WO2017193956A1 - 一种双特异性抗原结合构建体及其制备方法和应用 - Google Patents

一种双特异性抗原结合构建体及其制备方法和应用 Download PDF

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WO2017193956A1
WO2017193956A1 PCT/CN2017/083961 CN2017083961W WO2017193956A1 WO 2017193956 A1 WO2017193956 A1 WO 2017193956A1 CN 2017083961 W CN2017083961 W CN 2017083961W WO 2017193956 A1 WO2017193956 A1 WO 2017193956A1
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construct
amino acid
seq
acid sequence
antigen binding
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李华顺
任宝永
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李华顺
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3015—Breast
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • 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
    • C07K16/2809—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 against the T-cell receptor (TcR)-CD3 complex
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    • C07—ORGANIC CHEMISTRY
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    • 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
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    • C07—ORGANIC CHEMISTRY
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the invention relates to the technical field of immunology, in particular to a bispecific antigen binding construct and a preparation method and application thereof.
  • Robo is a transmembrane receptor protein that has been cloned into four Robo genes in mammals. From the perspective of species evolution, the extracellular portion of Robo1, 2, and 3 is very conserved. From Drosophila to human, it consists of five Ig-like functional regions and three Fibronectin III-type repeats. Robos has a short transmembrane region and a long intracellular region; according to the sequence conservation, the intracellular region is divided into four smaller regions, named: CC0, CC1, CC2, CC3. The extracellular IgG domains of Robos are thought to be required for binding to the ligand Slit.
  • the longer intracellular region interacts with some important signaling molecules and participates in signal transduction downstream of Slit/Robo, thereby completing the stimulation signal by the cell.
  • the mechanism analysis of the protein in the interaction region between Slit2 and Robo has been completed, and it is found that the second domain D2 of Slit2 binds to Ig1 of Robo1, thereby initiating signal transduction.
  • Robo1 is overexpressed in a variety of cancers, such as hepatocellular carcinoma, breast cancer, colon cancer, pancreatic cancer, prostate cancer, glioma, and the like. Studies have shown that Robo1 is abundantly expressed in liver cancer, but only a small amount is expressed in normal tissues, and 84.7% of liver cancer tissue samples are positively expressed; 80% of colon cancer patients have high expression of Robo1 mRNA, and 45% of patients are normal tissues. Four times, 15% of patients are 12 times more likely to be normal tissues, so Robo1 can be a new tumor-associated antigen and a potential therapeutic and diagnostic target.
  • the differentiation cluster 3 (CD3) molecule is only present on the surface of T cells, and often binds tightly to the T cell receptor (TCR) to form a TCR-CD3 complex, which has T cell activation signal transduction and stabilizes the function of the TCR structure.
  • the present invention can effectively activate resting T cells to achieve the purpose of killing diseased cells by providing a bispecific antigen binding construct having an antigen binding site which can bind to CD3 and Robo1, respectively.
  • It is an object of the present invention to provide a bispecific antigen binding construct comprising a first antigen binding unit unit and a second antigen binding unit, the first antigen binding unit being a specific binding immune cell
  • a surface antigen for example, a single-chain variable fragment (ScFV, also referred to as a single-chain antibody)
  • the second antigen-binding unit specifically binds to a tumor cell surface Slit2 of the antigen Robo1 or a fragment thereof (for example, a Slit2D2 fragment).
  • the Slit2D2 fragment has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: Amino acid sequence;
  • the Slit2D2 fragment has the amino acid sequence set forth in SEQ ID NO: 1.
  • the immune cells are selected from the group consisting of: T cells, NKT cells, and CIK cells; more preferably T cells;
  • the first antigen binding unit specifically binds to the immune cell surface antigen CD3;
  • the ScFV comprises a heavy chain variable region (VH) and a light chain variable region (VL); in the ScFV, the VH passes through the C-terminus Attached to the N-terminus of VL, or VH is linked to the C-terminus of VL via the N-terminus; the linkage may be a direct linkage, without any linker peptide, ie, by only forming a peptide bond; or by a linker peptide; Said linkage is achieved by a linker peptide which is a polypeptide having 1-20 amino acids, more preferably 10-16 amino acids, further preferably 14 amino acids;
  • the VH has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: Amino acid sequence;
  • the VH has the amino acid sequence set forth in SEQ ID NO: 2;
  • the VL has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: Amino acid sequence;
  • the VL has the amino acid sequence set forth in SEQ ID NO:3;
  • the ScFV has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: Amino acid sequence;
  • the ScFV has the amino acid sequence set forth in SEQ ID NO:4;
  • the ScFV has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: Amino acid sequence;
  • the ScFV has the amino acid sequence set forth in SEQ ID NO: 5;
  • Slit2D2 is linked to the N-terminus of ScFV through the C-terminus, or Slit2D2 is passed through the N-terminus and ScFV.
  • C-terminally linked; the linkage may be a direct linkage, without any linker peptide, ie, by only forming a peptide bond; or by a linker peptide; preferably, the linkage is achieved by a linker peptide, said
  • the linker peptide is an oligopeptide having 1-10 amino acids, more preferably 3-7 amino acids, further preferably 5 amino acids;
  • the construct of the construct comprises: VH-VL-Slit2D2, VL-VH-Slit2D2, Slit2D2-VH-VL, Slit2D2-VL-VH, "-" indicates a linkage or Link the peptide.
  • the construct has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100 of the amino acid sequence set forth in SEQ ID NO:6. % identical amino acid sequence.
  • the construct has the amino acid sequence set forth in SEQ ID NO: 6.
  • the construct has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100 of the amino acid sequence set forth in SEQ ID NO:7. % identical amino acid sequence.
  • the construct has the amino acid sequence set forth in SEQ ID NO: 7.
  • the construct has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100 of the amino acid sequence set forth in SEQ ID NO:8. % identical amino acid sequence.
  • the construct has the amino acid sequence set forth in SEQ ID NO: 8.
  • the construct has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100 of the amino acid sequence set forth in SEQ ID NO:9. % identical amino acid sequence.
  • the construct has the amino acid sequence set forth in SEQ ID NO: 9.
  • the Slit2D2 fragment, ScFV and bispecific antigen binding constructs of the invention further comprise variants thereof.
  • the variant may comprise or be substituted, deleted, inserted or added with one or more amino acids based on the specific amino acid sequence or the parent sequence disclosed herein and retains the activity of substitution, deletion, insertion or addition.
  • the amino acid sequence consists of.
  • a variant of a Slit2D2 fragment may comprise or consist of an amino acid sequence which has been substituted, deleted, inserted or added with one or several amino acids on SEQ ID NO: 1 and which retains the activity of substitution, deletion, insertion or addition, preferably, The activity is specific binding to the tumor cell surface antigen Robo1.
  • a variant of ScFV may comprise or consist of an amino acid sequence which has been substituted, deleted, inserted or added with one or several amino acids on SEQ ID NO: 4 or 5 and which retains the activity of substitution, deletion, insertion or addition,
  • the activity is to specifically bind to immune cell surface CD3 molecules and activate immune cells (eg, T cells, NKT cells, and CIK cells).
  • a variant of a bispecific antigen binding construct may comprise or be substituted, deleted, inserted or added with one or several amino acids on SEQ ID NO: 6, 7, 8 or 9 and retains substitutions, deletions, Insertion or addition of a pre-active amino acid sequence composition, preferably, the activity is killing of tumor cells (eg, breast cancer cells, liver cancer cells).
  • tumor cells eg, breast cancer cells, liver cancer cells.
  • substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence according to the present invention means substitution, deletion, insertion or addition at any position in the sequence and in one or several amino acid sequences. 1 or a few amino acid residues (eg 2, 3, 4, 5, 6, 7, 8, or 9), and 2 or 2 of the substitutions, deletions, insertions, and additions The above can happen at the same time.
  • substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence according to the present invention can be carried out by using "Molecular Cloning 3" and “Current Protocols in Molecular Biology” (Modern Molecular Biology Practice) ) Obtained by site-directed mutagenesis as described.
  • the invention also provides a nucleotide encoding the above construct protein.
  • the encoding nucleotide has the sequence set forth in SEQ ID NO: 10 or a complement thereof.
  • the coding nucleotide of the invention comprises variants thereof, for example hybridizing to the nucleotide sequence of SEQ NO: 10 or its complement under stringent conditions, and encoding the pair of tumor cells a polynucleotide of an amino acid sequence having a killing activity (eg, breast cancer cells, liver cancer cells); or
  • the "stringent conditions” described herein may be any of low stringent conditions, medium stringent conditions, and high stringency conditions, preferably high stringency conditions.
  • the "low stringency conditions” may be 30 ° C, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 52% formamide
  • “medium stringent conditions” may be 40 ° C, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 52% formamide
  • “high stringency conditions” may be 50 ° C, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 52% formamide.
  • the higher the temperature the more highly homologous polynucleotides can be obtained.
  • one skilled in the art can select a comprehensive result of a plurality of factors affecting the stringency of hybridization, probe concentration, probe length, ionic strength, time, salt concentration, etc. to achieve a corresponding stringency.
  • the invention also provides a vector comprising the above-described encoding nucleotide.
  • the vector may be a recombinant plasmid, recombinant cell, recombinant strain, recombinant virus comprising a nucleotide comprising the above-described coding construct protein.
  • the construct of the present invention can be artificially synthesized, or the coding gene can be synthesized first, and then obtained by biological expression.
  • the invention also provides a preparation method of the above construct, the specific steps comprising:
  • the step (1) described includes the following specific steps:
  • the gene fragment encoding the construct protein is obtained by whole gene synthesis, and the PCR product is obtained by PCR amplification, and the product is purified and recovered, cloned into a vector plasmid, and the resulting recombinant vector is transformed into the first host cell. After being transferred to a solid medium containing ampicillin (AMP) for propagation, screening positive clones, confirming the successful construction of the vector by sequencing and preserving the seed;
  • AMP ampicillin
  • the vector plasmid is a pCDNA plasmid, preferably pCDNA4.3;
  • the first host cell includes, but is not limited to, an E. coli strain, preferably a TOP10 strain.
  • the step (2) described includes the following specific steps:
  • the second host cell is cultured, and when the cell density reaches 2.5 ⁇ 10 6 cells/ml, the extracted recombinant vector is transfected, the second host cell successfully transfected with the target gene is a transformant, the transformant is selected, and the transformant is fermented. Collect the supernatant after 5 days;
  • the second host cell includes, but is not limited to, an ExpiCHO cell.
  • the step (3) described includes the following specific steps:
  • Such purification includes, but is not limited to, ion exchange chromatography to purify the protein.
  • step (4) described includes the following specific steps:
  • the molecular weight and purity of the fusion protein are identified.
  • the molecular weight of the fusion protein can be detected by general protein biochemical means, including but not limited to: SDS-PAGE, Western blotting, mass spectrometry MS and the like.
  • the purity of the fusion protein was detected by SEC-HPLC.
  • the invention also provides a pharmaceutical composition comprising the above bispecific antigen binding construct, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition of the present invention may be a tablet (including a sugar-coated tablet, a film-coated tablet, a sublingual tablet, an orally disintegrating tablet, an oral tablet, etc.), a pill, a powder, a granule, a capsule.
  • the pharmaceutical composition is an injection.
  • the pharmaceutically acceptable excipients of the present invention are preferably pharmaceutically acceptable injectable excipients, such as isotonic sterile saline solutions (sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, chlorination). Calcium, magnesium chloride, etc., or a mixture of the above salts, or dried, for example, a freeze-dried composition, suitably formed into an injectable solute by the addition of sterile water or physiological saline.
  • injectable excipients such as isotonic sterile saline solutions (sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, chlorination). Calcium, magnesium chloride, etc., or a mixture of the above salts, or dried, for example, a freeze-dried composition, suitably formed into an injectable solute by the addition of sterile water or physiological saline.
  • the invention also provides the use of the above bispecific antigen binding construct for the preparation of an antitumor drug.
  • the invention also provides the use of the above bispecific antigen binding construct in the screening and pharmacodynamic evaluation of antitumor drugs.
  • the tumor is a tumor and a related disease which express Robo1 high, and the high expression described herein means that the expression level of Robo1 in the tumor cell is higher than that in the normal cell.
  • the tumor disease is liver cancer or breast cancer.
  • domain refers to a region having a specific structure and an independent function in a biological macromolecule, for example, the Slit2 protein D2 domain refers to a second structure of four leucine-rich repeats in the Slit2 protein. area.
  • the bispecific antigen binding construct provided by the invention can simultaneously bind the surface antigen of the immune cell and the Robo1 molecule on the surface of the tumor cell, especially the surface CD3 molecule of the immune cell and the Robo1 molecule on the surface of the tumor cell, thereby narrowing the relationship between the tumor cell and the immune cell.
  • the distance effectively activates resting immune cells, especially T cells, to kill tumors. Compared with traditional antibodies, it has the advantages of small molecular weight and good tissue permeability, and has significant killing effect on high expression of Robo1 tumor cells. It can be used for the development of anti-tumor drugs.
  • VH-VL-Slit2D2, LV-VH-Slit2D2, Slit2D2-VL-VH, Slit2D2-VH-VL bind to breast cancer cells MDA-MB-231 and liver cancer cells SMCC7721.
  • MHCC97H has high killing activity.
  • Figure 1 is a schematic representation of the construction of a bispecific antigen binding construct of the invention.
  • Figure 2 is a map of the expression vector of the bispecific antigen binding construct of the present invention.
  • Figure 3 is a SDS-PAGE electropherogram of the bispecific antigen binding construct protein of Example 1, wherein M is the marker lane and 6 and 7 are the bispecific antigen binding construct protein lanes.
  • Figure 4 is a graph showing the results of a tumor killing test for detecting drug activity in Example 2.
  • Fig. 5 is a graph showing the results of growth changes of tumor volume in the treatment group and the control group in the MHCC97H+PBMC tumor model of Example 3.
  • Figure 6 is a graph showing changes in body weight of the experimental mice in the MHCC97H+PBMC tumor model of Example 3 as a function of treatment time.
  • the bispecific antigen binding construct prepared in this example is Slit2D2-VH-VL, and its schematic diagram is shown in FIG.
  • Cell line ExpiCHO-S TM cells (Gibco Catalog No. A29127);
  • the Slit2D2-ScFV gene fragment was obtained by whole gene synthesis, and the PCR product was amplified by PCR as a template, and the gel recovery reagent was used. The above product was recovered by cartridge purification. According to the principle of TA cloning, it was cloned into pCDNA3.4 vector. The recombinant plasmid map is shown in Figure 2, then transformed into E. coli TOP10, ampicillin was screened and positive clones were picked, and the vector was confirmed to be successfully constructed by sequencing. DNA extraction kit toxin without the plasmid DNA extracted, used to transfect cells ExpiCHO-S TM.
  • ExpiCHO-S TM culture cells culture conditions were: 37 °C, 8% incubator, shaking culture of CO 2, when transfected mL when density reached 2.5 ⁇ 10 6 cells /.
  • Cell culture ExpiCHO-S TM after transfection ExpiCHO TM Expression Medium the transfected culture supernatants were collected after 5 days, high-speed centrifugation, the supernatant retained for subsequent purification.
  • Binding Buffer 20 mM phosphate, 0.5 M NaCl, 20 mM imidazole, adjusted to pH 7.4;
  • Elution Buffer 20 mM phosphate, 0.5 M NaCl, 500 mM imidazole, adjusted to pH 7.4;
  • the dialysis bag was dialyzed to remove salt ions, and replaced with a PBS solution to preserve the protein.
  • the target protein was obtained by the above purification process, and its SDS-PAGE electrophoresis pattern is shown in FIG. As can be seen from Fig. 3, the molecular weight of the target protein is about 55 KD, and the purity is very high. No heteroprotein is detected, indicating that the recombinant expression vector of Slit2D2-VH-VL has been successfully constructed and its expression in the host cell is achieved.
  • Example 2 Tumor killing test for detecting drug activity
  • the killing activity of the drug on tumor cells was determined by lactate dehydrogenase (LDH) release method.
  • Test kit CytoTox Non-Radioactive Cytotoxicity Assay kit (promega);
  • Kit components 5vials Substrate Mix, 60ml Assay Buffer, 25 ⁇ l LDH Positive Control, 5ml Lysis Solution (10X), 65ml Stop Solution;
  • Target cells breast cancer cells MDA-MB-231, liver cancer cells SMCC7721, PBMC;
  • DMEM 10% FBS, 5% double antibody
  • Tibco T cell medium
  • Reagent preparation Assay Buffer was incubated at 37 ° C, and 12 ml Assay Buffer was added to the Substrate Mix bottle to make CytoTox. Reagent.
  • the drug was set to 3 gradients: 1.2 ⁇ g/ml, 0.12 ⁇ g/ml, and 0.012 ⁇ g/ml;
  • the formula for calculating the killing activity of tumor cells is:
  • Fig. 4 The experimental results are shown in Fig. 4.
  • the drug (Example 1 prepared purified bispecific antigen binding construct protein Slit2D2-VH-VL) on liver cancer cell SMCC-7721 and breast cancer cell MDA-MB-231
  • the cells all have significant killing activity, and their killing activity increases as the drug concentration increases.
  • the experiment was divided into three groups: 1. blank control group (excluding PBMC inoculation group), 2. vehicle control group, and 3. sample group.
  • the experimental design is shown in Table 1.
  • i.v tail vein administration
  • qd once a day
  • ZD016 Slit2D2-VH-VL, prepared in Example 1 and purified.
  • mice NOD/SCID mice, male, 5-6 weeks (the age of mice at the time of tumor cell inoculation), weighing 19.3-21.7 g, 20 rats. Purchased from Beijing Huakangkang Biotechnology Co., Ltd., animal certificate number: 11401300037778. Feeding environment: SPF level.
  • Vehicle control group PBS, stored at 4 ° C;
  • Sample group 0.554 ml of ZD016 was taken, mixed with 3.046 ml of PBS to obtain a 3.6 ml solution of 0.1 mg/ml, and filtered for use. All operations were performed on ice and stored at -80 °C.
  • MHCC97H cells were cultured in DMEM medium containing 10% fetal bovine serum. MHCC97H cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration and mixed with PBMC, 1:1, and an equal volume of matrigel was added for subcutaneous tumor inoculation in mice.
  • TGI Relative tumor inhibition rate
  • TGI 1 - T / C (%).
  • T/C% is the relative tumor growth rate, that is, the percentage of tumor volume relative to the treatment group and the control group at a certain time point; T and C are the relative tumor volumes of the treatment group and the control group at a certain time point respectively ( RTV).
  • T/C% T RTV /C RTV *100%
  • T RTV treatment group mean RTV
  • C RTV vehicle control group mean RTV
  • RTV V t /V 0
  • V 0 is the group when the animal the tumor volume
  • V t is the tumor volume after treatment of the animal).
  • a single animal has a tumor volume of more than 3000 mm 3 or a group of animals has an average tumor volume of more than 2000 mm 3 , or a single or whole group of animals are euthanized before the animal is dying.
  • the mean tumor value of the non-PBMC-inoculated group reached 1464 mm 3
  • the mean value of the vehicle in the vehicle control group reached 776 mm 3 .
  • the tumor growth of the treatment group and the control group is shown in Table 2 and Figure 5.
  • test drug ZD016 (1 mg/kg, qd ⁇ 7 days) had no animal death in the treatment group, showed no obvious drug toxicity, and was well tolerated during the treatment.

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Abstract

一种双特异性抗原结合构建体及其制备方法和应用，所述的构建体包括第一抗原结合单元和第二抗原结合单元，所述的第一抗原结合单元为特异性结合免疫细胞表面抗原的单链可变区抗体片段ScFV，所述的第二抗原结合单元为特异性结合肿瘤细胞表面抗原Robo1的Slit2D2蛋白片段。即所述的构建体能够同时结合免疫细胞表面抗原和肿瘤细胞表面Robo1分子，从而拉近肿瘤细胞和免疫细胞间的距离，有效激活静止的免疫细胞，起到杀伤肿瘤的效果，且具有分子量小、组织穿透性好等优点，对高表达Robo1肿瘤细胞具有显著的杀伤效果，可用于抗肿瘤药物的开发。

Description

一种双特异性抗原结合构建体及其制备方法和应用 技术领域
本发明涉及免疫学技术领域，具体涉及一种双特异性抗原结合构建体及其制备方法和应用。
背景技术
Robo是一次跨膜的受体蛋白，在哺乳动物中，已经克隆到4个Robo基因。从物种进化的角度看，Robo1,2,3的胞外部分非常保守，从果蝇到人类都是由5个Ig样功能区和3个FibronectinIII型重复序列组成。Robos有很短的跨膜区域和一个较长的胞内区；按照序列的保守性，胞内区被划分为4个更小的区域，分别命名为：CC0,CC1,CC2,CC3。Robos胞外的IgG domains被认为是与配体Slit结合所必需的，较长的胞内区域则和一些重要的信号分子相互作用，参与Slit/Robo下游的信号转导，从而完成刺激信号由细胞外部到内部骨架的传递。目前，已完成Slit2与Robo相互作用区域蛋白质的机构解析，发现Slit2的第二个结构域D2与Robo1的Ig1结合，进而启动信号传导。
组织病理学检测显示Robo1在多种癌症中过表达，如肝细胞癌、乳腺癌、结肠癌、胰腺癌、前列腺癌、神经胶质瘤等。有研究显示Robo1在肝癌中大量表达，而在正常组织中只有少量表达，且84.7％的肝癌组织样本为阳性表达；80％的结肠癌患者的癌组织高度表达Robo1mRNA，45％的患者是正常组织的4倍，15％的患者是正常组织的12倍，因此Robo1可以作为一种新的肿瘤相关抗原，是一种潜在的治疗和诊断靶标。
分化簇3(CD3)分子仅存在于T细胞表面，常与T细胞受体(T cell receptor，TCR)紧密结合形成TCR-CD3复合体，具有T细胞活化信号转导，稳定TCR结构的功能。
本发明通过提供一种具有分别可结合CD3和Robo1的抗原结合部位的双特异性抗原结合构建体，可有效地激活静止的T细胞，达到杀伤病变细胞的目的。
发明内容
本发明的一个目的是提供一种双特异性抗原结合构建体，所述的构建体包括第一抗原结合单元单元和第二抗原结合单元，所述的第一抗原结合单元为特异性结合免疫细胞表面抗原的抗体或抗体片段(例如，单链可变区抗体片段(single-chain variable fragment，ScFV，又称为单链抗体))；所述的第二抗原结合单元为特异性结合肿瘤细胞表面抗原Robo1的Slit2或其片段(例如Slit2D2片段)。
在本发明的一个实施例中，所述Slit2D2片段具有与SEQ ID NO：1所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列；
在本发明的一个优选实施例中，所述Slit2D2片段具有如SEQ ID NO：1所示的氨基酸序列。
优选的，所述的免疫细胞选自：T细胞、NKT细胞和CIK细胞；更优选为T细胞；
优选的，所述的第一抗原结合单元特异性结合免疫细胞表面抗原CD3；
优选的，所述的ScFV包括重链可变区结构域(heavy chain variable region，VH)和轻链可变区结构域(light chain variable region，VL)；所述的ScFV中，VH通过C末端与VL的N末端连接，或VH通过N末端与VL的C末端连接；所述的连接可为直接连接，无任何连接肽，即仅通过形成肽键实现连接；也可通过连接肽实现；优选的，所述的连接通过连接肽实现，所述的连接肽为含1-20个氨基酸的多肽，更优选为10-16个氨基酸，进一步优选为14个氨基酸；
在本发明的一个实施例中，所述的VH具有与SEQ ID NO：2所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列；
在本发明的一个优选实施例中，所述的VH具有如SEQ ID NO：2所示的氨基酸序列；
在本发明的一个实施例中，所述的VL具有与SEQ ID NO：3所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列；
在本发明的一个优选实施例中，所述的VL具有如SEQ ID NO：3所示的氨基酸序列；
在本发明的一个实施例中，所述的ScFV具有与SEQ ID NO：4所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列；
在本发明的一个优选实施例中，所述的ScFV具有如SEQ ID NO：4所示的氨基酸序列；
在本发明另一个实施例中，所述的ScFV具有与SEQ ID NO：5所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列；
在本发明的一个优选实施例中，所述的ScFV具有如SEQ ID NO：5所示的氨基酸序列；
优选的，所述的构建体中，Slit2D2通过C末端与ScFV的N末端连接，或Slit2D2通过N末端与ScFV 的C末端连接；所述的连接可为直接连接，无任何连接肽，即仅通过形成肽键实现连接；也可通过连接肽实现；优选的，所述的连接通过连接肽实现，所述的连接肽为含1-10个氨基酸的寡肽，更优选为3-7个氨基酸，进一步优选为5个氨基酸；
在本发明的一个实施例中，所述的构建体的构建方式包括：VH-VL-Slit2D2、VL-VH-Slit2D2、Slit2D2-VH-VL、Slit2D2-VL-VH，“-”表示连接键或连接肽。
在本发明的一个优选实施例中，所述的构建体具有与SEQ ID NO：6所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列。
在本发明的一个更优选实施例中，所述的构建体具有如SEQ ID NO：6所示的氨基酸序列。
在本发明的一个优选实施例中，所述的构建体具有与SEQ ID NO：7所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列。
在本发明的一个更优选实施例中，所述的构建体具有如SEQ ID NO：7所示的氨基酸序列。
在本发明的一个优选实施例中，所述的构建体具有与SEQ ID NO：8所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列。
在本发明的一个更优选实施例中，所述的构建体具有如SEQ ID NO：8所示的氨基酸序列。
在本发明的一个优选实施例中，所述的构建体具有与SEQ ID NO：9所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列。
在本发明的一个更优选实施例中，所述的构建体具有如SEQ ID NO：9所示的氨基酸序列。
在本发明的一个实施方式中，本发明的Slit2D2片段、ScFV和双特异性抗原结合构建体还包含其变体。示例性地，所述变体可包含或由本发明所公开的具体氨基酸序列或者母本序列的基础上经过取代、缺失、插入或添加一个或几个氨基酸且保留取代、缺失、插入或添加前活性的氨基酸序列组成。例如Slit2D2片段的变体可包含或由在SEQ ID NO：1上经过取代、缺失、插入或添加一个或几个氨基酸且保留取代、缺失、插入或添加前活性的氨基酸序列组成，优选地，所述活性为特异性结合肿瘤细胞表面抗原Robo1。
示例性地，ScFV的变体可包含或由在SEQ ID NO：4或5上经过取代、缺失、插入或添加一个或几个氨基酸且保留取代、缺失、插入或添加前活性的氨基酸序列组成，优选地，所述活性为特异性结合免疫细胞表面CD3分子并激活免疫细胞(例如T细胞、NKT细胞和CIK细胞)。
示例性地，双特异性抗原结合构建体的变体可包含或由在SEQ ID NO：6、7、8或9上经过取代、缺失、插入或添加一个或几个氨基酸且保留取代、缺失、插入或添加前活性的氨基酸序列组成，优选地，所述活性为对肿瘤细胞(例如乳腺癌细胞、肝癌细胞)的杀伤。
本发明所述的在氨基酸序列中取代、缺失、插入或添加1个或几个的氨基酸，是指在序列中的任意且1个或几个氨基酸序列中的位置上取代、缺失、插入或添加1个或几个氨基酸残基(例如2个、3个、4个、5个、6个、7个、8个或9个)，并且取代、缺失、插入和添加中的2种或两种以上可同时发生。
本发明所述的在氨基酸序列中取代、缺失、插入或添加1个或几个的氨基酸可通过使用《Molecular Cloning3》(分子克隆3)和《Current Protocols in Molecular Biology》(现代分子生物学操作规程)等记载的定点诱变法获得。
本发明还提供一种编码上述构建体蛋白的核苷酸。
在本发明的一个优选实施例中，所述编码核苷酸具有SEQ ID NO：10所示的序列或其互补序列。
在本发明的一个优选实施例中，本发明的编码核苷酸包括其变体，例如在严谨条件下与SEQ NO：10的核苷酸序列或其互补序列进行杂交、且编码具有对肿瘤细胞(例如乳腺癌细胞、肝癌细胞)杀伤活性的氨基酸序列的多核苷酸；或
本文所述的“严谨条件”，可以为低严谨条件、中严谨条件、高严谨条件中的任一种，优选为高严谨条件。示例性地，“低严谨条件”可为30℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件；“中严谨条件”可为40℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件；“高严谨条件”可为50℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件。本领域技术人员应当理解温度越高越能得到高同源性的多核苷酸。另外，本领域技术人员可以选择影响杂交的严谨度的温度、探针浓度、探针长度、离子强度、时间、盐浓度等多个因素形成的综合结果来实现相应的严谨度。
本发明还提供一种包含上述编码核苷酸的载体。
在本发明的一个实施例中，所述载体可为包括含有上述编码构建体蛋白的核苷酸的重组质粒、重组细胞、重组菌株、重组病毒。
本发明所述的构建体可人工合成，也可先合成其编码基因，再通过生物表达得到。
本发明还提供一种上述构建体的制备方法，其具体步骤包括：
(1)重组载体的构建；
(2)转化体的制备和发酵；
(3)构建体蛋白的分离和纯化；
任选的，(4)构建体蛋白的鉴定。
所述的步骤(1)包括以下具体步骤：
通过全基因合成得到编码构建体蛋白的基因片段，以其为模板，通过PCR扩增得到PCR产物，纯化回收上述产物，将其克隆至载体质粒中，将得到的重组载体转化到第一宿主细胞后转种到含有氨苄青霉素(AMP)的固体培养基中进行繁殖、筛选阳性克隆、通过测序确认载体构建成功并保种；
所述的载体质粒为pCDNA质粒，优选为pCDNA4.3；
所述的第一宿主细胞包括但不限于大肠杆菌菌株，优选为TOP10菌株。
所述的步骤(2)包括以下具体步骤：
在第一宿主细胞中提取重组载体；
培养第二宿主细胞，当细胞密度达到2.5×106细胞/毫升时转染已提取的重组载体，成功转染目标基因的第二宿主细胞为转化体，筛选转化体，进行转化体的发酵，5天后收集上清；
所述的第二宿主细胞包括但不限于ExpiCHO细胞。
所述的步骤(3)包括以下具体步骤：
取步骤(2)收集的培养液上清，高速离心，分离并纯化构建体蛋白；
所述的纯化包括但不限于离子交换层析纯化蛋白。
所述的步骤(4)包括以下具体步骤：
鉴定融合蛋白的分子量和纯度。
所述的融合蛋白的分子量鉴定可以通过一般的蛋白生化手段检测，包括但不限于：SDS-PAGE、Western blotting、质谱MS等。
所述融合蛋白的纯度通过SEC-HPLC检测蛋白。
本发明还提供一种药物组合物，包括上述双特异性抗原结合构建体，以及药学上可接受的辅料。
本发明所述的药物组合物可以为片剂(包括糖衣片剂，膜包衣片剂，舌下片剂，口腔崩解片，口腔片剂等等)，丸剂，粉剂，颗粒剂，胶囊剂(包括软胶囊，微胶囊)，锭剂，糖浆剂，液体，乳剂，混悬剂，控制释放制剂(例如，瞬时释放制剂，缓释制剂，缓释微囊)，气雾剂，膜剂(例如，口服崩解膜剂，口腔粘膜-粘附膜剂)，注射剂(例如，皮下注射，静脉注射，肌内注射，腹膜内注射)，静脉滴注剂，透皮吸收制剂，软膏剂，洗剂，粘附制剂，栓剂(例如，直肠栓剂，阴道栓剂)，小药丸，鼻制剂，肺制剂(吸入剂)，眼睛滴剂等等，口服或胃肠外制剂(例如，静脉内，肌内，皮下，器官内，鼻内，皮内，滴注，脑内，直肠内等给药形式，给药至肿瘤的附近和直接给药至病变处)。优选的，所述的药物组合物为注射剂。
本发明所述的药学上可接受的辅料优选为药学上可接受的注射剂辅料，例如等渗的无菌盐溶液(磷酸二氢钠、磷酸氢二钠、氯化钠、氯化钾、氯化钙、氯化镁等，或上述盐的混合物)，或干燥的例如是冷冻干燥的组合物，其适当地通过加入无菌水或生理盐水形成可注射溶质。
本发明还提供一种上述双特异性抗原结合构建体在制备抗肿瘤药物中的应用。
本发明还提供一种上述双特异性抗原结合构建体在抗肿瘤药物的筛选和药效评价中的应用。
优选的，所述的肿瘤为高表达Robo1的肿瘤及相关疾病，此处所述的高表达指肿瘤细胞中Robo1的表达水平高于正常细胞中其表达水平。
在本发明的一个实施例中，所述的肿瘤疾病为肝癌、乳腺癌。
本发明中术语“结构域”是指是生物大分子中具有特异结构和独立功能的区域，例如Slit2蛋白D2结构域是指Slit2蛋白中4个富含亮氨酸的重复序列中的第二结构域。
本发明提供的双特异性抗原结合构建体，能够同时结合免疫细胞表面抗原和肿瘤细胞表面Robo1分子，尤其是免疫细胞表面CD3分子和肿瘤细胞表面Robo1分子，从而拉近肿瘤细胞和免疫细胞间的距离，有效激活静止的免疫细胞，尤其是T细胞，起到杀伤肿瘤的效果，与传统抗体相比，具有分子量小、组织穿透性好等优点，对高表达Robo1肿瘤细胞具有显著的杀伤效果，可用于抗肿瘤药物的开发。实验表明，本发明的双特异性抗原结合构建体VH-VL-Slit2D2、LV-VH-Slit2D2、Slit2D2-VL-VH、Slit2D2-VH-VL对乳腺癌细胞MDA-MB-231、肝癌细胞SMCC7721、MHCC97H均具有较高的杀伤活性。
附图说明
图1为本发明的双特异性抗原结合构建体的构建示意图。
图2为本发明的双特异性抗原结合构建体表达载体图谱。
图3为实施例1中双特异性抗原结合构建体蛋白的SDS-PAGE电泳图，其中M为标志物泳道，6和7为双特异性抗原结合构建体蛋白泳道。
图4为实施例2中检测药物活性的肿瘤杀伤实验结果。
图5为实施例3中MHCC97H+PBMC肿瘤模型中治疗组和对照组小鼠肿瘤体积的生长变化结果。
图6为实施例3中MHCC97H+PBMC肿瘤模型中实验小鼠体重随治疗时间的变化结果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：双特异性抗原结合构建体Slit2D2-ScFV的制备
本实施例制备的双特异性抗原结合构建体为Slit2D2-VH-VL，其构建示意图如图1所示。
1.蛋白表达
1.1实验材料：
细胞株：ExpiCHO-STMcells(Gibco Catalog No.A29127)；
转染试剂盒：ExpiFectamineTMCHO Transfection Kit(Gibco Catalog No.A29129)OptiPROTMSFM(Gibco Catalog No.12309-050)；
培养基：ExpiCHOTMExpression Medium(Gibco Catalog No.A29100-01)；
pCDNA3.4质粒载体(Invitron)。
1.2实验过程：
依据设计的Slit2D2-ScFV的基因序列(如SEQ ID NO：10所示)，通过全基因合成得到Slit2D2-VH-VL基因片段，并以其为模板通过PCR扩增得到PCR产物，用胶回收试剂盒纯化回收上述产物。根据T-A克隆原理，将其克隆至pCDNA3.4载体中，重组质粒图谱如图2所示，然后转化至大肠杆菌TOP10中，氨苄青霉素筛选并挑取阳性克隆，并通过测序确认载体构建成功。用无内毒素DNA提取试剂盒提取质粒DNA，用于转染ExpiCHO-STM细胞。培养ExpiCHO-STM细胞，培养条件为：37℃、含8％CO2的培养箱，摇床培养，当密度达到2.5×106细胞/毫升时进行转染。在ExpiCHOTMExpression Medium中培养转染后的ExpiCHO-STM细胞，转染5天后收集培养液上清，高速离心，保留上清，用于后续纯化。
2.蛋白纯化
2.1实验仪器和材料：
AKTA蛋白纯化系统；
Binding Buffer：20mM磷酸盐、0.5M NaCl、20mM咪唑，调pH至7.4；
Elution Buffer：20mM磷酸盐、0.5M NaCl、500mM咪唑，调pH至7.4；
去离子水；
20％的乙醇溶液。
2.2实验过程：
(1)启动AKTA设备，将His柱与AKTA连接；
(2)使用3-5个柱体积的去离子水清洗His柱；
(3)使用5个柱体积的Binding Buffer平衡His柱；
(4)取出过滤的细胞上清液，依次流过His柱；
(5)使用Binding Buffer清洗杂蛋白，直至UV280处吸光值趋近于0；
(6)使用Elution Buffer洗脱目的蛋白，当UV280>400的时候开始收集洗脱液；
(7)PBS于4℃透析收集的蛋白洗脱液，透析后的蛋白保存于-80℃；
(8)蛋白收集结束后，使用10个柱体积的Binding Buffer冲洗His柱；
(9)使用10个柱体积的去离子水清洗His柱；
(10)使用10个柱体积的20％的乙醇清洗His柱，并将His柱保存于20％的乙醇中，4℃存放；
(11)透析袋透析去除盐离子，置换为PBS溶液保存蛋白。
注：a.纯化过程中所使用的试剂均需用0.45um滤膜过滤。
b.纯化过程中禁止空气进入His柱。
c.整个过程应在冰上操作，以防蛋白失活。
2.3实验结果
利用上述纯化过程获得目的蛋白，其SDS-PAGE电泳图如图3所示。由图3可知，目的蛋白分子量约为55KD，且纯度非常高，未检测到杂蛋白，表明已成功构建Slit2D2-VH-VL的重组表达载体，并实现其在宿主细胞中的表达。
实施例2肿瘤杀伤实验检测药物活性
采用乳酸脱氢酶(lactate dehydrogenase；LDH)释放法测定药物对肿瘤细胞的杀伤活性。
1.1实验材料：
检测试剂盒：CytoTox
Figure PCTCN2017083961-appb-000001
Non-Radioactive Cytotoxicity Assay kit(promega)；
试剂盒成分：5vials Substrate Mix、60ml Assay Buffer、25μl LDH Positive Control、5ml Lysis Solution(10X)、65ml Stop Solution；
靶细胞：乳腺癌细胞MDA-MB-231、肝癌细胞SMCC7721、PBMC；
药物：双特异性抗原结合构建体蛋白Slit2D2-VH-VL(实施例1制备纯化得到)；
培养基：DMEM(10％FBS，5％双抗)(Gibco)、T细胞培养基(Gibco)；
试剂准备：Assay Buffer在37℃下孵育，取12ml Assay Buffer加入Substrate Mix瓶中配制成CytoTox 
Figure PCTCN2017083961-appb-000002
Reagent。
1.2实验过程：
(1)取靶细胞(MDA-MB-231/SMCC7721)，按100μl/4×103/孔加入96孔板；
取效应细胞PBMC，按100μl/2×105/孔加入96孔板(即效靶比50:1)；
药物设为3个梯度：1.2μg/ml、0.12μg/ml、0.012μg/ml；
LDH释放对照组的设计(每组均设6个复孔)：
(a)培养液对照组：单纯无血清1640培养液；
(b)LDH低释放组：靶细胞4×103/孔；
(c)LDH高释放组：靶细胞4×103/孔(培养结束后处理)；
(d)效应细胞对照组：PBMC 2×105/孔；
(2)将已加入细胞的96孔板置于37℃、体积分数5％CO2的培养箱中培养过夜(18-24小时)；
(3)培养结束后，在LDH高释放组中加入细胞裂解液(Lysis Solution10X)，10μl/孔，37℃孵育45-60min；
(4)将每孔细胞分别转移到EP管中，1000rpm离心5min；
(5)取50μl上述离心所得的上清液，加入酶标板中，于每孔中加入50μl CytoTox
Figure PCTCN2017083961-appb-000003
Reagent，室温孵育30min；
(6)于每孔中加入50μl终止液(Stop Solution)终止反应；
(7)利用酶标仪检测步骤(6)得到的样品在492nm处的吸光度数值。
1.3实验结果：
对肿瘤细胞的杀伤活性的计算公式为：
Figure PCTCN2017083961-appb-000004
实验结果如图4所示，由图4可知，药物(实施例1制备纯化的双特异性抗原结合构建体蛋白Slit2D2-VH-VL)对肝癌细胞SMCC-7721和乳腺癌细胞MDA-MB-231细胞均具有显著的杀伤活性，且其杀伤活性随药物浓度升高而增强。
实施例3
1.1实验设计
实验分为三组：1.空白对照组(不含PBMC接种组)、2.溶媒对照组、3.样品组，实验设计如表1所示。
表1实验设计方案
Figure PCTCN2017083961-appb-000005
注：i.v：尾静脉给药；qd：每天一次给药；ZD016：Slit2D2-VH-VL，实施例1制备纯化得到。
1.2实验材料
1.2.1实验动物：NOD/SCID小鼠，雄性，5-6周(肿瘤细胞接种时的小鼠周龄)，体重19.3-21.7g，20只。购自北京华阜康生物科技有限公司，动物合格证编号：11401300037778。饲养环境：SPF级。
1.2.2实验样品：ZD016，包装规格：0.65mg/ml×0.5ml×3vial，-80℃密封保存。
1.2.3细胞：PBMC，包装规格：5ml×1vial，-80℃密封保存，复苏得9×107，细胞存活率94.2％。
1.3实验方法和步骤
1.3.1测试药品配制
溶媒对照组：PBS，4℃储存；
样品组：取0.554ml的ZD016，加入3.046ml的PBS漩涡混匀，得到3.6ml的0.1mg/ml的溶液，过滤备用，所有操作均在冰上操作，-80℃储存。
1.3.2细胞：MHCC97H细胞培养在含10％胎牛血清的DMEM培养液中。收集指数生长期的MHCC97H细胞，PBS重悬至适合浓度并与PBMC，1：1混合后，加入等体积的matrigel用于小鼠皮下肿瘤接种。
1.3.3动物造模和分组：20只雄性小鼠接种前一天，腹腔注射CD122(0.2mg/只小鼠)，接种当天于右侧 皮下接种5×106MHCC97H+5×106PBMC细胞。于接种当天开始分组给药，随机分组(见表1)。
1.3.4实验观察：肿瘤接种后，常规监测包括了肿瘤生长及治疗对动物正常行为的影响，具体内容有实验动物的活动性，摄食和饮水情况，体重增加或降低(体重每周测量3次)情况，眼睛、被毛及其它异常情况。实验过程中观察到的临床症状均记录在原始数据中。
本实验过程中动物实验的实验方案均通过动物伦理委员会审核并批准通过。实验过程中，动物实验操作均根据AAALAC的要求。
1.3.5实验结果判断标准
相对肿瘤抑制率TGI(％)：TGI＝1-T/C(％)。T/C％为相对肿瘤增值率，即在某一时间点，治疗组和对照组相对肿瘤体积的百分比值；T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积(RTV)。
计算公式如下：T/C％＝TRTV/CRTV*100％(TRTV：治疗组平均RTV；CRTV：溶媒对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)。
1.3.6实验终点
当单只动物瘤体积超过3000mm3或一组动物平均瘤体积超过2000mm3，或者在动物在濒临死亡前将单只或整组动物安乐死。
本实验在分组给药后第25天(接种后第25天)时，未加PBMC接种组肿瘤均值达到1464mm3，溶媒对照组肿瘤均值达到776mm3，量瘤记录后，安乐小鼠，结束实验，不收取组织。
1.4实验结果
1.4.1测试药ZD016在MHCC97H+PBMC肿瘤模型中的抗肿瘤作用研究结果
治疗组和对照组肿瘤生长情况见表2和图5。
完全空白对照组(不含PBMC接种组)在分组给药后第25天平均肿瘤体积为1464mm3，溶媒对照组小鼠在分组给药后第25天平均肿瘤体积为776mm3，测试药ZD016(1mg/kg，qd×7天)治疗组在分组给药后第25天平均肿瘤体积为421mm3，相对肿瘤抑制率TGI(％)为45.76％。
表2MHCC97H+PBMC肿瘤模型中各实验组TGI和T/C值
Figure PCTCN2017083961-appb-000006
1.4.2测试药ZD016在MHCC97H+PBMC肿瘤模型中的安全性研究结果
测试药ZD016(1mg/kg，qd×7天)治疗组无动物死亡，没有表现明显的药物毒性，治疗期间耐受良好。
治疗组和对照组给药后体重变化见图6。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
Figure PCTCN2017083961-appb-000007
Figure PCTCN2017083961-appb-000008
Figure PCTCN2017083961-appb-000009
Figure PCTCN2017083961-appb-000010
Figure PCTCN2017083961-appb-000011
Figure PCTCN2017083961-appb-000012
Figure PCTCN2017083961-appb-000013
Figure PCTCN2017083961-appb-000014
Figure PCTCN2017083961-appb-000015
Figure PCTCN2017083961-appb-000016
Figure PCTCN2017083961-appb-000017
Figure PCTCN2017083961-appb-000018

Claims (15)

  1. 一种双特异性抗原结合构建体，所述的构建体包括第一抗原结合单元和第二抗原结合单元，所述的第一抗原结合单元为特异性结合免疫细胞表面抗原的抗体或抗体片段(例如单链可变区抗体片段ScFV)；所述的第二抗原结合单元为特异性结合肿瘤细胞表面抗原Robo1的Slit2或其片段(例如Slit2D2片段)。
  2. 如权利要求1所述的构建体，其特征在于，所述的免疫细胞选自：T细胞、NKT细胞和CIK细胞。
  3. 如权利要求1所述的构建体，其特征在于，所述的Slit2D2片段具有与SEQ ID NO：1所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列。
  4. 如权利要求1所述的构建体，其特征在于，所述的第一抗原结合单元特异性结合免疫细胞表面抗原CD3。
  5. 如权利要求1所述的构建体，其特征在于，所述的单链可变区抗体片段ScFV包括VH和VL；
    所述的VH具有与SEQ ID NO：2所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列，和/或，所述的VL具有与SEQ ID NO：3所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列。
  6. 如权利要求1所述的构建体，其特征在于，所述的ScFV具有与选自SEQ ID NO：4和SEQ ID NO：5所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列。
  7. 如权利要求1所述的构建体，其特征在于，所述的构建体的构建方式包括：VH-VL-Slit2D2、VL-VH-Slit2D2、Slit2D2-VH-VL、Slit2D2-VL-VH。
  8. 如权利要求7所述的构建体，其特征在于，所述的构建体具有与选自SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的氨基酸序列至少80％、90％、95％、96％、97％、98％、99％或100％相同的氨基酸序列。
  9. 一种编码权利要求1-8任一项所述的构建体的核苷酸。
  10. 如权利要求9所述的编码核苷酸，其特征在于，所述的编码核苷酸包含具有如SEQ ID NO：10所示的序列或其互补序列。
  11. 一种包含权利要求9或10所述的编码核苷酸的载体。
  12. 一种药物组合物，包括权利要求1-8任一项所述的双特异性抗原结合构建体以及药学上可接受的辅料。
  13. 一种权利要求1-8任一项所述的双特异性抗原结合构建体在制备抗肿瘤药物中的应用。
  14. 如权利要求13所述的应用，其特征在于，所述的肿瘤为高表达Robo1的肿瘤及相关疾病。
  15. 如权利要求14所述的应用，其特征在于，所述的肿瘤疾病为肝癌、乳腺癌。
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