CN110248645B - 基于外来体的抗癌剂 - Google Patents

基于外来体的抗癌剂 Download PDF

Info

Publication number
CN110248645B
CN110248645B CN201780081782.8A CN201780081782A CN110248645B CN 110248645 B CN110248645 B CN 110248645B CN 201780081782 A CN201780081782 A CN 201780081782A CN 110248645 B CN110248645 B CN 110248645B
Authority
CN
China
Prior art keywords
thr
ser
val
gly
pro
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
CN201780081782.8A
Other languages
English (en)
Other versions
CN110248645A (zh
Inventor
高恩梨
李恩贞
梁有秀
金仁山
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.)
Hanshang Mobile Biochemical Co., Ltd.
Original Assignee
Korea Advanced Institute of Science and Technology KAIST
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 Korea Advanced Institute of Science and Technology KAIST filed Critical Korea Advanced Institute of Science and Technology KAIST
Publication of CN110248645A publication Critical patent/CN110248645A/zh
Application granted granted Critical
Publication of CN110248645B publication Critical patent/CN110248645B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A61K47/6913—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10001—Receptor protein-tyrosine kinase (2.7.10.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Wood Science & Technology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Dispersion Chemistry (AREA)
  • Biotechnology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明提供了具有在其表面上呈递的吞噬作用促进蛋白的重组外来体及其用途。

Description

基于外来体的抗癌剂
相关申请的交叉引用
本申请根据35 U.S.C. §119要求于2016年12月29日提交的韩国专利申请号10-2016-0182573的优先权，以及由其产生的所有权益，所述申请的内容通过引用以其整体并入。
技术领域
本发明涉及新型抗癌剂，且更具体地，涉及增加对癌细胞的吞噬作用的新型抗癌剂。
背景
肿瘤通过表达激活和抑制与免疫细胞表面上存在的受体相互作用的配体来避免免疫监视，从而存活和增殖。肿瘤和免疫细胞之间的这种相互作用阻止肿瘤被免疫系统杀死(Pardoll, D.M., Nat. Rev. Cancer. 12: 252-264, 2012)。肿瘤的免疫逃逸的机制之一是CD47的过表达，其允许肿瘤避免先天免疫监视。当CD47结合先天免疫细胞(诸如巨噬细胞)的信号调节蛋白α(SIRPα)时，先天免疫细胞激活“别吃我”信号传导，这导致避免肿瘤被先天免疫细胞本身的吞噬作用，表明CD47-SIRPα相互作用的治疗靶向的充分证据，因为在各种恶性肿瘤细胞中丰富的CD47表达导致癌症患者的存活率更低。由于SIRPα的N-末端含有与CD47的N-末端相互作用的免疫球蛋白超家族V样结构域，所以已开发几种竞争性拮抗剂来阻断所述相互作用。人CD47阻断性单克隆抗体(CD47 mAb)在各种临床前肿瘤模型中证明效力，并触发肿瘤的T细胞介导的免疫原性破坏(Tseng, D.等人, Proc. Natl. Acad. Sci. USA 110: 11103-11108, 2013)。此外，用重组SIRPα蛋白或SIRPα-FC融合蛋白，CD47-SIRPα相互作用可以表现出拮抗作用。然而，由于天然CD47和SIRPα之间的弱相互作用可能限制野生型SIRPα蛋白作为治疗性拮抗剂的有用性，所以开发了SIRPα的高亲和力突变体并且其成功地拮抗癌细胞中的CD47。然而，SIRPα突变体仅充当肿瘤特异性抗体的佐剂，但没有超出预期地刺激吞噬作用和抑制肿瘤生长(Sockolosky, J.T.等人, Proc. Natl. Acad. Sci. USA 113: E2646-E2654, 2016)。在这方面，美国专利公开号2015-0376288公开了通过施用降低受感染细胞中的CD47与宿主吞噬细胞上的SIRPα的结合的试剂来治疗病原体感染的治疗方法。
概述
然而，在上述现有技术的情况下，施用抗CD47剂用于治疗感染性疾病降低SIRPα的结合，这不适合作为化疗的治疗剂。
因此，已进行本发明以解决上述问题，并且本发明的一个目的是提供新型的基于外来体的抗癌剂，其通过增加巨噬细胞和树突细胞对癌细胞的吞噬作用而有效地用于化疗以去除癌细胞。然而，这些问题是示例性的，并且不限制本发明的范围。
在本发明的一个方面，提供了重组外来体，其中在其表面上呈递吞噬作用促进蛋白。
在本发明的另一个方面，提供了用于抗癌疗法的药物组合物，其包含治疗有效量的重组外来体和药学上可接受的载体。
发明效果
根据如上所述的本发明的一个实施方案，可以完成有效的基于外来体的抗癌剂的生产，其中所述抗癌剂的巨噬细胞和树突细胞针对癌细胞的吞噬活性得到改善。具体而言，根据本发明的一个实施方案，其中SIRP存在于其表面上的重组外来体可以通过使用仅少量的外来体阻断SIRP-CD47相互作用来更有效地刺激抗癌免疫应答，因为SIRP在外来体的表面上以脂筏的形式簇集，使得对CD47蛋白具有高结合活性，所述CD47蛋白也在癌细胞表面上以脂筏的形式簇集。然而，本发明的范围不受这些效果的限制。
附图简述
图1是代表根据本发明的一个实施方案的表达SIRPα突变体(SIRPα-外来体)的质粒DNA的结构的示意图。
图2显示：显示根据本发明的一个实施方案制备的重组外来体(SIRPα-外来体)的表达的Western印迹凝胶照片(A)；显示通过流式细胞术检测外来体表面上的SIRPα的表达的柱状图(B)；和显示SIRPα-外来体的图像的电子显微照片(C)。
图3显示：显示根据本发明的一个实施方案制备的SIRPα-外来体的大小的动态光散射(DLS)分析的图(A)；SIRPα-外来体的透射电子显微镜图像(B)；代表通过流式细胞术检测外来体表面上的SIRPα的表达的柱状图(C)；和代表对照-外来体的大小的动态光散射(DLS)分析的结果的图(D)。
图4显示：显示根据本发明的一个实施方案的纯化的重组SIRPα-Myc单体蛋白(mSIRPα)的表达的Western印迹照片(A)；和定量重组SIRPα-外来体的SIRPα的量的图(B)。
图5是显示根据本发明的一个实施方案通过细胞结合测定，分别在HT29(A)、Raji(B)和CT26.CL25细胞(C)的表面上的CD47表达的一系列图。
图6显示：显示根据本发明的一个实施方案通过重组SIRPα的CD47结合活性分析的HT29细胞的荧光强度的图(A)；显示流式细胞术分析的柱状图(B)；和代表分别在Raji和CT26.CL25细胞中检测到的平均荧光强度的图(C)。
图7是显示根据本发明的一个实施方案的重组SIRPα在HT29细胞中的有效结合的一系列荧光显微照片。
图8显示：代表根据本发明的一个实施方案的骨髓衍生的巨噬细胞(BMDM)的分化的FACS分析的柱状图(A)；和代表通过分析细胞的吞噬作用而获得的FACS数据的一系列柱状图(B) 。
图9显示：代表根据本发明的一个实施方案的SIRPα-外来体的吞噬作用增加的图(A)；代表骨髓衍生的巨噬细胞(BMDM)对Raji和CT26.CL25细胞的吞噬作用的分析的图(B)；显示吞噬作用指数(PI)的图(C)和代表被骨髓衍生的巨噬细胞(BMDM)吞噬的HT29细胞的外观的一系列荧光显微图像(D) 。
图10显示：显示施用根据本发明的一个实施方案SIRPα-外来体的携带肿瘤的免疫缺陷小鼠中的肿瘤生长的图(A)；显示携带肿瘤的免疫缺陷小鼠的切除肿瘤的重量的图(B)和显示切除肿瘤的一系列照片(C)。
图11代表根据本发明的一个实施方案的SIRPα-外来体的抗肿瘤作用的分析：(A)显示Cy5.5标记的外来体在携带HT29肿瘤的小鼠中的生物分布的一系列照片；(B)显示注射Cy5.5标记的外来体之后24小时切除的肿瘤的照片；和(C)代表携带HT29肿瘤的小鼠中的平均辐射效率变化的图。
图12是通过将根据本发明的一个实施方案的Cy5.5标记的外来体注射至小鼠中而获得的IVIS图像，其显示检测各个器官中的荧光的结果。
图13代表使用免疫缺陷和免疫活性小鼠的根据本发明的一个实施方案的SIRPα-外来体的抗肿瘤作用：(A)显示施用SIRPα-外来体的携带HT29肿瘤的免疫缺陷BALB/c小鼠中的肿瘤生长抑制的分析的图；(B)显示从免疫缺陷小鼠提取的切除肿瘤的重量的图；(C)显示施用SIRPα-外来体的携带CT26.CL25肿瘤的免疫活性BALB/c小鼠中的肿瘤生长抑制的分析的图；和显示来自携带CT26.CL25肿瘤的小鼠的切除肿瘤的重量的图(D) 。
图14代表根据本发明的一个实施方案的SIRPα-外来体的抗肿瘤作用的分析：(A)显示从HT29小鼠模型切除的肿瘤的一系列照片；和(B)显示从携带CT26.CL25肿瘤的小鼠模型切除的肿瘤的一系列照片。
图15代表根据本发明的一个实施方案的SIRPα-外来体的抗肿瘤作用的分析：(A)显示单体SIRPα蛋白与CD47的结合程度的图；(B)显示HT29细胞中单体SIRPα介导的吞噬活性的图；和(C)显示分别在Raji和CT26.CL25细胞中单体SIRPα介导的吞噬活性的图。
实施方案的详述
术语的定义
如本文所用，术语“外来体”是指由活生物体中的细胞分泌的脂质双层的小体，其执行特定功能，诸如凝血、细胞间信号传导和细胞废物管理。或者，外来体可以被称为“细胞外囊泡”或“细胞衍生的膜囊泡”。已知其在其中具有特定核酸和蛋白并释放至各种体液中。
如本文所用，术语“SIRP(信号调节蛋白)”是指调节性膜糖蛋白，其主要在骨髓细胞中表达并在干细胞或神经元中表达。迄今为止报道了四种SIRP，即SIRPα、SIRPβ、SIRPγ和SIRPδ。其中已知SIRPα和SIRPγ是抑制性受体并与CD47蛋白相互作用，所述CD47蛋白是在各种癌细胞中广泛表达的跨膜蛋白。SIRP和CD47之间的相互作用被称为“别吃我(don'teat me)”信号。这种相互作用负调节先天免疫细胞的效应功能，诸如它们对肿瘤细胞的吞噬作用。这类似于MHC I家族分子经由Ig样或Ly49受体提供的自身信号。过表达CD47的癌细胞激活SIRPα或SIRPγ以抑制巨噬细胞介导的破坏。最近的研究已显示，SIRPα的高亲和力突变体通过掩蔽癌细胞上的CD47来增加对癌细胞的吞噬作用(Weiskopf等人, Science341 (6141): 88-91, 2013)。
如本文所用，术语“受体酪氨酸激酶(RTK)”是指参与细胞增殖、分化、致癌、形态发生等的重要蛋白组，并且RTK包括例如表皮生长因子受体、神经生长因子受体、胰岛素受体和造血干细胞增殖因子受体。只有当与这些生长因子在细胞外结合时，所述受体才激活细胞内酪氨酸激酶并将信号转移至细胞中。
如本文所用，术语“CD-47结合结构域”是指能够结合CD47的N-末端结构域，并且所述N-末端结构域包含多至112a.a.。
发明详述
在本发明的一个方面，提供了重组外来体，其中在其表面上呈递吞噬作用促进蛋白。
根据重组外来体，所述吞噬作用促进蛋白可以是与受体酪氨酸激酶的跨膜结构域的N-末端连接的融合蛋白，并且所述受体酪氨酸激酶可以是血小板衍生的生长因子受体(PDGFR)、表皮生长因子受体(FGFR)、血管内皮生长因子受体(VEGFR)、肝细胞生长因子受体(HGFR)、原肌球蛋白受体激酶(IRK)、胰岛素受体(IR)、白细胞受体酪氨酸激酶、血管生成素受体、受体酪氨酸激酶样孤儿受体(DOR)、网柄菌凝素结构域受体(DDR)、转染期间重排受体(rearranged during transfection receptor, RETR)、与受体酪氨酸激酶相似的酪氨酸蛋白激酶(PTK)或MuSK(肌肉特异性激酶)。
外来体是由细胞产生的一种天然物质，其是生物相容性物质并且可以使免疫反应最小化。外来体的概念中包括“细胞外囊泡”或“细胞衍生的膜囊泡”。在细胞表面上表达的细胞膜蛋白诸如受体可以通过将其与细胞相同的方向定向而呈递于表面上。因此，它是一种在其表面上呈递细胞表面蛋白方面具有很大优势的物质。
根据重组外来体，所述吞噬作用促进蛋白可以是SIRP或SIRP的含有CD47结合结构域的片段，表面活性蛋白A，表面活性蛋白D或抗CD47抗体，并且SIRP可以是SIRPα、SIRPγ或其高亲和力变体。
根据重组外来体，SIRP可以包含SEQ ID NO:1至61中的任一个的氨基酸序列。根据重组外来体，可以进一步包括抗癌剂。所述抗癌剂可以是抗癌蛋白或抗癌化合物。具体而言，上述吞噬作用促进蛋白优选是抗CD47抗体或SIRP蛋白，其可以阻断由于CD47蛋白的簇集导致的信号转导。具体而言，由于血小板衍生的生长因子受体(PDGFR)的跨膜结构域，呈递在外来体表面上的SIRP在外来体表面上以脂筏的形式簇集，且因此对也在癌细胞表面上以脂筏的形式簇集的CD47蛋白具有高结合亲合力，由此使用少量外来体阻断SIRP-CD47相互作用，且更有效地刺激抗癌免疫应答。本发明人首先鉴定了负载在上述外来体上的SIRPα的协同作用。
在重组外来体中，抗癌蛋白可以是天冬酰胺酶、蛋白毒素、对癌抗原特异性的抗体、抗体的片段、肿瘤抑制蛋白或抗血管生成因子。所述蛋白毒素可以选自肉毒杆菌毒素、破伤风毒素、志贺毒素、白喉毒素(DT)、蓖麻毒素、假单胞菌外毒素(PE)、溶细胞素A(ClyA)和γ-白树毒蛋白。肿瘤抑制蛋白是抑制肿瘤发展的蛋白。肿瘤抑制蛋白的实例包括VHL(von Hippel Lindau)、APC (腺瘤性结肠息肉病)、CD95(分化簇95)、ST5(致瘤性抑制5)、YPEL3(Yippee样3)、ST7(致瘤性抑制7)和ST14(致瘤性抑制14)。
根据重组外来体，所述抗癌化合物选自甲氨蝶呤、嘧啶类似物、羟基脲、嘌呤类似物、烷化剂、免疫原性细胞死亡诱导剂、有丝分裂抑制剂、血管生成抑制剂、嵌入剂或放射性核素。
作为抗癌化合物，可以使用以下化合物：
(i)甲氨蝶呤；
(ii)嘧啶类似物
5-氟尿嘧啶、吉西他滨和阿拉伯糖基胞嘧啶；
(iii)羟基脲；
(iv)嘌呤类似物
巯嘌呤和硫鸟嘌呤；
(v)烷化剂
氮芥和环孢酰胺(cyclosporamide)；
(vi)抗生素
蒽环霉素、多柔比星、柔红霉素、伊达比星和放线菌素D；
(vii)有丝分裂抑制剂
长春新碱和紫杉醇；
(viii)抗血管生成剂
抗VEGF抗体、康普瑞汀A4、夫马菌素、除莠霉素A、2-甲氧基雌二醇、OGT 2115、TNP470、曲尼司特、XRP44X、沙利度胺、内皮抑素、salmosin、血管抑制素和纤溶酶原或载脂蛋白的三环结构域；
(ix)嵌入剂
卡铂和顺铂；和
(x)放射性核素
18F、90Y、188Re、32P、89Sr、165Dy、186Re、198Au、153Sm、131I、169Er、125I、99Tc和166Ho等。
根据重组外来体，免疫原性细胞死亡诱导剂可以选自蒽环霉素型抗癌剂、抗EGFR抗体、BK通道激动剂、硼替佐米、强心苷+非免疫原性细胞死亡诱导剂、环磷酰胺、GADD34/PP1抑制剂+丝裂霉素、LV-tSMAC、麻疹病毒和奥沙利铂。根据重组外来体，所述蒽环霉素型抗癌剂可以是柔红霉素、多柔比星、表柔比星、伊达比星、匹克生琼、萨巴比星或戊柔比星。
在本发明的另一个方面，提供了用于治疗受试者中的癌症的药物组合物，其包含治疗有效量的重组外来体和药学上可接受的载体。
根据药物组合物，可以进一步包括一种或多种抗癌剂。含有药学上可接受的载体的组合物可以是各种口服或肠胃外制剂，但其优选是用于肠胃外施用的制剂。为了制备制剂，可以使用稀释剂或赋形剂，诸如填充剂、增量剂、粘合剂、保湿剂、崩解剂、表面活性剂等。用于口服施用的固体制剂可以包括片剂、丸剂、粉剂、颗粒剂、胶囊剂等，并且这些固体制剂可以通过添加至少一种赋形剂(例如淀粉、碳酸钙、蔗糖或乳糖、明胶等)来制备。此外，除了简单的赋形剂以外，还可以使用润滑剂，诸如硬脂酸镁、滑石等。用于口服施用的液体制剂可以包括混悬剂、内用的液体药物、乳剂、糖浆剂等，并且除了常用的简单稀释剂诸如水和液体石蜡以外，还可以使用各种赋形剂，诸如保湿剂、甜味剂、芳香剂和防腐剂。用于肠胃外施用的制剂可以包括无菌水溶液、非水溶剂、混悬剂、乳剂、冻干制剂、栓剂等。非水溶剂和混悬剂的实例可以包括植物油，诸如丙二醇、聚乙二醇和橄榄油；可注射的酯，诸如油酸乙酯；等。栓剂的基质的实例可以包括Witepsol、聚乙二醇、Tween 61、可可脂、laurinum、甘油明胶等。
所述药物组合物可以具有一种选自以下的制剂：片剂、丸剂、粉剂、颗粒剂、胶囊剂、混悬剂、溶液、乳剂、糖浆剂、无菌水溶液、非水溶液、冻干制剂和栓剂。
本公开的药物组合物可以口服或胃肠外施用。当肠胃外施用时，所述药物组合物可以经由各种途径(包括静脉内注射、鼻内吸入、肌肉内施用、腹膜内施用、经皮吸收等)施用。
可以以治疗有效量施用本公开的组合物。
如本文所用，术语“治疗有效量”是指足以以适用于医学治疗的合理利益/风险比治疗疾病的量，并且有效剂量的水平可以基于包括受试者的种类、疾病的严重程度、年龄、性别、药物活性、药物敏感性、施用时间、施用途径和溶解速率、治疗持续时间的因素，包括待同时组合使用的药物的因素，以及在医学领域中众所周知的其他因素来确定。本公开的药物组合物可以以0.1 mg/kg至1 g/kg且更优选地1 mg/kg至500 mg/kg的量施用。同时，可以根据患者的年龄、性别和健康状况适当地调节施用剂量。
本公开的组合物可以作为单独的治疗剂施用，与用于糖尿病或肌肉疾病的其他治疗剂组合施用，或与常规治疗剂依次或同时施用，并且可以施用一次或多次。考虑到上述所有因素，重要的是施用以最小量获得最大效果而没有不利影响的量，并且这些因素可以由本领域普通技术人员容易地确定。
实施本发明的最佳方式
在下文中，将详细描述本公开的优选实施方案。然而，本公开不限于本文中解释的实施方案，但可以在各个方面中指明。反而，提供实施方案以便本公开的构思充分地向本领域技术人员转达其中介绍的彻底和完整的内容。
实施例1：质粒DNA构建
根据本发明的一个实施方案，构建表达SIRPα变体(SIRPα-外来体)的质粒DNA，所述SIRPα变体(SIRPα-外来体)阻断肿瘤细胞表面上的CD47并增加对肿瘤细胞的吞噬作用。具体地，通过基因合成服务(Cosmo Genetech Co.)获得SIRPα突变基因，并将编码SIRPα变体(SEQ ID NO:1)的DNA序列(SEQ ID NO:62)插入血小板衍生生长因子受体(PDGFR)的N-末端信号肽和pDisplay载体的膜锚之间的框架中(图1)。
实施例2：外来体的分离
根据本发明的一个实施方案，将HEK293T细胞(6x106)在补充有10% FBS和1%抗生素的Dulbecco氏改良的Eagle氏培养基(DMEM，4,500 mg/L葡萄糖)中培养，并将细胞培养物维持在37℃和5% CO2下，并在15-cm培养皿中在80-90%汇合度的浓度添加胰岛素-转铁蛋白-硒(Gibco)，并用无血清DMEM替换。从培养基替换起2小时后，根据制造商的说明书，使用转染试剂(Lipofectamine 3000, Invitrogen)，用编码SIRPα变体的质粒DNA(20 μg)转染细胞。转染48小时之后，通过差速离心获得细胞培养上清液以分离外来体，并且详细程序如下：
首先，为了除去含有外来体的培养基中的细胞碎片和其他细胞组分，依次进行以300 xg离心10分钟，以2,000 xg离心10分钟，且以10,000 xg离心30分钟，并且用0.22 μm过滤器过滤培养物，并使用70Ti转子(Beckman Instruments)以36,900 rpm实施超离心2小时。分别地，将所得重组外来体(SIRPα-外来体)重悬浮于含有蛋白酶抑制剂(Roche)的PBS中，并使用BCA蛋白测定试剂盒(Bio-Rad)测量分离的外来体的蛋白浓度。
实施例3：重组外来体的表征
通过western印迹(WB)、流式细胞术、动态光散射(DLS)和透射电子显微镜(TEM)测定根据本发明的一个实施方案制备的重组外来体(SIRPα-外来体)的质量和特征。
首先，对于Western印迹分析，使用含有蛋白酶抑制剂混合物(Calbiochem)的RIPA缓冲液(Cell Signaling Technology)溶解超离心的重组外来体沉淀，并通过SDS-PAGE分析等量的外来体蛋白(10 μg)并转移至硝酸纤维素膜。然后，将抗Myc抗体(1:3000, Abcam,ab9106)和抗HA抗体(1:500, Santa Cruz, sc-805)添加至BLAD以检测SIRPα表达，(1:500,Santa Cruz, sc-99010)，抗Tsg101抗体(1:500, Santa Cruz, sc-22774)和抗CD63抗体(1:500, Santa Cruz, sc-15363)用作外来体标志物。然后将HRP缀合的二抗(1:4,000,Sigma-Aldrich)添加至膜中并通过化学发光可视化。此外，通过流式细胞术分析外来体表面上SIRPα的表达。首先，在室温下将10 μg外来体插入4 μl乙醛/硫酸盐乳胶珠(Invitrogen)中，终体积为1 ml PBS，持续2小时，然后用补充有0.5% BSA的PBS洗涤两次，并通过与Alexa fluor 488-缀合的二抗(1:800, Jackson ImmunoResearch)和抗Myc抗体(1:400, Abcam, ab9106)在4℃下孵育1小时，将SIRPα染色。使用AccuriTM C6流式细胞术分析仪(BD biosciences)和FlowJo_V10软件(FlowJo)分析荧光信号。
另外，如下分析重组外来体的形状：首先，将重组外来体置于配备有碳膜的铜网格(Electron microscopy science)上，并用铀酰乙酸溶液负染。通过免疫电子显微镜证实SIRPα在外来体表面上的呈递。具体地，使用抗Myc抗体(1:100, Abcam, ab9106)和金缀合的抗体(1:50, Aurion)捕获表面SIRPα，并使用透射电子显微镜(Tecnai)获得图像。最后，使用Zetasizer Nano ZS (Malvern Instruments, Ltd., UK)通过动态光散射(DLS)分析重组外来体的大小分布，并且在25℃，173°的固定角度下测量外来体大小(Z-平均值)。
作为结果，观察到纯化的SIRPα-外来体在其膜上具有SIRPα变体并含有外来体标志物蛋白(图2)。而且，透射电子显微镜(TEM)图像和动态光散射(DLS)分析证实重组外来体的平均大小为100 nm，具有圆形形状(图3)。
实施例4：单体SIRPα蛋白的表达和纯化
为了获得根据本发明的一个实施方案的单体SIPRα蛋白(mSIRPα)，使用编码NH2-Nde I-SIRPα变体-Myc-Hind III-COOH)的引物通过PCR扩增获得基因克隆，并将基因克隆用pET-28a质粒载体连接，以表达具有N-末端组氨酸标签的SIRPα。
随后，将转化的细菌细胞在含有卡那霉素的LB培养基中在37℃下培养，直至OD600达到0.5，并添加0.5 mM异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Bioneer, Korea)以诱导蛋白表达。在20℃下孵育18小时之后，将细胞以6,000 xg离心10分钟，使用超声研磨机匀浆，并将沉淀重悬浮于裂解缓冲液(1 M Tris-HCl pH 8.0, 150 mM NaCl)中，并通过Ni-NTA亲和柱色谱纯化mSIRPα，并将抗Myc抗体(1:5,000, Abcam, ab9106)和HRP-缀合的二抗(1:4,000, Sigma Aldrich)用于western印迹分析。
作为结果，从HEK293T细胞获得100 μg总蛋白和2x109 SIRPα-外来体，并且使用来自纯化的重组SIRPα-Myc单体蛋白(mSIRPα)的western印迹图像的标准曲线定量SIRPα的量。定量的SIRPα-外来体的量小于5 ng/1 μg外来体(图4)。关于PCR扩增中使用的引物的信息概述于下表1中。
表 1：用于PCR扩增的引物
引物 核苷酸序列(5’ → 3’) SEQ ID NO
正向 AAA CATATG GAA GAG GAG CTG CAG 67
反向 AAA AAGCTT TCA ATT CAG ATC CTC TTC 68
实施例5：细胞结合测定
通过在补充有10% FBS和1%抗生素的RPMI-1640培养基中培养HT29人结肠腺癌(ATCC)、Raji人B细胞淋巴瘤(ATCC)和CT26.CL25小鼠结肠癌细胞(ATCC)来实施根据本发明的一个实施方案的细胞结合分析，并将培养物维持在37℃和5% CO2下。然后通过添加抗人CD47抗体(B6H12.2, Abcam, ab3283)来培养HT29和Raji细胞，并将CT26.CL25细胞与抗小鼠CD47抗体(Santa Cruz, sc-12731)一起孵育。对于细胞结合测定，将HT29、Raji和CT26.CL25细胞(1x106)在分别添加PBS、外来体或mSIRPα的情况下在4℃下孵育30分钟。然后将细胞与抗Myc抗体(1:400, Abcam, ab9106)一起孵育，并通过添加Alexa fluor 488-缀合的二抗(1:800, Jackson ImmunoResearch)来检测。然后使用AccuriTM C6流式细胞仪(BD Biosciences)测量细胞，并使用FlowJo_V10软件(FlowJo)分析。通过将细胞与抗人CD47抗体(1:100, Abcam, ab3283)预孵育，通过阻断实验分析SIRPα-外来体对CD47的结合特异性。
此外，对于荧光显微镜分析，将HT29细胞(2x105)接种在玻璃底4孔室中并与抗Myc抗体(1:400, Abcam, ab9106)和Alexa fluor 488-缀合的二抗(1:800, JacksonImmunoResearch)孵育。去除残余的非特异性信号之后，将细胞用Hoechst 33258在25℃下固定10分钟，随后在核染色后用4%多聚甲醛固定7分钟。通过荧光显微镜(Nikon EclipseTi, Nikon)检查外来体的细胞结合能力，并使用LAS AF Lite软件(Leica)进行分析。
作为结果，将肿瘤细胞与外来体一起培养以证实SIRPα-外来体拮抗CD47+人肿瘤细胞系中的细胞表面CD47的能力(图5)。此外，与对照组的外来体相比，SIRPα-外来体在HT29肿瘤细胞表面的结合中显示对CD47的更高亲和力，其效果取决于SIRPα-外来体的浓度。通过与抗人CD47抗体预孵育来降低SIRPα-外来体的结合，表明SIRPα-外来体特异性结合肿瘤细胞表面上的CD47。同样，对于SIRPα-外来体与CD47的结合，在人Raji Burkitt氏淋巴瘤(Raji)和小鼠CT26.CL25结肠癌细胞中观察到类似的结果。这些结果表明，外来体中的膜结合的SIRPα变体的表达具有结合癌细胞中的CD47的能力(图6和7)。
实施例6：吞噬作用的分析
根据本发明的一个实施方案，进行吞噬作用的分析以观察具有肿瘤细胞的拮抗活性的CD47的掩蔽是否增加对肿瘤细胞的吞噬作用。具体地，处死BALB/c小鼠，并从腿骨分离骨髓细胞以产生骨髓衍生的巨噬细胞(BMDM)用于体外吞噬作用分析。将分离的BMDM维持在补充有10% FBS和1%抗生素的RPMI培养基中，并用巨噬细胞集落刺激因子(M-CSF)分化7天。通过将BMDM和癌细胞在无血清RPMI培养基中共培养4小时来分析吞噬作用。对于流式细胞术，将分化的巨噬细胞(2.5x105)用0.5 μM CellTracker™ Green染色，并将外来体或mSIRPα蛋白与癌细胞预孵育，且然后将BMDM与混合物孵育4小时。使用AccuriTM C6流式细胞分析仪(BD biosciences)和FlowJo_V10软件(FlowJo)评估作为双阳性信号的百分比的吞噬作用比率。
对于用于测量吞噬作用指数(PI)的荧光显微镜分析，将用CellTrackerTM Green(Thermo fisher scientific)染色的BMDM以2.5x105的密度接种在35 mm玻璃底皿上，并将HT29细胞的混合物(1x106)用pHrodo Deep red (Thermo fisher scientific)染色，并用巨噬细胞处理外来体。在共培养4小时之后，通过荧光显微镜(Nikon Eclipse Ti, Nikon)分析巨噬细胞对HT29细胞的吞噬，作为与巨噬细胞中的吞噬细胞形成相关的红色阳性信号。
作为结果，与对照组(PBS处理或对照外来体处理)相比，在SIRPα-外来体处理组中发现具有双阳性信号(深红色和绿色)的巨噬细胞的吞噬作用以浓度依赖性方式增加(图8)，并且结果在用SIRPα-外来体处理的Raji和CT26.CL25结肠癌细胞中相似。此外，SIRPα-外来体的处理增加对肿瘤细胞的吞噬作用，表明PI在SIRPα-外来体处理组中显著增加(图9)。因此，使用SIRPα-外来体阻断CD47-SIRPα相互作用与骨髓衍生的巨噬细胞(BMDM)对各种癌细胞的吞噬作用的增加相关。
实施例7：体内分布的研究
为了检查根据本发明的一个实施方案的SIRPα-外来体的体内分布，用Cy5.5-NHS标记外来体。在以Cy5.5-NHS染料(1 μg)对100 μg外来体处理之后，将外来体在室温下孵育2小时，并使用空气离心机(airfase centrifuge)(Beckman coulter)离心45分钟。然后在洗涤两次以除去未结合的染料之后将洗涤的外来体沉淀重悬浮于PBS中，并使用荧光微板阅读器(Infinite M200 Pro, TECAN)测量荧光强度并归一化。此外，将Cy5.5标记的外来体(500 μg)、游离染料和PBS分别静脉内施用于携带HT29肿瘤的BALB/c裸小鼠，并将所有样品的荧光强度针对使用用荧光微板阅读器获得的数据的相同值归一化。使用IVIS成像系统(Caliper Life Sciences)在各个时间点(5分钟，2小时，4小时，8小时，16小时和24小时)进行小鼠的体内全身成像。此外，为了分析肿瘤的荧光强度，使用Analysis Workstation软件(Advanced Research Technologies Inc.)计算ROI中每平方厘米每球面度的总光子数。在注射时处死小鼠并且切除肿瘤和主要器官，包括肝、肺、脾、肾和心脏，并以与上述相同的方式分析。
作为结果，在SIRPα-外来体处理组中切除的肿瘤的平均重量显著低于对照组中，与观察到的肿瘤生长的消退一致(图10)。
实施例8：抗肿瘤效应测定
用于根据本发明的一个实施方案的体内实验的免疫缺陷型BALB/c裸小鼠和免疫活性BALB/c小鼠在7周龄时用作异种移植肿瘤模型动物，并在韩国科学技术研究所(KIST)的住宿设施中进行管理。随后，将HT29细胞(1x107)皮下接种在BALB/c裸小鼠的左腿上，并使肿瘤生长一周，且然后分别每3天用对照-外来体、SIRPα-外来体和对照PBS注射五次。然后，为了分析局部抗肿瘤作用，将100 μg外来体注射至小鼠的肿瘤中。对于SIRPα-外来体对肿瘤生长的全身作用，每3天经由尾静脉将外来体(200μg)和PBS静脉内注射至携带HT29肿瘤的小鼠中，进行5次，并且当肿瘤生长至1000 mm3时切除肿瘤并称重。此外，将CT26.CL25细胞(1x106)皮下植入免疫活性BALB/c小鼠的左腿中，并且一周后，通过上述小鼠各自的尾静脉注射200 μg外来体、200 μg SIRPα-外来体、1μg mSIRPα(对应于200 μg SIRPα-外来体中的SIRPα的量)或PBS(分别为每组n=7)，并切除肿瘤并称重。
作为结果，用Cy5.5染料标记的SIRPα-外来体在肿瘤部位连续累积，表明不仅与肿瘤细胞中过表达的CD47的相互作用得到改善，而且纳米颗粒样外来体的渗透性和保留作用也得到改善(图11)。此外，离体图像分析显示Cy5.5标记的外来体在小鼠的肝脏和肾脏中累积，支持整个动物成像分析的结果(图12)。进一步，将SIRPα-外来体在免疫缺陷小鼠中的体内抗癌作用与对照组的体内抗癌作用进行比较，但肿瘤生长没有显示显著差异。另一方面，除了免疫缺陷小鼠以外，还在免疫活性小鼠中分析SIRPα-外来体注射的抗肿瘤作用。作为结果，SIRPα-外来体略微减少肿瘤生长，但不显著，而在携带CT26.CL25肿瘤的免疫活性BALB/c小鼠中施用SIRPα-外来体显示非常好的抗肿瘤作用。这证实通过施用本发明的SIRPα-外体体，SIRPα-外来体经由免疫功能的显著的抗肿瘤作用(图13和14)。另一方面，重组SIRPα蛋白(mSIRPα)的施用没有适当地诱导肿瘤的吞噬作用(图15)，尽管它可以有效地结合癌细胞的CD47。
总之，根据本发明的一个实施方案制备的重组外来体(SIRPα-外来体)阻断CD47-SIRPα相互作用，且因此增加巨噬细胞和树突细胞对肿瘤细胞的吞噬作用，且因此它可以用作用于癌症治疗的新型抗癌剂。
已经参考实施方案描述了本公开内容，但应当理解，本文提供它们用于举例说明的目的，并且在不脱离本公开内容的范围的情况下，各种改变和等同实施方案对于本领域技术人员是可能的。因此，本公开内容的真正保护范围应当由所附权利要求的技术构思确定。
<110> Korea Institute of Science and Technology
<120> 新型的基于外来体的抗癌剂
<130> PO19-5195
<150> KR 10-2016-0182573
<151> 2016-12-29
<150> PCT/KR 2017/015784
<151> 2016-12-29
<160> 68
<170> KoPatentIn 3.0
<210> 1
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 1
Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro
115
<210> 2
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> 人野生型SIRPα
<400> 2
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro
115
<210> 3
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> 野生型SIRPγ
<400> 3
Glu Glu Glu Leu Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr
1 5 10 15
Val Gly Lys Thr Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro
20 25 30
Val Gly Pro Val Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser
65 70 75 80
Ile Thr Pro Ala Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Glu Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met
100 105 110
Ala Leu Gly Ala Lys Pro Ser
115
<210> 4
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SIRPγ变体1(V1)
<400> 4
Glu Glu Glu Leu Gln Ile Ile Gln Pro Glu Lys Leu Leu Leu Val Thr
1 5 10 15
Val Gly Lys Thr Ala Thr Leu His Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Val Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser
65 70 75 80
Ile Thr Pro Ala Asp Val Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Glu Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met
100 105 110
Ala Leu Gly Ala Lys Pro
115
<210> 5
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SIRPγ变体2(V2)
<400> 5
Glu Glu Glu Leu Gln Ile Ile Gln Pro Glu Lys Leu Leu Leu Val Thr
1 5 10 15
Val Gly Lys Thr Ala Thr Leu His Cys Thr Val Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Val Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser
65 70 75 80
Ile Thr Pro Ala Asp Val Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Glu Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met
100 105 110
Ala Leu Gly Ala Lys Pro
115
<210> 6
<211> 117
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 6
Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Ile Ser
50 55 60
Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro
115
<210> 7
<211> 117
<212> PRT
<213> 人工序列
<220>
<223> SiRPα D1结构域
<400> 7
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro
115
<210> 8
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 8
Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Ile Ser
50 55 60
Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 9
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<220>
<221> 变体
<222> (1)
<223> 未知氨基酸
<400> 9
Xaa Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe Gly Asn
1 5 10 15
Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala Gln Asn
20 25 30
Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr
35 40 45
Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser
50 55 60
Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu
65 70 75 80
Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys
85 90 95
Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys
100 105 110
Tyr Arg Val Val Ser Thr Arg
115
<210> 10
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 10
Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Ile Ser
50 55 60
Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 11
<211> 121
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<220>
<221> 变体
<222> (1)
<223> 未知氨基酸
<400> 11
Xaa Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe Gly Asn
1 5 10 15
Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala Gln Asn
20 25 30
Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr
35 40 45
Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser
50 55 60
Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu
65 70 75 80
Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys
85 90 95
Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys
100 105 110
Tyr Arg Val Val Ser Trp Ser Thr Arg
115 120
<210> 12
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 12
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Thr Arg
115 120
<210> 13
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 13
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Thr Arg
115 120
<210> 14
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 14
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Arg Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 15
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 15
Glu Glu Glu Val Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Gly Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 16
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 16
Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Val Ile Leu His Cys Thr Ile Thr Ser Leu Thr Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Leu Leu
35 40 45
Ile Tyr Asn Gln Arg Glu Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Leu Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 17
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 17
Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Ser Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Gly Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Leu Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 18
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 18
Glu Glu Glu Ile Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Val Ile Ile His Cys Thr Val Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Arg Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Gly Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Val Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 19
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 19
Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ile Ile Leu His Cys Thr Val Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Glu Gly Arg Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Gly Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Leu Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 20
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 20
Glu Glu Glu Val Gln Leu Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Glu Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Gly Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 21
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 21
Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser
115
<210> 22
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 22
Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Leu Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 23
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 23
Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Lys Gln Gly Pro Phe Pro Arg Val Thr Thr Ile Ser
50 55 60
Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 24
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 24
Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Thr Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Gly Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 25
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 25
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser
115
<210> 26
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 26
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 27
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 27
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu Leu Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 28
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 28
Glu Glu Gly Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser
115
<210> 29
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 29
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Phe Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser
115
<210> 30
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 30
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Pro Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser
115
<210> 31
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 31
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Gly Lys Pro Ser
115
<210> 32
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 32
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 33
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 33
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser
115
<210> 34
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 34
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 35
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<220>
<221> 变体
<222> (4)
<223> 未知氨基酸
<220>
<221> 变体
<222> (6)
<223> 未知氨基酸
<220>
<221> 变体
<222> (21)
<223> 未知氨基酸
<220>
<221> 变体
<222> (27)
<223> 未知氨基酸
<220>
<221> 变体
<222> (31)
<223> 未知氨基酸
<220>
<221> 变体
<222> (47)
<223> 未知氨基酸
<220>
<221> 变体
<222> (53)..(54)
<223> 未知氨基酸
<220>
<221> 变体
<222> (56)
<223> 未知氨基酸
<220>
<221> 变体
<222> (66)
<223> 未知氨基酸
<220>
<221> 变体
<222> (68)
<223> 未知氨基酸
<220>
<221> 变体
<222> (80)
<223> 未知氨基酸
<220>
<221> 变体
<222> (83)
<223> 未知氨基酸
<220>
<221> 变体
<222> (92)
<223> 未知氨基酸
<220>
<221> 变体
<222> (94)
<223> 未知氨基酸
<220>
<221> 变体
<222> (104)
<223> 未知氨基酸
<400> 35
Glu Glu Glu Xaa Gln Xaa Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Xaa Thr Leu Arg Cys Thr Xaa Thr Ser Leu Xaa Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Xaa Leu
35 40 45
Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Xaa Thr Xaa Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Xaa
65 70 75 80
Ile Thr Xaa Ala Asp Ala Gly Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Xaa Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser
115
<210> 36
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<220>
<221> 变体
<222> (4)
<223> 未知氨基酸
<220>
<221> 变体
<222> (6)
<223> 未知氨基酸
<220>
<221> 变体
<222> (21)
<223> 未知氨基酸
<220>
<221> 变体
<222> (27)
<223> 未知氨基酸
<220>
<221> 变体
<222> (31)
<223> 未知氨基酸
<220>
<221> 变体
<222> (47)
<223> 未知氨基酸
<220>
<221> 变体
<222> (53)..(54)
<223> 未知氨基酸
<220>
<221> 变体
<222> (56)
<223> 未知氨基酸
<220>
<221> 变体
<222> (66)
<223> 未知氨基酸
<220>
<221> 变体
<222> (68)
<223> 未知氨基酸
<220>
<221> 变体
<222> (80)
<223> 未知氨基酸
<220>
<221> 变体
<222> (83)
<223> 未知氨基酸
<220>
<221> 变体
<222> (92)
<223> 未知氨基酸
<220>
<221> 变体
<222> (94)
<223> 未知氨基酸
<220>
<221> 变体
<222> (103)
<223> 未知氨基酸
<400> 36
Glu Glu Glu Xaa Gln Xaa Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Xaa Ile Leu His Cys Thr Xaa Thr Ser Leu Xaa Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Xaa Leu
35 40 45
Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Xaa Thr Xaa Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Xaa
65 70 75 80
Ile Thr Xaa Ala Asp Ala Gly Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Xaa Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 37
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<220>
<221> 变体
<222> (4)
<223> 未知氨基酸
<220>
<221> 变体
<222> (6)
<223> 未知氨基酸
<220>
<221> 变体
<222> (21)
<223> 未知氨基酸
<220>
<221> 变体
<222> (27)
<223> 未知氨基酸
<220>
<221> 变体
<222> (31)
<223> 未知氨基酸
<220>
<221> 变体
<222> (47)
<223> 未知氨基酸
<220>
<221> 变体
<222> (53)..(54)
<223> 未知氨基酸
<220>
<221> 变体
<222> (56)
<223> 未知氨基酸
<220>
<221> 变体
<222> (66)
<223> 未知氨基酸
<220>
<221> 变体
<222> (68)
<223> 未知氨基酸
<220>
<221> 变体
<222> (80)
<223> 未知氨基酸
<220>
<221> 变体
<222> (83)
<223> 未知氨基酸
<220>
<221> 变体
<222> (92)
<223> 未知氨基酸
<220>
<221> 变体
<222> (94)
<223> 未知氨基酸
<220>
<221> 变体
<222> (103)
<223> 未知氨基酸
<400> 37
Glu Glu Glu Xaa Gln Xaa Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Xaa Ile Leu Leu Cys Thr Xaa Thr Ser Leu Xaa Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Xaa Leu
35 40 45
Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Xaa Thr Xaa Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Xaa
65 70 75 80
Ile Thr Xaa Ala Asp Ala Gly Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Xaa Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 38
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<220>
<221> 变体
<222> (4)
<223> 未知氨基酸
<220>
<221> 变体
<222> (6)
<223> 未知氨基酸
<220>
<221> 变体
<222> (21)
<223> 未知氨基酸
<220>
<221> 变体
<222> (27)
<223> 未知氨基酸
<220>
<221> 变体
<222> (31)
<223> 未知氨基酸
<220>
<221> 变体
<222> (47)
<223> 未知氨基酸
<220>
<221> 变体
<222> (53)..(54)
<223> 未知氨基酸
<220>
<221> 变体
<222> (56)
<223> 未知氨基酸
<220>
<221> 变体
<222> (66)
<223> 未知氨基酸
<220>
<221> 变体
<222> (68)
<223> 未知氨基酸
<220>
<221> 变体
<222> (80)
<223> 未知氨基酸
<220>
<221> 变体
<222> (83)
<223> 未知氨基酸
<220>
<221> 变体
<222> (92)
<223> 未知氨基酸
<220>
<221> 变体
<222> (94)
<223> 未知氨基酸
<220>
<221> 变体
<222> (104)
<223> 未知氨基酸
<400> 38
Glu Glu Gly Xaa Gln Xaa Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Xaa Ile Leu His Cys Thr Xaa Thr Ser Leu Xaa Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Xaa Leu
35 40 45
Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Xaa Thr Xaa Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Xaa
65 70 75 80
Ile Thr Xaa Ala Asp Ala Gly Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Xaa Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser
115
<210> 39
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<220>
<221> 变体
<222> (4)
<223> 未知氨基酸
<220>
<221> 变体
<222> (6)
<223> 未知氨基酸
<220>
<221> 变体
<222> (21)
<223> 未知氨基酸
<220>
<221> 变体
<222> (27)
<223> 未知氨基酸
<220>
<221> 变体
<222> (31)
<223> 未知氨基酸
<220>
<221> 变体
<222> (47)
<223> 未知氨基酸
<220>
<221> 变体
<222> (53)..(54)
<223> 未知氨基酸
<220>
<221> 变体
<222> (56)
<223> 未知氨基酸
<220>
<221> 变体
<222> (66)
<223> 未知氨基酸
<220>
<221> 变体
<222> (68)
<223> 未知氨基酸
<220>
<221> 变体
<222> (80)
<223> 未知氨基酸
<220>
<221> 变体
<222> (83)
<223> 未知氨基酸
<220>
<221> 变体
<222> (92)
<223> 未知氨基酸
<220>
<221> 变体
<222> (94)
<223> 未知氨基酸
<220>
<221> 变体
<222> (104)
<223> 未知氨基酸
<400> 39
Glu Glu Glu Xaa Gln Xaa Ile Gln Pro Asp Lys Phe Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Xaa Thr Leu Arg Cys Thr Xaa Thr Ser Leu Xaa Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Xaa Leu
35 40 45
Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Xaa Thr Xaa Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Xaa
65 70 75 80
Ile Thr Xaa Ala Asp Ala Gly Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Xaa Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser
115
<210> 40
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<220>
<221> 变体
<222> (4)
<223> 未知氨基酸
<220>
<221> 变体
<222> (6)
<223> 未知氨基酸
<220>
<221> 变体
<222> (21)
<223> 未知氨基酸
<220>
<221> 变体
<222> (27)
<223> 未知氨基酸
<220>
<221> 变体
<222> (31)
<223> 未知氨基酸
<220>
<221> 变体
<222> (47)
<223> 未知氨基酸
<220>
<221> 变体
<222> (53)..(54)
<223> 未知氨基酸
<220>
<221> 变体
<222> (56)
<223> 未知氨基酸
<220>
<221> 变体
<222> (66)
<223> 未知氨基酸
<220>
<221> 变体
<222> (68)
<223> 未知氨基酸
<220>
<221> 变体
<222> (80)
<223> 未知氨基酸
<220>
<221> 变体
<222> (83)
<223> 未知氨基酸
<220>
<221> 变体
<222> (92)
<223> 未知氨基酸
<220>
<221> 变体
<222> (94)
<223> 未知氨基酸
<220>
<221> 变体
<222> (104)
<223> 未知氨基酸
<400> 40
Glu Glu Glu Xaa Gln Xaa Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Xaa Thr Leu Arg Cys Thr Xaa Thr Ser Leu Xaa Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Xaa Leu
35 40 45
Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Xaa Thr Xaa Arg Asn Asn Met Asp Phe Pro Ile Arg Ile Gly Xaa
65 70 75 80
Ile Thr Xaa Ala Asp Ala Gly Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Xaa Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser
115
<210> 41
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<220>
<221> 变体
<222> (4)
<223> 未知氨基酸
<220>
<221> 变体
<222> (6)
<223> 未知氨基酸
<220>
<221> 变体
<222> (21)
<223> 未知氨基酸
<220>
<221> 变体
<222> (27)
<223> 未知氨基酸
<220>
<221> 变体
<222> (31)
<223> 未知氨基酸
<220>
<221> 变体
<222> (47)
<223> 未知氨基酸
<220>
<221> 变体
<222> (53)..(54)
<223> 未知氨基酸
<220>
<221> 变体
<222> (56)
<223> 未知氨基酸
<220>
<221> 变体
<222> (66)
<223> 未知氨基酸
<220>
<221> 变体
<222> (68)
<223> 未知氨基酸
<220>
<221> 变体
<222> (80)
<223> 未知氨基酸
<220>
<221> 变体
<222> (83)
<223> 未知氨基酸
<220>
<221> 变体
<222> (92)
<223> 未知氨基酸
<220>
<221> 变体
<222> (94)
<223> 未知氨基酸
<220>
<221> 变体
<222> (103)
<223> 未知氨基酸
<400> 41
Glu Glu Glu Xaa Gln Xaa Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Xaa Ile Leu His Cys Thr Xaa Thr Ser Leu Xaa Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Xaa Leu
35 40 45
Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Xaa Thr Xaa Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Xaa
65 70 75 80
Ile Thr Xaa Ala Asp Ala Gly Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Xaa Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Gly Lys Pro Ser
115
<210> 42
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<220>
<221> 变体
<222> (4)
<223> 未知氨基酸
<220>
<221> 变体
<222> (6)
<223> 未知氨基酸
<220>
<221> 变体
<222> (21)
<223> 未知氨基酸
<220>
<221> 变体
<222> (27)
<223> 未知氨基酸
<220>
<221> 变体
<222> (31)
<223> 未知氨基酸
<220>
<221> 变体
<222> (47)
<223> 未知氨基酸
<220>
<221> 变体
<222> (53)..(54)
<223> 未知氨基酸
<220>
<221> 变体
<222> (56)
<223> 未知氨基酸
<220>
<221> 变体
<222> (66)
<223> 未知氨基酸
<220>
<221> 变体
<222> (68)
<223> 未知氨基酸
<220>
<221> 变体
<222> (80)
<223> 未知氨基酸
<220>
<221> 变体
<222> (83)
<223> 未知氨基酸
<220>
<221> 变体
<222> (92)
<223> 未知氨基酸
<220>
<221> 变体
<222> (94)
<223> 未知氨基酸
<220>
<221> 变体
<222> (103)
<223> 未知氨基酸
<400> 42
Glu Glu Glu Xaa Gln Xaa Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Xaa Thr Leu Arg Cys Thr Xaa Thr Ser Leu Xaa Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Xaa Leu
35 40 45
Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Xaa Thr Xaa Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Xaa
65 70 75 80
Ile Thr Xaa Ala Asp Ala Gly Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Xaa Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 43
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<220>
<221> 变体
<222> (4)
<223> 未知氨基酸
<220>
<221> 变体
<222> (6)
<223> 未知氨基酸
<220>
<221> 变体
<222> (21)
<223> 未知氨基酸
<220>
<221> 变体
<222> (27)
<223> 未知氨基酸
<220>
<221> 变体
<222> (31)
<223> 未知氨基酸
<220>
<221> 变体
<222> (47)
<223> 未知氨基酸
<220>
<221> 变体
<222> (53)..(54)
<223> 未知氨基酸
<220>
<221> 变体
<222> (56)
<223> 未知氨基酸
<220>
<221> 变体
<222> (66)
<223> 未知氨基酸
<220>
<221> 变体
<222> (68)
<223> 未知氨基酸
<220>
<221> 变体
<222> (80)
<223> 未知氨基酸
<220>
<221> 变体
<222> (83)
<223> 未知氨基酸
<220>
<221> 变体
<222> (92)
<223> 未知氨基酸
<220>
<221> 变体
<222> (94)
<223> 未知氨基酸
<220>
<221> 变体
<222> (104)
<223> 未知氨基酸
<400> 43
Glu Glu Glu Xaa Gln Xaa Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Xaa Thr Leu Arg Cys Thr Xaa Thr Ser Leu Xaa Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Xaa Leu
35 40 45
Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Xaa Thr Xaa Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Xaa
65 70 75 80
Ile Thr Xaa Ala Asp Ala Gly Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Xaa Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser
115
<210> 44
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<220>
<221> 变体
<222> (4)
<223> 未知氨基酸
<220>
<221> 变体
<222> (6)
<223> 未知氨基酸
<220>
<221> 变体
<222> (21)
<223> 未知氨基酸
<220>
<221> 变体
<222> (27)
<223> 未知氨基酸
<220>
<221> 变体
<222> (31)
<223> 未知氨基酸
<220>
<221> 变体
<222> (47)
<223> 未知氨基酸
<220>
<221> 变体
<222> (53)..(54)
<223> 未知氨基酸
<220>
<221> 变体
<222> (56)
<223> 未知氨基酸
<220>
<221> 变体
<222> (66)
<223> 未知氨基酸
<220>
<221> 变体
<222> (68)
<223> 未知氨基酸
<220>
<221> 变体
<222> (80)
<223> 未知氨基酸
<220>
<221> 变体
<222> (83)
<223> 未知氨基酸
<220>
<221> 变体
<222> (92)
<223> 未知氨基酸
<220>
<221> 变体
<222> (94)
<223> 未知氨基酸
<220>
<221> 变体
<222> (103)
<223> 未知氨基酸
<400> 44
Glu Glu Glu Xaa Gln Xaa Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Xaa Ile Leu His Cys Thr Xaa Thr Ser Leu Xaa Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Xaa Leu
35 40 45
Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Xaa Thr Xaa Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Xaa
65 70 75 80
Ile Thr Xaa Ala Asp Ala Gly Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Xaa Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 45
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<220>
<221> 变体
<222> (3)..(4)
<223> 未知氨基酸
<220>
<221> 变体
<222> (6)
<223> 未知氨基酸
<220>
<221> 变体
<222> (12)
<223> 未知氨基酸
<220>
<221> 变体
<222> (14)
<223> 未知氨基酸
<220>
<221> 变体
<222> (20)..(22)
<223> 未知氨基酸
<220>
<221> 变体
<222> (24)
<223> 未知氨基酸
<220>
<221> 变体
<222> (27)
<223> 未知氨基酸
<220>
<221> 变体
<222> (31)
<223> 未知氨基酸
<220>
<221> 变体
<222> (45)
<223> 未知氨基酸
<220>
<221> 变体
<222> (47)
<223> 未知氨基酸
<220>
<221> 变体
<222> (53)..(54)
<223> 未知氨基酸
<220>
<221> 变体
<222> (56)
<223> 未知氨基酸
<220>
<221> 变体
<222> (65)..(66)
<223> 未知氨基酸
<220>
<221> 变体
<222> (68)
<223> 未知氨基酸
<220>
<221> 变体
<222> (70)
<223> 未知氨基酸
<220>
<221> 变体
<222> (75)
<223> 未知氨基酸
<220>
<221> 变体
<222> (77)
<223> 未知氨基酸
<220>
<221> 变体
<222> (79)..(80)
<223> 未知氨基酸
<220>
<221> 变体
<222> (83)
<223> 未知氨基酸
<220>
<221> 变体
<222> (92)
<223> 未知氨基酸
<220>
<221> 变体
<222> (94)
<223> 未知氨基酸
<220>
<221> 变体
<222> (101)..(102)
<223> 未知氨基酸
<220>
<221> 变体
<222> (104)
<223> 未知氨基酸
<220>
<221> 变体
<222> (116)
<223> 未知氨基酸
<400> 45
Glu Glu Xaa Xaa Gln Xaa Ile Gln Pro Asp Lys Xaa Val Xaa Val Ala
1 5 10 15
Ala Gly Glu Xaa Xaa Xaa Leu Xaa Cys Thr Xaa Thr Ser Leu Xaa Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Xaa Arg Xaa Leu
35 40 45
Ile Tyr Asn Gln Xaa Xaa Gly Xaa Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Xaa Xaa Thr Xaa Arg Xaa Asn Met Asp Phe Xaa Ile Xaa Ile Xaa Xaa
65 70 75 80
Ile Thr Xaa Ala Asp Ala Gly Thr Tyr Tyr Cys Xaa Lys Xaa Arg Lys
85 90 95
Gly Ser Pro Asp Xaa Xaa Glu Xaa Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Xaa Lys Pro Ser
115
<210> 46
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 46
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Arg Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 47
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 47
Glu Glu Glu Val Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Gly Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 48
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 48
Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Val Ile Leu His Cys Thr Ile Thr Ser Leu Thr Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Leu Leu
35 40 45
Ile Tyr Asn Gln Arg Glu Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Leu Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 49
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 49
Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Ser Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Gly Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Leu Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 50
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 50
Glu Glu Glu Ile Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Val Ile Ile His Cys Thr Val Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Arg Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Gly Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Val Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 51
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 51
Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ile Ile Leu His Cys Thr Val Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Glu Gly Arg Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Gly Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Leu Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 52
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 52
Glu Glu Glu Val Gln Leu Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Glu Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Gly Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 53
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 53
Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser
115
<210> 54
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 54
Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Leu Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 55
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 55
Glu Glu Glu Val Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Lys Gln Gly Pro Phe Pro Arg Val Thr Thr Ile Ser
50 55 60
Glu Thr Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 56
<211> 118
<212> PRT
<213> 人工序列
<220>
<223> SiRPα变体
<400> 56
Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Thr Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Gly Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 57
<211> 474
<212> PRT
<213> 人工序列
<220>
<223> 人野生型SIRP-α(h-wa)完全序列
<400> 57
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala
115 120 125
Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly
130 135 140
Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu
145 150 155 160
Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser
165 170 175
Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val
180 185 190
His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp
195 200 205
Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro
210 215 220
Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn
225 230 235 240
Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr
245 250 255
Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val
260 265 270
Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val
275 280 285
Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu
290 295 300
His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser
305 310 315 320
Ala His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly
325 330 335
Ser Asn Glu Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr Leu
340 345 350
Leu Val Ala Leu Leu Met Ala Ala Leu Tyr Leu Val Arg Ile Arg Gln
355 360 365
Lys Lys Ala Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu
370 375 380
Lys Asn Ala Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala
385 390 395 400
Asp Leu Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu
405 410 415
Pro Asn Asn His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro
420 425 430
Ala Ser Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu
435 440 445
Asn Arg Thr Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser
450 455 460
Glu Tyr Ala Ser Val Gln Val Pro Arg Lys
465 470
<210> 58
<211> 474
<212> PRT
<213> 人工序列
<220>
<223> 变体SIRP-α(va)完全序列
<400> 58
Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala
115 120 125
Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly
130 135 140
Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu
145 150 155 160
Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser
165 170 175
Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val
180 185 190
His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp
195 200 205
Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro
210 215 220
Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn
225 230 235 240
Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr
245 250 255
Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val
260 265 270
Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val
275 280 285
Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu
290 295 300
His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser
305 310 315 320
Ala His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly
325 330 335
Ser Asn Glu Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr Leu
340 345 350
Leu Val Ala Leu Leu Met Ala Ala Leu Tyr Leu Val Arg Ile Arg Gln
355 360 365
Lys Lys Ala Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu
370 375 380
Lys Asn Ala Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala
385 390 395 400
Asp Leu Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu
405 410 415
Pro Asn Asn His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro
420 425 430
Ala Ser Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu
435 440 445
Asn Arg Thr Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser
450 455 460
Glu Tyr Ala Ser Val Gln Val Pro Arg Lys
465 470
<210> 59
<211> 359
<212> PRT
<213> 人工序列
<220>
<223> 野生型SIRP-γ(wg)完全序列
<400> 59
Glu Glu Glu Leu Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr
1 5 10 15
Val Gly Lys Thr Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro
20 25 30
Val Gly Pro Val Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser
65 70 75 80
Ile Thr Pro Ala Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Glu Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met
100 105 110
Ala Leu Gly Ala Lys Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala
115 120 125
Arg Thr Thr Pro Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly
130 135 140
Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu
145 150 155 160
Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala
165 170 175
Tyr Ser Ile Arg Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val
180 185 190
Arg Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp
195 200 205
Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro
210 215 220
Thr Leu Glu Val Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn
225 230 235 240
Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr
245 250 255
Trp Ser Glu Asn Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu
260 265 270
Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val
275 280 285
Asn Ile Ser Asp Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys
290 295 300
His Asp Gly Gln Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr
305 310 315 320
Val His Gln Lys Asp Gln Ser Ser Asp Ala Thr Pro Gly Pro Ala Ser
325 330 335
Ser Leu Thr Ala Leu Leu Leu Ile Ala Val Leu Leu Gly Pro Ile Tyr
340 345 350
Val Pro Trp Lys Gln Lys Thr
355
<210> 60
<211> 358
<212> PRT
<213> 人工序列
<220>
<223> 变体SIRP-γ-1 (vg-1)完全序列
<400> 60
Glu Glu Glu Leu Gln Ile Ile Gln Pro Glu Lys Leu Leu Leu Val Thr
1 5 10 15
Val Gly Lys Thr Ala Thr Leu His Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Val Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser
65 70 75 80
Ile Thr Pro Ala Asp Val Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Glu Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met
100 105 110
Ala Leu Gly Ala Lys Pro Ala Pro Val Val Leu Gly Pro Ala Ala Arg
115 120 125
Thr Thr Pro Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe
130 135 140
Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu
145 150 155 160
Ser Asp Phe Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr
165 170 175
Ser Ile Arg Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg
180 185 190
Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro
195 200 205
Leu Arg Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr
210 215 220
Leu Glu Val Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val
225 230 235 240
Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp
245 250 255
Ser Glu Asn Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr
260 265 270
Glu Asn Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn
275 280 285
Ile Ser Asp Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His
290 295 300
Asp Gly Gln Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val
305 310 315 320
His Gln Lys Asp Gln Ser Ser Asp Ala Thr Pro Gly Pro Ala Ser Ser
325 330 335
Leu Thr Ala Leu Leu Leu Ile Ala Val Leu Leu Gly Pro Ile Tyr Val
340 345 350
Pro Trp Lys Gln Lys Thr
355
<210> 61
<211> 358
<212> PRT
<213> 人工序列
<220>
<223> 变体SIRP-γ-2 (vg-2)完全序列
<400> 61
Glu Glu Glu Leu Gln Ile Ile Gln Pro Glu Lys Leu Leu Leu Val Thr
1 5 10 15
Val Gly Lys Thr Ala Thr Leu His Cys Thr Val Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Val Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser
65 70 75 80
Ile Thr Pro Ala Asp Val Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Glu Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met
100 105 110
Ala Leu Gly Ala Lys Pro Ala Pro Val Val Leu Gly Pro Ala Ala Arg
115 120 125
Thr Thr Pro Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe
130 135 140
Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu
145 150 155 160
Ser Asp Phe Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr
165 170 175
Ser Ile Arg Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg
180 185 190
Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro
195 200 205
Leu Arg Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr
210 215 220
Leu Glu Val Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val
225 230 235 240
Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp
245 250 255
Ser Glu Asn Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr
260 265 270
Glu Asn Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn
275 280 285
Ile Ser Asp Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His
290 295 300
Asp Gly Gln Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val
305 310 315 320
His Gln Lys Asp Gln Ser Ser Asp Ala Thr Pro Gly Pro Ala Ser Ser
325 330 335
Leu Thr Ala Leu Leu Leu Ile Ala Val Leu Leu Gly Pro Ile Tyr Val
340 345 350
Pro Trp Lys Gln Lys Thr
355
<210> 62
<211> 357
<212> DNA
<213> 人工序列
<220>
<223> 编码SiRPα变体的多核苷酸
<400> 62
gaagaggagc tgcagatcat ccagcctgac aagtccgtgc tggtcgctgc tggtgaaact 60
gccactctgc gttgtacgat taccagcctg ttcccggtgg gtccaatcca gtggttccgt 120
ggtgctggtc cgggtcgtgt tctgatctac aaccagcgtc aaggtccgtt cccgcgtgta 180
actaccgtta gcgataccac gaagcgtaac aacatggact tttccatccg cattggcaat 240
attaccccgg ccgacgcggg cacctactat tgcatcaaat ttcgcaaagg ctccccggat 300
gatgtagaat ttaaatctgg cgcaggcacc gaactgtctg ttcgcgcaaa accgtaa 357
<210> 63
<211> 357
<212> DNA
<213> 人工序列
<220>
<223> 人野生型SIRPα
<400> 63
gaggaggaat tacaggtcat tcaaccagat aaatcggtct tagtagcagc cggagagaca 60
gctacattga gatgtacggc gacaagcctt attcccgtgg ggccgatcca atggtttcgc 120
ggggcaggcc ccggaagaga attgatttac aaccagaagg agggtcattt ccctcgcgtg 180
acgacggtca gcgacttaac taagcgtaat aacatggatt tttcaataag aataggcaat 240
ataactccgg ccgacgcagg gacgtactac tgtgttaaat tccggaaggg atctccggat 300
gatgtcgagt tcaaatctgg ggcgggtaca gaattgagcg ttcgggcaaa gccctaa 357
<210> 64
<211> 354
<212> DNA
<213> 人工序列
<220>
<223> 野生型SIRPγ
<400> 64
gaggaagaat tgcaaatgat ccagccggaa aaattattac tggttaccgt gggaaaaacg 60
gcgacccttc attgcacagt cacgtccctg ttgccggtag gtccagtttt gtggttccgg 120
ggggttggac cagggcgtga actgatctat aatcaaaagg aaggtcattt tccgcgcgtg 180
accacagtga gcgatttgac taaacggaac aatatggact tctcgatccg catttctagt 240
attacaccgg cggacgttgg cacttattat tgcgtcaagt tccgcaaagg aagtcctgag 300
aacgtagagt tcaagtccgg tcctggcact gagatggctt tgggtgctaa accc 354
<210> 65
<211> 354
<212> DNA
<213> 人工序列
<220>
<223> 编码SIRPγ变体1的多核苷酸
<400> 65
gaagaggagc tgcagatcat ccagcctgac aagtccgtgc tggtcgctgc tggtgaaact 60
gccactctgc gttgtacgat taccagcctg ttcccggtgg gtccaatcca gtggttccgt 120
ggtgctggtc cgggtcgtgt tctgatctac aaccagcgtc aaggtccgtt cccgcgtgta 180
actaccgtta gcgataccac gaagcgtaac aacatggact tttccatccg cattggcaat 240
attaccccgg ccgacgcggg cacctactat tgcatcaaat ttcgcaaagg ctccccggat 300
gatgtagaat ttaaatctgg cgcaggcacc gaactgtctg ttcgcgcaaa accg 354
<210> 66
<211> 354
<212> DNA
<213> 人工序列
<220>
<223> 编码SIRPγ变体2的多核苷酸
<400> 66
gaagaggaat tacaaatcat acaacctgaa aagctgttat tggtcaccgt aggcaaaacc 60
gctactctgc actgcactgt gacgtccctt tttcctgttg gtcctgtctt atggtttcgt 120
ggagtcggtc cgggtcgggt tcttatctat aaccagcggc aaggaccatt cccacgggtt 180
accacggttt cggacacaac gaaacgcaat aacatggatt tttccattcg gatttcaagc 240
atcactccgg ccgacgttgg aacttattac tgcataaagt ttagaaaggg atctccggag 300
aacgtagaat ttaagtctgg tccaggtact gagatggccc ttggagcgaa gccg 354
<210> 67
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 正向引物
<400> 67
aaacatatgg aagaggagct gcag 24
<210> 68
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 反向引物
<400> 68
aaaaagcttt caattcagat cctcttc 27

Claims (12)

1.重组外来体，其中在其表面上呈递吞噬作用促进蛋白，其中所述吞噬作用促进蛋白是SIRPα、SIRPγ、SIRPα或SIRPγ的包含CD47结合结构域的片段或抗CD47抗体。
2.根据权利要求1所述的重组外来体，其中所述吞噬作用促进蛋白是与受体酪氨酸激酶的跨膜结构域的N-末端连接的融合蛋白。
3.根据权利要求2所述的重组外来体，其中所述受体酪氨酸激酶是血小板衍生的生长因子受体(PDGFR)、表皮生长因子受体(EGFR)、成纤维细胞生长因子受体(FGFR)、血管内皮生长因子受体(VEGFR)、肝细胞生长因子受体原肌球蛋白受体激酶、IR(胰岛素受体)、LTK(白细胞受体酪氨酸激酶)、血管生成素受体、ROR (受体酪氨酸激酶样孤儿受体)、DDR (网柄菌凝素结构域受体)、RETR转染期间受体、酪氨酸蛋白激酶样(PTK)、受体酪氨酸激酶相关(RYK)或肌肉特异性激酶(MuSK)。
4.根据权利要求1所述的重组外来体，其中所述SIRPα或SIRPγ由SEQ ID NO:1至61中任一个的氨基酸序列构成。
5.根据权利要求4所述的重组外来体，其中所述外来体在其中进一步包含抗癌剂。
6.根据权利要求5所述的重组外来体，其中所述抗癌剂是抗癌蛋白、抗癌化合物或放射性核素。
7.根据权利要求6所述的重组外来体，其中所述抗癌蛋白是天冬酰胺酶、蛋白毒素、对癌抗原特异性的抗体、或抗血管生成因子。
8.根据权利要求6所述的重组外来体，其中所述抗癌化合物选自甲氨蝶呤、选自5-氟尿嘧啶、吉西他滨和阿拉伯糖基胞嘧啶的嘧啶类似物、羟基脲、选自巯嘌呤和硫鸟嘌呤的嘌呤类似物、烷化剂、免疫原性凋亡诱导剂、有丝分裂抑制剂、新生血管抑制剂、或嵌入剂。
9.根据权利要求8所述的重组外来体，其中免疫原性细胞死亡诱导剂选自蒽环霉素抗癌剂、抗EGFR抗体、BK通道激动剂、硼替佐米、强心苷+非免疫原性细胞死亡诱导剂、环磷酰胺抗癌剂、GADD34/PP1抑制剂+丝裂霉素、LV-tSMAC、麻疹病毒或奥沙利铂。
10.根据权利要求9所述的重组外来体，其中所述蒽环霉素抗癌剂是选自以下的至少一种：柔红霉素、多柔比星、表柔比星、伊达比星、匹克生琼、萨巴比星和戊柔比星。
11.用于治疗受试者中的癌症的药物组合物，其包含治疗有效量的权利要求1-10中任一项的重组外来体和药学上可接受的载体。
12.根据权利要求11所述的药物组合物，其进一步包含至少一种抗癌剂。
CN201780081782.8A 2016-12-29 2017-12-29 基于外来体的抗癌剂 Active CN110248645B (zh)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
KR20160182573 2016-12-29
KR10-2016-0182573 2016-12-29
PCT/KR2017/015784 WO2018124835A1 (ko) 2016-12-29 2017-12-29 신규 엑소좀 계열 항암제

Publications (2)

Publication Number Publication Date
CN110248645A CN110248645A (zh) 2019-09-17
CN110248645B true CN110248645B (zh) 2022-04-26

Family

ID=62709619

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201780081782.8A Active CN110248645B (zh) 2016-12-29 2017-12-29 基于外来体的抗癌剂

Country Status (6)

Country Link
US (2) US11319360B2 (zh)
EP (1) EP3563835A4 (zh)
JP (1) JP6857736B2 (zh)
KR (1) KR102129067B1 (zh)
CN (1) CN110248645B (zh)
WO (1) WO2018124835A1 (zh)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6857736B2 (ja) * 2016-12-29 2021-04-14 コリア・インスティテュート・オブ・サイエンス・アンド・テクノロジー 新規なエクソソーム系抗癌剤
CN114173807B (zh) * 2019-09-02 2024-03-19 庆北大学校产学协力团 包含il-2表面表达-细胞外囊泡作为活性成分的用于预防或治疗癌症的组合物
KR102521664B1 (ko) * 2019-09-02 2023-04-14 경북대학교 산학협력단 Il-2 표면 발현 세포외 소포체를 유효성분으로 포함하는 암 질환 예방 또는 치료용 조성물
EP4197559A4 (en) * 2020-08-14 2024-04-24 Korea Inst Sci & Tech ARNSI/IMMUNOMODULATING PROTEIN COMPLEX WITH ANTI-CANCER ACTIVITY
EP4209586A1 (en) * 2020-09-04 2023-07-12 Daegu Gyeongbuk Institute Of Science and Technology Extracellular vesicle expressing cytokine and antibody, method for producing same, and use thereof
TW202219069A (zh) * 2020-09-04 2022-05-16 大陸商江蘇恆瑞醫藥股份有限公司 ＳＩＲＰγ變體及其融合蛋白
WO2022211473A1 (ko) 2021-03-30 2022-10-06 (주)카이노스메드 Faf1(fas-associated factor 1)이 탑재된 엑소좀 및 이의 항암제로서의 용도
KR102462745B1 (ko) * 2021-04-29 2022-11-04 재단법인 아산사회복지재단 형광 상관 분광법을 이용한 세포외소포체에 표지된 형광 염료의 정량 분석 방법 및 이의 용도
KR20230021248A (ko) * 2021-08-05 2023-02-14 경북대학교 산학협력단 T 세포의 세포외 소포체 및 항암제를 유효성분으로 포함하는 병용 투여용 약학적 조성물
CN114366821A (zh) * 2022-02-09 2022-04-19 台州学院 一种表达受体蛋白的细胞膜纳米囊泡及其制备方法和应用
WO2024025264A1 (en) * 2022-07-24 2024-02-01 Shiftbio Co., Ltd. Method for treating cancer and inflammatory diseases using stem cell- derived extracellular vesicles comprising sirp
KR20240045411A (ko) * 2022-09-29 2024-04-08 (주)카이노스메드 Faf1 엑소좀을 고수율로 생산하는 세포주 및 이의 제조방법
WO2024071924A1 (ko) * 2022-09-29 2024-04-04 (주)카이노스메드 종양을 예방하거나 치료하기 위한 엑소좀 및 이의 용도
CN117866890A (zh) * 2024-03-12 2024-04-12 山东翰康医学科技集团有限公司 一种脂肪间充质干细胞外泌体提取和纯化的制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102272159A (zh) * 2008-12-05 2011-12-07 阿布拉西斯生物科学有限责任公司 结合SPARC的ScFc
CN102671196A (zh) * 2006-04-05 2012-09-19 诺瓦提斯公司 用于治疗癌症的治疗剂的组合
CN105073780A (zh) * 2012-12-17 2015-11-18 特里姆治疗公司 用SIRPα-Fc融合蛋白处理CD47+疾病细胞
CN105163724A (zh) * 2013-02-07 2015-12-16 葛兰素史密丝克莱恩生物有限公司 包含囊泡的药物组合物
WO2016201323A1 (en) * 2015-06-10 2016-12-15 Board Of Regents, The University Of Texas System Use of exosomes for the treatment of disease

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4224586B2 (ja) * 2004-04-28 2009-02-18 国立大学法人群馬大学 マクロファージ活性化剤並びにその製造方法及びスクリーニング方法
CN104136037B (zh) * 2012-01-17 2018-02-23 小利兰·斯坦福大学托管委员会 高亲和力SIRP‑α试剂
DK3311824T3 (da) 2013-02-05 2020-04-14 Univ Leland Stanford Junior Cd47 målrettede terapier til behandlingen af infektionssygdomme
WO2017075708A1 (en) * 2015-11-05 2017-05-11 Exerkine Corporation Exosomes useful to treat cystic fibrosis
KR102052204B1 (ko) * 2016-07-15 2019-12-04 주식회사 탠덤 신규 재조합 엑소좀 및 그의 용도
WO2018075825A1 (en) * 2016-10-19 2018-04-26 Northwestern University Extracellular vesicle-based diagnostics and engineered exosomes for targeted therapeutics against cancer
JP6857736B2 (ja) * 2016-12-29 2021-04-14 コリア・インスティテュート・オブ・サイエンス・アンド・テクノロジー 新規なエクソソーム系抗癌剤

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102671196A (zh) * 2006-04-05 2012-09-19 诺瓦提斯公司 用于治疗癌症的治疗剂的组合
CN102272159A (zh) * 2008-12-05 2011-12-07 阿布拉西斯生物科学有限责任公司 结合SPARC的ScFc
CN105073780A (zh) * 2012-12-17 2015-11-18 特里姆治疗公司 用SIRPα-Fc融合蛋白处理CD47+疾病细胞
CN105163724A (zh) * 2013-02-07 2015-12-16 葛兰素史密丝克莱恩生物有限公司 包含囊泡的药物组合物
WO2016201323A1 (en) * 2015-06-10 2016-12-15 Board Of Regents, The University Of Texas System Use of exosomes for the treatment of disease

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy;Yanhua Tian等;《Biomaterials》;20131215;第35卷;第2383-2390页 *
Engineered SIRPα variants as immunotherapeutic adjuvants to anti-cancer antibodies;Kipp Weiskopf等;《Science》;20130705;第341卷;第1-13页 *
Exosome-SIRPa, a CD47 blockade increases cancer cell phagocytosis;Eunee Koh等;《Biomaterials》;20170103;第121卷;第121-129页 *
新型融合蛋白pro－SIRPα的构建及体外活性研究;赵一蓉，等;《现代免疫学》;20160331;第36卷(第2期);第89-95页 *

Also Published As

Publication number Publication date
US11952412B2 (en) 2024-04-09
EP3563835A1 (en) 2019-11-06
JP6857736B2 (ja) 2021-04-14
US20220251170A1 (en) 2022-08-11
US11319360B2 (en) 2022-05-03
WO2018124835A1 (ko) 2018-07-05
CN110248645A (zh) 2019-09-17
KR102129067B1 (ko) 2020-07-08
EP3563835A4 (en) 2020-12-30
KR20180078173A (ko) 2018-07-09
JP2020504613A (ja) 2020-02-13
US20200148746A1 (en) 2020-05-14

Similar Documents

Publication Publication Date Title
CN110248645B (zh) 基于外来体的抗癌剂
US10905774B2 (en) Nanocage and use thereof
US10130680B2 (en) Methods for treating cancer with EGFR nanobodies linked to DR5 binding moieties
JP2022130596A (ja) 臨床のための、改変されたＮＫ－９２ ｈａＮＫ００３細胞
Wu et al. HER2-specific chimeric antigen receptor-engineered natural killer cells combined with apatinib for the treatment of gastric cancer
CN113416260B (zh) 靶向Claudin18.2的特异性嵌合抗原受体细胞及其制备方法和应用
US11215618B2 (en) Articles and methods directed to personalized therapy of cancer
JP2022513372A (ja) シアリルルイスａを標的とするキメラ抗原受容体およびその使用
KR102324242B1 (ko) 신규한 앱타머 및 이의 용도
US10155024B2 (en) Composition for preventing or treating B-cell lymphoma comprising IL-21 expressing mesenchymal stem cells
CN109789183A (zh) 治疗性多靶向构建体及其用途
WO2020000634A1 (zh) 一种与cd105特异性结合的多肽及其应用
US20230277668A1 (en) Chimeric antigen receptor and use thereof
US20220281998A1 (en) Bifidobacterium spp. expressing and secreting diabody-type bsab
CN108392492A (zh) Ldlr过表达在nk细胞过继治疗中的应用
US20230374113A1 (en) Ferritin nanocage fused with pd-l1-binding peptide 1 and use thereof as anticancer immunotherapy agent
US20220018845A1 (en) Articles and methods directed to personalized therapy of cancer
WO2023016419A1 (zh) 重组灵芝免疫调节蛋白新突变体及其应用
WO2022268238A1 (en) Urine progenitor cells for use in cancer therapy

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
TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20220812

Address after: Seoul, South Kerean

Patentee after: Hanshang Mobile Biochemical Co., Ltd.

Address before: Seoul, South Kerean

Patentee before: KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY