WO2015184941A1 - 一种cd7纳米抗体、其编码序列及应用 - Google Patents

一种cd7纳米抗体、其编码序列及应用 Download PDF

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WO2015184941A1
WO2015184941A1 PCT/CN2015/077854 CN2015077854W WO2015184941A1 WO 2015184941 A1 WO2015184941 A1 WO 2015184941A1 CN 2015077854 W CN2015077854 W CN 2015077854W WO 2015184941 A1 WO2015184941 A1 WO 2015184941A1
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seq
nanobody
human
cells
antibody
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PCT/CN2015/077854
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French (fr)
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杨林
汤金乐
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博生吉医药科技(苏州)有限公司
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Priority to US15/316,256 priority Critical patent/US10106609B2/en
Publication of WO2015184941A1 publication Critical patent/WO2015184941A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the invention belongs to the technical field of biomedicine or biopharmaceutical, and relates to a nanobody directed against a human CD7 molecule, a coding sequence thereof and an application thereof.
  • HCAbs heavy chain antibodies
  • sdAb single domain antibody
  • Nanobodies Compared with conventional antibodies, Nanobodies have many unique properties: 1) the sequence encoded by Nanobodies has high homology with human VH family 3 and 4, making it weakly immunogenic; 2) The molecular weight of Nanobodies is small, only about 15kDa, Simple structure, easy to express in microorganisms, easy to purify; 3) Nanobodies can recognize a large number of epitopes, including some epitopes hidden in molecular cracks; 4) Due to the small molecular weight of Nanobodies, they are Easy to penetrate tissue, reach the site where conventional antibodies are difficult to reach; 5) Nano-antibodies have high solubility and stability in denaturing or high temperature environments.
  • the human CD7 molecule is a cell surface glycoprotein with a molecular weight of approximately 40 kDa belonging to the immunoglobulin superfamily.
  • the CD7 molecule is mainly expressed on the surface of most thymocytes, more than 85% of the surface of peripheral blood T lymphocytes and the surface of natural killer cells.
  • current studies indicate that the specific function of the CD7 molecule is not well understood, experiments have shown that CD7-deficient murine T lymphocytes respond normally to stimulation and to cell growth and proliferation when antibodies bind to CD7 molecules on human T lymphocytes. No effect.
  • an important property of the CD7 molecule is its rapid endocytosis when it binds to its antibody.
  • the technical problem to be solved by the present invention is to provide a Nanobody directed against a human CD7 molecule, while providing the coding sequence of the Nanobody and the use of the Nanobody in preparing a test (reagent or kit).
  • a VHH chain of a human CD7 Nanobody comprising a framework region FR and a complementarity determining region CDR, wherein the framework region FR is selected from the following amino acid sequences of FR1 to FR4 :
  • FR1 shown in SEQ ID NO. 1 FR2 shown in SEQ ID NO. 2
  • FR1 shown in SEQ ID NO. 5 FR2 shown in SEQ ID NO. 6, FR3 shown in SEQ ID NO. 7, FR4 shown in SEQ ID NO.
  • FR1 shown in SEQ ID NO. 5 FR2 shown in SEQ ID NO. 10, FR3 shown in SEQ ID NO. 11, FR4 shown in SEQ ID NO.
  • FR1 shown in SEQ ID NO. 13 FR2 shown in SEQ ID NO. 14, FR3 shown in SEQ ID NO. 15, FR4 shown in SEQ ID NO.
  • the complementarity determining region CDRs are selected from the following amino acid sequences of CDR1 to CDR3:
  • it has the amino acid sequence set forth in SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 or SEQ ID NO.
  • the invention provides a human CD7 Nanobody, which is a Nanobody directed against a human CD7 molecular epitope, comprising SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID The VHH chain of the amino acid sequence shown in NO. 35, SEQ ID NO. 36 or SEQ ID NO.
  • a DNA molecule encoding a protein selected from the group consisting of the VHH chain of the human CD7 Nanobody of claim 1 or 2, or the human CD7 Nanobody of claim 3.
  • the DNA molecule has a DNA sequence selected from the group consisting of SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42 or SEQ. ID NO.43.
  • an expression vector comprising SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO.
  • SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. The nucleotide sequence shown in SEQ ID NO.
  • a host cell characterized in that it expresses a Nanobody against human CD7.
  • a human CD7 Nanobody of the invention for detecting a human CD7 molecule.
  • a human CD7 Nanobody of the invention for flow detection and cellular immunofluorescence experiments.
  • the use is for non-diagnostic purposes.
  • a heavy chain variable region of an anti-human CD7 antibody wherein the heavy chain variable region comprises three complementarity determining regions CDR1, CDR2, and CDR3, wherein
  • CDR1 is selected from the group consisting of: SEQ ID NO.: 17, 20, 25, 28, and 30;
  • CDR2 is selected from the group consisting of: SEQ ID NO.: 18, 21, 23, 26, 29, and 31;
  • CDR3 is selected from the group consisting of: SEQ ID NO.: 19, 22, 24, and 27.
  • the heavy chain variable region comprises:
  • it has the amino acid sequence set forth in SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 or SEQ ID NO.
  • an antibody comprising: the heavy chain variable region of the eighth aspect of the invention.
  • the antibody has the amino acid set forth in SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 or SEQ ID NO. Sequence of VHH chains.
  • the antibody is an antibody specific for an anti-CD7 protein.
  • the antibody is a Nanobody.
  • a recombinant protein having:
  • the tag sequence includes a 6His tag and an HA tag.
  • the recombinant protein specifically binds to a CD7 protein.
  • an immunoconjugate comprising:
  • a coupling moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
  • the conjugate is selected from the group consisting of: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computer tomography) contrast agent, or is capable of producing a detectable agent
  • Product enzymes radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, pre- A drug activating enzyme (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (eg, cisplatin) or any form of nanoparticles, and the like.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the coupling moiety is a Pseudomonas exotoxin or a fragment thereof.
  • the immunoconjugate comprises: a multivalent (e.g., bivalent) VHH chain of a human CD7 Nanobody according to the first aspect of the invention, a human of the second aspect of the invention A CD7 Nanobody, a heavy chain variable region according to the eighth aspect of the invention, an antibody according to the ninth aspect of the invention, or a recombinant protein according to the tenth aspect of the invention.
  • the multivalent means that a plurality of repeated VHH chains of the human CD7 Nanobody according to the first aspect of the present invention are contained in the amino acid sequence of the immunoconjugate, such as The human CD7 Nanobody of the second aspect of the invention, the heavy chain variable region of the eighth aspect of the invention, the antibody of the ninth aspect of the invention, or the recombinant protein of the tenth aspect of the invention .
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition is in the form of an injection.
  • the pharmaceutical composition is used for preparing a medicament for treating a tumor, the tumor being selected from the group consisting of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, Colorectal cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor, or bladder tumor.
  • a thirteenth aspect of the invention the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the antibody according to the fifth aspect of the invention,
  • the reagent, the detection plate or the kit is used for: detecting the CD7 protein in the sample;
  • the agent is for treating or preventing a tumor expressing a CD7 protein.
  • the tumor comprises: gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, or adrenal gland Tumor.
  • a fourteenth aspect of the invention there is provided a method of detecting a CD7 protein in a sample, the method comprising the steps of:
  • a method for preparing a recombinant polypeptide comprising:
  • a method of treating a disease comprising administering a Nanobody or immunoconjugate of the invention to a subject in need thereof.
  • the present invention firstly selects a cancer cell line highly expressing human CD7 molecule by flow detection method, and after treatment, makes it immunogenic, and then uses the cell line to immunize camel, and extracts lymphocytes from camel peripheral blood to prepare nanometer.
  • the antibody was used to immunize the gene pool, and finally the CD7 Nanobody was screened on the human kidney epithelial cell line (293T cell line) to obtain a human CD7-specific Nanobody gene.
  • This gene was cloned into a prokaryotic expression vector and transformed into E. coli to establish a Nanobody strain capable of high expression in E. coli.
  • the present invention immunizes camels using cells highly expressing the antigen of interest, and the obtained immunological library is relatively abundant, in addition to screening for antigens of interest, and other highly expressed molecules on the immune cells can also be It is more cost-effective, time-consuming, and labor-intensive to produce a single type of antibody against a selected antigen than to use a polypeptide or protein as an antigen.
  • the present invention uses cells to perform screening of the prepared Nanobody library for human CD7 molecular nano-antibody, and can obtain a human CD7 molecule which specifically recognizes natural activity, and the specific Nano-antibody can be used for streaming. Detection and cellular immunofluorescence experiments.
  • Figure 1 is a first-round PCR product agarose gel electrophoresis map, cut into 650 ⁇ 750bp fragment;
  • 2 is a second round of PCR product agarose gel electrophoresis pattern, and the gel is recovered to recover a fragment of about 500 bp;
  • Figure 3 is an electrophoresis diagram of the SpeI and SacI double-enzyme phage vector pComb3XSS agarose gel, and the vector fragment of about 3200 bp is recovered by gel cutting;
  • Figure 4 is a random selection of 24 clones for colony PCR, agarose gel electrophoresis
  • Figure 5 is a result of screening specific single positive clones on cells by ELISA using phage enzyme-linked immunosorbent assay (ELISA);
  • Figure 7 is a graph showing the binding curve and affinity assay results of Nanobodies VHH6 and VHH10 and high expression CD7Jurkat cell lines;
  • Figure 8 is a result of staining CD7 positive cells Jurkat simultaneously with a commercialized CD7 antibody and the obtained high-purity Nanobody, and then analyzing the results by flow cytometry;
  • Figure 9 is a result of staining CD7-negative cell RPMI8226 with a commercialized CD7 antibody and the obtained high-purity Nanobody, and then analyzing the result by flow cytometry;
  • Figure 10 is a result of transfection of the expression CD7 plasmid pcDNA3.1-CD7 into the CD7-negative cell line H460, followed by staining with a commercial CD7 antibody, and analysis by flow cytometry;
  • Figure 11 is a diagram showing the same batch of H460 cells transfected with CD7 using the obtained high-purity Nanobody for cell immunofluorescence analysis.
  • Figure 12 is a staining of Jurkat and RPMI8226 cells with the obtained high-purity Nanobody for immunofluorescence analysis.
  • Figure 13 is a design and characterization of PG001 (VHH-6-ETA).
  • A A simplified schematic of the construction of PG001. 6His, His tag; VHH-6 is a CD7-specific Nanobody; 4 (G 4 S) is a linker chain consisting of glycine and serine; ETA is composed of Pseudomonas exotoxin domain II and domain III; KDEL is the endoplasmic reticulum retention sequence. The molecular weights are estimated based on their amino acid sequences.
  • B Coomassie blue staining analysis of the purity of the nickel column affinity purified recombinant immunotoxin PG001.
  • C Immunoblot analysis of recombinant immunotoxin PG001.
  • Figure 14 is a result of staining CD7 positive cells Jurkat, CEM and negative cells RPMI8226, H460 with the obtained high-purity coupled toxin antibody PG001, and then analyzing the results by flow cytometry;
  • Figure 15 is a treatment of (A) CD7-positive Jurkat cells (black lattice) and CD7-negative RPMI8226 cells (white lattice) and (B) CD7-positive CEM cells (black lattice) using the obtained high-purity coupled toxin antibody PG001. ) and CD7-negative H46 cells (white grid) for 72 hours. Cell growth inhibition was measured by WST-8. The mean values were from three independent experiments (Jurkat, CEM, RPMI 8226, H460). The standard deviation is expressed by the error.
  • Figure 16 shows the killing of Jurkat, CEM and RPMI8226 cells with the obtained high-purity coupled toxin antibody PG001150ng/ml PG001, and staining with Annexin V and 7-AAD at 24h, 48h and 72h respectively.
  • the cell population in the lower right quadrant represents early apoptotic cells (Annexin V positive and 7-AAD negative), and the cell population in the upper right quadrant represents dead cells (Annexin V and 7-AAD double positive).
  • the data shown is from three independent experiments.
  • Figure 17 is a treatment of CD7-positive Jurkat (C) and CEM (D) cells with a single dose of 150 ng/ml PG001. (in the figure), or the cells were treated in the presence of 100-fold parental Nanobody VHH6 (right), and after 48 hours, the treated cells were stained with Annexin V and 7-AAD.
  • the cell population in the lower right quadrant represents early apoptotic cells (Annexin V positive and 7-AAD negative), and the cell population in the upper right quadrant represents dead cells (Annexin V and 7-AAD double positive).
  • the data shown is from three independent experiments.
  • Figure 18 shows the results of treatment of JURkat, CEM and RPMI8226, H460 cells with the obtained high-purity coupled toxin antibody PG001150 ng/ml PG001 for 48 hours, and then immunoblot detection of PARP molecular cleavage.
  • Figure 19 is a preparation of the bivalent Nanobody immunotoxin PG002 and the toxicological effect of PG002.
  • A A simplified schematic diagram of the construction of PG002.
  • VHH-6 is a CD7-specific Nanobody; 4 (G4S) is a linking chain composed of glycine and serine.
  • B Affinity Purified Nanobody Immunotoxin PG001 was analyzed by Coomassie brilliant blue staining.
  • C CD7-positive Jurkat cells (black grid) and CD7-positive CEM cells (grey grid) and CD7-negative RPMI8226 cells (white grid) were treated with different concentrations of PG002 for 72 hours. Cell growth inhibition was detected by the WST-8 kit. The mean values were from three independent experiments (Jurkat, CEM, RPMI 8226). The standard deviation is expressed by the error.
  • Figure 20 is a diagram showing the effect of tail vein injection of NOD/SCID mice with the obtained high-purity coupled toxin antibody PG001, followed by monitoring the anti-CEM leukemia cells in vivo.
  • the inventors have successfully obtained a class of anti-CD7 Nanobodies through extensive and intensive research.
  • the results of the experiments show that the Nanobody is not only highly specific, but also can significantly differentiate with cell lines and T lymphocytes expressing CD7 molecules.
  • the efficient binding of CD7 molecules on cells or natural killer cells NK can be based on the modification of this type of antibody to deliver functional molecules (toxins or small RNAs) to kill CD7 molecule positive cells or other functional studies.
  • the present invention has been completed.
  • the present invention firstly immunizes a Xinjiang Bactrian camel with a CD7 high expression cell line Jurkat cells (5 ⁇ 10 6 ), and extracts the peripheral blood lymphocytes of the Bactrian camel after 7 consecutive immunizations and successfully constructs a single domain heavy chain antibody library. . Then 293T-CD7 - (293T raw not expressing CD7) and 293T-CD7 + (293T-CD7 stably transfected cell lines) for panning CD7 specific Nanobodies on the cell lines, thereby finally obtained efficiently in E. coli Expressed Nanobody strains.
  • antibody or "immunoglobulin” is an isotetrameric glycoprotein of about 150,000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
  • single domain antibody VHH
  • nanobody a single domain antibody consisting of only one heavy chain variable region.
  • VHH single domain antibody
  • CH1 light chain and heavy chain constant region 1
  • variable means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conserved portion of the variable region is referred to as the framework region (FR). Day The variable regions of the heavy and light chains each comprise four FR regions which are substantially in a ⁇ -sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial beta sheet structure.
  • CDR complementarity determining region
  • FR framework region
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Publ. No. 91-3242, Vol. I, pp. 647-669). (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules that are combined with the antibodies or fragments thereof of the invention to form Conjugate.
  • the invention also encompasses cell surface markers or antigens that bind to the anti-CD7 protein antibody or fragment thereof.
  • variable region are used interchangeably with “complementarity determining region (CDR).
  • the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3, wherein
  • CDR1 is selected from the group consisting of: SEQ ID NO.: 17, 20, 25, 28, and 30;
  • CDR2 is selected from the group consisting of: SEQ ID NOs: 18, 21, 23, 26, 29, and 31;
  • CDR3 is selected from the group consisting of: SEQ ID NO.: 19, 22, 24, and 27.
  • the heavy chain variable region has SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 or SEQ ID NO. The amino acid sequence shown.
  • the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above.
  • the terms "antibody of the invention”, “protein of the invention”, or “polypeptide of the invention” are used interchangeably and refer to a polypeptide which specifically binds to a CD7 protein, for example having a heavy chain variable region (eg SEQ ID) A protein or polypeptide of NO.: amino acid sequence of one of 32-37. They may or may not contain an initial methionine.
  • the invention also provides other proteins or fusion expression products having the antibodies of the invention.
  • the invention encompasses any protein or protein conjugate having a heavy chain comprising a variable region and a fusion expression product (ie, an immunoconjugate and a fusion expression product), so long as the variable region is linked to the heavy chain of an antibody of the invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are divided into four framework regions (FR), four FR amino acids.
  • FR framework regions
  • the sequence is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen.
  • the invention includes those molecules having an antibody heavy chain variable region with a CDR, as long as the CDRs thereof have 90% or more (preferably 95% or more, optimally 98% or more) homology to the CDRs identified herein. Sex.
  • the present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of an antibody of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues May or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a mature polypeptide a polypeptide formed by fusing a compound with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or a secretory sequence or To purify the sequence or proprotein sequence of this polypeptide, or a fusion protein
  • the antibody of the present invention refers to a polypeptide comprising the above CDR regions having CD7 protein binding activity.
  • the term also encompasses variant forms of a polypeptide comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include, but are not limited to, one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions , Insertion and/or Substitution, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • the function of the protein is generally not altered.
  • the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA capable of hybridizing to the DNA encoding the antibody of the present invention under high or low stringency conditions.
  • the encoded protein, and the polypeptide or protein obtained using an antiserum against the antibody of the present invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof.
  • the invention also includes fragments of the antibodies of the invention.
  • the fragment will have at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
  • “conservative variant of the antibody of the present invention” means having up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3, compared to the amino acid sequence of the antibody of the present invention. Amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table I.
  • the present invention also provides a polynucleotide molecule encoding the above antibody or a fragment thereof or a fusion protein thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • Polynucleotides encoding mature polypeptides of the invention include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optionally additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 90% identity between the two sequences, more It is good that hybridization occurs more than 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown by one of SEQ ID NO.: 32-37.
  • the full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • One possible method is to synthesize related sequences by artificial synthesis, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecule (nucleic acid, protein, etc.) to which the present invention relates includes biomolecules existing in an isolated form.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, and the like.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2.
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If necessary, it can be separated by various separation methods using its physical, chemical and other properties. And purifying the recombinant protein. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the invention may be used alone or in combination or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (electron computed tomography) contrast agents, or capable of producing detectable products Enzyme.
  • Therapeutic agents that can be bound or conjugated to the antibodies of the invention include, but are not limited to: 1. Radionuclides (Koppe et al, 2005, Cancer metastasis reviews 24, 539); 2. Biotoxicity (Chaudhary et al, 1989) , Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines such as IL-2, etc. (Gillies et al., 1992, National Academy of Sciences (PNAS) 89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4.
  • Gold nanoparticles / Nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 5. Viral particles (Peng et al, 2004) , Gene therapy 11, 1234); 6. Liposomes (Mamot et al, 2005, Cancer research 65, 11631); 7. Nanomagnetic particles; 8. Prodrug activating enzymes (eg, DT) - diaphorase (DTD) or biphenyl hydrolase-like egg Mass (BPHL));. 10 chemotherapeutic agents (e.g., cisplatin) or any other form of nanoparticles.
  • DTD Prodrug activating enzymes
  • BPHL biphenyl hydrolase-like egg Mass
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used for binding to a CD7 protein molecule, and thus can be used for the prevention and treatment of tumors.
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-described Nanobody of the present invention (or a conjugate thereof) and pharmaceutically An acceptable carrier or excipient.
  • a safe and effective amount e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the polypeptides of the invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the Nanobody is provided with a detectable label. More preferably, the label is selected from the group consisting of a colloidal gold label, a colored label or a fluorescent label.
  • the colloidal gold label can be carried out by methods known to those skilled in the art.
  • the monoclonal antibody to the CD7 protein is labeled with colloidal gold to provide a colloidal gold-labeled monoclonal antibody.
  • the CD7 protein monoclonal antibody of the present invention has good specificity and high potency.
  • the present invention relates to a method for detecting a CD7 protein in a sample that is solubilized in cells and/or tissues.
  • the method steps are substantially as follows: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of CD7 protein in the dissolved sample.
  • the sample used in the method of the invention may be any sample comprising cells present in the cell preservation solution, as used in liquid-based cell assays.
  • the invention also provides a kit comprising an antibody (or a fragment thereof) of the invention or a detection plate of the invention, in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, buffer Agents, etc.
  • the invention further designs a detection kit for detecting CD7 levels, the kit comprising an antibody recognizing a CD7 protein, a lysis medium for dissolving a sample, a detection of a desired universal reagent and a buffer, such as various buffers, detection markers , detection of substrates, etc.
  • the test kit can be an in vitro diagnostic device.
  • E. coli TOP10 was used to amplify plasmids and clones
  • E. coli XL1-Blue (Stratagene) was used to amplify a library of nanophage antibodies
  • E. coli BL-21 (DE3) was used to express proteins.
  • the phage plasmid pCOMB 3XSS was used to prepare a phage library
  • pcDNA3.1 was used to construct a eukaryotic expression plasmid
  • pET28a was used for prokaryotic expression.
  • the lentiviral vector Red-OG2 was used to construct a stable cell line.
  • blood or bone marrow of patients with T-ALL and T-AML is extracted to prepare primary cells. Isolation of monocytes with Ficol was performed according to standard procedures. Primary cells were cultured in 20% FBS RPMI1640 medium with the addition of a double antibody.
  • the leukemia cell line Jurkat, CEM, and the lung cancer cell line H460 were cultured with 10% FBS RPMI1640 medium and a double antibody was added.
  • the myeloma cell line RPMI8226 was cultured in 10% FBS IMDM medium and a double antibody was added.
  • the 293T cell line was cultured in 10% FBS DMEM medium and a double antibody was added.
  • the obtained positive clone Nanobody sequence was cloned into the prokaryotic expression vector pET28a, and the His tag and the HA tag were left at the C-terminus for purification and identification, and then transformed into the expression strain BL-21, and expression was induced by IPTG.
  • the prokaryotic expression of the Nanobody was purified by affinity purification to obtain a pure Nanobody.
  • the protein purity was identified by Coomassie blue staining, and the protein concentration was detected by the BCA method.
  • the whole gene was synthesized and the Pseudomonas toxin truncated PE38 and the Nanobody VHH-6 were inserted into the pET28a vector, and the 6-His tag was left at the N-terminus for purification and detection, and was also transformed into the expression strain BL-21. Expression was induced using IPTG. Prokaryotic expression of Nanobody immunotoxins obtained by affinity purification to obtain pure nano The antibody and protein purity were identified by Coomassie blue staining, and the protein concentration was detected by BCA method. The bivalent immunotoxin construct was inserted into the VHH-6 sequence by the PCR method in the same manner as the monovalent immunotoxin, and prokaryotic expression was carried out, followed by purification and identification.
  • the protocol was as follows: To assess the affinity of the screened Nanobodies, Jurkat cells were incubated on ice with different concentrations of Nanobodies. Wash twice with PBS and incubate with APC-labeled anti-HA monoclonal antibody (Miltenyi Biotec, CHN) in PBS containing 2% BSA for 1 h on ice. The PBS was washed twice, and the fluorescence intensity was measured using a FACSCalibur. In combination with the saturation curve, the nonlinear regression curve and Scatchard plots were fabricated using the software Graph Pad Prism (Graph Pad Software, Inc).
  • Nanobody immunotoxins The binding specificity of the nanobodies and Nanobody immunotoxins was detected by flow cytometry. Approximately 3 ⁇ 10 5 (Jurkat, CEM, H460, RPMI8226) cells were washed twice with PBS and then resuspended in a system of 50 ⁇ l PBS of nanoantibody and nanobody immunotoxin at a final concentration of 5 ⁇ g/ml, placed on ice.
  • the specificity of the Nanobody was detected by cellular immunofluorescence.
  • Jurkat and RPMI 8226 cells were seeded in six-well plates with polylysine-treated slides overnight at 37 ° C in a cell culture incubator. The slides were removed, washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, then blocked with 3% BSA for 1 hour, and finally incubated with Nanobodies for 1 hour at room temperature, washed twice with PBS and then added with anti-HA sheep. Incubate for 1 hour at room temperature, wash twice with PBS, add anti-gobe monoclonal antibody Alexa488 (CST) for 1 hour at room temperature, wash 3 times with PBS, and shoot with confocal fluorescence microscope.
  • CST anti-gobe monoclonal antibody Alexa488
  • Lipo2000 was transfected into H460 cells of pcDNA3.1 and pcDNA3.1-CD7, respectively, and subjected to cellular immunofluorescence assay in the same manner as above.
  • the concentration of the nanobody immunotoxin on the target cells is determined by the lazy cytotoxic effect using the WST-8 kit.
  • the cells were seeded at 1*10 4 cells/well in 96-well plates incubated with different concentrations of immunotoxins (H460 cells were seeded 3*10 3 ), and after 72 hours, 10 ⁇ l of WST-8 reagent was added and incubated at 37 ° C until The maximum absorbance reading is taken at 450 nm.
  • 10 ⁇ l of WST-8 reagent was added and incubated at 37 ° C until The maximum absorbance reading is taken at 450 nm.
  • 2.5 ⁇ 10 5 cells are seeded in a 24-well plate, and 150 ng/ml of toxin is added. After treatment for different time, the treated cells are labeled with FITC. Annexin V and 7-AAD staining were performed according to the kit protocol.
  • the blocking experiment was performed by adding 50-fold of the parent antibody 1 hour before the addition of the monovalent immunotoxin (150 ng/ml).
  • CEM cells were washed 1 times with PBS, and on day 0, each NOD / SCID mice were injected with 2 ⁇ 10 6 CEM cells through the tail vein. On day 5, each mouse was injected with 5 ⁇ g of PG001, and administered once every other day for 3 times. Monitor the daily health of the mice. The dying mice were sacrificed according to the protocol. The survival time of the mice was evaluated by Karl-Meier analysis to assess the therapeutic effect of the toxin and to detect median survival.
  • the anti-CD7 protein nano-antibody of the invention has high specificity, strong affinity, good thermal stability, and can be prepared in a large amount, and the quality is easy to control.
  • the Nanobody of the anti-CD7 protein of the present invention can efficiently induce apoptosis of a CD7-positive T lymphocyte leukemia cell line by conjugated with a Pseudomonas exotoxin.
  • Upstream primer GTCCTGGCTCTCTTCTACAAGG (SEQ ID NO.: 45)
  • Downstream primer GGTACGTGCTGTTGAACTGTTC (SEQ ID NO.: 46)
  • a fragment between the heavy chain antibody-directed peptide and the antibody CH2 was amplified, annealed at 55 ° C, and 32 cycles; agarose gel electrophoresis, and a DNA fragment of 650 bp to 750 bp in size was recovered by gelatinization, as shown in FIG.
  • Upstream primer CGAGCTCATGGATGTGCAGCTGCAGGAGTCTGGAGGAGG (SEQ ID NO.: 47)
  • Downstream primer GGACTAGTGATGGAGACGGTGACCTGGGT (SEQ ID NO.: 48)
  • Example 3 Screening for specific single positive clones using phage enzyme-linked immunosorbent assay (ELISA) (whole cell ELISA):
  • the gene sequences of each clone were analyzed, and the strains with the same CDR1, CDR2, and CDR3 sequences were regarded as the same clone, and the strains with different sequences were regarded as different clones, and finally, 6 strains of high affinity were obtained. Highly specific antibodies.
  • the preferred cloned sequence information screened by the present invention is as follows:
  • Example 4 Nanobody expression and purification in host strain Escherichia coli
  • Example 5 Analysis of the affinity of two Nanobodies for CD7-positive Jurkat cells by flow cytometry
  • Flow cytometric analysis was performed on CD7 positive cell line Jurkat cells with high purity Nanobody (clone 2, clone VHH-6) and compared with commercial CD7 antibody.
  • the experimental procedure was as follows: after incubating for 1 hour at room temperature, washing with PBS for 3 times, adding anti-HA-tag antibody (rabbit anti-) to incubate for 1 hour at room temperature, washing 3 times with PBS, and adding anti-rabbit Alexa.
  • the 488 fluorescently labeled antibody was incubated at room temperature for 1 hour, washed 3 times with PBS, and finally detected by flow cytometry. The results are shown in Fig.
  • the ratio of the obtained nanobody-binding positive cells and the commercial antibody (purchased from BD, As a conventional monoclonal antibody, the antibody can bind to cells expressing CD7 molecules on the surface of human cells, including some tumor cells expressing CD7 molecules and primary cells highly expressing CD7 such as T lymphocytes, natural killer cells.
  • Flow cytometry was performed on CD7-negative cell line RPMI8226 cells with high purity Nanobody to detect specificity. The experimental procedure was the same as above, and the results are shown in Figure 9, indicating that the obtained Nanobody specificity is better.
  • the upper left panel is the control group corresponding to the commercial antibody staining, and the upper right is the commercial PE labeled CD7 positive cell line (Jurkat).
  • the flow pattern shows that the CD7+ positive rate is 96.6%; The corresponding control group was stained for VHH antibody, and Jurkat was labeled with VHH-6 antibody at the bottom right.
  • the flow rate showed that the positive rate of CD7+ was 91.8%; the positive rate of both was similar.
  • the negative cell line RPMI-8226 the positive rate of commercialization of antibodies and VHH-6 antibodies was below 3% (respectively 0.648%, 2.82%).
  • the pcDNA3.1-CD7 plasmid was transfected into the CD7-negative cell line H460 cells with lipofectamine2000 transfection reagent. After 48 hours, the H460 cells were digested with trypsin, and half of the culture was continued for 24 hours before being used for flow detection (commercial antibody) assay transfection. The effect, the other half of the cells were used for cell climbing, and cellular immunofluorescence was performed 24 hours later.
  • the flow cytometry method was as follows: the transfected H460 cells were removed, washed with PBS for 3 times, and commercialized CD7 flow-type antibody was added. After incubation at room temperature for 1 hour, the PBS was washed 3 times for flow cytometry. The results are shown in Fig. 10.
  • the transfection efficiency is around 18%.
  • the steps of immunofluorescence detection were as follows: the slides were taken out, washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, then blocked with 3% BSA for 1 hour, and finally stained with antibody, in the same manner as in Example 5, and finally Using a confocal fluorescence microscope, the results are shown in Figure 10, indicating similar staining rates with commercial antibodies, and better specificity. Immunofluorescence staining was also performed on the Jurkat cell line expressing the CD7 molecule and the CD7-negative cell line RPMI-8226. It was shown that VHH6 specifically binds to Jurkat cells, while no fluorescence is detected on the surface of RPMI-8226 cells.
  • FIG. 10 Transfection of H460 cells with CD7 protein granules (pcDNA3.1-CD7) and flow cytometry with commercial PE-labeled CD7 molecule antibody at a transfection efficiency of 18.8%; likewise, using screened Nanobodies (this A representative antibody clone 6 of the invention, clone VHH-6) was subjected to immunofluorescence analysis of H460 cells transfected with pcDNA3.1-CD7 plasmid (Fig. 11), and the proportion of total cells in the field of view in a single field of view was analyzed. The positive rate is about 20%, indicating that the antibody effect of the present invention is similar to that of commercial antibodies.
  • Figure 12 analysis found that CD7 expression was detected on the surface of Jurkat cells, and RPMI8226 was used as a negative control.
  • the Pseudomonas toxin truncated PE38 and Nanobody VHH-6 sequences were codon-optimized and inserted into the vector pET28a, leaving a subsequently purified HIS tag at the N-terminus, and the C-terminal REDLK sequence was replaced with a KDEL sequence.
  • Figure 13A The vector was transformed into E. coli BL21 (DE3), and after expression and affinity purification, a highly purified Nanobody immunotoxin VHH-6-PE38 was obtained, designated as PG001 (Fig. 13B and C). The results showed that 1 liter of E. coli produced approximately 10 mg of purified PG001.
  • Specificity analysis of PG001 revealed that it was still able to bind to CD7 positive cell lines Jurkat and CEM cells with high specificity, but not to CD7 negative cell lines RPMI8226 and H460 cells (Fig. 14).
  • the coding sequence information of the punctuator PE38 of Pseudomonas toxin is SEQ ID NO.: 44)
  • the toxic effect of PG001 was detected by WST-8 kit.
  • the results showed that PG001 can strongly inhibit cell proliferation of CEM (EC50 ⁇ 0.5nM) and Jurkat (EC50 ⁇ 1.0nM) at a very low concentration, while negative for CD7.
  • the growth of RPMI8226 and H460 cells had no effect ( Figure 15).
  • To detect whether cell proliferation inhibition by PG001 occurred through apoptosis cells treated with PG001 were detected by Annexin V and 7-AAD staining.
  • the results showed that CEM and Jurkat proliferation inhibition of two CD7 positive cells was caused by PG001-induced apoptosis and was time-dependent, but did not cause apoptosis of RPMI 8226 cells under the same concentration conditions (Fig. 16).
  • the monovalent Nanobody has a smaller molecular weight and a smaller immunotoxin, it has a shorter half-life in vivo and is also intended to further enhance the binding of the immunotoxin to the target cells.
  • the inventors conducted the construction of a bivalent Nanobody by further reducing the amount of the immunotoxin.
  • the Pseudomonas toxin truncated PE38 and the bivalent Nanobody VHH-6 sequence were codon-optimized and inserted into the vector pET28a, leaving a subsequently purified HIS tag at the N-terminus, and the C-terminal REDLK sequence was replaced with a KDEL sequence.
  • the structure is as shown in (Fig. 19A).
  • the constructed vector was transformed into E. coli BL21, and after expression and affinity purification, a high-purity bivalent Nanobody immunotoxin (VHH-6) 2-PE38 was obtained, which was named PG002 (Fig. 19B).
  • the present inventors have found that 1 liter of E. coli can produce about 5 mg of purified PG002, and the toxic effect of PG002 is detected by WST-8 kit.
  • the results show that PG002 can strongly inhibit CD7 at a lower concentration (picomolar).
  • mice were injected per 2x10 6 CEM cells through the tail vein. After 5 days, 5 ⁇ g of immunotoxin PG001 was injected through the tail vein, injected every other day for a total of 3 times, or unconjugated forms of VHH-6 and PE38 (VHH-6+PE38).
  • the results showed that the survival period of the PG001 mice in the administration group was significantly longer than that of the control group PBS and VHH-6+PE38.
  • the median survival time of the PBS group was 28.5 days and the median survival time of the PG001 administration group was 37 days. The experiment found that the mice in the drug-administered group developed symptoms of disability and weight loss in the hind legs (Fig. 20).
  • the present inventors constructed immunogenic toxins PG001 and PG002 for monovalent and bivalent Nanobody-conjugated Pseudomonas exotoxin truncates against CD7 by screening specific CD7 Nanobodies from camelids. These two toxins are capable of efficiently and specifically inducing apoptosis in acute leukemia cell lines in nanomolar and picomolar concentrations, respectively, and are capable of efficiently inducing apoptosis in primary cells of T-ALL and T-AML patients. . Moreover, the monovalent nanobody immunotoxin PG001 is effective in inhibiting the growth of human leukemia cells transplanted into NOD/SCID mice.
  • the present inventors' research has the following new findings: a) The two Nanobody immunotoxins constructed by the present inventors, like the scFv fragments, do not have an Fc fragment, thus greatly reducing binding to non-target cells; b) the present invention
  • the human monovalent Nanobody immunotoxin PG001 has an IC50 concentration on the T-ALL cell line (Jurkat and CEM) at the nanomolar level, whereas the inventors constructed the bivalent Nanobody immunotoxin PG002 against the T-ALL cell line (Jurkat and CEM).
  • the IC50 concentration at the picomolar level increased the killing effect by about 30 times; c) the high-solubility, high-purity yield of the two immunotoxins constructed with Nanobodies using the prokaryotic expression of E. coli BL-21 (DE3) About 10mg and 5mg per liter of bacterial liquid, respectively, the yield is about 50 to 100 times higher than the prior art, which makes the production cost of such immunotoxins greatly reduced, and has more clinical application value;
  • the present inventors performed flow cytometry analysis of immunotoxin-treated cells by Annexin V and 7-AAD, and detected the cleavage of PARP molecules by Western blot, indicating that the inventors of the immunotoxin are induced by fine Apoptosis causes cell death; e) The Nanobody immunotoxin of the present inventors can not only effectively kill T-ALL cell line at a low concentration, but also the divalent Nanobody immunotoxin constructed by the present inventors can be lower.
  • VHH-6 antibody clones are representative, and other clones provided by the present invention (such as VHH-10) have similar biological activities as VHH-6 antibody clones.
  • the monovalent and bivalent Nanobody CD7 immunotoxins constructed by the present inventors are capable of efficiently scavenging CD7-positive T-ALL cell lines and capable of killing T-ALL in an antigen-specific manner at nanomolar and lower concentrations.

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Abstract

提供了一种针对人CD7分子的纳米抗体及其编码DNA序列，所述纳米抗体能够在大肠杆菌内高效表达，可用于制备检测CD7分子与靶向治疗的试剂。

Description

一种CD7纳米抗体、其编码序列及应用 技术领域
本发明属于生物医学或生物制药技术领域，涉及一种针对于人CD7分子的纳米抗体、其编码序列及应用。
背景技术
1993年，Hamers-Casterman和他的同事在骆驼科动物体内发现了一种特殊类型的抗体，即天然缺失轻链的重链抗体(Heavy chain antibodies,HCAbs)，克隆其可变区得到只由一个重链可变区组成的单域抗体(single domain antibody,sdAb)，其晶体结构呈椭圆形,直径2.5nm,高度4nm,所以又被称为纳米抗体(Nanobodies，Nb；15kDa)，它是现阶段最小的功能性抗原结合片段。纳米抗体和常规抗体相比具有许多独特的性质：1)纳米抗体编码的序列与人VH家族3和4同源性高，使得它免疫原性弱；2)纳米抗体分子量小，仅15kDa左右,结构简单，很容易在微生物中大量表达，易于纯化；3)纳米抗体可以识别大量的抗原表位，包括一些藏在分子裂缝中的表位都能识别；4)由于纳米抗体分子量小，使得它们易于穿透组织，到达常规抗体难以到达的部位；5)在变性或者高温环境下纳米抗体具有高可溶性和稳点性。
人CD7分子是一个分子量约40kDa细胞表面糖蛋白属于免疫球蛋白超家族中的一员。CD7分子主要表达在大多数的胸腺细胞表面，85％以上的外周血T淋巴细胞表面以及自然杀伤细胞表面。尽管目前的研究表明CD7分子的具体功能还不太清楚，但实验显示CD7缺陷的鼠T淋巴细胞对刺激反应正常以及当抗体与人T淋巴细胞上的CD7分子结合后对细胞的生长和增殖并没有影响。同时，CD7分子的一个重要性质是当它和它的抗体结合后会快速的发生内吞作用。在这个重要性质的基础上，已经有几项研究通过在CD7分子上偶联免疫毒素来靶向递送到人白血病以及淋巴癌细胞，从而达到治疗疾病的目的以及偶联免疫毒素来治疗急性移植物抗宿主病。这些实验都已进行临床实验阶段。同时有研究通过在CD7分子上偶联蛋白来靶向递送siRNA到T淋巴细胞内，来治疗HIV感染。但是这些实验都是在常规抗体中分离出来的单链抗体(scFv；30kDa)上进行的。由于单链抗体相对纳米抗体分子量较大，导致其侵入组织和细胞中困难，而纳米抗体由于分子量很小同时单链抗体在原核表达系统中很难可溶表达出来，而纳米抗体在原核表达系统中易可溶表达且易复性。所以制备人CD7纳米抗体来进行疾病治疗研究可能是更加有效的且研究成本较低的备选方案之一。
目前，也没有针对人CD7抗原表位为靶标的特异性纳米抗体的研究报道。
发明内容
发明目的：本发明所要解决的技术问题是提供一种针对人CD7分子的纳米抗体，同时提供该纳米抗体的编码序列及该纳米抗体在制备检测(试剂或试剂盒中)的应用。
技术方案：为实现上述目的，本发明的第一方面，一种人CD7纳米抗体的VHH链，包括框架区FR和互补决定区CDR，所述框架区FR选自以下的FR1～FR4的氨基酸序列：
SEQ ID NO.1所示的FR1，SEQ ID NO.2所示的FR2，SEQ ID NO.3所示的FR3，SEQ ID NO.4所示的FR4；
或SEQ ID NO.5所示的FR1，SEQIDNO.6所示的FR2，SEQIDNO.7所示的FR3，SEQ ID NO.8所示的FR4；
或SEQ ID NO.1所示的氨基酸序列FR1，SEQ ID NO.9所示的FR2，SEQ ID NO.3所示的FR3，SEQ ID NO.4所示的FR4；
SEQ ID NO.5所示的FR1，SEQ ID NO.10所示的FR2，SEQ ID NO.11所示的FR3，SEQ ID NO.12所示的FR4；
或SEQ ID NO.13所示的FR1，SEQIDNO.14所示的FR2，SEQIDNO.15所示的FR3，SEQ ID NO.8所示的FR4；
或SEQ ID NO.5所示的氨基酸序列FR1，SEQ ID NO.14所示的FR2，SEQ ID NO.16所示的FR3，SEQ ID NO.8所示的FR4；
所述的互补决定区CDR选自以下的CDR1～CDR3的氨基酸序列：
SEQ ID NO.17所示的CDR1，SEQ ID NO.18所示的CDR2，SEQ ID NO.19所示的CDR3；
或SEQ ID NO.20所示的CDR1，SEQ ID NO.21所示的CDR2，SEQ ID NO.22所示的CDR3；
或SEQ ID NO.17所示的CDR1，SEQ ID NO.23所示的CDR2，SEQ ID NO.24所示的CDR3；
或SEQ ID NO.25所示的CDR1，SEQ ID NO.26所示的CDR2，SEQ ID NO.27所示的CDR3；
或SEQ ID NO.28所示的CDR1，SEQ ID NO.29所示的CDR2，SEQ ID NO.22所示的CDR3；
或SEQ ID NO.30所示的CDR1，SEQ ID NO.31所示的CDR2，SEQ ID NO.22所示的CDR3。
优选地，它具有SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36或SEQ ID NO.37所示的氨基酸序列。
本发明第二方面，提供了一种人CD7纳米抗体，它是针对人CD7分子表位的的纳米抗体，包括具有SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36或SEQ ID NO.37所示的氨基酸序列的VHH链。
本发明第三方面，提供了一种DNA分子，它编码选自下组的蛋白质：权利要求1或2所示的人CD7纳米抗体的VHH链，或权利要求3所示的人CD7纳米抗体。
优选地，所述的DNA分子,它具有选自下组的DNA序列：SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42或SEQ ID NO.43。
本发明的第四方面，提供了一种表达载体,其特征在于,它含SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42或SEQ ID NO.43所示的核苷酸序列。
本发明的第五方面，提供了一种宿主细胞，其特征在于，它表达针对人CD7的纳米抗体。
本发明的第六方面，提供了本发明所述的人CD7纳米抗体用于检测人CD7分子的用途。
本发明的第七方面，提供了本发明所述的人CD7纳米抗体用于流式检测和细胞免疫荧光实验的用途。
在另一优选例中，所述用途为非诊断目的。
本发明的第八方面，提供了一种抗人CD7抗体的重链可变区，所述的重链可变区包括三个互补决定区CDR1、CDR2、和CDR3，其中
CDR1选自：SEQ ID NO.:17、20、25、28、和30；
CDR2选自：SEQ ID NO.:18、21、23、26、29、和31；
CDR3选自：SEQ ID NO.:19、22、24、和27。
在另一优选例中，所述重链可变区包括：
SEQ ID NO.17所示的CDR1，SEQ ID NO.18所示的CDR2，SEQ ID NO.19所示的CDR3；
或SEQ ID NO.20所示的CDR1，SEQ ID NO.21所示的CDR2，SEQ ID NO.22所示的CDR3；
或SEQ ID NO.17所示的CDR1，SEQ ID NO.23所示的CDR2，SEQ ID NO.24所示的CDR3；
或SEQ ID NO.25所示的CDR1，SEQ ID NO.26所示的CDR2，SEQ ID NO.27所示的CDR3；
或SEQ ID NO.28所示的CDR1，SEQ ID NO.29所示的CDR2，SEQ ID NO.22所示的CDR3；
或SEQ ID NO.30所示的CDR1，SEQ ID NO.31所示的CDR2，SEQ ID NO.22所示的CDR3。
优选地，它具有SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36或SEQ ID NO.37所示的氨基酸序列。
本发明的第九方面，提供了一种抗体，所述抗体具有：如本发明第八方面所述的重链可变区。
在另一优选例中，所述抗体具有SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36或SEQ ID NO.37所示的氨基酸序列的VHH链。
在另一优选例中，所述的抗体为特异性抗CD7蛋白的抗体。
在另一优选例中，所述抗体为纳米抗体。
本发明的第十方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第八方面所述的重链可变区的序列或如本发明第九方面所述的抗体的序列；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签和HA标签
在另一优选例中，所述的重组蛋白特异性结合CD7蛋白。
本发明的第十一方面，提供了一种免疫偶联物，该免疫偶联物含有：
(a)如本发明第一方面所述的人CD7纳米抗体的VHH链、如本发明第二方面所述的人CD7纳米抗体、如本发明第八方面所述的重链可变区、如本发明第九方面所述的抗体、或如本发明第十方面所述的重组蛋白；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
在另一优选例中，所述偶联部分为假单胞杆菌外毒素或其片段。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明第一方面所述的人CD7纳米抗体的VHH链、如本发明第二方面所述的人CD7纳米抗体、如本发明第八方面所述的重链可变区、如本发明第九方面所述的抗体、或如本发明第十方面所述的重组蛋白。所述多价是指，在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明第一方面所述的人CD7纳米抗体的VHH链、如本 发明第二方面所述的人CD7纳米抗体、如本发明第八方面所述的重链可变区、如本发明第九方面所述的抗体、或如本发明第十方面所述的重组蛋白。
本发明的第十二方面，提供了一种药物组合物，它含有：
(i)如本发明第八方面所述的重链可变区、如本发明第九方面所述的抗体、如本发明第十方面所述的重组蛋白、或如本发明第十一方面所述的免疫偶联物；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为注射剂型。
在另一优选例中，所述的药物组合物用于制备治疗肿瘤的药物，所述的肿瘤选自下组：胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、宫颈癌、淋巴癌、肾上腺肿瘤、或膀胱肿瘤。
本发明的第十三方面，提供了如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、或如本发明第十方面所述的免疫偶联物的用途，用于制备药剂、试剂、检测板或试剂盒；
所述试剂、检测板或试剂盒用于：检测样品中CD7蛋白；
所述药剂用于治疗或预防表达CD7蛋白的肿瘤。
在另一优选例中，所述肿瘤包括：胃癌、淋巴瘤、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、或肾上腺肿瘤。
本发明的第十四方面，提供了一种检测样品中CD7蛋白的方法，所述方法包括步骤：
(1)将样品与本发明第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CD7蛋白。
本发明的第十五方面，提供了一种重组多肽的制备方法，该方法包含：
(a)在适合表达的条件下，培养本发明第九方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是本发明第五方面所述的抗体或本发明第六方面所述的重组蛋白。
本发明的第十六方面，提供了一种治疗疾病的方法，所述方法包括，给需要的对象施用本发明的纳米抗体或免疫偶联物。
有益效果：
(1)本发明首先用流式检测方法，选择高表达人CD7分子的癌细胞系，经处理后使其具有免疫原性，然后用该细胞系免疫骆驼，取骆驼外周血提取淋巴细胞制备纳米抗体免疫基因库，最后在人肾上皮细胞系(293T cell line)上进行筛选CD7纳米抗体，从而获得了人CD7特异性的纳米抗体基因。将此基因克隆至原核表达载体并转化到大肠杆菌中，从而建立了能在大肠杆菌中高效表达的纳米抗体株。
(2)本发明使用高表达目的抗原的细胞免疫骆驼，获得的免疫库比较丰富，除了可以筛选目的抗原抗体，同时在所免疫细胞上的其它高表达分子同样可以 拿来被筛选，比用多肽或者蛋白作为抗原一次免疫只能产生一种类型的针对所选抗原的抗体更能够节约成本、时间以及人力；
(3)本发明使用细胞来对制备的纳米抗体库进行淘筛人CD7分子纳米抗体，能够获得特异性识别天然活性的人CD7分子，这种特异性的纳米抗体可以被拿来用于流式检测和细胞免疫荧光实验。
附图说明
图1是第一轮PCR产物琼脂糖凝胶电泳图，切胶回收650～750bp片段；
图2是第二轮PCR产物琼脂糖凝胶电泳图，切胶回收500bp左右的片段；
图3是SpeI和SacI双酶切噬菌体载体pComb3XSS琼脂糖凝胶电泳图，切胶回收3200bp左右的载体片段；
图4是随机的选取24个克隆做菌落PCR，琼脂糖凝胶电泳图；
图5是用噬菌体的酶联免疫方法(ELISA)在细胞上筛选特异性单个阳性克隆结果；
图6是纳米抗体经过原核表达用镍柱离子亲和层析进行纯化，对纯化收集的4管纳米抗体进行SDS-PAGE电泳后考马斯亮蓝染色图(克隆VHH6,克隆VHH10)；
图7是纳米抗体VHH6和VHH10与高表达CD7Jurkat细胞系的结合曲线和亲和力测定结果；
图8是用商业化CD7抗体和所获高纯度纳米抗体同时对CD7阳性细胞Jurkat染色，然后用流式细胞仪检测分析后所得结果；
图9是用商业化CD7抗体和所获高纯度纳米抗体同时对CD7阴性细胞RPMI8226染色，然后用流式细胞仪检测分析后所得结果；
图10是转染表达CD7质粒pcDNA3.1-CD7到CD7阴性细胞系H460中，然后用商业化CD7抗体染色，流式细胞仪检测分析后所得结果；
图11是用所获高纯度纳米抗体去染同一批转染CD7的H460细胞，用于细胞免疫荧光分析图。
图12是用所获高纯度纳米抗体对Jurkat以及RPMI8226细胞进行染色，用于免疫荧光分析图。
图13是PG001(VHH-6-ETA)设计以及性质鉴定。(A)PG001构建的简化示意图。6His,His标签；VHH-6是CD7特异性的纳米抗体；4(G4S)是连接链，由甘氨酸和丝氨酸组成；ETA是由假单胞杆菌外毒素结构域II和结构域III组成；KDEL是内质网滞留序列。分子量大小是根据它们的氨基酸序列估算的。(B)考马斯亮蓝染色分析镍柱亲和纯化的重组免疫毒素PG001的纯度。(C)免疫印迹分析重组免疫毒素PG001。
图14是用所获高纯度偶联毒素抗体PG001同时对CD7阳性细胞Jurkat，CEM以及阴性细胞RPMI8226，H460染色，然后用流式细胞仪检测分析后所得结果；
图15是用所获高纯度偶联毒素抗体PG001，设置浓度梯度分别处理(A)CD7阳性Jurkat细胞(黑色格子)和CD7阴性RPMI8226细胞(白色格子)和(B)CD7阳性CEM细胞(黑色格子)和CD7阴性H46细胞(白色格子)72小时。细胞生长抑制情况通过WST-8来测定。平均值来自三个(Jurkat,CEM,RPMI8226,H460)独立的实验。标准差用误差把表示。
图16是用所获高纯度偶联毒素抗体PG001150ng/ml PG001杀伤Jurkat，CEM和RPMI8226细胞，分别选取24h、48h、72h三个时间点用Annexin V和7-AAD染色处理。右下象限中的细胞群代表早期凋亡细胞(Annexin V阳性和7-AAD阴性)，右上象限中的细胞群代表死亡细胞(Annexin V和7-AAD双阳性)。所示数据来自三个独立实验。
图17是用单次计量的150ng/ml PG001处理CD7阳性Jurkat(C)和CEM(D)细胞 (图中)，或者在100倍亲本纳米抗体VHH6(图右)存在条件下处理上述细胞，48小时后，处理的细胞用Annexin V和7-AAD染色的结果。右下象限中的细胞群代表早期凋亡细胞(Annexin V阳性和7-AAD阴性)，右上象限中的细胞群代表死亡细胞(Annexin V和7-AAD双阳性)。所示数据来自三个独立实验。
图18是用所获高纯度偶联毒素抗体PG001150ng/ml PG001处理Jurkat，CEM以及RPMI8226，H460细胞48小时后，然后用免疫印迹检测PARP分子切割情况的结果。
图19是双价的纳米抗体免疫毒素PG002的制备以及PG002的毒理效应检测。(A)PG002的构建简易示意图。VHH-6是CD7特异性的纳米抗体；4(G4S)是连接链，由甘氨酸和丝氨酸组成。(B)亲和纯化的纳米抗体免疫毒素PG001用考马斯亮蓝染色实验分析。(C)CD7阳性Jurkat细胞(黑色格子)和CD7阳性CEM细胞(灰色格子)以及CD7阴性RPMI8226细胞(白色格子)经过不同浓度的PG002处理72小时。细胞生长抑制情况由WST-8试剂盒检测。平均值来自三个(Jurkat,CEM,RPMI8226)独立的实验。标准差用误差把表示。
图20是用所获高纯度偶联毒素抗体PG001尾静脉注射NOD/SCID小鼠，然后监测其体内抗CEM白血病细胞的效果。
具体实施方式
下面结合附图对本发明作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。
本发明人通过广泛而深入的研究，经过大量的筛选，成功的获得一类抗CD7纳米抗体，实验结果表明，该纳米抗体不仅特异性高，而且能够显著与表达CD7分子的细胞株和T淋巴细胞或自然杀伤细胞NK上的CD7分子进行高效结合，可以通过对这一类抗体改造来递送功能性分子(毒素或者小分子RNA)对CD7分子阳性细胞进行杀伤或者其他功能研究，在此基础上完成了本发明。
本发明首先用CD7高表达细胞系Jurkat cells(5×106)免疫一只新疆双峰驼，经过连续7次免疫之后提取该双峰驼外周血淋巴细胞并成功构建了单域重链抗体文库。然后在293T-CD7－(293T原始不表达CD7)和293T-CD7+(293T-CD7稳转细胞株)细胞系上进行淘筛CD7特异性的纳米抗体，从而最终获得了能在大肠杆菌中高效表达的纳米抗体菌株。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“单域抗体(VHH)”、“纳米抗体”(nanobody)具有相同的含义，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天 然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。本发明还包括与所述的抗CD7蛋白抗体或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括包括三个互补决定区CDR1、CDR2、和CDR3，其中
CDR1选自：SEQ ID NO.:17、20、25、28、和30；
CDR2选自：SEQ ID NO:18、21、23、26、29、和31；
CDR3选自：SEQ ID NO.:19、22、24、和27。
在另一优选例中，所述重链可变区具有SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36或SEQ ID NO.37所示的氨基酸序列。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合CD7蛋白的多肽，例如具有重链可变区(如SEQ ID NO.:32-37之一的氨基酸序列)的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽 与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有CD7蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含人抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表I进行氨基酸替换而产生。
表I
Figure PCTCN2015077854-appb-000001
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上,更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与SEQ ID NO.:32-37之一所示的成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离 和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素(Koppe等，2005，癌转移评论(Cancer metastasis reviews)24，539)；2.生物毒(Chaudhary等，1989，自然(Nature)339，394；Epel等，2002，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)51，565)；3.细胞因子如IL-2等(Gillies等，1992，美国国家科学院院刊(PNAS)89，1428；Card等，2004，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)53，345；Halin等，2003，癌症研究(Cancer Research)63，3202)；4.金纳米颗粒/纳米棒(Lapotko等，2005，癌症通信(Cancer letters)239，36；Huang等，2006，美国化学学会杂志(Journal of the American Chemical Society)128，2115)；5.病毒颗粒(Peng等，2004，基因治疗(Gene therapy)11，1234)；6.脂质体(Mamot等，2005，癌症研究(Cancer research)65，11631)；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；10.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合CD7蛋白分子，因而可用于预防和治疗肿瘤。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的纳米抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
标记的免疫球蛋白
在本发明的一个优选例中，所述纳米抗体带有可检测标记物。更佳地，所述的标记物选自下组：胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，CD7蛋白的单克隆抗体用胶体金标记，得到胶体金标记的单克隆抗体。
本发明的CD7蛋白单克隆抗体具有很好的特异性，很高的效价。
方法和样本
本发明涉及用于在以细胞和/或组织溶解的样本检测CD7蛋白的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中CD7蛋白的水平。本发明方法所使用的样本可以是存在于细胞保存液中的包括细胞的任何样本，正如在液基细胞检测法中所使用的。
试剂盒
本发明还提供了一种指含有本发明的抗体(或其片段)或本发明的检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明进一步设计用于检测CD7水平的检测试剂盒，该试剂盒包括识别CD7蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
材料和方法
1.菌株和质粒
大肠杆菌TOP10用来扩增质粒和和克隆，大肠杆菌XL1-Blue(Stratagene)用来扩增纳米噬菌体抗体库，大肠杆菌BL-21(DE3)用来表达蛋白。噬菌体质粒pCOMB 3XSS用来制备噬菌体库，pcDNA3.1用来构建真核表达质粒，pET28a用于原核表达。慢病毒载体Red-OG2用来构建稳转细胞系。
2.病人样本和细胞系
经过伦理委员会批准后，提取T-ALL和T-AML病人血液或骨髓以制备原代细胞。用Ficol分离单核细胞按照标准操作程序进行。原代细胞用加20％FBS RPMI1640培养基培养，同时加入并加入双抗。
白血病细胞系Jurkat、CEM，以及肺癌细胞系H460用加10％FBS RPMI1640培养基培养，并加入双抗。骨髓瘤细胞系RPMI8226使用10％FBS IMDM培养基培养，并加入双抗。293T细胞系使用10％FBS DMEM培养基培养，并加入双抗。
3.纳米抗体筛选
用CD7阳性的Jurkat细胞免疫骆驼，提取骆驼外周血，提取RNA，建立噬菌体抗体库。利用细胞淘洗的方法筛选获得CD7特异性的纳米抗体，简要操作流程如下：用慢病毒转染的方法构建293T-CD7稳定细胞系。新鲜制备的噬菌体纳米抗体悬液先后在293T和293T-CD7细胞上淘洗，反复淘筛3轮后，挑取单克隆，再在293T和293T-CD7细胞上进行全细胞酶联免疫吸附实验。选择阳性克隆进行测序。
4.CD7纳米抗体表达和纯化
把获得的阳性克隆纳米抗体序列克隆到原核表达载体pET28a，在C端留有His标签和HA标签用于纯化和鉴定，然后转化进入表达菌BL-21,利用IPTG诱导表达。原核表达的纳米抗体经过亲和纯化获得纯的纳米抗体，蛋白纯度经过考马斯亮蓝染色鉴定，蛋白浓度用BCA方法检测。
5.CD7纳米抗体免疫毒素的构建，表达和纯化
全基因合成假单胞杆菌毒素截短体PE38和纳米抗体VHH-6，然后插入pET28a载体中，在N端留有6-His标签用于纯化和检测，同样被转化进入表达菌BL-21，利用IPTG诱导表达。原核表达的纳米抗体免疫毒素经过亲和纯化获得纯的纳米 抗体，蛋白纯度经过考马斯亮蓝染色鉴定，蛋白浓度用BCA方法检测。二价的免疫毒素构建和一价免疫毒素一样通过PCR的方法在VHH-6序列之前插入VHH-6序列，并进行原核表达，进而纯化和鉴定。
6.对本发明的纳米抗体的亲和力进行了测定：
实验方案如下：为了评估筛选出的纳米抗体亲和力，用不同浓度的纳米抗体在冰上孵育Jurkat细胞。PBS洗两次，用APC标记的抗HA单抗(Miltenyi Biotec,CHN)在含有2％BSA的PBS中冰上孵育1h。PBS洗两次，利用FACSCalibur检测荧光强度。结合饱和度曲线，非线性回归曲线以及Scatchard plots利用软件Graph Pad Prism(Graph Pad Software,Inc)制作.
7.流式分析
通过流式细胞检测纳米抗体和纳米抗体免疫毒素的结合特异性。大约3×105个(Jurkat、CEM、H460、RPMI8226)细胞用PBS洗两遍然后，重悬在终浓度分别为5μg/ml的纳米抗体和纳米抗体免疫毒素的50μlPBS的体系中，冰上放置1小时，用PBS洗两遍，于PBS中重悬后加入抗HA羊多抗或者抗His鼠单抗1μg，冰上放置1小时，然后洗细胞两遍，分别用抗羊单抗Alexa488(CST)和抗鼠单抗Alexa647(CST)冰上孵育1小时，最后用PBS洗3遍，进行流式检测。Lipo2000转染pcDNA3.1-CD7的肺癌细胞系H460的CD7表达水平用商业化抗体进行细胞流式检测。大约3×105个被转染细胞经PBS洗两遍后，加入商业化抗体(BD CD7-PE)冰上孵育1个小时，PBS洗涤2遍，最后进行流式细胞检测。
8.细胞免疫荧光
通过细胞免疫荧光检测纳米抗体的特异性。
Jurkat和RPMI 8226细胞分别接种在放有多聚赖氨酸处理过的载玻片的六孔板中，37摄氏度细胞培养箱过夜。把载玻片取出，用PBS洗2遍后用4％多聚甲醛固定15分钟，然后用3％BSA封闭1小时，最后加纳米抗体室温孵育1小时，PBS洗2遍再加抗HA羊多抗室温孵育1小时，PBS再洗2遍后加入抗羊单抗Alexa488(CST)室温孵育1小时，PBS洗3遍后用共聚焦荧光显微镜拍摄。
Lipo2000分别转染pcDNA3.1和pcDNA3.1-CD7的H460细胞，和上述相同方法进行细胞免疫荧光检测。
9.CD7-纳米抗体免疫毒素的细胞毒效应
纳米抗体免疫毒素对靶细胞的浓度依懒性细胞毒效应用WST-8试剂盒检测。
按照1*104个细胞/孔将细胞接种在孵育了不同浓度的免疫毒素的96孔板中(其中H460细胞接种3*103)，72小时后加入10μl WST-8试剂，37摄氏度孵育直到在450nm出最大吸收值读数。为了检测纳米抗体免疫毒素引起的细胞生长抑制是否由凋亡导致的，2.5×105个细胞种在24孔板中，加入150ng/ml毒素，处理不同时间后，对处理的细胞用FITC标记的Annexin V和7-AAD染色，根据试剂盒方案进行。封闭实验是在加入单价免疫毒素(150ng/ml)之前1小时加入50倍的亲本抗体。
10.聚丙烯酰胺凝胶电泳和免疫印迹
SDS-PAGE实验按照标准的实验操作规程进行。凝胶用考马斯亮蓝R-250染色。免疫印迹使用HRP标记的二抗。增强发光显色试剂盒用于检测。纳米抗体免疫毒素用抗His标签检测。全长的PARP和它的特异性剪切产物用兔抗人PARP抗体。
11.毒素在NOD/SCID小鼠体内抗肿瘤活性
CEM细胞用PBS洗1遍，在第0天，每只NOD/SCID小鼠通过尾静脉注射2×106CEM细胞。第5天给每只小鼠分别注射5μg PG001，隔一天给药一次连续给药3次。监测小鼠日常健康状况。垂死的小鼠按照规程处死。小鼠的生存时间用卡尔-梅耶分析来评估毒素的治疗效果以及检测中位生存期。
本发明的主要优点在于：
(1)本发明抗CD7蛋白的纳米抗体，特异性高，亲和力强，具有良好的热稳定性，并且可以大量制备，质量容易控制。
(2)本发明抗CD7蛋白的纳米抗体通过偶联假单胞杆菌外毒素，能够高效诱导CD7阳性的T淋巴细胞白血病细胞系的凋亡。
下面结合具体实施例，进一步阐述本发明。
实施例1：针对于人CD7的纳米抗体文库的构建：
(1)用CD7高表达细胞系免疫骆驼：5×106个CD7高表达Jurkat细胞系(购自ATCC公司)免疫一只新疆双峰驼(大众养殖集团公司)，每周一次，共连续免疫7次，免疫过程中刺激B细胞表达抗原特异性的纳米抗体；(2)7次免疫结束后，提取骆驼外周血淋巴细胞50ml并提取总RNA(Trizol法)；(3)按照Thermo Scientfic K1621\K1622试剂盒说明书，将提取的RNA反转录成cDNA，然后利用PCR方法扩增VHH链，第一轮PCR：
上游引物：GTCCTGGCTCTCTTCTACAAGG(SEQ ID NO.:45)
下游引物：GGTACGTGCTGTTGAACTGTTC(SEQ ID NO.:46)
扩增重链抗体引导肽和抗体CH2之间的片段，55℃退火，32个循环；琼脂糖凝胶电泳，切胶回收大小在650bp～750bp的DNA片段，如图1所示。
第二轮PCR:
以第一轮PCR回收产物作为模板，
上游引物：CGAGCTCATGGATGTGCAGCTGCAGGAGTCTGGAGGAGG(SEQ ID NO.:47)
下游引物：GGACTAGTGATGGAGACGGTGACCTGGGT(SEQ ID NO.:48)
扩增重链抗体FR1区和FR4区，61℃退火，35个循环，回收大小在500bp左右的目的片段，结果如图2所示；(4)使用限制性的内切酶(购自NEB公司)Spe I及Sac I酶切10μg pComb3XSS噬菌体展示载体(购自Creative Biogene)如图3所示，以及双酶切10μg VHH，并用T4DNA连接酶(购自NEB公司)连接两个酶切片段；(5)将连接产物纯化后电转化至电转感受态细胞XL1-Blue(购自2ndLabTM)中，构建CD7的纳米抗体噬菌体展示文库并测定库容，库容的大小约为7.3×107；与此同时，通过菌落PCR检测所建文库的插入率检测结果，图4显示菌落PCR结果，随机的选取24颗克隆做菌落PCR，结果显示插入率达到96％。
实施例2：针对CD7的纳米抗体筛选过程：
(1)向293T细胞中加入3％BSA\PBS,室温孵育30min,去除3％BSA\PBS，并用PBS洗两遍后，立即加入新鲜制备的噬菌体纳米抗体悬液，37℃培养1h,并同时微摇培养皿；(2)向293T-CD7+细胞中加3％BSA\PBS,室温孵育30min；去除3％BSA\PBS，并用PBS洗两遍后，立即加入从(1)中吸取的上清悬液，37℃孵育1h,并同时微摇培养皿；(3)去上清，洗下细胞，并用PBS\Tween-20洗细胞3遍，然后加入甘氨酸-盐酸洗脱缓冲液(pH 2.2),37℃孵育30min,按比例加入Tris\HCl缓冲液(pH 7.4),中和pH值，离心，去细胞，上清中含有噬菌体悬液；(4)上一步中的噬菌体悬液进一步感染对数生长期的XL1-Blue大肠杆菌，产生并纯化噬菌体进行下一轮细胞筛选，反复进行3轮，噬菌体逐步得到淘筛。
实施例3：用噬菌体的酶联免疫方法(ELISA)筛选特异性单个阳性克隆(全细胞ELISA法)：
1.在微量滴定板中表达噬菌体抗体
(1)用无菌牙签挑取32个单个克隆，分别置于每孔含100μl 2×TY/amp/glu的96孔微量滴定板板孔中，振荡(300r/min)培养过夜，放在微量滴定板架上；(2)每孔加入50μl 2×TY/amp/glu/gly并储存于-70℃；(3)使用96孔无菌转移设备或移液器，从主板每孔中吸取2μl，接种到一块每孔含150μl 2×TY/amp/glu的96孔板中，37℃振荡，直到A600值接近0.5(2.5小时)；(4)每孔加入50μl含2X109pfu/m1辅助噬菌体(在储存料中稀释)的2×TY/amp/glu，噬菌体与细菌的比例接近20：1，37℃下孵育孔板30分钟；(5)以2700r/min离心孔板10分钟，用多道移液器或吸取设备移除上清液；(6)用150μl 2×TY/amp/kan重悬每孔的细菌沉淀，让噬菌体纳米抗体表达，37℃振荡(300r/min)培养孔板过夜；(7)次日，以2700r/min离心孔板10分钟；每孔取50μl上清液用来进行噬菌体ELISA。
2.全细胞ELISA
(1)在96孔板中分别加入293T-CD7+细胞(5×105个)和293T-CD7－细胞(5×105个)，每个克隆各取出50μl噬菌体悬液加入到96孔板中，并对应编号；室温孵育1h,离心96孔板，去除上清液，每孔细胞用PBS洗两遍；(2)向每孔中加入HRP标记的抗HA-Tag抗体，室温孵育1h,离心去除上清液，PBS洗细胞3遍，去尽上清，用TMB显色法进行显色；(3)用酶标仪在450nm处读板，并保存数据；(4)处理数据，分析实验结果；(5)当样品孔OD值大于对照孔OD值2.5倍以上时，判为阳性克隆孔，结果如图5所示；(6)将对应的阳性克隆孔的菌转摇在含有3毫升的LB液体中以便提取质粒并进行测序分析。
根据序列比对软件DNAMAN分析各个克隆株的基因序列，把CDR1，CDR2，CDR3序列相同的株视为同一克隆株，而其序列不同的株视为不同克隆株，最终共有6株高亲和性、高特异性的抗体。
本发明筛选到的较佳的克隆株序列信息如下：
克隆1：
其氨基酸序列为，
Figure PCTCN2015077854-appb-000002
其编码核苷酸序列为，
Figure PCTCN2015077854-appb-000003
克隆2：
其氨基酸序列为，
Figure PCTCN2015077854-appb-000004
其编码核苷酸序列为，
Figure PCTCN2015077854-appb-000005
Figure PCTCN2015077854-appb-000006
克隆3：
其氨基酸序列为，
Figure PCTCN2015077854-appb-000007
其编码核苷酸序列为，
Figure PCTCN2015077854-appb-000008
克隆4：
其氨基酸序列为，
Figure PCTCN2015077854-appb-000009
其编码核苷酸序列为，
Figure PCTCN2015077854-appb-000010
克隆5：
其氨基酸序列为，
Figure PCTCN2015077854-appb-000011
其编码核苷酸序列为，
Figure PCTCN2015077854-appb-000012
克隆6：
其氨基酸序列为，
Figure PCTCN2015077854-appb-000013
其编码核苷酸序列为，
Figure PCTCN2015077854-appb-000014
表1本发明中筛选出的较佳的克隆株序列信息
Figure PCTCN2015077854-appb-000015
Figure PCTCN2015077854-appb-000016
实施例4：纳米抗体在宿主菌大肠杆菌中表达、纯化
(1)将前面测序分析所获得6种纳米抗体亚克隆至表达载体PET27b(+)中，并将测序鉴定正确的重组质粒转化到表达型宿主菌BL(DE3)中，将其涂布在含有50μg/ml卡那霉素的LB固体培养基平板上，37℃过夜；(2)挑选单个菌落接种在3毫升含有卡那霉素的LB培养液中，37℃摇床培养过夜；(3)接种1ml的过夜菌种至250mlLB培养基中，37℃摇床培养，培养到OD值达到0.6～1时，加入IPTG，37℃摇床培养过夜；(4)第二天，离心收菌；(5)将菌体破碎以获得抗体粗提液；(6)经镍柱离子亲和层析纯化抗体蛋白，获得高纯度的纳米抗体，如图6所示，为其中一种纳米抗体(本发明的代表抗体克隆2，即克隆VHH-10)纯化后收集的连续4管纳米抗体，经过SDS-PAGE后，考马斯亮蓝染色图。克隆VHH-6经考马斯亮蓝染色具有同样大小的条带。
实施例5：利用流式细胞仪分析两株纳米抗体对CD7阳性的Jurkat细胞的亲和力
结果表明：本发明的两株纳米抗体VHH6，VHH10的亲和力分别为：15.37nM,29.63nM，如图7所示。
实施例6：用流式细胞仪检测所获得的纳米抗体活性
用高纯度的纳米抗体(克隆2，即克隆VHH-6)在CD7阳性的细胞系Jurkat细胞上进行流式细胞分析，并用商业化CD7抗体做对比。实验步骤如下：常温孵育1小时后用PBS洗3遍，加入抗HA-tag抗体(兔抗)常温孵育1小时，PBS洗3遍，加入抗兔Alexa
Figure PCTCN2015077854-appb-000017
488荧光标记抗体常温孵育1小时后，用PBS洗3遍，最后用流式细胞仪检测，结果如图8表示，表明所获的纳米抗体结合阳性细胞比例和商业化抗体(购自BD公司，为常规单克隆抗体，该抗体可以和人细胞表面表达CD7分子的细胞进行结合，包括一些表达CD7分子的肿瘤细胞以及高表达CD7的原代细胞如T淋巴细胞，自然杀伤细胞)相似。用高纯度的纳米抗体在CD7阴性的细胞系RPMI8226细胞上进行流式细胞分析，检测特异性。实验步骤同上，结果如图9所示，表明所获的纳米抗体特异性较好。
从图8CD7+阳性细胞株中可以看出，左上图为商业化抗体染色对应的对照组，右上为商业化PE标记CD7阳性细胞株(Jurkat)，流式结果显示其CD7+阳性率为96.6％；左下为VHH抗体染色对应的对照组，右下为用VHH-6抗体标记Jurkat，流式结果显示其CD7+阳性率为91.8％；两者的阳性率相近。同样在阴性细胞株RPMI-8226中，商业化抗体与VHH-6抗体对其染色阳性率都在3％以下(分别为 0.648％，2.82％)。
实施例7：用细胞免疫荧光法检测所获得的纳米抗体活性
用lipofectamine2000转染试剂转染pcDNA3.1-CD7质粒到CD7阴性细胞系H460细胞，48小时候后用胰酶消化H460细胞后，一半继续培养24小时后用于流式检测(商业抗体)检验转染效果，另一半细胞用于细胞爬片，24小时后进行细胞免疫荧光检测。流式细胞检测方法步骤如下：取出转染的H460细胞，PBS洗3遍，加入商业化CD7流式抗体，常温孵育1小时后PBS洗3遍，进行流式细胞仪检测，检测结果如图10所示，转染效率在18％左右。细胞免疫荧光检测步骤如下：把载玻片取出，用PBS洗2遍后用4％多聚甲醛固定15分钟，然后用3％BSA封闭1小时，最后加抗体染色，方法同实施例5，最后用共聚焦荧光显微镜拍摄，结果如图10所示，表明同商业化抗体染色比例相似，以及特异性较好。同样在表达CD7分子的Jurkat细胞系以及CD7-阴性细胞系RPMI-8226上进行免疫荧光染色验证。表明VHH6可特异性与Jurkat细胞进行结合，而在RPMI-8226细胞表面未检测到荧光。
图10，对H460细胞转染CD7蛋白质粒(pcDNA3.1-CD7)，并用商业化PE标记CD7分子抗体进行流式细胞术检测，转染效率为18.8％；同样，利用筛选的纳米抗体(本发明代表性的抗体克隆6，即克隆VHH-6)对转有pcDNA3.1-CD7质粒的H460细胞进行免疫荧光分析(图11)，分析单个视野中阳性细胞占视野中总细胞比例，得到的阳性率约为20％，以此说明本发明的抗体效果与商业化抗体相似。图12分析发现可在Jurkat细胞表面检测到CD7的表达，RPMI8226作为阴性对照。
实施例8 CD7纳米抗体免疫毒素的制备以及生物活性
1.构建单价纳米抗体免疫毒素及其亲和力和特异性分析
假单胞杆菌毒素的截短体PE38和纳米抗体VHH-6序列经密码子优化后插入载体pET28a中，在N末端留有后续纯化的HIS标签，C末端REDLK序列被替换成KDEL序列，序列结构如图13A所示。载体转化到大肠杆菌BL21(DE3)，经表达和亲和纯化后，获得纯度较高的纳米抗体免疫毒素VHH-6-PE38，命名为PG001(图13B和C)。结果表明，1升大肠杆菌约产出10毫克纯化的PG001。对PG001的特异性分析发现其仍然能够高特异性的结合CD7阳性细胞系Jurkat和CEM细胞，但结合不上CD7阴性细胞系RPMI8226和H460细胞(图14)。
假单胞杆菌毒素的截短体PE38的编码序列信息如SEQ ID NO.:44)
2.PG001对CD7+T淋巴细胞系的特异性毒性效应
通过WST-8试剂盒检测PG001的毒性效应，结果显示PG001能够在很低的浓度条件下强有力的抑制CEM(EC50≈0.5nM)和Jurkat(EC50≈1.0nM)的细胞增殖，而对CD7阴性的RPMI8226和H460细胞的生长没有影响(图15)。为了检测PG001引起的细胞增殖抑制是否经过凋亡发生的，对PG001处理的细胞用Annexin V和7-AAD染色检测。实验结果显示两种CD7阳性细胞CEM和Jurkat增值抑制是由于PG001诱导的细胞凋亡而导致的，并且具有时间依赖性，但是在相同的浓度条件下并没有导致RPMI 8226细胞的凋亡发生(图16)。同时，PG001对Jurkat和CEM细胞处理48小时引起的细胞毒效应是可以被提前1小时加入的浓度为其50倍的亲本抗体共孵育所阻抑(图17)。除此之外，PG001处理Jurkat和CEM细胞48小时后，可以诱导PARP剪切。众所周知，PARP的剪切是细胞发生凋亡的标志，而PG001处理RPMI8226和H460细胞48小时后，PARP剪切却没有发生(图18)。因此以上结果表明，单价的纳米抗体免疫毒素PG001 能够在纳摩尔浓度条件下特异性的诱导CD7阳性细胞系发生凋亡，而这种凋亡是由受体介导的纳米抗体免疫毒素内吞引起的。
3.二价纳米抗体免疫毒素的(VHH-6)2-PE38的构建及其对CD7+T淋巴细胞
系的特异性毒性效应
鉴于PG001优越的选择性和杀伤能力，但考虑到单价纳米抗体由于分子量较小，构建的免疫毒素也相应较小，因此在体内半衰期较短，同时也是为了进一步提高免疫毒素与靶细胞的结合力、进一步降低免疫毒素的用量，本发明人进行了二价纳米抗体的构建。假单胞杆菌毒素的截短体PE38和二价纳米抗体VHH-6序列经密码子优化后插入载体pET28a中，在N末端留有后续纯化的HIS标签，C末端REDLK序列被替换成KDEL序列，结构如(图19A)。构建好的载体转化到大肠杆菌BL21，经表达和亲和纯化后，获得高纯度的二价纳米抗体免疫毒素(VHH-6)2-PE38，命名为PG002(图19B)。本发明人发现1升大肠杆菌约能产出5毫克纯化的PG002，通过WST-8试剂盒检测PG002的毒性效应，结果显示PG002能够在更低的浓度(皮摩尔)条件下强有力的抑制CD7阳性的CEM(EC50＝23pM)和Jurkat(EC50＝30pM)细胞的增殖，而对CD7阴性的RPMI8226细胞的生长没有影响(图19C)。
4.PG001在NOD/SCID小鼠体内抗CEM白血病细胞的效果
为了检测纳米抗体免疫毒素在动物模型中的抗白血病潜力，本发明人首次使用PG001免疫毒素在NOD/SCID小鼠体内来评估其抗CEM白血病细胞系的生长情况。在第0天，每只小鼠通过尾静脉注射2x106CEM细胞。5天后，通过尾静脉注射5μg免疫毒素PG001，隔天注射，总共给药3次，或者非偶联形式的VHH-6和PE38(VHH-6+PE38)。结果显示给药组PG001小鼠的生存周期与对照组PBS和VHH-6+PE38相比能够显著延长，PBS组中位生存期为28.5天而PG001给药组的中位生存期为37天。实验发现，给药组小鼠后腿会发生残疾以及体重减轻的症状(图20)。
讨论
本发明人通过从骆驼体内筛选到特异性的CD7纳米抗体，从而构建了针对CD7的单价和二价的纳米抗体偶联假单胞杆菌外毒素截短体的免疫毒素PG001和PG002。这两种毒素分别能够以纳摩尔和皮摩尔浓度在体外高效且特异性的诱导急性白血病细胞系发生凋亡，同时能够高效诱导T-ALL和T-AML病人原代细胞特异性地发生凋亡。而且，单价的纳米抗体免疫毒素PG001能够有效的抑制移植入NOD/SCID小鼠体内的人白血病细胞的生长。
本发明人的研究有以下这些新的发现：a)本发明人构建的两种纳米抗体免疫毒素和scFv片段一样不带有Fc片段，因此大大减少了和非靶细胞的结合；b)本发明人的单价纳米抗体免疫毒素PG001对T-ALL细胞系(Jurkat和CEM)的IC50浓度在纳摩尔级别，然而本发明人构建的二价纳米抗体免疫毒素PG002对T-ALL细胞系(Jurkat和CEM)的IC50浓度在皮摩尔级别，杀伤效果提升了30倍左右；c)用纳米抗体构建的这两种免疫毒素利用大肠杆菌BL-21(DE3)原核表达所获得的高可溶性、高纯度的产量分别约为10mg和5mg每升菌液，在产量上与现有技术相比有了大约有50到100倍的提高，这使得这类免疫毒素的生产成本大大降低，更具临床应用价值；d)本发明人通过Annexin V和7-AAD对免疫毒素处理的细胞染色进行流式分析以及用Western blot方法检测PARP分子的切割情况，表明本发明人的免疫毒素是通过诱导细胞凋亡引起细胞死亡的；e)本发明人的纳米抗体免疫毒素不仅能够在低浓度下有效的对T-ALL细胞系进行杀伤，同时本发明人构建的二价纳米抗体免疫毒素能够以更低的浓度条件下在体外高效的杀伤T-ALL和T-AML病人原代细胞；f)本发明人对单价的纳米抗体 免疫毒素PG001在NOD/SCID小鼠体内进行抗人白血病细胞实验，结果显示PG001能够显著延长小鼠生存时间，这是首次运用纳米抗体免疫毒素在体内进行抗白血病研究。
本发明实施例中以VHH-6抗体克隆为代表，经实验验证，本发明提供的其它克隆(如VHH-10)具有与VHH-6抗体克隆类似的生物活性。
总之，本发明人构建的单价和二价纳米抗体CD7免疫毒素能够高效清除CD7阳性的T-ALL细胞系，并且能够在纳摩尔及更低浓度条件下以抗原特异性的方式杀死T-ALL和T-AML病人原代细胞。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变形，这些改进和变形也应视为本发明的保护范围。

Claims (12)

  1. 一种人CD7纳米抗体的VHH链，包括框架区FR和互补决定区CDR，其特征在于，所述框架区FR选自以下的FR1～FR4的氨基酸序列：
    SEQ ID NO.13所示的FR1，SEQIDNO.14所示的FR2，SEQIDNO.15所示的FR3，SEQ ID NO.8所示的FR4；
    或SEQ ID NO.5所示的FR1，SEQIDNO.6所示的FR2，SEQIDNO.7所示的FR3，SEQ ID NO.8所示的FR4；
    或SEQ ID NO.5所示的氨基酸序列FR1，SEQ ID NO.14所示的FR2，SEQ ID NO.16所示的FR3，SEQ ID NO.8所示的FR4；
    或SEQ ID NO.1所示的FR1，SEQ ID NO.2所示的FR2，SEQ ID NO.3所示的FR3，SEQ ID NO.4所示的FR4；
    或SEQ ID NO.1所示的氨基酸序列FR1，SEQ ID NO.9所示的FR2，SEQ ID NO.3所示的FR3，SEQ ID NO.4所示的FR4；
    SEQ ID NO.5所示的FR1，SEQ ID NO.10所示的FR2，SEQ ID NO.11所示的FR3，SEQ ID NO.12所示的FR4；
    所述的互补决定区CDR选自以下的CDR1～CDR3的氨基酸序列：
    SEQ ID NO.28所示的CDR1，SEQ ID NO.29所示的CDR2，SEQ ID NO.22所示的CDR3；
    或SEQ ID NO.20所示的CDR1，SEQ ID NO.21所示的CDR2，SEQ ID NO.22所示的CDR3；
    或SEQ ID NO.30所示的CDR1，SEQ ID NO.31所示的CDR2，SEQ ID NO.22所示的CDR3；
    或SEQ ID NO.17所示的CDR1，SEQ ID NO.18所示的CDR2，SEQ ID NO.19所示的CDR3；
    或SEQ ID NO.17所示的CDR1，SEQ ID NO.23所示的CDR2，SEQ ID NO.24所示的CDR3；
    或SEQ ID NO.25所示的CDR1，SEQ ID NO.26所示的CDR2，SEQ ID NO.27所示的CDR3。
  2. 根据权利要求1所述的人CD7纳米抗体的VHH链,其特征在于,它具有SEQ ID NO.36、SEQ ID NO.33、SEQ ID NO.37、SEQ ID NO.32、SEQ ID NO.34、或SEQ ID NO.35所示的氨基酸序列。
  3. 一种人CD7纳米抗体，其特征在于，它是针对人CD7分子表位的纳米抗体，包括具有SEQ ID NO.36、SEQ ID NO.33、SEQ ID NO.37、、SEQ ID NO.32、SEQ ID NO.34、或SEQ ID NO.35所示的氨基酸序列的VHH链。
  4. 一种DNA分子，其特征在于，它编码选自下组的蛋白质：权利要求1或2所示的人CD7纳米抗体的VHH链，或权利要求3所示的人CD7纳米抗体。
  5. 根据权利要求4所述的DNA分子，其特征在于，它具有选自下组的DNA序列：SEQ ID NO.42、SEQ ID NO.39、SEQ ID NO.43、SEQ ID NO.38、SEQ ID NO.40、或SEQ ID NO.41。
  6. —种表达载体,其特征在于,它含SEQ ID NO.42、SEQ ID NO.39、SEQ ID NO.38、SEQ ID NO.40、SEQ ID NO.41、或SEQ ID NO.43所示的核苷酸序列。
  7. 一种宿主细胞，其特征在于，它表达针对人CD7的纳米抗体。
  8. 权利要求3所述的人CD7纳米抗体用于检测人CD7分子的用途。
  9. 权利要求3所述的人CD7纳米抗体用于流式检测和细胞免疫荧光实验的用途。
  10. 一种免疫偶联物，其特征在于，该免疫偶联物含有：
    (a)如权利要求1所述的人CD7纳米抗体的VHH链、或如权利要求3所述的人CD7纳米抗体；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  11. 如权利要求10所述的免疫偶联物，其特征在于，所述偶联部分为假单胞杆菌外毒素或其片段。
  12. 如权利要求10所述的免疫偶联物，其特征在于，该免疫偶联物含有：多价(如二价)的如权利要求1所述的人CD7纳米抗体的VHH链、或如权利要求3所述的人CD7纳米抗体。
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CN106928358B (zh) * 2015-12-30 2020-09-29 广西医科大学 一种CD105纳米抗体Nb168
WO2020097193A1 (en) 2018-11-06 2020-05-14 The Regents Of The University Of California Chimeric antigen receptors for phagocytosis
CN109652379B (zh) * 2018-12-29 2022-08-16 博生吉医药科技(苏州)有限公司 Cd7嵌合抗原受体修饰的nk-92mi细胞及其应用
US11026973B2 (en) 2019-04-30 2021-06-08 Myeloid Therapeutics, Inc. Engineered phagocytic receptor compositions and methods of use thereof
GB2628935A (en) 2019-09-03 2024-10-09 Myeloid Therapeutics Inc Methods and compositions for genomic integration
CN110760007B (zh) * 2019-11-21 2022-08-26 博生吉医药科技(苏州)有限公司 Cd7-car-t细胞及其制备和应用
US10980836B1 (en) 2019-12-11 2021-04-20 Myeloid Therapeutics, Inc. Therapeutic cell compositions and methods of manufacturing and use thereof
JP2023549140A (ja) 2020-11-04 2023-11-22 マイエロイド・セラピューティクス，インコーポレーテッド 操作されたキメラ融合タンパク質組成物およびその使用方法
CN114702575B (zh) * 2022-01-24 2023-05-23 广东医科大学 抗SARS-CoV-2 S蛋白的纳米抗体、重组纳米抗体、重组载体、重组菌及应用
CN114560943B (zh) * 2022-02-28 2022-12-16 先进生物(苏州)有限公司 Cd7-car-t细胞及其制备方法和应用
CN114685662B (zh) * 2022-03-30 2022-12-27 河北森朗生物科技有限公司 抗cd7纳米抗体、衍生物及其在肿瘤治疗中的应用
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102812042A (zh) * 2010-02-03 2012-12-05 Ucb医药有限公司 用于获得抗体的方法
CN104004095A (zh) * 2014-06-04 2014-08-27 博生吉医药科技(苏州)有限公司 一种cd7纳米抗体、其编码序列及应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103333248B (zh) * 2013-06-07 2014-07-09 东南大学 一种cd25纳米抗体、其编码序列及应用
CN103421115B (zh) * 2013-09-02 2015-06-03 东南大学 一种cd38纳米抗体及应用
CN105384825B (zh) * 2015-08-11 2018-06-01 南京传奇生物科技有限公司 一种基于单域抗体的双特异性嵌合抗原受体及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102812042A (zh) * 2010-02-03 2012-12-05 Ucb医药有限公司 用于获得抗体的方法
CN104004095A (zh) * 2014-06-04 2014-08-27 博生吉医药科技(苏州)有限公司 一种cd7纳米抗体、其编码序列及应用

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023133595A2 (en) 2022-01-10 2023-07-13 Sana Biotechnology, Inc. Methods of ex vivo dosing and administration of lipid particles or viral vectors and related systems and uses
CN115991776A (zh) * 2022-10-26 2023-04-21 上海驯鹿生物技术有限公司 一种靶向cd7的全人源抗体及其应用
WO2024119157A1 (en) 2022-12-02 2024-06-06 Sana Biotechnology, Inc. Lipid particles with cofusogens and methods of producing and using the same

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