CN115724968B - VEGF binding molecules and uses thereof - Google Patents

VEGF binding molecules and uses thereof Download PDF

Info

Publication number
CN115724968B
CN115724968B CN202110995278.7A CN202110995278A CN115724968B CN 115724968 B CN115724968 B CN 115724968B CN 202110995278 A CN202110995278 A CN 202110995278A CN 115724968 B CN115724968 B CN 115724968B
Authority
CN
China
Prior art keywords
ser
leu
val
gly
thr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110995278.7A
Other languages
Chinese (zh)
Other versions
CN115724968A (en
Inventor
刘婵娟
郎国竣
闫鑫甜
闫闰
孔超
张文海
胡宇豪
周蕴华
许彩云
王立燕
顾佳惠
司远青
时霄霄
孙兴鲁
江茹兰
林紫绮
王文蓉
韩慢慢
戴珊珊
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sanyou Biopharmaceuticals Co Ltd
Original Assignee
Sanyou Biopharmaceuticals Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sanyou Biopharmaceuticals Co Ltd filed Critical Sanyou Biopharmaceuticals Co Ltd
Priority to CN202110995278.7A priority Critical patent/CN115724968B/en
Priority to PCT/CN2022/115030 priority patent/WO2023025273A1/en
Publication of CN115724968A publication Critical patent/CN115724968A/en
Application granted granted Critical
Publication of CN115724968B publication Critical patent/CN115724968B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The present disclosure provides VEGF binding molecules comprising immunoglobulin single variable domains. The present disclosure also provides nucleic acid molecules encoding the VEGF binding molecules, expression vectors and host cells for expressing the VEGF binding molecules. The present disclosure further provides methods of producing the VEGF binding molecules and their use for treating or preventing conditions associated with pathological angiogenesis.

Description

VEGF binding molecules and uses thereof
Sequence listing
The present disclosure includes a sequence listing, and is incorporated by reference in its entirety.
Technical Field
The present disclosure relates generally to the field of molecular biology. In particular, the disclosure relates to VEGF binding molecules. More specifically, the disclosure relates to antibody molecules, such as nanobodies, that specifically bind to VEGF, methods of making the same, and uses thereof.
Background
Normal angiogenesis plays a very important role in organisms and is capable of transporting nutrients and oxygen to various tissues and organs of the body, and simultaneously transporting metabolic wastes to metabolic organs, thereby self-cleaning. However, abnormal angiogenesis is associated with the development and progression of a variety of diseases, including solid tumors and vascular proliferation-related eye diseases. The development and the metastasis of the solid tumor can not leave the continuous formation of new blood vessels, so that enough nutrient substances and oxygen are provided for rapidly proliferating tumor cells, and a vascular system with high permeability is provided for infiltration and metastasis of the tumor cells; age-related macular degeneration (AMD), diabetic Macular Edema (DME), and the like are ocular diseases caused by abnormal hyperplasia of blood vessels, and there is a great market demand for drugs for both of these diseases (Apte, r.s., et al (2019), "VEGF in Signaling and Disease: beyond Discovery and development," Cell 176 (6): 1248-1264). Therefore, for diseases with pathological vascular proliferation, the angiogenesis is inhibited, the blood vessel is normalized, and the effect of curing or delaying the disease progress can be achieved.
The key cytokines that promote angiogenesis are vascular endothelial growth factor (Vascular Endothelial-derived Growth Factor, VEGF). Receptors for VEGF include VEGFR1 and VEGFR2, etc., which are expressed in vascular endothelial cells. VEGFR2 is the primary receptor tyrosine kinase receptor mediating angiogenesis, and VEGF activates VEGFR2 to promote vascular endothelial cell mitosis and increased vascular permeability, thereby promoting neovascular sprouting. Thus, targeting VEGF or VEGFR2 can effectively inhibit aberrant angiogenesis.
The anti-VEGF monoclonal antibody, bevacizumab, has relatively high curative effect and target safety in treating tumor and treating eye diseases related to abnormal vascular proliferation.
Although monoclonal antibody drugs targeting VEGF targets are currently available, there is still an urgent need to continue to develop better-potency antibodies targeting VEGF as therapeutic agents. It is desirable in the art to develop novel antibodies targeting VEGF, in particular single domain antibodies that specifically recognize VEGF.
Disclosure of Invention
Broadly, the present disclosure provides novel antibodies, methods of preparation, compositions and articles of manufacture. The benefits provided by the present disclosure are broadly applicable to the fields of antibody therapy and diagnosis. More specifically, the present disclosure provides single domain antibodies that target VEGF, as well as methods of making the antibodies, expression vectors and host cells for expressing the antibodies, and the like. Antibodies of the present disclosure provide methods of treating or preventing conditions associated with abnormal angiogenesis and uses thereof.
The inventors have discovered molecules that specifically bind to VEGF, including VEGF binding molecules that are capable of specifically binding to human VEGF (e.g., single domain antibodies that target VEGF).
The present disclosure includes at least the following embodiments, which are ordered and enumerated by way of "N" (where "N" represents a number), respectively. The following list is not exhaustive and the skilled person can combine different solutions.
A vegf binding molecule, wherein CDR1 hybridizes to SEQ ID NO: 7. 24, 25, 26 or 27, there is a difference in amino acid addition, deletion or substitution of not more than 2 amino acids; CDR2 hybridizes in amino acid sequence to SEQ ID NO: 8. 16, 28, 29 or 30, there is a difference in amino acid addition, deletion or substitution of not more than 2 amino acids; and/or CDR3 hybridizes in amino acid sequence to SEQ ID NO: 9. 17, 31 or 32, there is a difference in amino acid addition, deletion or substitution of not more than 2 amino acids.
2. The VEGF binding molecule of embodiment 1, wherein the VEGF binding molecule is an antibody or antigen-binding fragment thereof directed against VEGF.
3. A VEGF binding molecule of any one of the preceding embodiments, wherein the immunoglobulin single variable domain is a VHH; for example VHH from a camelid (e.g. alpaca).
4. A VEGF binding molecule of any one of the preceding embodiments, wherein the immunoglobulin single variable domain comprises:
i) As GX 1 X 2 CDR1 of the amino acid sequence shown in LDYYAIG, wherein X 1 F, Q or H, and X 2 T, G, R, K or a;
ii) CIGSSY as shown 1 Y 2 Y 3 Y 4 Y 5 CDR2 of the amino acid sequence shown, wherein Y 1 N, S or H, Y 2 S, K or I, Y 3 Is T or E, Y 4 Is T or E, and Y 5 Is N or T; and/or
iii) Such as GSPLCLISLQZ 1 Z 2 Z 3 Z 4 CDR3 of the amino acid sequence shown in LYEYDY, wherein Z 1 Is D or V, Z 2 M, H or P, Z 3 Is D or Y, and Z 4 Is S or G.
5. A VEGF binding molecule of any one of the preceding embodiments, wherein the immunoglobulin single variable domain comprises:
i) As GX 1 X 2 CDR1 of the amino acid sequence shown in LDYYAIG, wherein X 1 F, Q or H, and X 2 G, R or K;
ii) CIGSSY as shown 1 Y 2 CDR2 of the amino acid sequence shown in ETN, wherein Y 1 S, N or H, Y 2 Is K or I; and/or
iii) Such as GSPLCLISLQZ 1 Z 2 CDR3 of the amino acid sequence shown in YGLYEYDY wherein Z 1 Is D or V, Z 2 H, M or P.
6. A VEGF binding molecule of any one of the preceding embodiments, wherein the immunoglobulin single variable domain comprises:
i) As set forth in SEQ ID NO: 7. 24, 25, 26 or 27;
ii) the sequence as set forth in SEQ ID NO: 8. 16, 28, 29 and 30; and/or
iii) As set forth in SEQ ID NO: 9. 17, 31 or 32.
7. A VEGF binding molecule of any one of the preceding embodiments, wherein the immunoglobulin single variable domain comprises:
(a) As set forth in SEQ ID NO:7, as set forth in SEQ ID NO:8 and CDR2 of the amino acid sequence shown as SEQ ID NO:9, CDR3 of the amino acid sequence depicted;
(b) As set forth in SEQ ID NO:7, as set forth in SEQ ID NO:16 and CDR2 of the amino acid sequence shown as SEQ ID NO:17, CDR3 of the amino acid sequence depicted;
(c) As set forth in SEQ ID NO:24, as set forth in SEQ ID NO:28 and CDR2 of the amino acid sequence shown as SEQ ID NO:31, CDR3 of the amino acid sequence depicted;
(d) As set forth in SEQ ID NO:25, as set forth in SEQ ID NO:16 and CDR2 of the amino acid sequence shown as SEQ ID NO:32, CDR3 of the amino acid sequence shown in seq id no;
(e) As set forth in SEQ ID NO:26, as set forth in SEQ ID NO:29 and CDR2 of the amino acid sequence shown as SEQ ID NO:17, CDR3 of the amino acid sequence depicted; or (b)
(f) As set forth in SEQ ID NO:27, as set forth in SEQ ID NO:30 and CDR2 of the amino acid sequence shown as SEQ ID NO:17, and CDR3 of the amino acid sequence depicted in seq id no.
8. A VEGF binding molecule according to any one of the preceding embodiments, wherein the immunoglobulin single variable domain comprises or consists of:
(A) SEQ ID NO: 6. 12, 15, 20, 21, 22 or 23;
(B) And SEQ ID NO: 6. 12, 15, 20, 21, 22, or 23, at least 80%, 85%, 90%, or 95% identical; or (b)
(C) And SEQ ID NO: 6. 12, 15, 20, 21, 22, or 23, an amino acid sequence having additions, deletions, and/or substitutions of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids; preferably, the additions, deletions and/or substitutions do not occur in the CDR regions.
9. A VEGF binding molecule according to any one of the preceding embodiments, wherein the immunoglobulin single variable domain is modified (e.g., amino acid substitutions and/or additions) at one or more of the following positions corresponding to SEQ ID No. 6: amino acid 1, 87 or 88; preferably, the modification is a humanized modification; more preferably, the modification is Q1E, K R or P88A.
10. The VEGF binding molecule of any one of the preceding embodiments, wherein the VEGF binding molecule is a single domain antibody, e.g., a heavy chain single domain antibody, a chimeric antibody, or a humanized antibody.
11. A VEGF binding molecule according to any one of the preceding embodiments, wherein the immunoglobulin single variable domain is fused to another molecule, such as an Fc domain of an immunoglobulin (e.g., igG) or a fluorescent protein.
12. The VEGF binding molecule of embodiment 11, wherein the VEGF binding molecule is a chimeric antibody comprising a VHH from a camelid and an Fc domain of a human IgG (e.g., human IgG1 or IgG 4).
13. The VEGF binding molecule of embodiment 12, wherein the VEGF binding molecule is a chimeric antibody comprising a VHH from alpaca and an Fc domain of human IgG 1; preferably, the VEGF binding molecule comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 10. 13, 18, 33, 34, 35 or 36.
14. A VEGF binding molecule of any one of the preceding embodiments, wherein the VEGF binding molecule binds human VEGF.
A VEGF binding molecule that competes for the same epitope with a VEGF binding molecule of any one of the preceding embodiments.
16. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a VEGF binding molecule of any one of the preceding embodiments.
17. The isolated nucleic acid molecule of embodiment 16, comprising or consisting of the sequence: 11, 14, 19, 37, 38, 39 or 40.
18. An expression vector comprising the isolated nucleic acid molecule of embodiment 16 or 17.
19. A host cell comprising the expression vector of embodiment 18.
20. The host cell of embodiment 19, which is a bacterial cell (e.g., E.coli), a fungal cell (e.g., yeast), or a mammalian cell.
21. A pharmaceutical composition comprising at least one VEGF binding molecule of any one of embodiments 1-15 and a pharmaceutically acceptable carrier.
22. A method of making a VEGF binding molecule of any one of embodiments 1-15, comprising the steps of:
expressing the VEGF binding molecule of any one of embodiments 1-15 in the host cell of embodiment 19 or 20; and
isolating the VEGF binding molecule from the host cell.
23. Use of a VEGF binding molecule of any one of embodiments 1-15 in the manufacture of a medicament for treating or preventing a disorder associated with pathological angiogenesis.
24. A kit for treating or diagnosing a disorder associated with pathological angiogenesis comprising a container comprising a VEGF binding molecule of any one of embodiments 1-15.
25. A method of treating or preventing a disorder associated with pathological angiogenesis comprising administering to a subject in need thereof a VEGF binding molecule of any one of embodiments 1-15.
26. A VEGF binding molecule of any one of embodiments 1 to 15 for use in the treatment or prevention of a disorder associated with pathological angiogenesis.
The foregoing is a general description and may include simplifications, generalizations, and omissions of detail as necessary. Accordingly, those skilled in the art will recognize that this general description is merely illustrative and is not intended to be limiting in any way. Other aspects, features, and advantages of the methods, compositions, and/or devices described herein and/or other subject matter will become apparent in the teachings shown herein. The general description is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. The above summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. Further, the contents of all references, patents and published patent applications cited throughout this disclosure are incorporated herein by reference in their entirety.
Drawings
FIGS. 1A-1C show the binding activity of hVEGF-His, hVEGF-Fc and mVEGF-His to the positive control antibody Bevacizumab. Fig. 1A: binding activity of hVEGF-His to the positive control antibody Bevacizumab at a plate concentration of 2 μg/ml; fig. 1B: binding activity of hVEGF-Fc to the positive control antibody Bevacizumab under the conditions of a wrapper concentration experiment of 2 μg/ml; fig. 1C: binding activity of mVEGF-His to the positive control antibody Bevacizumab at a plate concentration of 2 μg/ml.
FIG. 2 shows that hVEGFR2-His protein has good binding activity to hVEGF-Fc.
FIG. 3 shows SDS-PAGE results of fusion protein P30 molecules under non-denaturing conditions and denaturing conditions.
FIG. 4 shows the protein monomer ratio of the fusion protein P30 molecule in the samples of the tested batches.
FIGS. 5A-5B show ELISA binding activity of fusion protein P30 molecules to human and mouse VEGF. Fig. 5A: binding affinity of fusion protein P30 molecule and human VEGF protein at ELISA level; fig. 5B: binding affinity of fusion protein P30 molecule and mouse VEGF protein at ELISA level.
FIG. 6 shows that fusion protein P30 molecule is capable of blocking VEGF-VEGFR2 binding and blocking activity is superior to Bevacizumab.
FIG. 7 shows the binding affinity of the fusion protein P30 molecule and its humanized engineered molecule P30-huVH5 to the antigen VEGF at ELISA level.
Figures 8A-8G show antigen-antibody binding dissociation graphs. Fig. 8A: affinity kinetic data for P30-huVH5 molecules; fig. 8B: affinity kinetic data for P30-10 molecule (P30-10-WT); fig. 8C: affinity kinetic data of Bevacizumab molecule (same batch and same experimental conditions as P30); fig. 8D: affinity kinetic data for P30-10-26 molecules; fig. 8E: affinity kinetic data for P30-10-9 molecules; fig. 8F: affinity kinetic data for P30-10-16 molecules; fig. 8G: affinity kinetic data for P30-10-25 molecules.
FIGS. 9A-9E show binding affinities to mouse VEGF protein at ELISA level. Wherein FIG. 9A shows the binding affinity of the engineered molecule P30-10-9, the engineered molecule P30-10-WT and the fusion protein P30 to the mouse VEGF protein at ELISA level; FIG. 9B shows the binding affinity of engineered molecule P30-huVH5 to mouse VEGF protein at ELISA level; FIG. 9C shows the binding affinity of engineered molecule P30-10-26 to mouse VEGF protein at ELISA level; FIG. 9D shows the binding affinity of engineered molecule P30-10-16 to mouse VEGF protein at ELISA level; FIG. 9E shows the binding affinity of engineered molecule P30-10-25 to mouse VEGF protein at ELISA level.
FIGS. 10A-10C show the VEGF neutralizing activity of various engineered molecules. Fig. 10A: effects of engineered molecule P30-huVH5, engineered molecule P30-10-WT, engineered molecule P30-10-26, bevacizumab neutralizing VEGF-VEGFR2 signaling pathway; fig. 10B: the modified molecules P30-10-16, P30-10-9 and Bevacizumab neutralize the effect of VEGF-VEGF2 signaling pathway; fig. 10C: effects of engineered molecules P30-10-25, P30-10-26 and Bevacizumab on neutralizing VEGF-VEGF2 signaling pathway.
FIG. 11A is a graph of animal model abnormal angiogenesis scoring criteria. Score 0 represents no vascular proliferation; 1/1.5 is divided into blood vessels and ear roots to start to turn red; 3/3.5 is divided into the root of the auricle which is deepened and forms a circular outline; the 4/4.5 part is that the auricle turns into dark red, the outline is clear, and the area is enlarged.
FIG. 11B shows the effect of engineered molecule P30-10-26 on inhibiting VEGF-mediated angiogenesis in animal models. If P <0.001, the two sets of data are shown to have very significant differences, denoted by "; if the p value of 0.001< 0.01, it is stated that the two sets of data have very significant differences, expressed as "; if 0.01< p value <0.05, it is stated that the two sets of data have significant differences, expressed as "; if the P value is > 0.05, it is stated that the two sets of data do not differ significantly, denoted by "ns".
FIG. 12A shows tumor volume change in an animal model of colon cancer tumor after administration of engineered molecule P30-10-26.
FIG. 12B shows the change in mouse body weight on an animal model of colon cancer tumor after administration of engineered molecule P30-10-26.
FIG. 12C shows tumor weight changes in colon cancer tumor animal models following administration of engineered molecule P30-10-26.
Detailed Description
While this invention may be embodied in many different forms, there are disclosed herein specific illustrative embodiments which are indicative of the principles of the invention. It should be emphasized that the present invention is not limited to the specific embodiments illustrated. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention will have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, terms in the singular shall include the plural and terms in the plural shall include the singular.
Generally, terms related to cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein, and techniques thereof, are well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout the present specification. See, e.g., abbas et al Cellular and Molecular Immunology,6th ed., w.b. samunders Company (2010); sambrook J. & Russell d.molecular Cloning: A Laboratory Manual,3rd ed., cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (2000); ausubel et al Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, wiley, john & Sons, inc. (2002); harlow and Lane Using Antibodies: A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (1998); and Coligan et al, short Protocols in Protein Science, wiley, john & Sons, inc. (2003). Terms related to analytical chemistry, synthetic organic chemistry and pharmaceutical chemistry, as well as laboratory procedures and techniques, described herein are terms well known and commonly used in the art. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Definition of the definition
For a better understanding of the present invention, definitions and explanations of related terms are provided below.
As used herein, the term "antibody" or "Ab" generally refers to any form of antibody that exhibits the desired biological or binding activity. It includes, but is not limited to, humanized antibodies, fully human antibodies, chimeric antibodies, and single domain antibodies. Antibodies may comprise a heavy chain and a light chain. Heavy chains can be divided into μ, δ, γ, α and ε, which define the isotype of antibodies as IgM, igD, igG, igA and IgE, respectively. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). VH and VL regions can be further divided into relatively conserved regions, known as Framework Regions (FR), and hypervariable regions, known as Complementarity Determining Regions (CDRs), separated by FR. Each VH and VL is composed of 3 CDRs and 4 FRs in the order from N-terminus to C-terminus of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The distribution of amino acids in various regions or domains follows the definition of AbM. Antibodies may have different antibody isotypes, for example IgG (including for example IgG1, igG2, igG3 or IgG4 subtypes), igA1, igA2, igD, igE or IgM antibodies.
The term "humanized antibody" as used herein refers to an antibody in which CDR sequences derived from the germline of another non-human mammal are grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequence.
The term "chimeric antibody" as used herein refers broadly to engineered antibodies that contain one or more regions from one antibody and one or more regions from one or more other antibodies. In particular, chimeric antibodies comprise a variable region derived from a non-human animal antibody and a constant region of another antibody, for example comprising a variable region of camel origin and a constant region of human origin. Chimeric antibodies may also refer to multispecific antibodies that are specific for at least two different antigens.
The term "nanobody" refers to a natural camel-derived heavy chain antibody or a heavy chain antibody containing no Fc fragment, etc., and the nanobody may be derived from a camelid such as alpaca. It may be used interchangeably with the terms "VHH", "VHH antibody", "VHH domain", "VHH antibody fragment", "VHH" or "nanobody", etc. VHH molecules from camelidae antibodies are one of the smallest known complete antigen binding domains (about 15KDa, or 1/10 of conventional IgG) and are therefore well suited for delivery to dense tissues and into the limited space between macromolecules.
The term "engineered molecule" refers to an antibody engineered molecule that has been engineered to humanize a natural antibody, to have been engineered to have been altered in a pharmaceutical manner, to have been altered in affinity maturation, and the like.
The term "fusion protein" refers to a fusion protein expressed by fusing genes encoding the variable region fragment of an antibody and the human Fc fragment. May be used interchangeably herein with chimeric antibodies.
The term "VEGF binding molecule" as used herein means a molecule that specifically binds VEGF.
The terms "VEGF antibody", "antibody against VEGF", "antibody that specifically binds to VEGF", "antibody that specifically targets VEGF", "antibody that specifically recognizes VEGF" as used herein are used interchangeably to mean an antibody capable of specifically binding to VEGF. In particular, in a specific embodiment, an antibody that specifically binds to human VEGF is meant.
The term "vascular endothelial growth factor" or "VEGF" refers to vascular endothelial growth factor protein A (VEGF-A). In addition to human derived VEGF, the term "VEGF" refers to VEGF derived from a non-human species (e.g., mouse, rat or primate). VEGF from a particular species is expressed by terms such as: hVEGF represents human VEGF, mVEGF represents mouse VEGF, and so on. The amino acid sequences of human VEGF (GenBank accession number: NP-001165097) and mouse VEGF (GenBank accession number: NP-001273986.1) used in the examples of the present disclosure are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
VEGF receptors include three major classes: tyrosine kinase receptors, neuropilins (NRP), and heparin sulfate proteoglycans (Heparan Sulfate Proteoglycans, HSPG). Tyrosine kinase receptors include VEGFR1, VEGFR2 and VEGFR3, wherein VEGFR2 is the predominant receptor tyrosine kinase receptor that mediates angiogenesis. VEGF activates VEGFR2 to promote vascular endothelial cell mitosis and increased vascular permeability, thereby promoting neovascular sprouting. Thus, without being bound by theory, targeting VEGF or VEGFR2 is able to effectively inhibit aberrant angiogenesis (Ferrara, n. (2010)). The amino acid sequence of human VEGFR2 (Ala 20-Glu 764) is shown in SEQ ID NO. 3.
The term "immunoglobulin single variable domain" or "ISV" as used herein is generally defined herein as the amino acid sequence: comprising an immunoglobulin fold or being capable of forming an immunoglobulin fold (i.e., by folding) under suitable conditions (e.g., physiological conditions), i.e., thereby forming an immunoglobulin variable domain (e.g., a VH, VL, or VHH domain); and forming (or under suitable conditions capable of forming) an immunoglobulin variable domain comprising a functional antigen binding site (in the sense that it does not require interaction with another immunoglobulin variable domain, such as VH-VL interactions, to form a functional antigen binding site).
The term "Ka" as used herein is intended to denote the rate of association of a particular antibody-antigen interaction, while the term "Kd" as used herein is intended to denote the rate of dissociation of a particular antibody-antigen interaction. As used herein, the term "KD" or "KD value" is intended to mean the dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of KD to Ka (i.e., KD/Ka) and expressed as molar concentration (M). The KD values of antibodies can be determined using well established methods in the art. A preferred method of determining antibody KD is by using surface plasmon resonance, preferably using a biosensor system such asThe system.
The term "specific binding" or "specifically binding to" as used herein refers to a non-random binding reaction between two molecules, e.g., between an antibody and an antigen.
As used herein, "ability to inhibit binding," "block binding," or "compete for the same epitope" refers to the ability of an antibody to inhibit the binding of two molecules to any detectable extent. In some embodiments, an antibody that blocks binding between two molecules inhibits binding interactions between two molecules by at least 50%. In some embodiments, the inhibition may be greater than 60%, greater than 70%, greater than 80% or greater than 90%.
The term "high affinity" antibody as used herein refers to an antibody having 1 x 10 binding to a target antigen -7 M or less, more preferably 5X 10 -8 M or less, even more preferably 1X 10 -8 M or less, even more preferably 5X 10 -9 M or less, and even more preferably 1X 10 -9 Antibodies with KD values of M or lower.
The term "IC50", also referred to as "half-inhibitory concentration", as used herein, refers to the concentration of inhibitor (e.g., VEGF antibody) required to inhibit 50% of a target such as an enzyme, cell, receptor, cytokine, etc.
As used herein, the term "isolated" refers to a state of a substance or component obtained from a natural state by artificial means. If a certain "isolated" substance or component occurs naturally, it may be due to a change in the natural environment in which it is located, or the separation of the substance or component from the natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and the same high purity polynucleotide or polypeptide that is isolated from that natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances nor other impure substances that do not affect the activity of the isolated substances.
As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. In addition, the isolated antibodies may be substantially free of other cellular material and/or chemicals.
As used herein, the term "vector" refers to a nucleic acid vector into which a polynucleotide may be inserted. When a vector allows expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector may be transformed, transduced or transfected into a host cell to express the carried genetic material element in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1 derived artificial chromosome (PAC), phages such as lambda phage or M13 phage, and animal viruses. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV 40). The vector may contain a number of elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may comprise an origin of replication.
As used herein, the term "host cell" refers to any kind of cell system into which a vector can be introduced, including, but not limited to, prokaryotic cells such as e.coli (e.coli) or bacillus subtilis (Bacillus subtilis), fungal cells such as yeast cells or Aspergillus (Aspergillus), insect cells such as S2 drosophila cells or Sf9, and animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK293 cells or human cells.
Methods for producing antibodies of the invention using host cells are conventional in the art and include expressing the antibodies in prokaryotic or eukaryotic cells, then isolating the antibodies, and generally purifying to a pharmaceutically acceptable purity. As in some embodiments, nucleic acids encoding antibodies are inserted into expression vectors and the expression vectors are introduced into suitable prokaryotic or eukaryotic host cells by standard techniques known in the art, the host cells are cultured, such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, per.c6 (R) cells, yeast or e.coli cells, under conditions and for a time sufficient to produce the antibodies or functional fragments thereof of the invention, and the antibodies are recovered from the cells (supernatant or cells after lysis) and purified. Conventional methods for producing antibodies are known in the art and are described, for example, in Makrides, s.c., protein expr.purif.17 (1999) 183-202; geisse, S.et al Protein expr. Purif.8 (1996) 271-282; kaufman, R.J., mol.Biotechnol.16 (2000) 151-160; fferner, r.g., drug res.48 (1998) review articles 870-880.
The term "subject" includes any human or non-human animal, preferably a human.
The term "treatment" as used herein in the context of treating a condition generally relates to the treatment and therapy of a human or animal in which some desired therapeutic effect is achieved, e.g., inhibiting the progression of the condition, including a decrease in the rate of progression, a arrest in the rate of progression, regression of the condition, improvement of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cell, or some combination thereof. For a tumor, "treating" includes removing all or part of the tumor, inhibiting or slowing the growth and metastasis of the tumor, preventing or delaying the progression of the tumor, or some combination thereof.
As used herein, the term "therapeutically effective amount" refers to an amount of an active compound or a material, composition or dosage form comprising an active compound that is effective for producing certain desired therapeutic effects commensurate with a reasonable benefit/risk ratio when administered according to a desired therapeutic regimen. In particular, a "therapeutically effective amount" means an amount or concentration of an antibody, or antigen-binding portion thereof, effective to treat a disorder associated with pathological angiogenesis.
As used herein, the term "pharmaceutically acceptable" means that the carrier, diluent, excipient, and/or salt thereof is chemically and/or physically compatible with the other ingredients in the formulation, and physiologically compatible with the recipient.
As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active agent, which is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to, pH modifiers, surfactants, adjuvants, and ionic strength enhancers. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.
VEGF binding molecules
In some aspects, the disclosure provides VEGF binding molecules.
In general, the VEGF binding molecule may comprise any molecule that specifically binds VEGF. In some cases, a "VEGF binding molecule" may include a "VEGF antagonist. The VEGF binding molecule or VEGF antagonist may be a polypeptide or protein, such as an antibody, more specifically an antibody that specifically binds VEGF (e.g., human VEGF).
Antibodies include, but are not limited to, chimeric, humanized or single domain antibodies, antibody engineered derivatives of these antibodies, and the like. In particular embodiments, the VEGF binding molecule is a single domain antibody, which generally refers to an antibody consisting of a single monomer variable antibody domain. Like full length antibodies, single domain antibodies are capable of selectively binding to a particular antigen.
In some embodiments, the VHH in the VEGF binding molecule is fused to an Fc domain of an antibody (e.g., an Fc domain of an IgG (e.g., igG1 or IgG 4)). By fusing VHH to Fc domains, effector functions can be more effectively recruited. Furthermore, fusion of VHH to Fc domains can help the VEGF binding molecules form dimers, and can also help to extend the in vivo half-life of the VEGF binding molecules.
For ease of description, the VEGF binding molecules are described hereinafter as VEGF antibodies.
VEGF antibodies capable of specifically binding VEGF
In one aspect, the disclosure relates to single domain antibodies that specifically bind VEGF.
In some embodiments, the inventors have discovered a VEGF binding molecule (e.g., a single domain antibody that targets VEGF) that is capable of specifically binding to human VEGF. As previously mentioned, the sequences of human VEGF and mouse VEGF used in the present disclosure are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
In a given heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et al, (1989) Nature 342:877-883, al-Lazikani et al, "Standardconformations for the canonical structures of immunoglobulins", journal of Molecular Biology,273,927-948 (1997)) based on the three-dimensional structure of antibodies and topology of CDR loops, kabat (Kabat et al, sequences of Proteins ofImmunological Interest, 4 th edition, U.S. Pat. No. of Health and Human Services, nationalInstitutes of Health (1987)), abM (University of Bath), contact (University College London), international ImMunoGeneTics database (IMGT) (world Wide Web IMGT. Cines. Fr /), and North CDR definitions based on neighbor-propagating clusters (affinity propagation clustering) using a large number of crystal structures. The VEGF antibodies of the present disclosure employ the AbM approach to defining the amino acid sequence of the CDR regions, but such definition is not exclusive and limiting and does not even suggest that the present disclosure only protects the antibody molecules represented by the CDR regions defined using the AbM approach. Based on the aforementioned various antibody CDR-assignment systems conventional in the art, the VEGF antibody molecules of the present disclosure can also be characterized in a variety of other CDR region definition manners. It should be noted that the boundaries of CDRs of the variable regions of the same antibody obtained based on different assignment systems may differ, i.e., CDR sequences of the same antibody variable regions defined under different assignment systems may differ. The CDR sequences defined under these different assignment systems and their characterized antibody molecules are also intended to be protected by the present disclosure.
The variable regions and CDRs in an antibody sequence can be identified according to general rules that have been developed in the art (as described above, e.g., the AbM numbering system) or by aligning the sequences with a database of known variable regions. Sequences are preferably analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT and Protein Databases (PDB) with structural data from PDB, see Protein Sequence and Structure Analysis of Antibody Variable domains in the book by dr. Andrew c.r.martin: antibody Engineering Lab Manual (Ed.: duebel, s.and Kontermann, r., springer-Verlag, heidelberg, ISBN-13:978-3540413547, also available on the website bioin for. The Abysis database website also includes general rules that have been developed for identifying CDRs that can be used in accordance with the teachings herein.
VEGF antibodies comprising CDRs with amino acid additions, deletions or substitutions
In some embodiments, a VEGF antibody of the disclosure comprises at least one immunoglobulin single variable domain (e.g., a VHH), wherein the VHH comprises CDR1, CDR2, and CDR3, and wherein CDR1 differs in amino acid sequence from the sequence shown in SEQ ID NO. 7, 24, 25, 26, or 27 by NO more than 2 (e.g., 0, 1, or 2) amino acid additions, deletions, or substitutions; CDR2 differs in amino acid sequence from the sequences shown in SEQ ID NOs 8, 16, 28, 29 or 30 by amino acid additions, deletions or substitutions of NO more than 2 (e.g., 0, 1 or 2) amino acids; and/or CDR3 differs in amino acid sequence from the sequence shown in SEQ ID NO 9, 17, 31 or 32 by amino acid additions, deletions or substitutions of not more than 2 (e.g.0, 1 or 2) amino acids. For example, CDR1, CDR2 and CDR3 differ from the amino acid sequences shown in SEQ ID NO:7, 24, 25, 26 or 27, SEQ ID NO:8, 16, 28, 29 or 30 and SEQ ID NO:9, 17, 31 or 32, respectively, by amino acid additions, deletions or substitutions of only one amino acid.
In some embodiments, CDR1 differs in amino acid sequence from the sequence shown in SEQ ID NO. 7 by NO more than 2 (e.g., 0, 1 or 2) amino acid additions, deletions or substitutions; CDR2 differs in amino acid sequence from the sequence shown in SEQ ID NO. 8 by amino acid additions, deletions or substitutions of NO more than 2 (e.g., 0, 1 or 2) amino acids; and/or CDR3 differs in amino acid sequence from the sequence shown in SEQ ID NO 9 by amino acid additions, deletions or substitutions of not more than 2 (e.g.0, 1 or 2) amino acids.
In some embodiments, CDR1 differs in amino acid sequence from the sequence shown in SEQ ID NO. 7 by NO more than 2 (e.g., 0, 1 or 2) amino acid additions, deletions or substitutions; CDR2 differs in amino acid sequence from the sequence shown in SEQ ID NO. 16 by amino acid additions, deletions or substitutions of NO more than 2 (e.g., 0, 1 or 2) amino acids; and/or CDR3 differs in amino acid sequence from the sequence shown in SEQ ID NO. 17 by amino acid additions, deletions or substitutions of not more than 2 (e.g.0, 1 or 2) amino acids.
In some embodiments, CDR1 differs in amino acid sequence from the sequence shown in SEQ ID NO. 24 by NO more than 2 (e.g., 0, 1 or 2) amino acid additions, deletions or substitutions; CDR2 differs in amino acid sequence from the sequence shown in SEQ ID NO. 28 by amino acid additions, deletions or substitutions of NO more than 2 (e.g., 0, 1 or 2) amino acids; and/or CDR3 differs in amino acid sequence from the sequence shown in SEQ ID NO. 31 by amino acid additions, deletions or substitutions of not more than 2 (e.g.0, 1 or 2) amino acids.
In some embodiments, CDR1 differs in amino acid sequence from the sequence shown in SEQ ID NO. 25 by NO more than 2 (e.g., 0, 1 or 2) amino acid additions, deletions or substitutions; CDR2 differs in amino acid sequence from the sequence shown in SEQ ID NO. 16 by amino acid additions, deletions or substitutions of NO more than 2 (e.g., 0, 1 or 2) amino acids; and/or CDR3 differs in amino acid sequence from the sequence shown in SEQ ID NO. 32 by amino acid additions, deletions or substitutions of not more than 2 (e.g., 0, 1 or 2) amino acids.
In some embodiments, CDR1 differs in amino acid sequence from the sequence shown in SEQ ID NO. 26 by NO more than 2 (e.g., 0, 1 or 2) amino acid additions, deletions or substitutions; CDR2 differs in amino acid sequence from the sequence shown in SEQ ID No. 29 by amino acid additions, deletions or substitutions of NO more than 2 (e.g., 0, 1 or 2) amino acids; and/or CDR3 differs in amino acid sequence from the sequence shown in SEQ ID NO. 17 by amino acid additions, deletions or substitutions of not more than 2 (e.g.0, 1 or 2) amino acids.
In some embodiments, CDR1 differs in amino acid sequence from the sequence shown in SEQ ID NO. 27 by NO more than 2 (e.g., 0, 1 or 2) amino acid additions, deletions or substitutions; CDR2 differs in amino acid sequence from the sequence shown in SEQ ID NO. 30 by amino acid additions, deletions or substitutions of NO more than 2 (e.g., 0, 1 or 2) amino acids; and/or CDR3 differs in amino acid sequence from the sequence shown in SEQ ID NO. 17 by amino acid additions, deletions or substitutions of not more than 2 (e.g.0, 1 or 2) amino acids.
Preferably, the CDRs of an isolated antibody or antigen binding portion thereof contain conservative substitutions of no more than 2 amino acids or no more than 1 amino acid. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the basic properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is substituted by another amino acid residue having a similar side chain, e.g., a residue that is physically or functionally similar (e.g., of similar size, shape, charge, chemical nature including the ability to form covalent or hydrogen bonds, etc.) is substituted by the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al, protein Eng.12 (10): 879-884 (1999); and Burks et al, proc. Natl. Acad. Sci. USA 94:412-417 (1997), which is incorporated herein by reference).
In certain embodiments, the immunoglobulin single variable domain of a VEGF antibody comprises: i) As GX 1 X 2 CDR1 of the amino acid sequence shown in LDYYAIG, wherein X 1 F, Q or H, and X 2 T, G, R, K or a; ii) CIGSSY as shown 1 Y 2 Y 3 Y 4 Y 5 CDR2 of the amino acid sequence shown, wherein Y 1 N, S or H, Y 2 S, K or I, Y 3 Is T or E, Y 4 Is T or E, and Y 5 Is N or T; and/or iii) a compound of formula GSPLCLISLQZ 1 Z 2 Z 3 Z 4 CDR3 of the amino acid sequence shown in LYEYDY, wherein Z 1 Is D or V, Z 2 M, H or P, Z 3 Is D or Y, and Z 4 Is S or G.
In certain embodiments, the immunoglobulin single variable domain of a VEGF antibody comprises: i) As GX 1 X 2 CDR1 of the amino acid sequence shown in LDYYAIG, wherein X 1 F, Q or H, and X 2 G, R or K; ii) CIGSSY as shown 1 Y 2 CDR2 of the amino acid sequence shown in ETN, wherein Y 1 S, N or H, Y 2 K or I; and/or iii) a compound of formula GSPLCLISLQZ 1 Z 2 CDR3 of the amino acid sequence shown in YGLYEYDY wherein Z 1 Is D or V, Z 2 H, M or P.
VEGF antibodies comprising CDRs
In some embodiments, a VEGF antibody of the disclosure comprises at least one immunoglobulin single variable domain (e.g., a VHH), wherein the VHH comprises CDR1, CDR2, and CDR3, and wherein CDR1, CDR2, and CDR3 are selected from the group consisting of i) a polypeptide as set forth in SEQ ID NO: 7. 24, 25, 26 or 27; ii) the sequence as set forth in SEQ ID NO: 8. 16, 28, 29 and 30; and/or iii) a polypeptide as set forth in SEQ ID NO: 9. 17, 31 or 32.
In some embodiments, a VEGF antibody of the disclosure comprises at least one immunoglobulin single variable domain (e.g., a VHH), wherein the VHH comprises CDR1, CDR2, and CDR3, and wherein CDR1, CDR2, and CDR3 are selected from the group consisting of:
(a) As set forth in SEQ ID NO:7, as set forth in SEQ ID NO:8 and CDR2 of the amino acid sequence shown as SEQ ID NO:9, CDR3 of the amino acid sequence depicted;
(b) As set forth in SEQ ID NO:7, as set forth in SEQ ID NO:16 and CDR2 of the amino acid sequence shown as SEQ ID NO:17, CDR3 of the amino acid sequence depicted;
(c) As set forth in SEQ ID NO:24, as set forth in SEQ ID NO:28 and CDR2 of the amino acid sequence shown as SEQ ID NO:31, CDR3 of the amino acid sequence depicted;
(d) As set forth in SEQ ID NO:25, as set forth in SEQ ID NO:16 and CDR2 of the amino acid sequence shown as SEQ ID NO:32, CDR3 of the amino acid sequence shown in seq id no;
(e) As set forth in SEQ ID NO:26, as set forth in SEQ ID NO:29 and CDR2 of the amino acid sequence shown as SEQ ID NO:17, CDR3 of the amino acid sequence depicted; or (b)
(f) As set forth in SEQ ID NO:27, as set forth in SEQ ID NO:30 and CDR2 of the amino acid sequence shown as SEQ ID NO:17, and CDR3 of the amino acid sequence depicted in seq id no.
VEGF antibodies defined by VHH sequences
In some embodiments, a VEGF antibody of the disclosure comprises at least one (e.g., one) immunoglobulin single variable domain (e.g., a VHH), wherein the VHH comprises:
(A) The amino acid sequence shown in SEQ ID NO. 6, 12, 15, 20, 21, 22 or 23;
(B) An amino acid sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical to SEQ ID No. 6, 12, 15, 20, 21, 22 or 23; or (b)
(C) Amino acid sequences having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids compared to SEQ ID NOs 6, 12, 15, 20, 21, 22, or 23, preferably, the additions, deletions, and/or substitutions do not occur in the CDR regions.
In other embodiments, the amino acid sequence of the VHH may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to each of the above sequences.
In some further embodiments, the VEGF antibodies of the disclosure may comprise conservative substitutions or modifications of amino acids in the heavy chain variable region. It is understood in the art that certain conservative sequence modifications may be made that do not eliminate antigen binding properties. See, for example, brummel et al (1993) Biochem 32:1180-8; de Wildt et al (1997) Prot.Eng.10:835-41; komissarov et al (1997) J.biol. Chem.272:26864-26870; hall et al (1992) J.Immunol.149:1605-12; kelley and O' Connell (1993) biochem.32:6862-35; adib-Conquy et al (1998) int. Immunol.10:341-6 and beer et al (2000) Clin. Can. Res.6:2835-43.
In some embodiments, the VEGF antibodies of the disclosure may include humanized modifications or removal of glycosylation sites. In some embodiments, VEGF antibodies of the disclosure are modified (e.g., amino acid substitutions and/or additions) at one or more of the following positions corresponding to SEQ ID NO: 6: amino acid 1, 87 or 88, preferably the modification is a humanised modification, preferably the modification is Q1E, K87R or P88A.
Nucleic acid molecules encoding antibodies of the present disclosure and methods of producing VEGF antibodies
In some aspects, the disclosure relates to isolated nucleic acid molecules comprising a nucleic acid sequence encoding a VEGF antibody of the disclosure.
In one embodiment of the present disclosure, the vector may be a mammalian expression vector pcdna3.3 (Invitrogen).
Pharmaceutical composition
In some aspects, the disclosure relates to pharmaceutical compositions comprising at least one VEGF binding molecule as disclosed herein (e.g., a VEGF antibody of the disclosure) and a pharmaceutically acceptable carrier.
Components of pharmaceutical compositions
The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the present disclosure may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the anti-VEGF antibody enhances the immune response of the subject to the vaccine. Pharmaceutically acceptable carriers can include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, nonaqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, combinations of various components known in the art or more.
Administration, formulation and dosage
The pharmaceutical compositions of the present disclosure may be administered to a subject in need thereof in vivo by a variety of routes including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The pharmaceutical compositions of the present disclosure may be formulated as solid, semi-solid, liquid or gaseous forms of formulation; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. The appropriate formulation and route of administration may be selected depending upon the intended application and treatment regimen.
Regardless, the antibodies of the disclosure, or antigen-binding portions thereof, are preferably administered to a subject in need thereof, as desired. The frequency of administration can be determined by one of skill in the art, for example, based on considerations by the treating disorder, the age of the subject being treated, the severity of the disorder being treated, the general health of the subject being treated, and the like.
Application of the invention
The VEGF binding molecules of the invention have a number of in vitro and in vivo uses.
Treatment of disease
As used herein, "angiogenesis" refers to the process by which new blood vessels are formed from pre-existing blood vessels. Disorders associated with pathological angiogenesis may be treated by the compositions and methods of the invention. These include non-neoplastic disorders, cell proliferation disorders, and disorders associated with unwanted vascular permeability, among others.
Non-neoplastic disorders include, but are not limited to, ocular disorders (non-limiting ocular disorders include, for example, retinopathies including proliferative diabetic retinopathy, choroidal Neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemia-related retinopathies, diabetic Macular Edema (DME).
In some embodiments, the disorder associated with pathological angiogenesis is an ocular disorder. In some embodiments, the ocular disorder is AMD, DME, DR, or RVO. In some embodiments, the ocular disorder is AMD. In some embodiments, the AMD is wet AMD.
In some embodiments, the disorder associated with pathological angiogenesis is a cell proliferation disorder. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with a degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is cancer. The term "cancer disease" or "cancer" refers to or describes a physiological condition in an individual that is generally characterized by unregulated cell growth.
It should be appreciated that the above classifications are not mutually exclusive and that a disorder may fall into multiple categories.
Advantages of the invention
The antibody developed in the invention can be a nano antibody targeting VEGF, and the effect of neutralizing VEGF activity in vitro is better than that of Bevacizumab on the marketIn addition, because the anti-VEGF antibody molecular form of the disclosure comprises nano antibodies, compared with Bevacizumab monoclonal antibodies in IgG form, the anti-VEGF antibody molecular form has the characteristics of small molecular weight, no light chain sequence and the like, can solve the problem of light chain mismatch in the aspect of development of bispecific antibodies in the future, and can be combined with anti-other target antibodies in various molecular forms to form bispecific antibodiesTherefore, the monoclonal antibody has very wide application prospect no matter being used as monoclonal antibody medicine, combined medicine and bispecific antibody development.
In some embodiments of the disclosure, antibodies to VEGF are obtained by alpaca immune repertoire screening, and are humanized, glycosylation site removed, and engineered. The current results of the partial candidate antibodies in the experiments to neutralize VEGF show better or at least comparable activity than the control antibodies.
Examples
The invention generally described herein will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. These examples are not intended to be an indication that the experiments below are all or only experiments performed.
Example 1 preparation of raw materials such as antigen, receptor and Positive control antibody
In this example, VEGF antigen protein and its receptor protein VEGFR2 were designed and produced by Sanyou biomedical (Shanghai) Inc. (hereinafter referred to as "Sanyou organism"). Meanwhile, bevacizumab developed by genetec corporation (Genentech) was prepared as a positive control antibody according to the antibody sequence information provided in U.S. patent application (US 20170369564 A1). The following 3 antigen proteins are used together in the present disclosure: human VEGF-Fc (Met 1-Arg 191), human VEGF-His (Met 1-Arg 191) and mouse VEGF-His (Met 1-Arg 190) were all three-major biosignals and preparations, and furthermore, the monkey VEGF antigen sequence was identical to human VEGF, so that no additional preparation was necessary. The ligand proteins used were 1 of the following: human-VEGFR 2-His (Ala 20-Glu 764). The preparation process is as follows: amino acid sequences of human VEGF, mouse VEGF, and human VEGFR2 were obtained from NCBI, wherein the human VEGF sequence was obtained from NCBI Gene ID 7422, the mouse VEGF sequence was obtained from NCBI Gene ID 16542, and the human VEGFR2 sequence was obtained from NCBI Gene ID 3791. The positive control antibody Bevacizumab sequence was obtained from US patent application US20170369564A1. The protein sequences (SEQ ID NOS: 1-5) were obtained according to the amino acid fragment positions, respectively, and after conversion into gene sequences, the target fragment gene synthesis was performed by general biosciences, inc. Each fragment of interest was PCR amplified and then constructed by homologous recombination to eukaryotic expression vector pcdna3.3 (Invitrogen) for expression of the subsequent recombinant protein.
1) Plasmid preparation
And respectively transforming the constructed recombinant protein expression vectors into escherichia coli SS320, culturing overnight at 37 ℃, and then carrying out plasmid extraction by using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain each endotoxin-free plasmid for eukaryotic expression.
2) Protein expression purification
Human VEGF-Fc (Met 1-Arg 191), human VEGF-His (Met 1-Arg 191) and mouse VEGF-His (Met 1-Arg 190) were all expressed by the Expi293 transient expression system (ThermoFisher, A14635). For specific methods, see the transient expression method described in example 1 of chinese patent application CN 111690058A.
3) Protein quality detection
The antigen proteins human-VEGF-Fc (abbreviated as hVEGF-Fc), human-VEGF-His (abbreviated as hVEGF-His) and mouse-VEGF-His (abbreviated as mVEGF-His) prepared in 1.1.2 above were subjected to an inter-binding activity assay with the positive control antibody Bevacizumab by ELISA assay, and the results are shown in FIGS. 1A-1C. As shown in FIGS. 1A-1C, all of hVEGF-His, hVEGF-Fc and mVEGF-His have good binding activity to the control antibody Bevacizumab, and it is confirmed that each antigen protein and the control antibody have good activity. The prepared receptor protein human-VEGFR 2-His (abbreviated as hVEGFR 2-His) and antigen protein hVEGF-Fc were assayed for binding activity by ELISA assay, and the results are shown in FIG. 2. As shown in FIG. 2, hVEGFR2-His has good binding activity to hVEGF-Fc, wherein the binding activity is best at hVEGFR2-His protein coated plate concentrations of 4 μg/ml and 8 μg/ml.
EXAMPLE 2HEK293 VEGFR2/NFAT luciferase reporter cell line construction
In this example, the constructed HEK293 VEGFR2/NFAT luciferase reporter cell line was used to screen candidate antibody molecules capable of neutralizing VEGF activity. A HEK293 NFAT luciferase reporter stable transgenic cell strain prepared by Sanyou organisms is adopted, and on the basis, a full-length Gene sequence (NCBI Gene ID: 3791) of a VEGFR2 receptor is stably transformed, and a monoclonal cell strain is screened. VEGF recombinant protein is added into the cell strain culture system, the transcription and expression of an intracellular NFAT luciferase reporter gene are activated through a VEGF-VEGFR2 signal pathway, and a fluorescent signal is generated through a catalytic substrate added with luciferase. The HEK293 VEGFR2/NFAT luciferase reporter cell strain was prepared as follows:
construction of plasmid expressing full Length human VEGFR2 (Met 1-Val 1356): DNA fragments containing the human VEGFR2 protein sequences were synthesized by gene synthesis techniques and cloned into expression vectors. Coli is introduced by chemical transfection. And (3) sequencing after the E.coli monoclonal is picked to obtain correct plasmid clone, extracting the plasmid and sequencing again for confirmation. Electrotransfection method: HEK293 cells were cultured using DMEM serum-free medium (Gibco, cat# 12634010). The day before electrotransfection, cells were passaged to 2X 10 5 The constructed plasmids were introduced into HEK293 cells at each mL, the next day using an electrotransformation kit (Invitrogen, cat# MPK 10096) and electrotransformation apparatus (Invitrogen, cat# MP 922947). The cells after electrotransformation were transferred to DMEM medium and placed in a 37 ℃ cell incubator for 48 hours. Cell plating after electrotransformation: HEK293 cells after electrotransformation were plated into 96-well plates at 1000 cells/well, puromycin was added at a final concentration of 2. Mu.g/mL, and the plates were placed in a 37℃carbon dioxide incubator for 14 days, followed by supplementation with 2. Mu.g/mL puromycin medium. Clone selection, cell expansion culture and FACS identification: single cell clones grown in 96 well plates were picked, transferred to 24 well plates for continued expansion culture, and then human VEGFR2 stably transformed cell lines were identified by FACS.
EXAMPLE 3VEGF antigen protein immunization of alpaca
In this example, alpaca (Nanchang Dajia technologies Co., ltd.) was immunized by subcutaneous injection and the immunizing antigen used was the recombinant protein hVEGF-His (obtained according to example 1) which was prepared and qualified by three major organisms. The single immunization dose is 500 mug, CFA/IFA (Freund's complete adjuvant and Freund's incomplete adjuvant) is supplemented, immunization is carried out 1 time every 2 weeks, total immunization is carried out 4 times, and the titers of the targeted VEGF recombinant protein antibodies in serum are respectively sampled and detected after the end of 2 times, 3 times and 4 times of immunization. The results are shown in Table 1, and the antibody titer of the targeted VEGF recombinant protein is 12.8 ten thousand at the end of 4 immunizations, so that the immune effect is good, and the targeted VEGF recombinant protein can be used for blood sampling and banking.
Table 1 alpaca serum titers test table
Example 4 phage display library construction and VEGF-targeting nanobody screening
In the embodiment, the alpaca peripheral blood B cell antibody genes immunized by the antigen protein hVEGF-His are cloned, a nanobody gene phage display library is constructed, and recombinant VEGF-His and VEGF-Fc proteins are used as screening antigens to screen the library, so that a plurality of nanobodies which specifically bind the VEGF protein are obtained.
1) Construction of Gene library of camel-derived nanobodies
The method for constructing the nano antibody library is described in example 3 of Chinese patent application CN 111978402A.
2) Screening of antibody Gene phage display libraries
Screening of antibody gene phage display library by magnetic bead method
The screening by the magnetic bead method is based on labeling antigen protein (VEGF) with biotin and then combining with streptavidin-coupled magnetic beads, and the antigen-coupled magnetic beads and antibody gene phage display library are subjected to a panning process of incubation, washing and elution, and are usually subjected to 3-4 rounds of panning, so that specific monoclonal antibodies against the antigen can be enriched in large quantities. In this example, biotin-labeled VEGF-His protein was used for phage display library screening, and after 3 rounds of panning, monoclonal antibody primary screening against VEGF protein was performed. The specific implementation method of antibody screening is as follows:
The biotin-labeled VEGF-His protein was first incubated with streptavidin-conjugated magnetic beads such that the biotin-labeled VEGF-His protein was bound to the magnetic beads. The magnetic beads bound to the biotin-labeled VEGF-His protein and the constructed phage library were incubated for 2h at room temperature. After washing 6-8 times with PBST (phosphate tween buffer), the non-specifically adsorbed phage was removed, pancreatin digest (Gibco, 25200072) was added, gently mixed and reacted for 20min to elute the specifically bound antibody-displaying phage. Subsequently, SS320 cells (Lucigen, MC 1061F) in log phase were infected with the eluted phage and allowed to stand for 30min, then incubated for 1h at 220rpm, VSCM13 helper phage was added and allowed to stand for 30min, further incubation for 1h at 220rpm was continued, centrifuged and replaced into c+/k+2-YT medium (c+k+ is double resistance to carbenicillin 50 μg/mL/kanamycin 40 μg/mL) and the phage finally obtained was continued for the next round of panning.
Screening of antibody Gene phage display library by Immunotubular method
The aim of the immune tube method and the magnetic bead method is to enrich specific antibodies against antigens, and the method is two mutually complementary and verified experimental methods. The principle of the immune tube screening is that VEGF protein is coated on the surface of an immune tube with high adsorption capacity, and a phage display antibody library is added into the immune tube and subjected to a panning process of incubation, washing and elution with antigen protein adsorbed on the surface of the immune tube, and then subjected to 2-4 rounds of panning, and finally, the monoclonal antibody specific to the antigen is enriched. The specific implementation method is as follows:
In the first round of screening, 1mL of 30. Mu.g/mL VEGF-Fc was added to the immune tube, the coating was left overnight at 4 ℃, the coating was discarded the next day, PBS buffer containing 5% skimmed milk was added to seal for 2h, PBS was added after two washes, phage stock containing VEGF nanobody display was added, incubated for 2h, washed 8 times with PBS, then washed 2 times with PBST to remove non-specifically bound phage, then 0.8mL of pancreatin digest containing 0.05% EDTA was added to the immune tube for elution of phage specifically binding to the antigen of interest, then it was infected with logarithmic phase SS320 bacterial cells (Lucigen, 60512-1), allowed to stand for 30min at 37℃and then at 220rpm, VSCM13 helper phage was added, allowed to stand for 30min, further incubation for 1h at 220rpm, centrifugation and displacement into C+/K+2-YT medium, and further incubation was continued overnight at 30℃and 220 rpm. The following day phages were precipitated for the following 2-4 rounds of screening. The antigen coating concentration used for the second and third rounds of phage selection was sequentially decreased, 10. Mu.g/mL and 2. Mu.g/mL, respectively; in addition, the PBS rinse strength was gradually increased, and the PBS elution times were 12 times and 16 times in order.
ELISA detection is carried out on phage pools eluted in each round to evaluate enrichment effect, and a large number of monoclonal antibodies are selected for ELISA primary screening aiming at round with better enrichment, so that 5 nano antibody molecules with higher sequence diversity and VEGF antigen binding are finally obtained.
3) Monoclonal selection
After a total round of screening, the clones obtained in the third round were selected for positive clone screening by ELISA method. After sequencing analysis and ELISA binding analysis, sequences of a plurality of clones are selected to construct a full-length antibody for further experiments, and through verification of the full-length construct molecules, the nano-antibody with the clone number of P30 is selected for subsequent experiments.
EXAMPLE 5 fusion protein construction, expression, and purification
In this example, the nanobody of clone number P30 obtained in example 4 was constructed as a fusion protein containing a human IgG1 subtype Fc fragment by adding an Fc fragment, the fusion protein having no light chain sequence.
1) Plasmid construction
From the nanobody (VHH) strain containing clone number P30 obtained by screening, a nanobody fragment was obtained by PCR amplification. The complete VHH-Fc full-length gene was constructed by homologous recombination onto an engineered eukaryotic expression vector plasmid pcDNA3.3 (Invitrogen, cat. K830001) containing a human heavy chain Fc fragment. SEQ ID NO:6-11 are the variable region amino acid sequence, CDR amino acid sequence, fusion protein full length amino acid sequence and fusion protein full length nucleotide sequence encoding the P30 fusion protein.
2) Plasmid preparation
The constructed P30 VHH-Fc vector was transformed into E.coli SS320 and cultured overnight at 37 ℃. Plasmid extraction was performed using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free antibody plasmids for eukaryotic expression.
3) Expression purification of fusion proteins
The fusion protein clone No. P30 was expressed by the ExpiCHO transient expression system (Thermo Fisher, A29133) as described below in example 4 of the Chinese patent application CN 111978402A.
Example 6 physicochemical Property identification of fusion proteins
In this example, the fusion protein clone No. P30 (hereinafter abbreviated as P30 antibody or candidate antibody P30) prepared in example 5 was examined for its relative molecular weight and purity by SDS-PAGE and SEC-HPLC.
1) SDS-PAGE identification of fusion protein with clone number P30
Sample solution preparation
Preparation of non-reducing solution: clone number P30 fusion protein, control antibody and quality control IPI (IPI is the abbreviation of Ipilimumab, available from BMS company, used as quality control of physicochemical properties such as SDS-PAGE, SEC-HPLC) 1 μg added with 5 XSDS loading buffer and 40mM iodoacetamide, 75 ℃ dry bath heating for 10min, cooling to room temperature, 12000rpm centrifugation for 5min, and collecting supernatant.
Preparation of a reduction solution: the fusion protein with clone number P30, control antibody and quality control IPI 2. Mu.g were added with 5 XSDS loading buffer and 5mM DTT, heated in a dry bath at 100℃for 10min, cooled to room temperature, and centrifuged at 12000rpm for 5min to obtain the supernatant.
Experimental procedure
And (3) carrying out protein electrophoresis by using Bis-tris 4-12% gradient gel (gold Rui, M00654), setting the electrophoresis voltage to be constant voltage of 110V, stopping running when coomassie brilliant blue migrates to the bottom of the gel, taking out the gel sheet, placing the gel sheet into coomassie brilliant blue staining solution for 1-2h, discarding the staining solution, adding a decolorizing solution, replacing the decolorizing solution for 2-3 times as required, decolorizing until the gel background is transparent, and storing in deionized water.
Experimental results
The results are shown in fig. 3. The result shows that the bands of the fusion protein with clone number P30 and the quality control IPI are about 80kD and 150kD respectively, the band of the P30 reducing gum is about 40kD, the quality control IPI is about 55kD and 25kD respectively, the expected sizes are met, the purities are both more than 95%, and the purity of the P30 fusion protein batch sample is 96.10%.
2) Identification of the purity of the fusion protein monomer with clone number P30
In this example, SEC-HPLC was used to detect the monomer purity of the fusion protein clone number P30.
Material preparation
Mobile phase: 150mmol/L phosphate buffer, pH 7.4.
Sample preparation: the fusion protein, the control antibody and the quality control IPI are diluted to 0.5mg/mL by using a mobile phase solution.
Experimental procedure
Agilent HPLC 1100 column (XBIridge BEH SEC 3.5 μm,7.8mm I.D. times.30 cm, waters) flow rate was set at 0.8mL/min, sample volume 20. Mu.L, VWD detector wavelengths 280nm and 214nm. And sequentially feeding a blank solution, an IPI quality control product solution and a sample solution.
Experimental results
The percentage of high molecular weight polymer, fusion protein monomer and low molecular weight material in the sample was calculated according to the area normalization method, and the results are shown in fig. 4. The results show that the purity of the monomer in the fusion protein batch sample is 98.73%, and the purity of the fusion protein is very high.
Example 7 determination of affinity and blocking Activity of fusion proteins
In this example, ELISA-based methods validated the affinity of the fusion protein clone number P30 for human and mouse VEGF antigen proteins, and ELISA-based methods validated the effect of fusion protein P30 blocking VEGF and VEGFR2 binding.
1) ELISA-based detection of the affinity of the fusion protein clone No. P30 to human and mouse VEGF
Human VEGF-His and mouse VEGF-His were coated on 96-well ELISA plates, 2. Mu.g/mL, 30. Mu.L/well, respectively, overnight at 4 ℃. The following day, the well plate was blocked with 5% skim milk for 2h after 3 washes with PBST, and after 3 washes with PBST, gradient diluted fusion protein P30 and positive control antibody Bevacizumab were added and incubated for 1h. After 3 washes with PBST, secondary antibodies (anti-human-IgG-Fc-HRP) were added and incubated for 1h. After incubation, PBST plates were washed six times and developed by addition of TMB (SurModics, TMBS-1000-01). Based on the color development results, the reaction was stopped by adding 2M HCl and the plate read by a microplate reader (Molecular Devices, specterMax 190) at OD450, the results are shown in FIGS. 5A and 5B. The result shows that the fusion protein with clone number P30 has better binding activity with human VEGF and mouse VEGF, and has better affinity with human VEGF than the control antibody Bevacizumab.
2) ELISA-based detection of fusion protein clone number P30 blocks VEGF and VEGFR2 binding
In this example, ELISA plates were incubated overnight at 4deg.C with human VEGFR2-His protein at a dose of 4 μg/mL,30 μl/well. The next day, the well plates were blocked with 5% skim milk for 2h after 3 washes with PBST buffer. Then, the fusion protein P30 or the positive control antibody Bevacizumab is diluted in a gradient way, and is premixed with the biotin-marked human VEGF-Fc for 0.5h in advance, and after the milk blocking of the previous step is finished and the plate washing is finished, the premixed liquid prepared in the step is added into a 96-well ELISA plate, and the incubation is carried out for 1h. After 3 washes with PBST, secondary antibodies (Neutravidin-HRP, therofisther, 31001) were added and incubated for 1h. After incubation, PBST was washed six times with TMB (SurModics, TMBS-1000-01) and the reaction was stopped by adding 2M HCl based on the color development results, and the results are shown in FIG. 6 by reading the plate at OD450 with a microplate reader (Molecular Devices, specterMax 190) which showed that the clone number P30 fusion protein had an effect of blocking human VEGF and VEGFR2 binding superior to the Bevacizumab antibody.
EXAMPLE 8 humanized engineering of clone number P30 fusion protein
In this example, in order to reduce the immunogenicity that may be caused by the camel-derived fusion protein clone number P30, the framework region of the variable region VHH of the P30 fusion protein was subjected to humanized mutation design, and the degree of humanization of the antibody sequence was increased by back mutation.
1) Humanized reconstruction process of fusion protein with clone number P30
Comparing the fusion protein sequence with clone number P30 with a database of human antibody germ line genes (Germline), finding 1-3 germ line genes Germline with relatively high homology with P30, simultaneously taking into account the drug formation of the germ line genes Germline, selecting a proper Germline template for comparison, and finally selecting IGHV3-23 germ line genes Germline. The total number of non-human sites in the framework region of the P30 fusion protein was counted to be 9. Homology modeling is performed on the P30 fusion protein, and the homology modeling refers to a fusion protein result model of a PDB database (http:// www.rcsb.org /). Combining the structure model of P30 and the non-human site situation, carrying out combined back mutation design, avoiding introducing potential post-translational modification sites by the back mutation design, and designing 5 modified molecular sequences with different humanization degrees aiming at P30. Specific methods are described in examples 5-7 by antibody plasmid construction and expression purification procedures, physicochemical properties and ELISA affinity assays.
2) Humanized modification result of fusion protein clone No. P30
Through the detection process of example 8.1, the modified molecule P30-huVH5 is preferably used as a humanized modified molecule, the humanized degree reaches 95.35%, and the physicochemical property and the affinity effect are better than those of the parent P30. Table 2 shows the degree of humanization, relative expression comparison, SDS-PAGE and SEC purity data for the designed fusion proteins. Therefore, the humanization degree, the expression quantity, the monomer purity and the like of the modified molecule P30-huVH5 are greatly improved after the humanization modification; FIG. 7 shows the affinity of the engineered molecule after humanization and the antigen ELISA levels, and shows that the affinity of the engineered molecule P30-huVH5 was increased by more than 2 orders of magnitude over the fusion protein P30 prior to humanization. P30-huVH5 was selected as the preferred humanized engineered molecule, combining degree of humanization and affinity. SEQ ID NO: 12. 7-9, 13-14 respectively encode the heavy chain variable region amino acid sequence, CDR amino acid sequence, fusion protein full length amino acid sequence and fusion protein full length nucleotide sequence of the fusion Fc of the engineered molecule P30-huVH 5.
Table 2P30 fusion protein humanized designs and data results
/>
Example 9 detection of humanized altered molecular neutralization VEGF Activity
In this example, to determine the function of the candidate molecule to neutralize VEGF-VEGFR2 signaling pathway, HEK293VEGFR2/NFAT luciferase reporter cell lines were used as materials to test the ability of the candidate molecule to neutralize VEGF-VEGFR2 binding and thereby block downstream NFAT luciferase reporter expression. The specific implementation mode is as follows:
resuscitating HEK293VEGFR2/NFAT luciferase reporter gene cell strain, subjecting cells with good growth state and passage 2-4 times to experiment, re-suspending cells after pancreatin digestion, centrifuging at normal temperature and 300g to remove supernatant, re-suspending cells with DMEM culture medium, counting, and adjusting cell density to 4×10 5 Each cell/mL was added at 100. Mu.L per well to a fresh 96-well cell culture plate and placed in a 37℃cell incubator. Meanwhile, the antibody P30-huVH5 to be detected and the positive control Bevacizumab are diluted in a gradient manner by using a DMEM culture medium, 60ng/ml VEGF-Fc protein is added, and the mixture is incubated for 30min at room temperature after mixing. Subsequently, the co-incubated mixture of the gradient diluted antibody and VEGF-Fc protein was added to a 96-well cell plate and incubated in an incubator at 37℃for 18 hours. After the culture, 30 mu L of luciferase substrate Bright-Lite (Vazyme, DD 1204-03) is added into each well, the fluorescence value of the 96-well plate is detected after shaking for 2min, and the result shows that the effect of the humanized modified molecule P30-huVH5 for neutralizing VEGF is not shown to be superior to that of a positive control antibody Bevacizumab.
For this case, a round of antibody engineering was designed with the aim of optimizing the neutralising activity of the humanised engineered molecule.
EXAMPLE 10 engineering of humanized engineered molecular antibodies
In view of the results of example 9, this example further antibody engineering was performed on the humanized engineered molecule P30-huVH5 to increase the neutralizing activity of the engineered molecule. The modified molecule obtained by modifying the P30-huVH5 molecule in the pharmacy is named as P30-10 (also called as P30-10-WT), and the pharmacy is improved on the basis of maintaining the affinity of the modified molecule. The amino acid sequence of the variable region of the modified molecule P30-10, the amino acid sequence and the nucleotide sequence of the fusion protein of the fusion Fc are shown as SEQ ID NO: 15. 18 and 19. Then, the engineering of the antibody is carried out based on the P30-10 molecular sequence. The transformation method is based on phage display technology, and comprises library design and construction, screening, functional verification of the transformed molecules and molecular selection.
1) Antibody engineering library design and construction
The antibody engineering library is designed to carry out mutation design aiming at the variable region sequence of the fusion protein, and the mutation of the variable region sequence of different fusion proteins is combined to construct a mutation combination library.
2) Screening of antibody engineering libraries
The specific operation method of library screening is shown in the part of screening of the Chinese library in the example 4, and the screening, the initial screening, the affinity sequencing and the sequence analysis of the library are carried out, and the neutralization activity detection is carried out on 4 clone bacteria expressed VHH supernatants. The results are detailed in Table 3, which shows the ability of the antibody to neutralize VEGF-VEGFR2 signal axis at 5 dilution concentration points ranging from 2 to 0.003. Mu.g/ml, and the HPLC detection value is the expression level detection data of the candidate antibody expression supernatant, and 4 preferred modified molecules P30-10-9, modified molecules P30-10-26, modified molecules P30-10-16 and modified molecules P30-10-25 were selected for sample preparation and functional screening, respectively, in combination with the neutralization activity detection data and sequences.
The sequence SEQ ID NO: 22. 35 and 39 are the amino acid sequence of the modified molecule P30-10-9, the full-length amino acid sequence of the fusion protein of the fusion Fc and the full-length nucleotide sequence of the fusion protein, respectively. The sequence SEQ ID NO: 20. 33, 37 are the amino acid sequence encoding the engineered molecule P30-10-26, the full length amino acid sequence of the fusion protein of the fusion Fc and the full length nucleotide sequence of the fusion protein, respectively. The sequence SEQ ID NO:21 and 23, 34 and 36, 38 and 40 are the fusion protein amino acid sequences of the two engineered molecules, the fusion protein full-length amino acid sequence of the fusion Fc and the fusion protein full-length nucleotide sequence of the fusion protein, encoding the two engineered molecules P30-10-16 and P30-10-25, respectively.
As can be seen from Table 3, under the same protein concentration condition, the modified molecule P30-10-9 blocks the VEGF-VEGFR2 signal path to cause more reduction in fluorescence value, that is, the modified molecule P30-10-9 has stronger capability of neutralizing the signal path, and the modified P30-10-9 antibody has better drug effect than the control antibody Bevacizumab.
Under the condition of the same protein concentration, the fluorescence value caused by blocking the VEGF-VEGFR2 signal path by the modified molecule P30-10-26 is more reduced, namely the capability of neutralizing the signal path by the modified molecule P30-10-26 is stronger, and the drug effect of the modified molecule P30-10-26 antibody is better than that of a control antibody Bevacizumab.
Under the same protein concentration condition, compared with a positive control, the modified molecules P30-10-16 and P30-10-25 block the VEGF-VEGFR2 signal path, namely the modified molecules P30-10-16 and P30-10-25 have stronger capability of neutralizing the signal path, and the modified molecules P30-10-16 and P30-10-25 are better than the control antibody Bevacizumab.
Table 3 antibody engineered 4 post-engineered molecular neutralization VEGF activity assays
3) Engineered molecule preparation after antibody engineering, physicochemical property evaluation, affinity and neutralization activity evaluation
The preparation of the modified molecules and the methods for assessing physicochemical properties are detailed in examples 5 and 6.
Specific procedures for evaluating the affinity of the modified molecular ELISA levels for cross-binding to murine VEGF protein are detailed in example 7.
Evaluation of neutralizing Activity of molecules after modification specific procedures are detailed in example 9.
The molecular affinity kinetics detection method and the molecular affinity kinetics detection result after modification are as follows:
the affinity of the engineered molecules after antibody engineering to VEGF protein was detected using the Fortebio BLItz instrument.
Material preparation
10g of Bovine Serum Albumin (BSA) was weighed, 5mL of Tween 20 was measured, 1000mL of 10 XPBS was added thereto, and the mixture was mixed uniformly to prepare a 10 XKB buffer. Filtering, and packaging for storage. 0.1mL of a glycine solution of 0.1M and pH=2.0 was taken up in 0.9mL of ultrapure water, and mixed well to prepare a sensor regeneration buffer. VEGF-His as antigen was diluted to 10. Mu.g/mL in 10 XKB buffer and the antibody was diluted 2-fold in 10 XKB, followed by 50, 25, 12.5, 0nM.
Experimental procedure
Under dark conditions, a 10 XKB buffer prewetting sensor (Anti-Penta-HIS, HIS1K, fortebio, calif.) was used, and after at least 10min, the sample plate (Greinier Bio, PN 655209) was started and tested error free and then following the pre-set procedure. Firstly, combining the modified molecule and a protein A sensor for 120s, after the combination is completed and the balance is continued for 30s in a 10 XKB buffer solution, transferring the sensor combined with the modified molecule into antigen diluents with different concentrations for combining for 120s, transferring the sensor into the 10 XKB buffer solution after the signal is stable, enabling the dissociation time to be 120s, and finally obtaining K through fitting of the combination dissociation data of the modified molecule with different concentrations D 、K on And K off . The graphs of antigen-antibody binding dissociation are detailed in FIGS. 8A-8G, respectively. As shown, fig. 8A: affinity kinetic data for P30-huVH5 molecules; fig. 8B: affinity kinetic data for engineered molecule P30-10 (P30-10-WT); fig. 8C: affinity kinetic data of Bevacizumab molecule (same batch and same experimental conditions as P30); fig. 8D: affinity kinetic data for P30-10-26 molecules; fig. 8E: affinity kinetic data for P30-10-9 molecules; fig. 8F: affinity kinetic data for P30-10-16 molecules; fig. 8G: affinity kinetic data for P30-10-25 molecules. The affinities of the modified molecule P30-10-WT, the humanized modified molecule P30-huVH5 and the 3 affinity modified molecules P30-10-26, P30-10-16 and P30-10-9 are basically similar or equivalent to those of the control antibody Bevacizumab.
4) Analysis of antibody engineering results
The data of the evaluation of each index of the modified molecule after affinity maturation are detailed in the following table. Based on the affinity of the engineered molecules and the effect of neutralizing VEGF, 4 engineered molecules were selected. The data are summarized in Table 4, with binding affinities to murine VEGF protein shown in FIGS. 9A-9E and VEGF neutralization activity shown in FIGS. 10A-10C. The results are shown below: FIGS. 9A-9B show that the modified molecules P30-10-9 and P30-10-WT and the modified molecules P30-huVH5 have obvious cross-binding activity to the murine VEGF protein, FIG. 9C shows that the modified molecules P30-10-26 have obvious cross-binding to the murine VEGF protein at ELISA level, and FIGS. 9D and 9E show that both the modified molecules P30-10-16 and the modified molecules P30-10-25 have good cross-binding activity to the murine VEGF protein, respectively.
FIG. 10A shows the effect of neutralizing VEGF-VEGFR2 signaling pathway by antibody molecule P30-huVH5, glycosylation site engineered molecule P30-10-WT, antibody engineered molecule P30-10-26, and Bevacizumab, showing that P30-huVH5, glycosylation site engineered molecule P30-10-WT has weaker neutralizing activity, while P30-10-26 neutralizing activity is superior to control antibody Bevacizumab; FIG. 10B shows the effects of engineered antibodies P30-10-16, P30-10-9 and Bevacizumab on neutralizing VEGF, showing that P30-10-16 neutralizing activity is superior to Bevacizumab, while P30-10-9 neutralizing activity is comparable to Bevacizumab; FIG. 10C shows the effects of engineered antibodies P30-10-26, P30-10-25 and Bevacizumab on neutralizing VEGF, showing that P30-10-26 has better neutralizing activity than Bevacizumab and P30-10-25 has weaker neutralizing activity than Bevacizumab. The IC50 values in Table 4 also show that the neutralization activity of the P30-10-26 and P30-10-16 molecules is better than Bevacizumab. The IC50 of the two modified molecules P30-10-26 and P30-10-16 is improved by about 1 time compared with the IC50 value of the control antibody Bevacizumab, and the P30-10-9 molecule is slightly better than the Bevacizumab.
TABLE 4 post-engineering molecular evaluation data after affinity maturation
Example 11 animal model evaluation of the effects of the modified molecules on inhibiting angiogenesis
In this example, 4-6 week old female nude mice (Beijing Vitrending laboratory animal technologies Co., ltd.) were randomly divided into 4 groups of 5 animals each. By intradermal injection 10 into the right ear of mice 7 The pfu Ad-hVEGF165 or equivalent Ad-GFP (and Meta Biotechnology (Shanghai) Inc.) constructs a mouse angiogenesis animal model. After 30 minutes, PBS group B was administered intraperitoneallyThe evacizumab 1mg/kg group, the P30-10-26.53 mg/kg group and the P30-10-26 1mg/kg group are administered in a volume of 10mL/kg for a period of 2 times/week for 4 times. The right ear of the mouse was photographed 1 time/week. After the end of the experiment, animals were euthanized.
And (3) result statistics: at the end of Day14 experiments, scoring reference criteria were set according to abnormal morphology of angiogenesis, scoring was 1-5 points, and abnormal angiogenesis was graded from mild to severe from 1 point to 5 points, and the scoring criteria are shown in fig. 11A. To count the severity of abnormal angiogenesis, 3 technicians were randomly selected to score each mouse experimental condition, and the average score was taken for 3 persons as the final statistics. Compared with the PBS of the control group, the Bevacizumab monoclonal antibody or the P30-10-26 can obviously inhibit the ear angiogenesis of the mice (P < 0.05), and the effect of inhibiting abnormal angiogenesis of the P30-10-26 is obviously better than that of the Bevacizumab under the condition of the same 1mg/kg administration dose, and the result is shown in figure 11B in detail. FIG. 11B shows the effect of engineered molecule P30-10-26 on inhibiting VEGF-mediated angiogenesis in animal models.
Example 12 detection of efficacy of animal model of molecular inhibition Colo205 tumor cells after modification
In this example, the tumor inhibiting effect of 1 engineered molecule P30-10-26 and the positive control antibody Bevacizumab in animals was verified, and the tumor cells used were colon cancer cells Colo205 (ATCC: CCL-222) TM ). Male nude mice (Peking Violet laboratory animal technologies Co., ltd.) of about 20g were used 6-8 weeks old and each nude mouse was subcutaneously injected 5X 10 6 Colo205 cells with tumor volume up to 100mm 3 When the medicine is left and right, the operation of dividing the medicine into component cages and administering medicine is carried out. Each group of 8 tumor-bearing nude mice, 3 groups, including 1 post-engineering molecular group, 1 negative control group, and 1 positive control antibody Bevacizumab group. The administration mode is intraperitoneal injection, the dosage is 3mg/kg, the administration is carried out once every 3-4 days, the administration is carried out 2 times a week, the tumor volume is measured 2 times, and the administration is carried out 9 times/4.5 weeks. Tumor volume (V) calculation mode: v=l×w2/2 (where L is the longest of the tumor diameters and W is the shortest of the tumor diameters). Mice were euthanized 1 week after dosing was completed, tumors were removed and tumor weights were measured. Analysis of tumor volume, tumor weight and mouse weightChange data, calculate tumor inhibition rate, tumor inhibition rate TGI%) = (average tumor volume of 1-experimental group/average tumor volume of PBS control group) ×100%.
The results are shown in FIGS. 12A-12C and Table 5, respectively: the weights of the mice in each group are not obviously different, the tumor growth of the mice in the PBS negative control group is fastest, and compared with the PBS group, the PBS negative control group has obvious tumor inhibiting effect; the tumor volume and tumor weight of the modified molecular P30-10-26 group mice are lower than those of the positive control antibody Bevacizumab group, and the modified molecular P30-10-26 group mice show better tumor inhibition effect.
TABLE 5 tumor rejection percentage TGI (%)
Time (d) PBS Bevacizumab P30-10-26
13 0 22.8858 12.8562
19 0 42.1620 47.7678
22 0 36.8322 54.3292
25 0 47.0534 59.0647
28 0 39.8551 58.2719
32 0 51.5894 64.9276
35 0 49.9552 67.8967
39 0 53.8794 74.3391
42 0 48.6967 73.3581
46 0 48.8583 75.7403
49 0 51.4243 74.2836
TABLE 6 CDR amino acid sequences of antibodies of the disclosure
Table 7 overview of sequences shown in the sequence listing
Those skilled in the art will further recognize that the invention may be embodied in other specific forms without departing from the spirit or central characteristics thereof. Since the foregoing description of the present disclosure discloses only exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims for indicating the scope and content of the invention.
Sequence listing
<110> Sanyou biomedical (Shanghai) Co., ltd
<120> VEGF binding molecules and uses thereof
<130> I2021TC6143CS
<160> 40
<170> PatentIn version 3.5
<210> 1
<211> 191
<212> PRT
<213> Homo sapiens
<400> 1
Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu
1 5 10 15
Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly
20 25 30
Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln
35 40 45
Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu
50 55 60
Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu
65 70 75 80
Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro
85 90 95
Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His
100 105 110
Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys
115 120 125
Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly
130 135 140
Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr
145 150 155 160
Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln
165 170 175
Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg
180 185 190
<210> 2
<211> 190
<212> PRT
<213> Mus musculus
<400> 2
Met Asn Phe Leu Leu Ser Trp Val His Trp Thr Leu Ala Leu Leu Leu
1 5 10 15
Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Thr Thr Glu Gly
20 25 30
Glu Gln Lys Ser His Glu Val Ile Lys Phe Met Asp Val Tyr Gln Arg
35 40 45
Ser Tyr Cys Arg Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr
50 55 60
Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met
65 70 75 80
Arg Cys Ala Gly Cys Cys Asn Asp Glu Ala Leu Glu Cys Val Pro Thr
85 90 95
Ser Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln
100 105 110
Ser Gln His Ile Gly Glu Met Ser Phe Leu Gln His Ser Arg Cys Glu
115 120 125
Cys Arg Pro Lys Lys Asp Arg Thr Lys Pro Glu Asn His Cys Glu Pro
130 135 140
Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr Cys
145 150 155 160
Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu
165 170 175
Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg
180 185 190
<210> 3
<211> 745
<212> PRT
<213> Homo sapiens
<400> 3
Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro Arg Leu Ser
1 5 10 15
Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr Leu Gln Ile
20 25 30
Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro Asn Asn Gln
35 40 45
Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser Asp Gly Leu
50 55 60
Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn Asp Thr Gly
65 70 75 80
Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser Val Ile Tyr
85 90 95
Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp
100 105 110
Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val
115 120 125
Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala
130 135 140
Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp
145 150 155 160
Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile Ser Tyr Ala
165 170 175
Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser
180 185 190
Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val
195 200 205
Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val
210 215 220
Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn
225 230 235 240
Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg
245 250 255
Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr
260 265 270
Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys
275 280 285
Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg
290 295 300
Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu
305 310 315 320
Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu
325 330 335
Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro Leu
340 345 350
Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr Ile Met Glu
355 360 365
Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu Thr Asn Pro
370 375 380
Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val Val Tyr Val
385 390 395 400
Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val Asp Ser Tyr
405 410 415
Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr Ala Ile Pro
420 425 430
Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu Glu Cys Ala
435 440 445
Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr Pro Cys Glu
450 455 460
Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys Ile Glu Val
465 470 475 480
Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys Thr Val Ser
485 490 495
Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr Lys Cys Glu
500 505 510
Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser Phe His Val
515 520 525
Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln Pro Thr Glu
530 535 540
Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser Thr Phe Glu
545 550 555 560
Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro Ile His Val
565 570 575
Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr Leu Trp Lys
580 585 590
Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile Met
595 600 605
Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr Val Cys Leu
610 615 620
Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val Arg Gln Leu
625 630 635 640
Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn Leu Glu Asn
645 650 655
Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys Thr Ala Ser
660 665 670
Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn Glu Thr Leu
675 680 685
Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg Asn Leu Thr
690 695 700
Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr Cys Gln Ala
705 710 715 720
Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe Ile Ile Glu
725 730 735
Gly Ala Gln Glu Lys Thr Asn Leu Glu
740 745
<210> 4
<211> 453
<212> PRT
<213> Artificial Sequence
<220>
<223> Bevacizumab-HC
<400> 4
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe
50 55 60
Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
225 230 235 240
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
305 310 315 320
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
355 360 365
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
385 390 395 400
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
405 410 415
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
435 440 445
Leu Ser Pro Gly Lys
450
<210> 5
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> Bevacizumab-LC
<400> 5
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile
35 40 45
Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 6
<211> 129
<212> PRT
<213> Artificial Sequence
<220>
<223> P30 variable region
<400> 6
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Asn Ser Thr Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp Met Asp Ser
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 7
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> P30 CDR1
<400> 7
Gly Phe Thr Leu Asp Tyr Tyr Ala Ile Gly
1 5 10
<210> 8
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> P30 CDR2
<400> 8
Cys Ile Gly Ser Ser Asn Ser Thr Thr Asn
1 5 10
<210> 9
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> P30 CDR3
<400> 9
Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp Met Asp Ser Leu Tyr
1 5 10 15
Glu Tyr Asp Tyr
20
<210> 10
<211> 361
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-Fc
<400> 10
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Asn Ser Thr Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp Met Asp Ser
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
130 135 140
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155 160
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
165 170 175
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
180 185 190
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
195 200 205
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
210 215 220
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
225 230 235 240
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
245 250 255
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
260 265 270
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
275 280 285
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
305 310 315 320
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
325 330 335
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
340 345 350
Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 11
<211> 1083
<212> DNA
<213> Artificial Sequence
<220>
<223> P30-Fc nucleotide sequence
<400> 11
caggtccagt tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggatt cactttggat tattatgcca taggctggtt ccgccaggcc 120
ccagggaagg agcgtgaggg ggtctcatgt attggtagta gtaatagtac cacaaactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acagccgttt attactgtac agcaggctcc 300
cccctctgtc ttattagtct tcaggacatg gattccctct atgagtacga ctactggggc 360
caggggaccc tggtcaccgt ctcctcagag cctaagagct gcgacaagac ccacacctgt 420
cctccatgtc ctgctccaga actgctcggc ggaccttccg tgttcctgtt tcctccaaag 480
cctaaggaca ccctgatgat cagcagaacc cctgaagtga cctgcgtggt ggtggatgtg 540
tcccacgagg atcccgaagt gaagttcaat tggtacgtgg acggcgtgga agtgcacaac 600
gccaagacca agcctagaga ggaacagtac aacagcacct acagagtggt gtccgtgctg 660
accgtgctgc accaggattg gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 720
gccctgcctg ctcctatcga gaaaaccatc agcaaggcca agggccagcc tagggaaccc 780
caggtttaca cactgcctcc aagcagggac gagctgacca agaatcaggt gtccctgacc 840
tgcctggtca agggcttcta cccttccgat atcgccgtgg aatgggagag caatggccag 900
cctgagaaca actacaagac aacccctcct gtgctggaca gcgacggctc attcttcctg 960
tacagcaagc tgacagtgga caagagcaga tggcagcagg gcaacgtgtt cagctgcagc 1020
gtgatgcacg aggccctgca caaccactac acccagaagt ccctgagcct gtctcctggc 1080
aaa 1083
<210> 12
<211> 129
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-hVH5 variable region
<400> 12
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Asn Ser Thr Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp Met Asp Ser
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 13
<211> 361
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-hVH5-Fc
<400> 13
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Asn Ser Thr Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp Met Asp Ser
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
130 135 140
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155 160
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
165 170 175
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
180 185 190
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
195 200 205
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
210 215 220
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
225 230 235 240
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
245 250 255
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
260 265 270
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
275 280 285
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
305 310 315 320
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
325 330 335
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
340 345 350
Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 14
<211> 1083
<212> DNA
<213> Artificial Sequence
<220>
<223> P30-hVH5-Fc nucleotide sequence
<400> 14
gaggtgcagc tggtggagtc cggaggagga ctggtgcagc ctggcggcag cctgaggctg 60
tcttgcgccg cttccggctt caccctggac tactatgcca tcggctggtt tagacaggct 120
cctggcaagg agagggaggg cgtgagctgt atcggctcca gcaactctac cacaaattat 180
gccgactccg tgaagggcag gttcaccatc agccgggata acgctaagaa tacagtgtac 240
ctgcagatga actctctgag agccgaggac accgccgtgt actattgcac agccggcagc 300
ccactgtgcc tgatctctct gcaggacatg gattccctgt acgagtatga ttactggggc 360
cagggcaccc tggtgacagt gtcttccgag cctaagagct gcgacaagac ccacacctgt 420
cctccatgtc ctgctccaga actgctcggc ggaccttccg tgttcctgtt tcctccaaag 480
cctaaggaca ccctgatgat cagcagaacc cctgaagtga cctgcgtggt ggtggatgtg 540
tcccacgagg atcccgaagt gaagttcaat tggtacgtgg acggcgtgga agtgcacaac 600
gccaagacca agcctagaga ggaacagtac aacagcacct acagagtggt gtccgtgctg 660
accgtgctgc accaggattg gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 720
gccctgcctg ctcctatcga gaaaaccatc agcaaggcca agggccagcc tagggaaccc 780
caggtttaca cactgcctcc aagcagggac gagctgacca agaatcaggt gtccctgacc 840
tgcctggtca agggcttcta cccttccgat atcgccgtgg aatgggagag caatggccag 900
cctgagaaca actacaagac aacccctcct gtgctggaca gcgacggctc attcttcctg 960
tacagcaagc tgacagtgga caagagcaga tggcagcagg gcaacgtgtt cagctgcagc 1020
gtgatgcacg aggccctgca caaccactac acccagaagt ccctgagcct gtctcctggc 1080
aaa 1083
<210> 15
<211> 129
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10 variable region
<400> 15
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Asn Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp Met Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 16
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10 CDR2
<400> 16
Cys Ile Gly Ser Ser Asn Lys Glu Thr Asn
1 5 10
<210> 17
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10 CDR3
<400> 17
Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp Met Tyr Gly Leu Tyr
1 5 10 15
Glu Tyr Asp Tyr
20
<210> 18
<211> 361
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-Fc
<400> 18
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Asn Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp Met Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
130 135 140
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155 160
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
165 170 175
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
180 185 190
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
195 200 205
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
210 215 220
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
225 230 235 240
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
245 250 255
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
260 265 270
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
275 280 285
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
305 310 315 320
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
325 330 335
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
340 345 350
Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 19
<211> 1083
<212> DNA
<213> Artificial Sequence
<220>
<223> P30-10-Fc nucleotide sequence
<400> 19
gaggtgcagc tggttgaatc tggcggagga ttggttcagc ctggcggctc tctgagactg 60
tcttgtgccg cttctggctt caccctggac tactacgcca tcggctggtt cagacaggcc 120
cctggcaaag agagagaggg cgtttcctgt atcggctcct ccaacaagga gaccaactac 180
gccgactccg tgaagggcag attcaccatc tccagagaca acgccaagaa caccgtgtac 240
ctgcagatga actccctgag agccgaggac accgccgtgt actactgtac cgctggctct 300
cctctgtgcc tgatctccct gcaagacatg tatgggctgt acgagtacga ctactggggc 360
cagggcacac tggtcacagt ctcttctgag cctaagagct gcgacaagac ccacacctgt 420
cctccatgtc ctgctccaga actgctcggc ggaccttccg tgttcctgtt tcctccaaag 480
cctaaggaca ccctgatgat cagcagaacc cctgaagtga cctgcgtggt ggtggatgtg 540
tcccacgagg atcccgaagt gaagttcaat tggtacgtgg acggcgtgga agtgcacaac 600
gccaagacca agcctagaga ggaacagtac aacagcacct acagagtggt gtccgtgctg 660
accgtgctgc accaggattg gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 720
gccctgcctg ctcctatcga gaaaaccatc agcaaggcca agggccagcc tagggaaccc 780
caggtttaca cactgcctcc aagcagggac gagctgacca agaatcaggt gtccctgacc 840
tgcctggtca agggcttcta cccttccgat atcgccgtgg aatgggagag caatggccag 900
cctgagaaca actacaagac aacccctcct gtgctggaca gcgacggctc attcttcctg 960
tacagcaagc tgacagtgga caagagcaga tggcagcagg gcaacgtgtt cagctgcagc 1020
gtgatgcacg aggccctgca caaccactac acccagaagt ccctgagcct gtctcctggc 1080
aaa 1083
<210> 20
<211> 129
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-26-variable region
<400> 20
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 21
<211> 129
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-16 variable region
<400> 21
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gln Arg Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Asn Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Val Pro Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 22
<211> 129
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-9 variable region
<400> 22
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly His Lys Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser His Ile Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp Met Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 23
<211> 129
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-25 variable region
<400> 23
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Asn Lys Glu Glu Thr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp Met Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 24
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-26 CDR1
<400> 24
Gly Phe Gly Leu Asp Tyr Tyr Ala Ile Gly
1 5 10
<210> 25
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-16 CDR1
<400> 25
Gly Gln Arg Leu Asp Tyr Tyr Ala Ile Gly
1 5 10
<210> 26
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-9 CDR1
<400> 26
Gly His Lys Leu Asp Tyr Tyr Ala Ile Gly
1 5 10
<210> 27
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-25 CDR1
<400> 27
Gly Phe Ala Leu Asp Tyr Tyr Ala Ile Gly
1 5 10
<210> 28
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-26 CDR2
<400> 28
Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn
1 5 10
<210> 29
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-9 CDR2
<400> 29
Cys Ile Gly Ser Ser His Ile Glu Thr Asn
1 5 10
<210> 30
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-25 CDR2
<400> 30
Cys Ile Gly Ser Ser Asn Lys Glu Glu Thr
1 5 10
<210> 31
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-26 CDR3
<400> 31
Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly Leu Tyr
1 5 10 15
Glu Tyr Asp Tyr
20
<210> 32
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-16 CDR3
<400> 32
Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Val Pro Tyr Gly Leu Tyr
1 5 10 15
Glu Tyr Asp Tyr
20
<210> 33
<211> 361
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-26-Fc
<400> 33
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
130 135 140
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155 160
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
165 170 175
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
180 185 190
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
195 200 205
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
210 215 220
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
225 230 235 240
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
245 250 255
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
260 265 270
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
275 280 285
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
305 310 315 320
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
325 330 335
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
340 345 350
Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 34
<211> 361
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-16-Fc
<400> 34
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gln Arg Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Asn Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Val Pro Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
130 135 140
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155 160
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
165 170 175
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
180 185 190
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
195 200 205
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
210 215 220
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
225 230 235 240
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
245 250 255
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
260 265 270
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
275 280 285
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
305 310 315 320
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
325 330 335
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
340 345 350
Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 35
<211> 361
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-9-Fc
<400> 35
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly His Lys Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser His Ile Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp Met Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
130 135 140
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155 160
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
165 170 175
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
180 185 190
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
195 200 205
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
210 215 220
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
225 230 235 240
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
245 250 255
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
260 265 270
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
275 280 285
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
305 310 315 320
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
325 330 335
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
340 345 350
Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 36
<211> 361
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-25-Fc
<400> 36
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Asn Lys Glu Glu Thr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp Met Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
130 135 140
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155 160
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
165 170 175
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
180 185 190
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
195 200 205
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
210 215 220
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
225 230 235 240
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
245 250 255
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
260 265 270
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
275 280 285
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
305 310 315 320
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
325 330 335
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
340 345 350
Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 37
<211> 1083
<212> DNA
<213> Artificial Sequence
<220>
<223> P30-10-26-Fc nucleotide sequence
<400> 37
gaggtgcagc tggttgaatc tggcggagga ttggttcagc ctggcggctc tctgagactg 60
tcttgtgccg cttctggctt cggtctggac tactacgcca tcggctggtt cagacaggcc 120
cctggcaaag agagagaggg cgtttcctgt atcggctcct cctcgaagga gaccaactac 180
gccgactccg tgaagggcag attcaccatc tccagagaca acgccaagaa caccgtgtac 240
ctgcagatga actccctgag agccgaggac accgccgtgt actactgtac cgctggctct 300
cctctgtgcc tgatctccct gcaagaccat tatgggctgt acgagtacga ctactggggc 360
cagggcacac tggtcacagt ctcttctgag cctaagagct gcgacaagac ccacacctgt 420
cctccatgtc ctgctccaga actgctcggc ggaccttccg tgttcctgtt tcctccaaag 480
cctaaggaca ccctgatgat cagcagaacc cctgaagtga cctgcgtggt ggtggatgtg 540
tcccacgagg atcccgaagt gaagttcaat tggtacgtgg acggcgtgga agtgcacaac 600
gccaagacca agcctagaga ggaacagtac aacagcacct acagagtggt gtccgtgctg 660
accgtgctgc accaggattg gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 720
gccctgcctg ctcctatcga gaaaaccatc agcaaggcca agggccagcc tagggaaccc 780
caggtttaca cactgcctcc aagcagggac gagctgacca agaatcaggt gtccctgacc 840
tgcctggtca agggcttcta cccttccgat atcgccgtgg aatgggagag caatggccag 900
cctgagaaca actacaagac aacccctcct gtgctggaca gcgacggctc attcttcctg 960
tacagcaagc tgacagtgga caagagcaga tggcagcagg gcaacgtgtt cagctgcagc 1020
gtgatgcacg aggccctgca caaccactac acccagaagt ccctgagcct gtctcctggc 1080
aaa 1083
<210> 38
<211> 1083
<212> DNA
<213> Artificial Sequence
<220>
<223> P30-10-16-Fc nucleotide sequence
<400> 38
gaggtgcagc tggttgaatc tggcggagga ttggttcagc ctggcggctc tctgagactg 60
tcttgtgccg cttctggcca gcggctggac tactacgcca tcggctggtt cagacaggcc 120
cctggcaaag agagagaggg cgtttcctgt atcggctcct ccaacaagga gaccaactac 180
gccgactccg tgaagggcag attcaccatc tccagagaca acgccaagaa caccgtgtac 240
ctgcagatga actccctgag agccgaggac accgccgtgt actactgtac cgctggctct 300
cctctgtgcc tgatctccct gcaagttcct tatgggctgt acgagtacga ctactggggc 360
cagggcacac tggtcacagt ctcttctgag cctaagagct gcgacaagac ccacacctgt 420
cctccatgtc ctgctccaga actgctcggc ggaccttccg tgttcctgtt tcctccaaag 480
cctaaggaca ccctgatgat cagcagaacc cctgaagtga cctgcgtggt ggtggatgtg 540
tcccacgagg atcccgaagt gaagttcaat tggtacgtgg acggcgtgga agtgcacaac 600
gccaagacca agcctagaga ggaacagtac aacagcacct acagagtggt gtccgtgctg 660
accgtgctgc accaggattg gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 720
gccctgcctg ctcctatcga gaaaaccatc agcaaggcca agggccagcc tagggaaccc 780
caggtttaca cactgcctcc aagcagggac gagctgacca agaatcaggt gtccctgacc 840
tgcctggtca agggcttcta cccttccgat atcgccgtgg aatgggagag caatggccag 900
cctgagaaca actacaagac aacccctcct gtgctggaca gcgacggctc attcttcctg 960
tacagcaagc tgacagtgga caagagcaga tggcagcagg gcaacgtgtt cagctgcagc 1020
gtgatgcacg aggccctgca caaccactac acccagaagt ccctgagcct gtctcctggc 1080
aaa 1083
<210> 39
<211> 1083
<212> DNA
<213> Artificial Sequence
<220>
<223> P30-10-9-Fc nucleotide sequence
<400> 39
gaggtgcagc tggttgaatc tggcggagga ttggttcagc ctggcggctc tctgagactg 60
tcttgtgccg cttctggcca taagctggac tactacgcca tcggctggtt cagacaggcc 120
cctggcaaag agagagaggg cgtttcctgt atcggctcct cccatattga gaccaactac 180
gccgactccg tgaagggcag attcaccatc tccagagaca acgccaagaa caccgtgtac 240
ctgcagatga actccctgag agccgaggac accgccgtgt actactgtac cgctggctct 300
cctctgtgcc tgatctccct gcaagacatg tatgggctgt acgagtacga ctactggggc 360
cagggcacac tggtcacagt ctcttctgag cctaagagct gcgacaagac ccacacctgt 420
cctccatgtc ctgctccaga actgctcggc ggaccttccg tgttcctgtt tcctccaaag 480
cctaaggaca ccctgatgat cagcagaacc cctgaagtga cctgcgtggt ggtggatgtg 540
tcccacgagg atcccgaagt gaagttcaat tggtacgtgg acggcgtgga agtgcacaac 600
gccaagacca agcctagaga ggaacagtac aacagcacct acagagtggt gtccgtgctg 660
accgtgctgc accaggattg gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 720
gccctgcctg ctcctatcga gaaaaccatc agcaaggcca agggccagcc tagggaaccc 780
caggtttaca cactgcctcc aagcagggac gagctgacca agaatcaggt gtccctgacc 840
tgcctggtca agggcttcta cccttccgat atcgccgtgg aatgggagag caatggccag 900
cctgagaaca actacaagac aacccctcct gtgctggaca gcgacggctc attcttcctg 960
tacagcaagc tgacagtgga caagagcaga tggcagcagg gcaacgtgtt cagctgcagc 1020
gtgatgcacg aggccctgca caaccactac acccagaagt ccctgagcct gtctcctggc 1080
aaa 1083
<210> 40
<211> 1083
<212> DNA
<213> Artificial Sequence
<220>
<223> P30-10-25-Fc nucleotide sequence
<400> 40
gaggtgcagc tggttgaatc tggcggagga ttggttcagc ctggcggctc tctgagactg 60
tcttgtgccg cttctggctt cgctctggac tactacgcca tcggctggtt cagacaggcc 120
cctggcaaag agagagaggg cgtttcctgt atcggctcct ccaacaagga ggagacgtac 180
gccgactccg tgaagggcag attcaccatc tccagagaca acgccaagaa caccgtgtac 240
ctgcagatga actccctgag agccgaggac accgccgtgt actactgtac cgctggctct 300
cctctgtgcc tgatctccct gcaagacatg tatgggctgt acgagtacga ctactggggc 360
cagggcacac tggtcacagt ctcttctgag cctaagagct gcgacaagac ccacacctgt 420
cctccatgtc ctgctccaga actgctcggc ggaccttccg tgttcctgtt tcctccaaag 480
cctaaggaca ccctgatgat cagcagaacc cctgaagtga cctgcgtggt ggtggatgtg 540
tcccacgagg atcccgaagt gaagttcaat tggtacgtgg acggcgtgga agtgcacaac 600
gccaagacca agcctagaga ggaacagtac aacagcacct acagagtggt gtccgtgctg 660
accgtgctgc accaggattg gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 720
gccctgcctg ctcctatcga gaaaaccatc agcaaggcca agggccagcc tagggaaccc 780
caggtttaca cactgcctcc aagcagggac gagctgacca agaatcaggt gtccctgacc 840
tgcctggtca agggcttcta cccttccgat atcgccgtgg aatgggagag caatggccag 900
cctgagaaca actacaagac aacccctcct gtgctggaca gcgacggctc attcttcctg 960
tacagcaagc tgacagtgga caagagcaga tggcagcagg gcaacgtgtt cagctgcagc 1020
gtgatgcacg aggccctgca caaccactac acccagaagt ccctgagcct gtctcctggc 1080
aaa 1083

Claims (29)

  1. A vegf binding molecule comprising at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises:
    (a) As set forth in SEQ ID NO:7, as set forth in SEQ ID NO:8 and CDR2 of the amino acid sequence shown as SEQ ID NO:9, CDR3 of the amino acid sequence depicted;
    (b) As set forth in SEQ ID NO:7, as set forth in SEQ ID NO:16 and CDR2 of the amino acid sequence shown as SEQ ID NO:17, CDR3 of the amino acid sequence depicted;
    (c) As set forth in SEQ ID NO:24, as set forth in SEQ ID NO:28 and CDR2 of the amino acid sequence shown as SEQ ID NO:31, CDR3 of the amino acid sequence depicted;
    (d) As set forth in SEQ ID NO:25, as set forth in SEQ ID NO:16 and CDR2 of the amino acid sequence shown as SEQ ID NO:32, CDR3 of the amino acid sequence shown in seq id no;
    (e) As set forth in SEQ ID NO:26, as set forth in SEQ ID NO:29 and CDR2 of the amino acid sequence shown as SEQ ID NO:17, CDR3 of the amino acid sequence depicted; or (b)
    (f) As set forth in SEQ ID NO:27, as set forth in SEQ ID NO:30 and CDR2 of the amino acid sequence shown as SEQ ID NO:17, and CDR3 of the amino acid sequence depicted in seq id no.
  2. 2. The VEGF binding molecule of claim 1, wherein the VEGF binding molecule is an antibody or antigen-binding fragment thereof directed against VEGF.
  3. 3. The VEGF binding molecule of claim 1, wherein the immunoglobulin single variable domain is a VHH.
  4. 4. The VEGF binding molecule of claim 1, wherein the immunoglobulin single variable domain is a VHH from a camelid.
  5. 5. The VEGF binding molecule of claim 1, wherein the immunoglobulin single variable domain is a VHH from alpaca.
  6. 6. The VEGF binding molecule of claim 1, wherein the immunoglobulin single variable domain comprises or consists of:
    (A) SEQ ID NO: 6. 12, 15, 20, 21, 22 or 23;
    (B) And SEQ ID NO: 6. 12, 15, 20, 21, 22, or 23, at least 80%, 85%, 90%, or 95% identical; or (b)
    (C) And SEQ ID NO: 6. 12, 15, 20, 21, 22 or 23, wherein the addition, deletion and/or substitution does not occur in the CDR regions.
  7. 7. The VEGF binding molecule of claim 1, wherein the immunoglobulin single variable domain comprises or consists of: and SEQ ID NO: 6. 12, 15, 20, 21, 22 or 23, wherein the additions, deletions and/or substitutions do not occur in the CDR regions, have an amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  8. 8. The VEGF binding molecule of claim 1, wherein the immunoglobulin single variable domain is modified at one or more of the following positions corresponding to SEQ ID No. 6: amino acid 1, 87 or 88, wherein the modification is substitution and/or addition of an amino acid.
  9. 9. The VEGF binding molecule of claim 8, wherein the modification is a humanized modification.
  10. 10. The VEGF binding molecule of claim 8, wherein the modification is Q1E, K87R or P88A.
  11. 11. The VEGF binding molecule of any one of claims 1 to 10, wherein the VEGF binding molecule is a single domain antibody, a chimeric antibody, or a humanized antibody.
  12. 12. The VEGF binding molecule of any one of claims 1 to 10, wherein the VEGF binding molecule is a heavy chain single domain antibody.
  13. 13. A VEGF binding molecule as claimed in any one of claims 1 to 10 wherein the immunoglobulin single variable domain is fused to another molecule which is an Fc domain of an immunoglobulin or a fluorescent protein.
  14. 14. A VEGF binding molecule as claimed in any one of claims 1 to 10 wherein the immunoglobulin single variable domain is fused to another molecule which is an Fc domain of IgG.
  15. 15. The VEGF binding molecule of claim 13, wherein the VEGF binding molecule is a chimeric antibody comprising VHH from a camelid and an Fc domain of human IgG.
  16. 16. The VEGF binding molecule of claim 13, wherein the VEGF binding molecule is a chimeric antibody comprising a VHH from a camelid and an Fc domain of human IgG1 or IgG 4.
  17. 17. The VEGF binding molecule of claim 15, wherein the VEGF binding molecule is a chimeric antibody comprising a VHH from alpaca and an Fc domain of human IgG 1.
  18. 18. The VEGF binding molecule of claim 17, wherein the VEGF binding molecule comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 10. 13, 18, 33, 34, 35 or 36.
  19. 19. The VEGF binding molecule of any one of claims 1 to 10, wherein the VEGF binding molecule binds human VEGF.
  20. 20. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the VEGF binding molecule of any one of claims 1 to 19.
  21. 21. The isolated nucleic acid molecule of claim 20, comprising or consisting of the sequence: 11, 14, 19, 37, 38, 39 or 40.
  22. 22. An expression vector comprising the isolated nucleic acid molecule of claim 20 or 21.
  23. 23. A host cell comprising the expression vector of claim 22.
  24. 24. The host cell of claim 23, which is a bacterial cell, a fungal cell, or a mammalian cell.
  25. 25. The host cell of claim 23, wherein the host cell is e.coli (e.coli) or yeast.
  26. 26. A pharmaceutical composition comprising at least one VEGF binding molecule of any one of claims 1-19 and a pharmaceutically acceptable carrier.
  27. 27. A method of preparing a VEGF binding molecule of any one of claims 1 to 19, comprising the steps of:
    -expressing the VEGF binding molecule of any one of claims 1 to 19 in a host cell of any one of claims 23 to 25; and
    -isolating the VEGF binding molecule from the host cell.
  28. 28. Use of a VEGF binding molecule of any one of claims 1-19 in the manufacture of a medicament for treating or preventing a disorder associated with pathological angiogenesis, wherein the disorder associated with pathological angiogenesis is selected from the group consisting of proliferative diabetic retinopathy, choroidal Neovascularization (CNV), age-related macular degeneration (AMD), retinal Vein Occlusion (RVO), diabetic Retinopathy (DR), diabetic Macular Edema (DME), and colon cancer.
  29. 29. A kit for treating or diagnosing a disorder associated with pathological angiogenesis comprising a container comprising the VEGF binding molecule of any one of claims 1-19, wherein the disorder associated with pathological angiogenesis is selected from the group consisting of proliferative diabetic retinopathy, choroidal Neovascularization (CNV), age-related macular degeneration (AMD), retinal Vein Occlusion (RVO), diabetic Retinopathy (DR), diabetic Macular Edema (DME), and colon cancer.
CN202110995278.7A 2021-08-27 2021-08-27 VEGF binding molecules and uses thereof Active CN115724968B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202110995278.7A CN115724968B (en) 2021-08-27 2021-08-27 VEGF binding molecules and uses thereof
PCT/CN2022/115030 WO2023025273A1 (en) 2021-08-27 2022-08-26 Vegf binding molecule and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110995278.7A CN115724968B (en) 2021-08-27 2021-08-27 VEGF binding molecules and uses thereof

Publications (2)

Publication Number Publication Date
CN115724968A CN115724968A (en) 2023-03-03
CN115724968B true CN115724968B (en) 2023-08-08

Family

ID=85290296

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110995278.7A Active CN115724968B (en) 2021-08-27 2021-08-27 VEGF binding molecules and uses thereof

Country Status (2)

Country Link
CN (1) CN115724968B (en)
WO (1) WO2023025273A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116773813B (en) * 2023-06-20 2024-01-30 北京健平金星生物医药有限公司 Preparation and application of VEGFR1 detection kit

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2012201667A1 (en) * 2003-08-01 2012-04-12 Genentech, Inc. Anti-VEGF antibodies
CN106674349A (en) * 2017-03-07 2017-05-17 北京东方百泰生物科技有限公司 Improved anti-VEGFR (vascular endothelial growth factor receptor)-2 monoclonal antibody
CN108101986A (en) * 2015-01-06 2018-06-01 珠海亿胜生物制药有限公司 Anti-VEGF antibody
CN108129564A (en) * 2017-12-17 2018-06-08 北京格根生物科技有限公司 A kind of anti-vegf single-chain antibody in full people source and its application

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7696320B2 (en) * 2004-08-24 2010-04-13 Domantis Limited Ligands that have binding specificity for VEGF and/or EGFR and methods of use therefor
KR20080077261A (en) * 2005-12-06 2008-08-21 도만티스 리미티드 Ligands that have binding specificity for egfr and/or vegf and methods of use therefor
UY32920A (en) * 2009-10-02 2011-04-29 Boehringer Ingelheim Int BISPECIFIC UNION MOLECULES FOR ANTI-ANGIOGENESIS THERAPY
US20120225081A1 (en) * 2010-09-03 2012-09-06 Boehringer Ingelheim International Gmbh Vegf-binding molecules
CN110452297B (en) * 2019-09-03 2020-04-14 上海洛启生物医药技术有限公司 anti-VEGF single domain antibody and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2012201667A1 (en) * 2003-08-01 2012-04-12 Genentech, Inc. Anti-VEGF antibodies
CN108101986A (en) * 2015-01-06 2018-06-01 珠海亿胜生物制药有限公司 Anti-VEGF antibody
CN106674349A (en) * 2017-03-07 2017-05-17 北京东方百泰生物科技有限公司 Improved anti-VEGFR (vascular endothelial growth factor receptor)-2 monoclonal antibody
CN108129564A (en) * 2017-12-17 2018-06-08 北京格根生物科技有限公司 A kind of anti-vegf single-chain antibody in full people source and its application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Production of novel vhh nanobody inhibiting angiogenesis by targeting binding site of VEGF;Walead Ebrahimizadeh wt al;《Applied Biochemistry and Biotechnology》;第176卷;全文 *

Also Published As

Publication number Publication date
WO2023025273A1 (en) 2023-03-02
CN115724968A (en) 2023-03-03

Similar Documents

Publication Publication Date Title
CN112601762B (en) anti-CD 47 antibodies and uses thereof
CN110366560B (en) anti-B7-H4 antibody, antigen binding fragment thereof and medical application thereof
US9840561B2 (en) Human anti-human epidermal growth factor receptor antibody and encoding gene and application thereof
CN112409483A (en) anti-PD-L1 nano antibody
WO2021155635A1 (en) Anti-cd3 and anti-cd123 bispecific antibody and use thereof
JP2020532285A (en) Polypeptides that bind to complement component C5 or serum albumin and their fusion proteins
CN112513088B (en) anti-OX 40 antibodies, antigen binding fragments thereof, and medical uses thereof
WO2022152144A1 (en) Cd73-binding protein and use thereof
WO2022105811A1 (en) Humanized cd19 antibody and use thereof
CN115724968B (en) VEGF binding molecules and uses thereof
WO2021143914A1 (en) Activated anti-ox40 antibody, production method therefor and application thereof
WO2022161454A1 (en) Antigen binding protein and use thereof
CN111662385B (en) Fully human anti-human GPC3 monoclonal antibody and application thereof
JP2023539453A (en) Single variable domain and antigen binding molecule that binds BCMA
KR20170099927A (en) Novel humanized adam17 antibody
KR101854529B1 (en) Antibodies cross-reactive to Human and Mouse Sema3A and Uses thereof
WO2024078558A1 (en) Anti-cd100 antibody and use thereof
EP4292661A1 (en) Anti-vegf antibody and use thereof
CN113164601B (en) Isolated antigen binding proteins and uses thereof
WO2024017326A1 (en) Anti-gprc5d nanobody and use thereof
CN113166264B (en) Isolated antigen binding proteins and uses thereof
WO2023143484A1 (en) Antigen-binding protein and use thereof
WO2022174808A1 (en) ANTIBODY AGAINST IL-13Rα2 AND USE THEREOF
US20180244776A1 (en) Antibody To Be Cross-Linked To Human SEMA3A And Use Thereof
TW202313693A (en) CDC platform antibody

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
PE01 Entry into force of the registration of the contract for pledge of patent right
PE01 Entry into force of the registration of the contract for pledge of patent right

Denomination of invention: VEGF Binding Molecules and Their Applications

Granted publication date: 20230808

Pledgee: Industrial Bank Co.,Ltd. Shanghai Pudong Sub branch

Pledgor: Sanyou biomedical (Shanghai) Co.,Ltd.

Registration number: Y2024310000408