthropomorphic antibody or binding fragment thereof having vWp specificity as disclosed in claim 44, wherein the activity induces platelet aggregation activity by sirtuin 46. , 14_17 and 35 of the ice (nstocetm-induced platelet agglutination activity) was measured. 〇 47·If the patent application scope is ^7-10, 14-1^24-35垣 by red, —rS ^ , —rS Wherein the binding fragment is 135 201041902 selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab,)2 and a diabody. 51. An anthropomorphic antibody having a vWF specificity or a binding fragment thereof according to claim 50, wherein the antibody fragment is not a Fab. 52. An isolated nucleic acid, which encodes an antibody or a binding fragment thereof according to any one of the above claims. And an isolated nucleic acid comprising a coding vector (vect〇〇(3) tearing nucleic acid sequence, the light chain, the vector GS264 is deposited with the DSMZ in the microorganism, and its accession number (accession No.) is DSM21059. An isolated nucleic acid comprising a light chain encoding a nucleic acid sequence f of a vector GS265, the vector GS265 being deposited with a DSMZ in a microorganism, the accession No. being DSM21060. 55. Isolated nucleic acid, encoding An anthropomorphic antibody or binding fragment thereof having vWF specificity, the humanized antibody or binding fragment thereof comprising the heavy chain variant region sequence as described in 19 and the light chain variant region sequence as set forth in SEQ ID NO: 28. 56. An isolated nucleic acid encoding a humanized antibody having a vWF specificity, or a binding fragment thereof, comprising the heavy chain sequence as described in SEqIDN〇:237 and SEQ ID NO: 238 The light chain sequence described in the above. Ο 57. A vector comprising the isolated nucleic acid of any one of claims 52-56. 58. a host (h〇st) cell comprising An isolated nucleic acid according to any one of the items in the scope of the invention, or a method for producing a humanized antibody or a binding fragment thereof, comprising: cultivating a host cell of claim 58 of the patent scope The nucleic acid is expressed and the antibody is produced.. 60. The method for preparing a humanized antibody or a binding fragment thereof according to claim 59 of the patent application, further comprising recovering the antibody from a host cell culture. The method of preparing a humanized antibody or a binding fragment thereof according to claim 59, wherein the anti-system is recovered from a host cell culture medium (meduim). 6Z, as described in claim 59, anthropomorphic antibody or a binding fragment thereof. 136 201041902 The method, the towel, before the scaly, the carrier of the nucleic acid containing the strand variant region and the vector containing the light chain to change the co-transfected. And, 63. - a composition comprising: a patented scope (4) of anthropomorphic antibody or a binding fragment thereof, and a pharmaceutically acceptable carrier (pharmaceutically Acceptable carrier ). 64. - A composition comprising: patent application scope g (4) ϊ 拟 personification (10) or its knot correction segment, and the second AJW200 65 in the A1 field combined with VW, such as application fine _ 64 a composition, wherein the second antibody is a therapeutic agent of a vWF vector disease or abnormality in a subject comprising: a humanized antibody of the subject Fragment. Bessie 67. A method of treatment of vWp in a subject of claim 66, which is to be tested for humans. Media field disease or difference 68. In the subject of the patent application of item 66, the treatment of the disease, wherein the VWF vector disease or abnormal f is a thrombus]: 匕〇 69. If the patent application scope 6 δ In the subject of the subject, the method of treatment, wherein the recorded disease or abnormality is a cardio-f disease or a tube, a 70. As in the subject of claim 69, the vWp is often Treatment method 'The cardiovascular disease is atherosclerosis, drink s /, ^to: 〇Sd_is), vascular reocclusion (earning nQsls), angina pectoris (heart, myocardial infarction (call me (d) dkl infarctiQn), acute coronary a coronary syndrome, or a heart-and-blood camp associated with diabetes. 71. In the subject of claim 68, the vWp median concussion method, wherein the thrombotic disease or different f is vascular inflammation, venous blood ^ knife-shaped golden ball disease, xenograft rejection (xen〇graftrejecti〇n), peripheral disease (peripheral vascular disease), embolic thrombocytopenic purple therapy S 137 201041902 (thrombotic toombocytopenic purpura ),bag Cystic, vascular dementia, Raymond's disease (R^_d, s disease), rheumatoid arthritis (rheumat〇id arthritis), or diabetes. A method of treating 2vWp vector disease or different suspension in a subject of claim 69, wherein the cerebrovascular disease is vascular dementia (er de dementia), ischemic stroke, or recurrence (recmTent) 73. A method for treating a vector disease or abnormality in a subject according to any one of claims 66-72, wherein the effective therapeutic amount is from a dose of 100 mg/kg. Ο Ο Patent Scope 73 The method of treating a vWF vector disease or a different suspension in a subject, wherein the effective therapeutic amount is from about 0002 to about 20 mg/kg. The method of treating a vWF vector disease or a different suspension, wherein the effective therapeutic amount is from about _2 to about 10 mg/kg. 76. The subject of any one of claims 66-75. Media) ι disease or abnormal treatment method 'where the single or multiple small doses d (10)) The invention relates to a vwp ΪΓ disease or an abnormal massage therapy method in a subject of any one of the claims 66.75, wherein the New Zealand treatment slave In order to inhibit platelet agglutination, but not enough to cause obvious clinical symptoms of bleeding.甘入78. ^ In the subject of the scope of the patent range 66_72 - = = method, wherein the effective treatment is from about 1 to about 250 times the ED, without causing obvious bleeding clinical symptoms . 79. An anthropomorphic fragment of any of the claims in the scope of the patent application, which is _ (medieament), comprising administering the anthropomorphic antibody or a binding fragment thereof at a therapeutic dose of 2:. Effective " gentleman's 8 corpse's 1 sacred eye 2 range Μ 3 of the anthropomorphic antibody or its mouth ^ _ _ in the preparation of vWp vector disease or abnormal treatment 4, 匕 3 - administering the anthropomorphic antibody or binding fragment thereof in a therapeutically effective amount. ..., 81. If the patent application scope is the first? The application of the nine-part patent scope (4) - 138 201041902, wherein the agent is used to treat a humanized antibody or a binding fragment thereof with a vWF vector disease or abnormality. The use of a humanized antibody or a binding fragment thereof, such as the thrombinous disease or abnormality, is the use of a humanized antibody or a binding fragment thereof, as claimed in paragraphs 1 to 43 of the Patent Publication No. 8 or 81 of the May. s t·If you apply for any of the patent applications in Sections 1-43 of the application scope of Article 82 of the patent scope, Or the use of a binding fragment thereof, wherein the thrombotic abnormality is a cardiovascular disease or a cerebrovascular disease. Ο s 84. The patent of claim No. I-43 of claim 83, wherein the heart and vascular disease are arteriosclerosis (athe iOS _is), Blood disorder (her _is), angina (angina), acute myocardial infarction (acutemy〇cardiaHnfarcti〇n), acute coronary artery disease (acutecoronary syndrome), or cardiovascular abnormalities associated with diabetes (diabetes). 85. If the application of the patent scope of the application of the scope of the patent scope 丨 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 肖 途 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖 肖Thrombosis, sickle cell disease, xen〇graft rejection, peripheral vascular disease, thrombocytosis thrombocytopenic purpura, cystic fibrosis, vascular dementia (vascular dememia), Raynaud s disease, rheumatoid arthritis (rheumat〇id, or diabetes. 86. As claimed in claim 83, claiming any one of the claims Use of anthropomorphic antibody or a binding fragment thereof, wherein the cerebrovascular disease is vascular dementia, ischemic stroke, or recurrent stroke. 87. Patent Application No. 79-81 The use of the anthropomorphic antibody or a binding fragment thereof according to any one of claims 1-43 of any one of the items 83-86] wherein the effective therapeutic amount is from about 0.001 Approx. 100 mg/kg. ^ 88. For the use of the anthropomorphic antibody or its binding fragment of any of the 139 201041902 claims in the scope of claim 87 of the patent application, from 0.002 to about 20 mg/kg The effective therapeutic amount of Bayi is from the use of anthropomorphic antibody or a binding fragment thereof in the application of claim 87, i.e., any one of 0.002 to about l〇mg/kg. I is an anthropomorphic antibody or its knot, which will be single- or multiple-dose (sub_dGse), in the case of the stipulations of the stipulations of the stipulations of the stipulations of the stipulations of the stipulations of the stipulations. The effective ''shooting line or its binding fragment is administered to the subject. ', Li. The anthropomorphic antibody Ο 治疗 S treatment amount is sufficient to inhibit blood small _, but not enough to be "displayed in the secret" | The effective therapeutic amount of the patent application is from the ED H body or a conjugate thereof, which is about 1 to about 250 times, without causing significant hemorrhagic anthropomorphic city or a binding fragment thereof, when it is used as _ ^, therapeutic amount of the anthropomorphic antibody or its combination # segment. Effective patent scope i_5, 7] G, m7, and 2 «5 items任-常常时的包括性的抗体性的抗体性的性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性性This agent is used in the treatment of vWF-mediated diseases or in the elderly. - σ is a segment of λ6 (four) 4 or 95 of the anthropomorphic anti-observation of the film ^ a disease or abnormality is a bloody disease or abnormality. (d) the range of the humanoid antibody or its binding fragment, The blood is a cardiovascular disease or a cerebrovascular disease. 98. The anthropomorphic antibody or a binding fragment thereof according to the scope of the patent application, wherein the cardiovascular disease is atherosclerosis in 140 201041902 ( a^er0scier0sis), restenosis, angina, acute myocardial infarction, acute coronary syndrome, or cardiovascular abnormalities associated with diabetes. The anthropomorphic antibody or its binding fragment of claim 96, wherein the blood test disease comprises: vascular inflammation, intravenous jk test, sickle blood cell disease, xenograft rejection, peripheral vascular disease (peripheral vascular disease) Disease ), embolic thrombocytopenic purpura (also thrombocytopenic purpura), cystic fibrosis, vascular Physic dementia, Raynaud's disease, rheumatoid arthritis, or diabetes. 100. Anthropomorphic antibody or a binding fragment thereof, in claim 97, wherein The cerebrovascular disease is a vascular dementja, an ischemic stroke, or a recurrent stroke. 101. The anthropomorphic antibody of any one of claims 95-100, or A binding fragment, wherein the effective therapeutic amount is from about 1 to about 1 mg/kg. 102. The anthropomorphic antibody or a binding fragment thereof according to claim 1, wherein the effective therapeutic amount is From about 0.002 to about 20 mg/kg. 103. An anthropomorphic antibody or a binding fragment thereof according to claim 2, wherein the effective therapeutic amount is from about 0.002 to about 10 mg/kg. The anthropomorphic antibody or a binding fragment thereof, according to any one of clauses 95, 97-100, 102-103, wherein the anthropomorphic treatment of the effective therapeutic amount of the single- or multiple-dose (d=b_d〇se) The antibody or its binding fragment is administered to the recipient 1. 〇 5. As claimed in claim 95, 97-100, 102-103, any of the L antibodies or their knots, wherein the therapeutic amount is sufficient to inhibit platelet aggregation, but not enough to cause obvious Clinical symptoms of bleeding. The anthropomorphic antibody or /,,, σ δ 片 slave of any of the 外 ^ 请 专利 专利 专利 专利 专利 , , , , 专利 专利 专利 专利 专利 专利 专利 专利 专利 专利 专利 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟 拟Does not cause obvious clinical symptoms of bleeding. '° ^ 1〇7_ — a human antibody with a specificity of von Willis factor (vWF) or its 141 201041902 = segment and an effective therapeutic amount of about 1 to about 250 times from ED10° plus and without causing obvious Bleeding clinical symptoms. 108. The human antibody or its binding in the human antibody of paragraph 1Q7 of the patent application, or the combination thereof, and the bioinjection of the human VWF, the m-species of the human VWF, will be any one of the patent scopes M3. The method of administering the antibiotics to the subject in need thereof, comprising: administering = the amount of the anthropomorphic antibody or a binding fragment thereof, which causes the clinical symptoms of the bleeding to be caused by the ankle. No. 110. In the subject of claim 66, 2VWF Ο Ο i::i green' wherein the anthropomorphic antibody or a binding fragment thereof is administered via subcutaneous 异常 abnormal disease or formula. 〃τ顾人An antibody or a binding fragment thereof is a VWF-mediated disease or a combination of cytokines in a subject of a genus in the veins; (4) Among the 66 items, the vwf-age disease or the amount passed from 3 to the person who recorded the H. The effective treatment of the knot is from about 1 to about 250 times of the ED100. - The museum l!t The bribe of the human mu-antibody or its binding fragment in the item M3 of the application patent of item 79, The anthropomorphic antibody or 〜f is administered at a dose of about i to about 250 times from the smear of the smear. The product comprising the ribs of each of the vWF ages is reduced by f, the product = such as the patent of 26 The anthropomorphic antibody or binding fragment thereof according to any one of the items M3, the set of V = the disease or abnormality, the binding fragment, the anthropomorphic antibody or the U7 of any one of the patent target - The use of the anthropomorphic antibody or its 142 201041902 binding fragment of any of the claims 1 to 43 of the patent application for its use in non-therapeutic applications. 118 • Patent application as claimed in claim 117 The quaternary _43 upper-term anthropomorphic antibody or a binding fragment thereof, wherein the non-therapeutic, diagnostic assay. ' 119. Anthropomorphic antibody or its human being as claimed in claim 93 The effective therapeutic amount is from about 〇·〇〇1 to about l〇〇mg/kg. α °又” 120. The anthropomorphic antibody or the therapeutically effective amount in the human body of claim 94 From about 0.001 to about 1 mg/kg. ..." 121. : Anthropomorphic antibody or its ligated ◎ in the scope of patent application No. 93 Ο = = single or multiple small doses (sub_d (10)) of the effective ^ ^ 匕 antibody or its knots The person in the I. The anthropomorphic IT single such as Lifan '94 humanized antibody or its binding fragment, the antibody or its binding fragment is administered to the subject. The anthropomorphic system in the treatment A humanized antibody of 9h6f or a binding fragment thereof, an antibody thereof or a combination thereof, or a humanized antibody thereof, wherein the anthropomorphic antibody or a binding fragment thereof, Clinical symptoms. Inhibition of platelet aggregation, but not enough to show bleeding in the invention of the invention, the anthropomorphic antibody of the 94th item of the patent or the clinical treatment of the human body is sufficient to inhibit platelet aggregation, but not enough to cause; Blood, the f% of the reading of the aunt _ Wei Wei section, the clinical symptoms. "Inhibition of platelet aggregation, but insufficient (four) invention of bleeding, (2) · If the patent application of the iqi item of anthropomorphic antibody or its binding fragment, 143 201041902 but not enough to trigger It is obvious that the effective therapeutic amount is sufficient to inhibit platelet aggregation and clinical symptoms of blood. U9. If the application for the scope of the application of the scope of the patent application is to apply for a personification, the anthropomorphic resistance of the article is to be paid in the middle of the law. The binding fragment is administered by a humanized antibody or a binding fragment thereof under the subcutaneous surface, wherein the human antibody or a binding fragment thereof is administered intravenously by I31. The patent scope of the article shall be subject to the scope of the patent. ^ 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任 任The scope of the patent application will be the scope of the application for the scope of the application of the mth term - the person to be enrolled in the case of the need to accept the need to receive the "Chinese law" where the anthropomorphic antibody or its binding fragment of the effective treatment of about 1 to about 250 times. ^^ nUl〇〇8, schema z ❹ 144