WO2022100585A1 - 抗Her-2抗体-趋化因子融合蛋白及其制法和应用 - Google Patents

抗Her-2抗体-趋化因子融合蛋白及其制法和应用 Download PDF

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WO2022100585A1
WO2022100585A1 PCT/CN2021/129639 CN2021129639W WO2022100585A1 WO 2022100585 A1 WO2022100585 A1 WO 2022100585A1 CN 2021129639 W CN2021129639 W CN 2021129639W WO 2022100585 A1 WO2022100585 A1 WO 2022100585A1
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fusion protein
antibody
ccl11
protein
trastuzumab
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PCT/CN2021/129639
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French (fr)
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蔡则玲
陈羿
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上海康岱生物医药技术股份有限公司
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
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Definitions

  • the present invention belongs to the field of biomedicine, and more particularly, the present invention relates to an anti-Her-2 antibody-chemokine fusion protein and its preparation method and application.
  • Her-2 is a proto-oncogene and belongs to the human epidermal growth factor receptor family. It inhibits cancer cell apoptosis and promotes its proliferation and invasion by regulating downstream signaling pathways. Her-2 amplification or overexpression accounts for the proportion of breast cancer patients. About 20%-30%, in addition, Her-2 positivity (Her-2+) is often detected in gastric cancer.
  • the representative drug of Her-2 target, trastuzumab has achieved good curative effect in Her-2+ breast and gastric cancer, but the resistance rate and recurrence rate of breast cancer to trastuzumab are increasing year by year. The final resistance rate is as high as 65%, including 70% of patients who were sensitive to trastuzumab at the beginning of treatment and eventually developed resistance. Therefore, new combinations are urgently needed to achieve effective control of Her-2+ tumors.
  • the purpose of the present invention is to provide an anti-Her-2 fusion protein that is safe, effective and precisely targeting tumors.
  • a fusion protein single chain comprising the following elements fused together:
  • the first protein element is a protein element of an anti-Her-2 antibody or an active fragment thereof;
  • the second protein element is a protein element selected from the CC family of chemokines.
  • the chemokine is selected from CCL-11 or CCL2.
  • the anti-Her-2 antibody or its active fragment is an active fragment containing F(ab), scFv, VH, CH, VL or VHH.
  • the anti-Her-2 antibody or its active fragment is selected from the active fragment of trastuzumab.
  • the linker element is a peptide bond or a peptide linker.
  • fusion protein composed of the fusion protein single chain described in the first aspect of the present invention, and the fusion protein has the dimer structure shown in the following formula I or II:
  • H-Chain--V-Chain is the protein element of anti-Her-2 antibody or its active fragment, wherein,
  • H-Chain is no, or heavy chain fusion protein in anti-Her-2 antibody or its active fragment
  • V-Chain is none, or the light chain fusion protein in anti-Her-2 antibody or its active fragment
  • CCL is a protein element selected from the CC family of chemokines
  • the heavy chain fusion protein includes or contains the heavy chain, VH, CH, VHH, Fc region or HCDR of an anti-Her-2 antibody.
  • the light chain fusion protein includes or contains the light chain, VL, CL or LCDR of the anti-Her-2 antibody.
  • the H-Chain is the heavy chain of trastuzumab.
  • the V-Chain is the light chain of trastuzumab.
  • CCL is preferably CCL-11.
  • the H-Chain or V-Chain and CCL are connected in a head-to-head, head-to-tail, or tail-to-tail manner.
  • the "head” refers to the N-terminus of a polypeptide or a fragment thereof, especially the N-terminus of a wild-type polypeptide or a fragment thereof.
  • the "tail” refers to the C-terminus of a polypeptide or a fragment thereof, especially the C-terminus of a wild-type polypeptide or a fragment thereof.
  • the length of the peptide linker is 0-20 amino acids, preferably 1-15 amino acids.
  • the H-Chain contains or has positions 1-449 in SEQ ID NO: 19, and the V-Chain contains or has positions 1-214 in SEQ ID NO: 22 , the CCL11 contains or has positions 450-523 in SEQ ID NO: 19, or positions 215-288 in SEQ ID NO: 22.
  • sequence of the peptide linker is positions 215-221 in SEQ ID NO:23.
  • sequence of the fusion protein is selected from the following group:
  • the amino acid sequence in (1) to (3) is formed by substitution, deletion or addition of one or more amino acid residues, and has the activity of simultaneously binding to Her-2 protein and binding to CCL-11 by ( 1) to (3) derived polypeptides.
  • the third aspect of the present invention provides an isolated polynucleotide encoding the fusion protein of the second aspect of the present invention.
  • nucleotide sequence of the fusion protein is selected from the following group:
  • the light chain nucleotide sequence is shown in SEQ ID NO: 17; and the heavy chain amino acid sequence is shown in SEQ ID NO: 16; or
  • the light chain nucleotide sequence is shown in SEQ ID NO:18; and the heavy chain amino acid sequence is shown in SEQ ID NO:16.
  • the fourth aspect of the present invention provides a vector, which contains the polynucleotide according to the third aspect of the present invention.
  • the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • the fifth aspect of the present invention provides a host cell comprising the vector of the fourth aspect of the present invention or the polynucleotide of the third aspect of the present invention integrated into the genome.
  • the host cells include prokaryotic cells and eukaryotic cells.
  • the host cells include mammalian cells.
  • the sixth aspect of the present invention provides a method for producing the fusion protein of the second aspect of the present invention, which comprises the steps:
  • a seventh aspect of the present invention provides a pharmaceutical composition, the composition comprising:
  • a pharmaceutically acceptable carrier is selected from:
  • the pharmaceutical composition further contains: additional active ingredients, preferably the active ingredients include: small molecule compounds, cytokines, antibodies (such as anti-PD-1 antibody, anti-OX40 antibody) , anti-CD137 antibody, anti-CD47 antibody, ADC, CAR-immune cells).
  • additional active ingredients include: small molecule compounds, cytokines, antibodies (such as anti-PD-1 antibody, anti-OX40 antibody) , anti-CD137 antibody, anti-CD47 antibody, ADC, CAR-immune cells).
  • the pharmaceutical composition is in the form of injection.
  • the eighth aspect of the present invention provides an immune cell carrying the fusion protein of the second aspect of the present invention.
  • a ninth aspect of the present invention provides a pharmaceutical composition, the composition comprising:
  • a pharmaceutically acceptable carrier is selected from:
  • the tenth aspect of the present invention provides a use of the fusion protein according to the second aspect of the present invention or the immune cell according to the eighth aspect of the present invention for preparing a medicament for treating tumors.
  • the tumor is a Her-2 positive tumor.
  • the tumors include: breast cancer tumors, gastric cancer tumors, bladder cancer tumors, pancreatic cancer tumors, colorectal cancer tumors, lung cancer tumors, liver cancer tumors, and melanoma tumors.
  • the drug for treating tumors can be used in combination with another tumor immunotherapy, including but not limited to: chemotherapy, anti-CD20 mAb, anti-TIM-3 mAb, anti-LAG-3 mAb, anti-CD73 mAb, anti-CD47 mAb, Anti-DLL3 mAb, anti-FRmAb mAb, anti-CTLA-4 antibody, anti-OX40 antibody, anti-CD137 antibody, anti-PD-1 antibody, PD-1/PD-L1 therapy, other immuno-oncology drugs, anti-angiogenic agents, radiotherapy, antibodies - Drug conjugates (ADCs), targeted therapy or other anticancer drugs.
  • ADCs Anti- Drug conjugates
  • An eleventh aspect of the present invention provides an immunoconjugate, the immunoconjugate comprising:
  • a conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
  • the conjugate moiety is selected from: fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or Detection of products of enzymes, radionuclides, biological toxins, cytokines (such as IL-2, etc.).
  • the immunoconjugate includes an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the twelfth aspect of the present invention provides a method for treating tumors, the method comprising administering the above-mentioned fusion protein, or an immunoconjugate thereof, or a pharmaceutical composition thereof to a subject in need thereof.
  • the tumors include: breast cancer tumors, gastric cancer tumors, bladder cancer tumors, pancreatic cancer tumors, colorectal cancer tumors, lung cancer tumors, liver cancer tumors, and melanoma tumors.
  • Figures 1A-1B show two schematic structural diagrams of embodiments of the anti-Her-2 monoclonal-chemokine fusion protein of the present invention.
  • Figure 1A is a schematic diagram of the structure of the trastuzumab HC-CCL11 fusion protein
  • Figure 1B is a schematic diagram of the structure of the trastuzumab LC-CCL11 (left) and trastuzumab LC-linker-CCL11 fusion proteins (right).
  • Figure 2 shows an SDS-PAGE electrophoresis analysis study of trastuzumab HC-CCL11 and trastuzumab LC-linker-CCL11 fusion proteins.
  • lanes 1, 3 and 5 are 4-12% non-reducing SDS-PAGE electrophoresis analysis of trastuzumab, trastuzumab HC-CCL11 and trastuzumab LC-linker-CCL11 fusion proteins, respectively.
  • Lanes 2, 4 and 6 are the respective 4-12% reducing SDS-PAGE electrophoretic analyses, respectively.
  • Single-chain HC-CCL11 is marked with an asterisk in lane 4
  • single-chain LC-linker-CCL11 is marked with an asterisk in lane 6.
  • Lane M is the protein molecular weight standard (kDa).
  • Figure 3 shows a flow cytometric analysis study of trastuzumab HC-CCL11 and trastuzumab LC-linker-CCL11 fusion proteins binding to Her-2 on cell membranes.
  • Human breast cancer cells BT-474 with high Her-2 expression were incubated with different concentrations of trastuzumab-CCL11 fusion protein or trastuzumab, and then detected with FITC-labeled goat anti-human IgG1 Fc antibody with Her-2 The bound antibody, FITC fluorescence intensity was detected by flow cytometry.
  • Figure 4 shows a flow cytometric analysis study of trastuzumab HC-CCL11 and trastuzumab LC-linker-CCL11 fusion proteins binding to the receptor CCR3 on the cell membrane.
  • Human embryonic lung cells MRC-5 with high CCR3 expression were incubated with different concentrations of trastuzumab-CCL11 fusion protein or trastuzumab, and then the antibody bound to CCR3 was detected with FITC-labeled goat anti-human IgG1 Fc antibody.
  • FITC fluorescence intensity was detected by flow cytometry.
  • Figure 5 shows the study of trastuzumab HC-CCL11 fusion protein inhibiting the growth of mouse melanoma cells B16 expressing human Her-2 in mice.
  • the stable cell line B16/Her-2 transfected with human Her-2 gene was inoculated on the back of mice, and PBS ( indicated), trastuzumab HC-CCL11 fusion protein ( indicated) and trastuzumab ( indicated), and then once a week for a total of 4 doses.
  • the drug doses were all 4 mg/kg.
  • the tumor volume was measured by (length ⁇ width ⁇ width/2).
  • the inventors have conducted extensive and in-depth research, and unexpectedly found that the fusion protein obtained by fusing (a) an anti-Her-2 antibody or its active fragment and (b) a protein of the chemokine CC family has a high-efficiency tumor cell killing activity , The synergistic effect of less toxic and side effects.
  • the fusion proteins of the present invention target Her-2-expressing tumors and are fused to biologically active chemokines. Specifically, the fusion protein of the present invention specifically recognizes human epidermal growth factor receptor (Her-2), and attracts and regulates the chemokine CCL11 of eosinophils.
  • the obtained fusion protein can specifically bind to Her-2 expressed in tumor tissue to inhibit tumor growth, and deliver chemokine CCL11 to targeted tumor tissue to enhance the tumor-killing effect of eosinophils. Therefore, the fusion protein of the present invention can be used for the treatment of Her-2+ tumors. On this basis, the inventors have completed the present invention.
  • the chemokine can be linked to the anti-Her-2 antibody or its active fragment in a head-to-head, head-to-tail, or tail-to-tail manner.
  • the anti-Her-2 antibody or its active fragment can be an active fragment containing F(ab), F(ab)2, scFv or VHH, and the chemokine can be CCL2, CCL11.
  • a preferred linking manner in the present invention is that the chemokine CCL11 is linked to the end of the heavy chain of trastuzumab.
  • the fusion protein of the present invention has a good synergistic effect, which is better than the effect of trastuzumab or chemokine CCL11 administered alone, and the effect of combined use of trastuzumab and chemokine CCL11.
  • fusion protein of the present invention can be used interchangeably, and all refer to the present invention.
  • the fusion protein mentioned in the first aspect can be used interchangeably, and all refer to the present invention.
  • the term “about” means that the value may vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • Fc refers to the Fc fragment of a human immunoglobulin.
  • immunoglobulin Fc region refers to the constant region of an immunoglobulin chain, particularly the carboxy-terminus or a part thereof, of the constant region of an immunoglobulin heavy chain, for example, an immunoglobulin Fc region may include both heavy chains CH1, CH2, CH3.
  • the combination of one or more domains and the immunoglobulin hinge region, in a preferred embodiment, the Fc region of the immunoglobulin used includes at least one immunoglobulin hinge region, one CH2 domain and one CH3 domain, preferably lacking CH1 domain.
  • the Fc region of a globulin is within the purview of those skilled in the art, and in a preferred example, the Fc region of an immunoglobulin can be selected to comprise a coding sequence comprising an Fc region of a human immunoglobulin IgG4 subclass, in which an immunoglobulin Fc region is deleted.
  • the globulin heavy chain 1 domain (CH1) but includes the hinge region and the coding sequences for the CH2, CH3, and two domains.
  • the fusion protein is an isolated protein, unrelated to other proteins, polypeptides or molecules, expressed in a recombinant host cell, or the product of isolation or purification.
  • the fusion protein constructed by the present invention consists of the following two parts:
  • a chemokine with biological activity in the chemotactic cytokine family such as CCL-11 or CCL2.
  • fusion protein contains anti-Her-2 antibody heavy chain, which contains or does not contain CH1 or CH2 or CH3 of heavy chain constant region, and its C-terminus is associated with active cell chemotaxis factor fusion.
  • an anti-Her-2 antibody-chemokine (such as CCL11) fusion protein can be produced, which can bind to tumors expressing Her-2 cells and can deliver chemokines to tumor sites.
  • the bioactive chemokine is fused with the anti-Her-2 single-chain antibody.
  • the complete fusion protein is a polypeptide chain, and each functional region is connected by a connecting peptide to ensure that the fusion protein has the correct spatial structure and maintains its biological activity.
  • the fusion proteins of the present invention are a new class of molecules with two biological functions: first, they can target tumor tissues expressing Her-2, and second, they can specifically deliver biologically active cytokines to tumors part. These cytokines have the function of attracting immune cells and regulating the activity of immune cells. Therefore, they can increase the infiltration of immune cells into tumor tissue and enhance the activity of immune cells, so that the growth of tumors, such as breast cancer and gastric cancer, is inhibited. Since chemokines are mainly confined to tumor tissue, the toxicity to patients is relatively small.
  • the antibody in the fusion protein of the present invention can be a full-length antibody or a certain key fragment of the antibody, such as scFv, F(ab)2 or VHH.
  • all antibodies that can bind to the Her-2 receptor on the tumor cell membrane are suitable for the construction of the antibody-chemokine fusion protein of the present invention (trastuzumab, lapatinib, and pertuzumab). monoclonal antibody).
  • trastuzumab is preferred.
  • the chemokine portion of the fusion protein of the present invention selected from the chemokines of the biologically active CC family, is linked to the antibody portion directly or through a peptide linker.
  • the present invention provides a fusion protein comprising the following elements:
  • a linker element (a) a protein element of an anti-Her-2 antibody or an active fragment thereof, (b) a protein element of the CC family of chemokines (eg, CCL11), and (c) a linker element.
  • a linker may or may not be included.
  • the fusion protein of the present invention not only has a longer half-life in vivo, but also can more effectively inhibit the concentration of immune disease-related antibodies (especially IgE) in serum.
  • the fusion protein of the present invention also includes its conservative variants, which means that compared with the amino acid sequence of the fusion protein of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, Optimally, up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.
  • conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.
  • the fusion protein of the present invention can easily prepare by various known methods. Such methods are, for example, but not limited to, recombinant DNA methods, artificial synthesis, etc. [see Murray KM, Dahl SLAnn; Pharmacother 1997 Nov; 31(11):1335-8].
  • a preferred fusion protein is trastuzumab HC-CCL11 fusion protein, the heavy chain nucleotide sequence is shown in SEQ ID NO:14, and the heavy chain amino acid sequence is shown in SEQ ID NO:19; Positions 1-449 in the chain amino acid (SEQ ID NO: 19) sequence are the amino acid sequence of trastuzumab; positions 450-523 are the amino acid sequence of CCL11.
  • a preferred fusion protein is trastuzumab LC-CCL11 fusion protein, and its light chain nucleotide sequence is shown in SEQ ID NO:17, and its light chain amino acid sequence is shown in SEQ ID NO:22;
  • the 1-214th position in the chain amino acid (SEQ ID NO:22) sequence is the light chain amino acid sequence of trastuzumab; the 215th-288th position is the CCL11 amino acid sequence.
  • a preferred fusion protein is trastuzumab LC-linker-CCL11 fusion protein, and its light chain nucleotide sequence is as shown in SEQ ID NO:18, and its light chain amino acid sequence is as shown in SEQ ID NO:23; wherein , the 1-214th position in the light chain amino acid (SEQ ID NO:23) sequence is the light chain amino acid sequence of trastuzumab; the 215th-221st position is the linker sequence; the 222nd-295th position is the CCL11 amino acid sequence.
  • the light chain nucleotide sequence of the trastuzumab HC-CCL11 fusion protein of the present invention is shown in SEQ ID NO: 15, and the light chain amino acid sequence is shown in SEQ ID NO: 20.
  • the heavy chain nucleotide sequence of the trastuzumab LC-CCL11 or LC-linker-CCL11 fusion protein of the present invention is shown in SEQ ID NO: 16, and the heavy chain amino acid sequence is shown in SEQ ID NO: :21.
  • isolated refers to the separation of a substance from its original environment (in the case of a natural substance, the original environment is the natural environment).
  • the original environment is the natural environment.
  • polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances present in the natural state.
  • an "isolated recombinant fusion protein” refers to a recombinant fusion protein that is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify recombinant fusion proteins using standard protein purification techniques. Substantially pure proteins produce a single major band on non-reducing polyacrylamide gels.
  • the polynucleotides 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.
  • DNA can be the coding or non-coding strand.
  • the present invention also relates to variants of the above-mentioned polynucleotides, which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but which does not substantially alter the function of the encoded polypeptide.
  • primer refers to a general term for oligonucleotides that, when paired with a template, can be used as a starting point to synthesize a DNA strand complementary to a template under the action of a DNA polymerase.
  • Primers can be natural RNA, DNA, or any form of natural nucleotides. Primers can even be non-natural nucleotides such as LNA or ZNA and the like.
  • a primer is “substantially” (or “substantially") complementary to a particular sequence on one strand of the template. The primer must be sufficiently complementary to one strand on the template to initiate extension, but the sequence of the primer does not have to be fully complementary to that of the template.
  • a primer for example, adding a sequence that is not complementary to the template to the 5' end of a primer whose 3' end is complementary to the template, such a primer is still substantially complementary to the template.
  • the primers of sufficient length can sufficiently bind to the template, non-completely complementary primers can also form primer-template complexes with the template to perform amplification.
  • the amino acid sequence provided by the present invention those skilled in the art can easily prepare the fusion protein of the present invention by various known methods. These methods include, but are not limited to, recombinant DNA methods, artificial synthesis, and the like.
  • the full-length nucleotide sequence of the element of the fusion protein of the present invention (eg, anti-Her-2 antibody active fragment or CCL) or its fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • primers can be designed according to the published relevant nucleotide sequences, especially the open reading frame sequences, and commercial cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as template, and the related sequences are amplified. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splicing the amplified fragments together in the correct order.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • synthetic methods can also be used to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • a method of amplifying DNA/RNA using PCR technology is preferred for obtaining the gene of the present invention.
  • Primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods.
  • Amplified DNA/RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • the present invention also relates to vectors comprising the polynucleotides of the present invention, as well as host cells genetically engineered with the vectors or fusion protein coding sequences of the present invention, and methods for recombinantly producing the proteins of the present invention.
  • polynucleotide sequences of the present invention can be used to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally there are the following steps:
  • Expression vectors containing DNA sequences encoding the proteins of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • Expression vectors also include a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or for tetracycline or ampicillin resistance in E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or for tetracycline or ampicillin resistance in E. coli.
  • Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, can be used to transform appropriate host cells so that they can express the protein.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • Representative examples are: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS, or 293 cells, and the like.
  • a particularly preferred cell is human and non-human mammalian cells, especially immune cells, including T cells, NK cells.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention.
  • the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • the protein in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, proteins can be isolated and purified by various separation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • chemokines are divided into several major families, such as CC, CXC, and CX3C.
  • chemokines are preferably selected from the CC family.
  • CCL11 and CC2 are the chemokines of choice.
  • the present invention provides an antibody-chemokine fusion protein, wherein the chemokine is selected from CCL11 in the CC family.
  • the bifunctional fusion proteins of the present invention may optionally contain or not contain a peptide linker.
  • Peptide linker size and complexity may affect protein activity.
  • the peptide linker should be of sufficient length and flexibility to ensure that the two proteins being joined have sufficient freedom in space to perform their function. At the same time, the influence of the formation of ⁇ helix or ⁇ sheet in the peptide linker on the stability of the fusion protein is avoided.
  • the length of the peptide linker is generally 0-20 amino acids, preferably 1-15 amino acids.
  • Examples of preferred peptide linkers include, but are not limited to: GSGGGGS (SEQ ID NO. 24), (G4S)3.
  • the amino acid sequence of the peptide linker is: positions 215-221 in the amino acid sequence of trastuzumab LC-linker-CCL11 (SEQ ID NO: 23).
  • compositions and methods of administration are provided.
  • the present invention also provides a composition
  • a composition comprising (a) an effective amount of the fusion protein of the present invention and/or an effective amount of the immune cell of the present invention, and a pharmaceutically acceptable carrier.
  • the fusion proteins of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably, the pH is about 6-8.
  • the term "effective amount” or “effective dose” refers to an amount that can produce a function or activity on humans and/or animals and can be accepted by humans/or animals, such as 0.001-99 wt%; preferably 0.01-95wt%; more preferably, 0.1-90wt%.
  • the “effective amount” or “effective dose” refers to 1 ⁇ 10 3 to 1 ⁇ 10 7 of the immune cells/ml.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation, and allergy), ie, a substance with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the fusion protein of the present invention and a pharmaceutically acceptable carrier.
  • Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by using normal saline or an aqueous solution containing glucose and other adjuvants by conventional methods.
  • the pharmaceutical compositions are preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the pharmaceutical preparation of the present invention can also be made into a sustained-release preparation.
  • the effective amount of the fusion protein of the present invention may vary with the mode of administration, the severity of the disease to be treated, and the like. Selection of the preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include, but are not limited to: the pharmacokinetic parameters of the fusion protein of the present invention such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the dosage of the drug. way etc. Generally, when the fusion protein of the present invention is administered at a dose of about 5 mg-20 mg/kg animal body weight (preferably 5 mg-10 mg/kg animal body weight) per day, satisfactory effects can be obtained. For example, several divided doses may be administered daily, or the dose may be proportionally reduced, as dictated by the exigencies of the therapeutic situation.
  • the fusion protein of the present invention is particularly suitable for treating diseases such as tumors.
  • Representative tumors include (but are not limited to): breast cancer tumors, gastric cancer tumors, bladder cancer tumors, pancreatic cancer tumors, colorectal cancer tumors, lung cancer tumors, liver cancer tumors, and melanoma tumors.
  • the fusion protein of Her-2 and CCL11 of the present invention has the advantages of precise identification, immunotherapy and controllable toxicity.
  • the fusion protein of Her-2 and CCL11 of the present invention can recognize and inhibit the growth of tumors that abnormally express Her-2, and at the same time, enhance the killing of eosinophils in the tumor microenvironment through CCL11-mediated chemotaxis , to further improve the efficacy of Her-2+ tumors.
  • the fusion protein of Her-2 and CCL11 of the present invention can accurately carry CCL11 to the tumor microenvironment through the characteristics of HER-2 targeting tumors, further enhance the local tumor killing mediated by eosinophils, and reduce the The incidence of normal tissue eosinophil-related adverse reactions (eosinophilic gastroenteritis, eosinophilic tracheitis, etc.)
  • trastuzumab as a Her-2 antibody is exemplified in Figure 1A.
  • the complete cDNAs encoding the heavy and light chains of trastuzumab were synthesized by GenScrip (USA) and cloned into the pUC57 vector, respectively.
  • the cDNA of human CCL11 was purchased from OpenBiosystems (USA).
  • the gene encoding the heavy chain of trastuzumab was linked to the gene encoding CCL11 using a two-step polymerase chain reaction (PCR) method.
  • the first step is to amplify the heavy chain gene using the PCR method (high-fidelity polymerase Pfx, Invitrogen) with artificially synthesized antibody heavy chain DNA as the substrate:
  • 5'-end primer M13-F (SEQ ID NO: 1): 5'-TGTAAAACGACGGCCAGT-3' on the pUC57 vector.
  • the 3'-end primer KDP004 (SEQ ID NO: 2): 5'-TCCTGGGGACAGTGACAGTG-3' is an antibody heavy chain gene specific primer.
  • 5'-TGGTGGTGTCTAGAGACTTATGGCTTTGGAGTTGG-3' is a specific primer for CCL11 gene.
  • the first 20 nucleotide sequences of the primer KDP008 are complementary to the nucleotide sequence of the primer KDP004, so that in the overlap extension PCR process of the second step, the two PCR fragments can be connected.
  • Primer M13-F SEQ ID NO: 1
  • KDP007 SEQ ID NO: 4
  • This mammalian cell expression vector is an improved pcDNA3.1 (Invitrogen).
  • the anti-neomycin (neomycin) gene in pcDNA3.1 is replaced by the DHFR (dihydrofolate reductase) gene.
  • the improved vector is suitable for screening for stable transfection. Mammalian cells with high protein expression.
  • the recombinant plasmid was transfected into DH5a competent bacteria, positive colonies containing the correct recombinant plasmid were identified by colony PCR, and the recombinant plasmid was purified.
  • the trastuzumab heavy chain-CCL11 recombinant gene had the correct sequence after restriction enzyme digestion and sequencing.
  • trastuzumab light chain cDNA was cloned into another modified pcDNA3.1 plasmid by subcloning with Not I and Xba I cloning enzymes.
  • CCL11 is linked directly to the C-terminus of the trastuzumab light chain (LC-CCL11), or via a linker peptide to the C-terminus of the trastuzumab light chain (LC-linker-CCL11).
  • LC-CCL11 linker peptide to the C-terminus of the trastuzumab light chain
  • the genes encoding trastuzumab single-chain LC-CCL11 were linked using a two-step polymerase chain reaction (PCR) method.
  • the first step is to amplify the light chain gene using the PCR method (high-fidelity polymerase Pfx, Invitrogen) with the synthetic antibody light chain DNA as the substrate:
  • 5'-end primer KDP068 (SEQ ID NO: 5): 5'-CTTTGGCAAAGAATTGGG-3', located on the vector.
  • the 3'-end primer KDP206 (SEQ ID NO: 6): 5'-ACATTCGCCACGATTAAAGGAT-3' is a specific primer for antibody light chain gene.
  • the first 19 nucleotide sequences of the primer KDP603 are complementary to the nucleotide sequence of the primer KDP206, so that the two PCR fragments can be connected together during the overlap extension PCR process in the second step.
  • 5'-end primer KDP066 (SEQ ID NO: 9): 5'-CGAACATCGATTGAATTCC-3';
  • the LC-CCL11 recombinant gene was cloned into a mammalian cell expression vector, and then a plasmid was prepared.
  • the first 20 nucleotide sequences of the primer KDP601 are complementary to the first 20 nucleotide sequences of the primer KDP602, so that the two PCR fragments can be connected during the overlap extension PCR process in the second step.
  • the LC-linker-CCL11 recombinant gene was cloned into a mammalian cell expression vector by the same method as the construction of the HC-CCL11 expression gene in Example 1, and then a plasmid was prepared.
  • trastuzumab heavy chain cDNA is cloned into mammalian cell expression vector, such as pcDNA3.1 plasmid, by subcloning method, and the cloning enzymes are Not I and Xba I.
  • Example 3 Establishment of a cell line stably expressing trastuzumab-CCL11 fusion protein
  • the host cell used to stably express the trastuzumab-CCL11 fusion protein is Chinese hamster ovary cell CHO-KS.
  • CHO-KS is a cultured CHO-K1 cell grown in a medium containing fetal bovine serum (FBS) that gradually reduces the FBS content in the medium until it is cultured in an FBS-free medium, and is finally acclimated to an FBS-free OptiCHO medium ( Invitrogen) cells grown in suspension.
  • FBS fetal bovine serum
  • the anti-neomycin gene in the pcDNA3.1 vector containing the fusion protein gene was replaced with the rat glutamine synthetase gene, and the heavy chain expression plasmid and light chain were expressed by electrotransfection (Bio-Rad, Gene Pulser Xcell).
  • the expression plasmids were co-transfected into CHO-KS cells, and after culturing the transfected cells for 24-48 hours, the transfected cells were screened and cultured on a 96-well culture plate by limiting dilution method.
  • the selection medium was OptiCHO, 5 ⁇ g/ml recombinant human insulin and 10 ⁇ M sulfoxide methionine (MSX).
  • Cells were cultured in a 37 °C, 8% CO 2 incubator. After 3 weeks, the cell culture medium of each well with a cell population was analyzed by ELISA method (alkaline phosphatase-conjugated goat anti-human IgG Fc antibody, Jackson ImmunoResearch Lab), and cells positive for fusion protein expression were identified. The population is further amplified, detected by ELISA, and then amplified, and finally a stable cell population with fusion protein expression is obtained.
  • ELISA method alkaline phosphatase-conjugated goat anti-human IgG Fc antibody, Jackson ImmunoResearch Lab
  • the plasmids used to express trastuzumab HC-CCL fusion protein are HC-CCL expression plasmid and LC expression plasmid
  • the plasmids used to express trastuzumab LC-linker-CCL11 fusion protein are HC expression plasmid and LC- linker-CCL11 expression plasmid.
  • the cell lines with high expression of the trastuzumab HC-CCL11 fusion protein and the trastuzumab HC-linker-CCL11 fusion protein obtained in Example 3 were cultured and expanded to 2 liters respectively. The culture supernatant was used to purify the antibody. Protein-A affinity chromatography (POROS MabCapture A, Life Tech) followed by purification with an anion column (flow through).
  • the 4-12% non-reducing SDS-PAGE electrophoresis gels of Figure 2 show the molecular weight averages of the intact trastuzumab HC-CCL11 fusion protein and the trastuzumab LC-linker-CCL11 fusion protein Slightly larger than trastuzumab, close to its theoretical value of 162kDa.
  • Human breast cancer cell BT-474 (purchased from the cell bank of the Chinese Academy of Sciences) is a tumor cell highly expressing Her-2. An appropriate amount of BT-474 cells was taken, adjusted to a cell density of 2 ⁇ 10 6 cells/ml with pre-cooled FACS working solution (PBS containing 0.1% FBS), aliquoted into 100 ⁇ L/tube, and blocked on ice for 1 hour.
  • trastuzumab, trastuzumab HC-CCL11 fusion protein and trastuzumab LC-linker-CCL11 fusion protein were serially diluted to 50 ⁇ g/mL, 10 ⁇ g/mL and 2 ⁇ g/mL with FACS working solution, and the 10 ⁇ L of serial dilutions were added to 100 ⁇ L of cell suspension to give final antibody concentrations of 5, 1 and 0.2 ⁇ g/mL, respectively.
  • Isotype IgG1 was used as a negative control.
  • trastuzumab was able to bind to Her-2 on the cell membrane, among which trastuzumab
  • the binding ability of the LC-linker-CCL11 fusion protein of trastuzumab was comparable to that of trastuzumab, and the binding ability of the HC-CCL11 fusion protein of trastuzumab to cell membrane Her-2 was slightly weaker (see Figure 3).
  • trastuzumab-CCL11 fusion proteins with different structures of the present invention basically keep the binding properties with Her-2 intact.
  • Human embryonic lung cells MRC-5 express the receptor membrane protein CCR3 of CCL11.
  • the binding of each trastuzumab-CCL11 fusion protein to cell MRC-5 was studied by flow cytometry (MRC-5 cells were purchased from the Cell Bank of the Chinese Academy of Sciences).
  • the experimental method refers to the method in Example 4.
  • the concentrations of each trastuzumab-CCL11 fusion protein were 0.2, 1, and 5 ⁇ g/mL, respectively.
  • Trastuzumab was used as a negative control.
  • trastuzumab HC-CCL11 fusion protein and trastuzumab LC-linker-CCL11 fusion protein can bind to MRC-5, while trastuzumab does not bind to MRC-5 It is proved that the trastuzumab HC-CCL11 fusion protein and the trastuzumab LC-linker-CCL11 fusion protein of the present invention completely retain the function of specifically binding to the CCL11 receptor CCR3 on the cell membrane.
  • the ability of the trastuzumab HC-CCL11 fusion protein of the present invention to bind to MRC-5 cells expressing CCR3 in vitro at a concentration of 5 ⁇ g/mL is significantly better than that of the trastuzumab LC-linker-CCL11 fusion protein and Trastuzumab.
  • Example 7 Construction of mouse tumor cell line stably expressing human Her-2
  • Mouse melanoma cells B16 were obtained from the Cell Bank of the Type Culture Collection, Chinese Academy of Sciences, and mouse ovarian cancer cells ID-8 were purchased from Shanghai Hongshun Biotechnology Co., Ltd.
  • the tumor cells were cultured in RPMI1640/10% FBS (Gibco) medium inside.
  • the human Her-2 expression gene was cloned in the expression vector pcDNA3.1 (Invitrogen), and the recombinant plasmid was transfected into mouse melanoma cell B16 and mouse ovarian cancer cell ID-8 with Lipofectmaine 3000 (Invitrogen), respectively.
  • Cells were cultured in RPMI/10% FBS medium containing G418 (Sigma) to obtain stable cell pools.
  • the monoclonal stable cell lines B16/Her-2 and ID8/Her-2 with high Her-2 expression were screened from the stable cell pool by flow cytometry (Influx, BD Biosciences).
  • Example 8 Trastuzumab-CCL11 fusion protein inhibits the growth of mouse melanoma B16 expressing human Her-2 in mice
  • C57BL/6 mice were from Shanghai Slack Co., Ltd. and were raised in an SPF environment.
  • mice in each group were divided into groups, 8-10 mice in each group, and 3 ⁇ 10 6 B16/Her-2 cells were injected into the armpits of mice by subcutaneous inoculation. Two days after cell inoculation, mice in each group were given PBS (control group), trastuzumab 4 mg/kg, CCL11 0.4 mg/kg (the same molar mass as trastuzumab), and trastuzumab via tail vein.
  • Monoclonal antibody+CCL11 4mg/kg (3.6mg/kg+0.4mg/kg, the weight percentage of CCL11 in trastuzumab-CCL11 fusion protein is 10%), trastuzumab HC-CCL11 fusion protein 4mg/kg , Trastuzumab LC-CCL11 fusion protein 4mg/kg and Trastuzumab HC-linker-CCL11 fusion protein 4mg/kg. It was then administered once a week for a total of 4 times. Tumor volume was measured at each dose and mice were weighed. The tumor volume was measured by (length ⁇ width ⁇ width/2).
  • Table 1 shows the inhibition rate of trastuzumab HC-CCL11 fusion protein and trastuzumab on B16/Her-2 tumor growth at different time points. The data indicated that the trastuzumab HC-CCL11 fusion protein was more effective in inhibiting tumor growth than trastuzumab.
  • FIG. 5 shows that compared with the average tumor volume of control mice, trastuzumab HC-CCL11 fusion protein can significantly inhibit the growth of B16/Her-2 tumors in mice, with statistical significance (p value ⁇ 0.01 ).
  • Trastuzumab had some effect on B16/Her-2 tumor growth, but there was no statistical difference compared to the control group.
  • each part of the trastuzumab-CCL11 fusion protein of the present invention has a synergistic effect, which is not only better than the effect of CCL11 or trastuzumab administered alone, but also better than the combined use of trastuzumab and CCL11 Effect.
  • eosinophils play a significant role in the inhibition of tumor growth, which can mediate antitumor responses through direct and indirect mechanisms, and eosinophils secrete cytotoxic proteins (basic protein MBP, eosinophilic protein). Eosinophil cationic protein ECP, eosinophil-derived neurotoxin EDN) to induce tumor cell death. In addition, eosinophils can indirectly mediate anti-tumor effects through IL-12, IL-10, NK cells, IFN ⁇ , CD8+T cells and other pathways. (Sharon Grisaru-Tal, A new dawn for eosinophils in the tumor microenvironment, Nat Rev Cancer.
  • CCL11 plays an important role in the regulation of eosinophils. It can be mainly divided into the following two ways: one is to induce the chemotaxis of eosinophils to the tumor microenvironment (Lorena, S Eotaxin expression in oral squamous cell carcinomas with and without tumor associated tissue eosinophilia. Oral. Dis. 9, 279–283 (2003) )); the second is to enhance eosinophil-mediated tumor killing (Simson, L. Regulation of carcinogenesis by IL-5 and CCL11: a potential role for eosinophils in tumor immune surveillance. J. Immunol 178, 4222–4229 (2007 )).
  • SEQ ID NO.14 Trastuzumab HC-CCL11 fusion protein heavy chain nucleotide sequence:

Abstract

本发明提供了一种抗Her-2抗体-趋化因子融合蛋白及其制法和应用。具体地，本发明提供了一种融合蛋白，所述融合蛋白包括抗Her-2抗体或其活性片段和趋化因子。本发明的融合蛋白在识别异常表达Her-2的肿瘤并抑制其生长的同时，通过CCL11介导的趋化作用增强嗜酸性粒细胞在肿瘤微环境中的杀伤，进一步提高对Her-2+肿瘤的杀伤力。

Description

抗Her-2抗体-趋化因子融合蛋白及其制法和应用 技术领域
本发明涉及属于生物医学领域，更具体地，本发明涉及一种抗Her-2抗体-趋化因子融合蛋白及其制法和应用。
背景技术
Her-2为原癌基因，属于人表皮生长因子受体家族，通过调节下游信号通路来抑制癌细胞凋亡，促进其增殖、侵袭，Her-2扩增或过表达在乳腺癌患者中占比约20％-30％，此外，胃癌中也常检测到Her-2阳性(Her-2+)。Her-2靶点的代表药物曲妥珠单抗目前在Her-2+的乳腺癌和胃癌中取得了不错的疗效，但是目前乳腺癌对曲妥珠单抗的耐药率及复发率逐年升高，其最终耐药率高达65％，这其中包括70％的患者在治疗初期对曲妥珠单抗治疗敏感，最终也出现耐药性。因此亟需新的组合来实现对Her-2+肿瘤的有效控制。
综上所述，本领域迫切需要开发一种更安全、有效、精准靶向肿瘤的抗Her-2的融合蛋白。
发明内容
本发明的目的在于提供一种安全、有效、精准靶向肿瘤的抗Her-2的融合蛋白。
在本发明的第一方面，提供了一种融合蛋白单链，所述融合蛋白单链包括融合在一起的以下元件：
(a)第一蛋白元件；
(b)第二蛋白元件；以及
(c)任选的位于第一蛋白元件和第二蛋白元件之间的接头元件；
其中，所述第一蛋白元件为抗Her-2抗体或其活性片段的蛋白元件；
第二蛋白元件为选自趋化因子CC家族的蛋白元件。
在另一优选例中，所述趋化因子选自CCL-11或CCL2。
在另一优选例中，所述抗Her-2抗体或其活性片段为含有F(ab)、scFv、VH、 CH、VL或VHH的活性片段。
在另一优选例中，所述抗Her-2抗体或其活性片段选自曲妥珠单抗的活性片段。
在另一优选例中，所述接头元件为肽键或肽接头。
在本发明的第二方面，提供了一种由本发明第一方面所述的融合蛋白单链组成的融合蛋白，所述融合蛋白具有下式I或II所示的二聚体结构：
Figure PCTCN2021129639-appb-000001
Figure PCTCN2021129639-appb-000002
式中，
H-Chain--V-Chain为抗Her-2抗体或其活性片段的蛋白元件，其中，
H-Chain为无、或抗Her-2抗体或其活性片段中的重链融合蛋白；
V-Chain为无、或抗Her-2抗体或其活性片段中的轻链融合蛋白；
CCL为选自趋化因子CC家族的蛋白元件；
Figure PCTCN2021129639-appb-000003
表示重链和轻链之间的二硫键；
“-”代表肽键或肽接头。
在另一优选例中，所述的重链融合蛋白包括或含有抗Her-2抗体中的重链、VH、CH、VHH、Fc区或HCDR。
在另一优选例中，所述的轻链融合蛋白包括或含有抗Her-2抗体中的轻链、VL、CL或LCDR。
在另一优选例中，H-Chain为曲妥珠单抗的重链。
在另一优选例中，V-Chain为曲妥珠单抗的轻链。
在另一优选例中，CCL优选为CCL-11。
在另一优选例中，在所述的融合蛋白中，所述的H-Chain或V-Chain与CCL以头-头、头-尾、或尾-尾方式相连。
在另一优选例中，所述的“头部”指多肽或其片段的N端，尤其是野生型多肽的或其片段的N端。
在另一优选例中，所述的“尾部”指多肽或其片段的C端，尤其是野生型多肽的或其片段的C端。
在另一优选例中，所述的肽接头的长度为0-20个氨基酸，较佳地1-15个氨基酸。
在另一优选例中，所述的H-Chain含有或具有SEQ ID NO:19中的第1-449位，所述的V-Chain含有或具有SEQ ID NO:22中的第1-214位，所述的CCL11含有或具有SEQ ID NO:19中的第450-523位，或SEQ ID NO:22中的第215-288位。
在另一优选例中，所述肽接头的序列为SEQ ID NO:23中的第215-221位。
在另一优选例中，所述融合蛋白的序列选自下组：
(1)轻链氨基酸序列如SEQ ID NO:20所示；和重链氨基酸序列如SEQ ID NO:19所示；
(2)轻链氨基酸序列如SEQ ID NO:22所示；和重链氨基酸序列如SEQ ID NO:21所示；或
(3)轻链氨基酸序列如SEQ ID NO:23所示；和重链氨基酸序列如SEQ ID NO:21所示；
(4)将(1)至(3)中的氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有同时结合Her-2蛋白以及结合CCL-11活性的由(1)至(3)衍生的多肽。
本发明的第三方面，提供了一种分离的多核苷酸，所述的多核苷酸编码本发明第二方面所述的融合蛋白。
在另一优选例中，所述融合蛋白的核苷酸序列选自下组：
(1)轻链核苷酸序列如SEQ ID NO:15所示；和重链氨基酸序列如SEQ ID NO:14所示；
(1)轻链核苷酸序列如SEQ ID NO:17所示；和重链氨基酸序列如SEQ ID NO:16所示；或
(1)轻链核苷酸序列如SEQ ID NO:18所示；和重链氨基酸序列如SEQ ID NO:16所示。
本发明的第四方面，提供了一种载体，它含有本发明的第三方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
本发明的第五方面，提供了一种宿主细胞，它含有本发明的第四方面所述的载体或基因组中整合有本发明的第三方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞和真核细胞。
在另一优选例中，所述的宿主细胞包括哺乳动物细胞。
本发明的第六方面，提供了一种产生本发明第二方面所述融合蛋白的方法，它包括步骤：
(1)在适合表达的条件下，培养本发明的第五方面所述的宿主细胞，从而表达出本发明的第二方面所述的融合蛋白；和
(2)任选地分离所述融合蛋白。
本发明的第七方面，提供了一种药物组合物，所述组合物包含：
本发明第二方面所述的融合蛋白，以及
药学上可接受的载体。
在另一优选例中，所述的药物组合物还含有：额外的活性成分，较佳地所述的活性成分包括：小分子化合物、细胞因子、抗体(如抗PD-1抗体、抗OX40抗体、抗CD137抗体、抗CD47抗体、ADC、CAR-免疫细胞)。
在另一优选例中，所述的药物组合物为注射剂型。
本发明的第八方面，提供了一种免疫细胞，所述的免疫细胞携带本发明第二方面所述的融合蛋白。
本发明的第九方面，提供了一种药物组合物，所述的组合物包含：
本发明的第八方面所述的免疫细胞，以及
药学上可接受的载体。
本发明第十方面，提供了一种如本发明第二方面所述的融合蛋白或本发明第八方面所述免疫细胞的用途，用于制备治疗肿瘤的药物。
在另一优选例中，所述的肿瘤为Her-2阳性的肿瘤。
在另一优选例中，所述的肿瘤包括：乳腺癌肿瘤、胃癌肿瘤、膀胱癌肿瘤、胰腺癌肿瘤、大肠癌肿瘤、肺癌肿瘤、肝癌肿瘤、黑色素肿瘤。
在另一优选例中，所述治疗肿瘤的药物可与另一种肿瘤免疫治疗联用，包括但不限于：化疗、抗CD20mAb、抗TIM-3mAb、抗LAG-3mAb、抗CD73mAb、抗CD47mAb、抗DLL3mAb、抗FRmAb mAb、抗CTLA-4抗体、抗OX40抗体、抗CD137抗体、抗PD-1抗体、PD-1/PD-L1治疗、其他免疫肿瘤药物、抗血管生成剂、放射治疗、抗体-药物偶联物(ADC)、靶向治疗或其他抗癌药物。
本发明第十一方面，提供了一种免疫偶联物，所述免疫偶联物包含：
(a)如上所述的融合蛋白；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，所述偶联物部分选自：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)。
在另一优选例中，所述的免疫偶联物包括抗体－药物偶联物(ADC)。
本发明第十二方面，提供了一种治疗肿瘤的方法，所述方法包括向有需要的受试者施用如上所述的融合蛋白、或其免疫偶联物、或其药物组合物。
在另一优选例中，所述的肿瘤包括：乳腺癌肿瘤、胃癌肿瘤、膀胱癌肿瘤、胰腺癌肿瘤、大肠癌肿瘤、肺癌肿瘤、肝癌肿瘤、黑色素肿瘤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1A-1B显示了本发明抗Her-2单克隆-趋化因子融合蛋白实施方式的两种结构示意图。图1A为曲妥珠单抗HC-CCL11融合蛋白结构示意图，图1B为曲妥珠单抗LC-CCL11(左)和曲妥珠单抗LC-linker-CCL11融合蛋白结构示意图(右)。
图2显示了曲妥珠单抗HC-CCL11和曲妥珠单抗LC-linker-CCL11融合蛋白的SDS-PAGE电泳分析研究。其中，泳道1、3和5分别是曲妥珠单抗、曲妥珠单抗HC-CCL11和曲妥珠单抗LC-linker-CCL11融合蛋白的4-12％非还原SDS-PAGE电泳分析。泳道2、4和6分别是各自的4-12％还原SDS-PAGE电泳分析。泳道4中星号标记的是单链HC-CCL11，泳道6中星号标记的是单链LC-linker-CCL11。泳道M为蛋白分子量标准(kDa)。
图3显示了曲妥珠单抗HC-CCL11和曲妥珠单抗LC-linker-CCL11融合蛋白与细胞膜上Her-2结合的流式细胞技术分析研究。Her-2高表达的人乳腺癌细胞BT-474与不同浓度的曲妥珠单抗-CCL11融合蛋白或曲妥珠单抗孵育，然后用FITC标记的羊抗人IgG1 Fc抗体检测与Her-2结合的抗体，FITC荧光强度用流式细胞仪检测。
图4显示了曲妥珠单抗HC-CCL11和曲妥珠单抗LC-linker-CCL11融合蛋白与细胞膜上受体CCR3结合的流式细胞技术分析研究。CCR3高表达的人胚肺细胞MRC-5与不同浓度的曲妥珠单抗-CCL11融合蛋白或曲妥珠单抗孵育，然后用FITC标记的羊抗人IgG1 Fc抗体检测与CCR3结合的抗体，FITC荧光强度用流式细胞仪检测。
图5显示了曲妥珠单抗HC-CCL11融合蛋白抑制表达人Her-2的小鼠黑色素瘤细胞B16在小鼠体内生长的研究。将人Her-2基因转染的稳定细胞株B16/Her-2接种在小鼠背部，3天后通过尾静脉注射分别给予PBS(
Figure PCTCN2021129639-appb-000004
表示)、曲妥珠单抗HC-CCL11融合蛋白(
Figure PCTCN2021129639-appb-000005
表示)和曲妥珠单抗(
Figure PCTCN2021129639-appb-000006
表示)，然后每周1次，共给药4次。药物剂量均为4mg/kg。肿瘤体积测量按(长×宽×宽/2)计算。
具体实施方式
本发明人过广泛而深入的研究，意外发现，将(a)抗Her-2抗体或其活性片段、(b)趋化因子CC家族的蛋白相融合，获得的融合蛋白具有高效杀伤肿瘤细胞活性、毒副作用小的协同作用。本发明的融合蛋白靶向Her-2表达的肿瘤，并 与有生物活性的趋化因子融合。具体地，本发明中的融合蛋白，特异性识别人表皮生长因子受体(Her-2)，并吸引和调节嗜酸性粒细胞的趋化因子CCL11。所得的融合蛋白可以特异性结合肿瘤组织表达的Her-2，抑制肿瘤生长；将趋化因子CCL11传递到靶向的肿瘤组织，增强嗜酸性粒细胞杀伤肿瘤的作用。因此，本发明中的融合蛋白可用于Her-2+肿瘤的治疗。在此基础上，发明人完成了本发明。
本发明设计的融合蛋白中，如图1A、1B所示，趋化因子可以以头-头、头-尾、或尾-尾方式与抗Her-2抗体或其活性片段连接。抗Her-2抗体或其活性片段可以是含有F(ab)、F(ab)2、scFv或VHH的活性片段，趋化因子可以是CCL2、CCL11。本发明中一种优选的连接方式为趋化因子CCL11连接在曲妥珠单抗的重链末端。实验证明，本发明的融合蛋白具有良好的协同作用，优于曲妥珠单抗或趋化因子CCL11单独施用的效果，优于曲妥珠单抗和趋化因子CCL11联合使用的效果。
术语
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
在本发明中，术语“本发明的融合蛋白”、“本发明的Her-2抗体-趋化因子融合蛋白”、“Her-2抗体-CCL11融合蛋白”可互换使用，皆指本发明中第一方面所提及的融合蛋白。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，除非另外说明，Fc是指人免疫球蛋白的Fc片段。术语“免疫球蛋白Fc区”指免疫球蛋白链恒定区，特别是免疫球蛋白重链恒定区的羧基端或其中的一部分，例如免疫球蛋白Fc区可包括重链CH1、CH2、CH3的两个或更多结构域与免疫球蛋白铰链区的组合，在优选例中，所用的免疫球蛋白的Fc区包括至少一个免疫球蛋白绞链区，一个CH2结构域和一个CH3结构域，优选缺少CH1结构域。
已知人免疫球蛋白有多种类别，如IgA、IgD、IgE、IgM及IgG(包括IgG1、IgG2、IgG3、IgG4四个亚类)，从特定的免疫球蛋白类别和亚类中选择特定的免疫球蛋白Fc区是在本领域技术人员所掌握的范围之内的，在一个优选的实例 中，免疫球蛋白Fc区可选择包含有人免疫球蛋白IgG4亚类Fc区的编码序列，其中缺失一个免疫球蛋白重链1结构域(CH1)，但包括了铰链区以及CH2、CH3、二个结构域的编码序列。
如本文所用，所述的“含有”，“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。
融合蛋白
如本文所用，除非另外说明，所述的融合蛋白是一种分离的蛋白，与其它蛋白、多肽或分子无联系，是重组宿主细胞所表达的，或经分离或纯化的产物。
本发明构建的融合蛋白，由以下二部分组成：
(1)识别肿瘤特异性抗原Her-2的全长单克隆抗体或最小识别抗原的部分；
(2)趋化性细胞因子家族中的一个具有生物活性的趋化因子，比如CCL-11或CCL2。
利用重组DNA技术构建融合蛋白编码核糖核苷酸，融合蛋白含有抗Her-2抗体重链，其含或不含重链恒定区的CH1或CH2或CH3，其C末端与有活性的细胞趋化因子融合。
当融合蛋白重链表达质粒与抗Her-2抗体轻链表达质粒共转染，可以产生抗Her-2抗体-趋化因子(如CCL11)融合蛋白，此融合蛋白能结合表达Her-2的肿瘤细胞，并能把趋化因子递送到肿瘤部位。
将有生物活性的趋化因子与抗Her-2的单链抗体融合，完整的融合蛋白是一条多肽链，各功能区域由连接肽连接，保证融合蛋白具有正确的空间结构，保持其生物活性。
本发明的融合蛋白是一类全新的分子，具有二种生物功能：第一，它们能靶向表达Her-2的肿瘤组织，第二，它们能把具有生物活性的细胞因子特异性递送到肿瘤部位。这些细胞因子具有吸引免疫细胞并调节免疫细胞活性的功能，因此，能增加免疫细胞肿瘤组织浸润以及增强免疫细胞活性，使肿瘤，例如乳腺癌、胃癌等，生长得到抑制。由于趋化因子主要局限在肿瘤组织部位，给患者造成的毒性相对较小。
本发明中融合蛋白里的抗体可以是全长抗体，也可以是抗体的某一关键片 段，比如，scFv、F(ab)2或VHH。从理论上来说，所有能结合肿瘤细胞膜上Her-2受体的抗体都适用于构建本发明的抗体-趋化因子融合蛋白(曲妥珠单抗、拉帕替尼单抗、和帕妥珠单抗)。在本发明中，优选曲妥珠单抗。
本发明的融合蛋白趋化因子部分，选自有生物活性的CC家族的趋化因子，直接或通过肽接头与抗体部分连接。
本发明提供了一种融合蛋白，包含以下元件：
(a)抗Her-2抗体或其活性片段的蛋白元件、(b)趋化因子CC家族(如CCL11)的蛋白元件、和(c)接头元件。本发明所述的融合蛋白中，所述的各元件之间(如元件a与元件b之间)，可以含有或不含有接头。
本发明的融合蛋白，不仅具有更长的体内半衰期，可以更有效地抑制血清中免疫疾病相关的抗体(尤其是IgE)的浓度。
在本发明中，本发明的融合蛋白还包括其保守性变异体，指与本发明的合蛋白的氨基酸序列相比，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A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
根据本发明提供的氨基酸序列，本技术领域人员可方便地用各种已知方法制得本发明的融合蛋白。这些方法例如但不限于：重组DNA法，人工合成，等[参见Murray KM,Dahl SLAnn；Pharmacother 1997Nov；31(11):1335-8]。
在得知了本发明的融合蛋白的氨基酸序列后，本领域人员可以方便地根据所述的氨基酸序列获得编码本发明的融合蛋白的基因序列。
一种优选的融合蛋白为曲妥珠单抗HC-CCL11融合蛋白，其重链核苷酸序列如SEQ ID NO:14所示，重链氨基酸序列如SEQ ID NO:19所示；其中，重链氨基酸(SEQ ID NO:19)序列中的1-449位为曲妥珠单抗的氨基酸序列；第450-523位为CCL11氨基酸序列。
一种优选的融合蛋白为曲妥珠单抗LC-CCL11融合蛋白，其轻链核苷酸序列如SEQ ID NO:17所示，轻链氨基酸序列如SEQ ID NO:22所示；其中，轻链氨基酸(SEQ ID NO:22)序列中的1-214位为曲妥珠单抗的轻链氨基酸序列；第215-288位为CCL11氨基酸序列。
一种优选的融合蛋白为曲妥珠单抗LC-linker-CCL11融合蛋白，其轻链核苷酸序列如SEQ ID NO:18所示，轻链氨基酸序列如SEQ ID NO:23所示；其中，轻链氨基酸(SEQ ID NO:23)序列中的1-214位为曲妥珠单抗的轻链氨基酸序列；第215-221位为linker序列；第222-295位为CCL11氨基酸序列。
在另一优选例中，本发明的曲妥珠单抗HC-CCL11融合蛋白的轻链核苷酸序列如SEQ ID NO:15所示，轻链氨基酸序列如SEQ ID NO:20所示。
在另一优选例中，本发明的曲妥珠单抗LC-CCL11或LC-linker-CCL11融合蛋白的重链核苷酸序列如SEQ ID NO:16所示，重链氨基酸序列如SEQ ID NO:21所示。
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多核苷酸和多肽是没有分离纯化的，但同样的多核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
如本文所用，“分离的重组融合蛋白”是指重组融合蛋白基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化重组融合蛋白。基本上纯的蛋白在非还原聚丙烯酰胺凝胶上能产生单一的主带。
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。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的蛋白质片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码多肽的功能。
如本文所用，术语“引物”指的是在与模板配对，在DNA聚合酶的作用下能以其为起点进行合成与模板互补的DNA链的寡居核苷酸的总称。引物可以是天然的RNA、DNA，也可以是任何形式的天然核苷酸。引物甚至可以是非天然的核苷酸如LNA或ZNA等。引物“大致上”(或“基本上”)与模板上一条链上的一个特殊的序列互补。引物必须与模板上的一条链充分互补才能开始延伸，但引物的序列不必与模板的序列完全互补。比如，在一个3'端与模板互补的引物的5'端加上一段与模板不互补的序列，这样的引物仍大致上与模板互补。只要有足够长的引物能与模板充分的结合，非完全互补的引物也可以与模板形成引物-模板复合物，从而进行扩增。
根据本发明提供的氨基酸序列，本技术领域人员可方便地用各种已知方法制得本发明的融合蛋白。这些方法例如但不限于：重组DNA法，人工合成等。
本发明融合蛋白的元件(如抗Her-2抗体活性片段或CCL)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。对于PCR扩增法，可根据已公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
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。
此外，还可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。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的基因。用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或融合蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述蛋白质的方法。
通过常规的重组DNA技术，可利用本发明的多核苷酸序列可用来表达或生产重组蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本领域的技术人员熟知的方法能用于构建含本发明蛋白的编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
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，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，链霉菌属的细菌细胞；真菌细胞如酵母；植物细胞；果蝇S2或Sf9的昆虫细胞；CHO、COS、或293细胞的动物细胞等。
一种特别优选的细胞为人和非人哺乳动物的细胞，尤其是免疫细胞，包括T细胞、NK细胞。
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CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。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。
在上面的方法中的蛋白质可在细胞内、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。
趋化因子
根据序列特征，趋化因子分为几个主要家族，比如CC、CXC和CX3C等。在本发明中，趋化因子优先选自CC家族。在CC家族中，CCL11和CC2是优先选择的趋化因子。
在一具体实施例中，本发明提供了一种抗体-趋化因子融合蛋白，其趋化因子选自CC家族中的CCL11。
肽接头
本发明的双功能融合蛋白可任选地含有或不含有肽接头。肽接头大小和复杂性可能会影响蛋白的活性。通常，肽接头应当具有足够的长度和柔韧性，以保证连接的两个蛋白在空间上有足够的自由度以发挥其功能。同时避免肽接头中形成α螺旋或β折叠等对融合蛋白的稳定性的影响。
肽接头的长度一般为0-20个氨基酸，较佳地1-15个氨基酸。
优选的肽接头的例子包括(但并不限于)：GSGGGGS(SEQ ID NO.24)、(G4S)3。
在本发明的一个具体实施例中，肽接头的氨基酸序列为：曲妥珠单抗LC-linker-CCL11氨基酸序列(SEQ ID NO:23)中的第215-221位。
药物组合物及施用方法
本发明还提供了一种组合物，它含有(a)有效量的本发明融合蛋白和/或有效量的本发明的免疫细胞，以及药学上可接受的载体。
通常，可将本发明的融合蛋白配制于无毒的、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。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量，如0.001-99wt％；较佳的0.01-95wt％；更佳的，0.1-90wt％。
当本发明的药物组合物含有免疫细胞时，“有效量”或“有效剂量”是指1×10 3-1×10 7个所述的免疫细胞/ml。
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
本发明的药物组合物含有安全有效量的本发明的融合蛋白以及药学上可接受的载体。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。通常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。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。本发明的药物制剂还可制成缓释制剂。
本发明融合蛋白的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：本发明融合蛋白的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本发明的融合蛋白每天以约5mg-20mg/kg动物体重(较佳的5mg-10mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
本发明融合蛋白特别适合用于治疗肿瘤等疾病。代表性的肿瘤包括(但并不限于)：乳腺癌肿瘤、胃癌肿瘤、膀胱癌肿瘤、胰腺癌肿瘤、大肠癌肿瘤、肺癌肿瘤、肝癌肿瘤、黑色素肿瘤。
本发明的主要优点包括：
(1)本发明的Her-2与CCL11的融合蛋白具有精确识别、免疫治疗、毒性可 控的优势。
(2)本发明的Her-2与CCL11的融合蛋白在识别异常表达Her-2的肿瘤并抑制其生长的同时，通过CCL11介导的趋化并增强嗜酸性粒细胞在肿瘤微环境中的杀伤，进一步提高对Her-2+肿瘤的疗效。
(3)本发明的Her-2与CCL11的融合蛋白，通过HER-2靶向肿瘤的特性，精准的将CCL11携带至肿瘤微环境，进一步强化嗜酸性粒细胞介导的局部肿瘤杀伤，同时降低了正常组织嗜酸性粒细胞相关的不良反应(嗜酸粒细胞性胃肠炎、嗜酸粒细胞气管炎等)的发生率。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅以阐释为目的而非限制本发明的范围。本领域技术人员可对本发明做出适当的修改、变动，这些修改和变动都在本发明的范围之内。
以下实施例中未注明具体条件的实验方法可采用本领域中的常规方法，例如参考《分子克隆实验指南》(第三版，纽约，冷泉港实验室出版社，New York：Cold Spring Harbor Laboratory Press，1989)或按照供应商所建议的条件。DNA的测序方法为本领域常规的方法，也可由商业公司提供测试。
实施例1.抗体-CCL11融合蛋白表达质粒的构建
1.曲妥珠单抗HC-CCL11融合蛋白表达质粒的构建
曲妥珠单抗作为Her-2抗体的以图1A示例。编码曲妥珠单抗重链和轻链的完整cDNA由GenScrip(USA)公司合成，分别克隆在pUC57载体上。人CCL11的cDNA购自OpenBiosystems(美国)。
有大量报道显示，在单克隆抗体表达和制备过程中，绝大部分抗体的重链C-末端赖氨酸被降解掉，所以在构建抗体-CCL11融合蛋白时，去掉了这个赖氨酸，使本发明的抗体融合蛋白能保持完整性。
编码曲妥珠单抗重链的基因与编码CCL11的基因用两步聚合酶链式反应技术(PCR)方法连接起来。第一步，用PCR方法(高保真聚合酶Pfx，Invitrogen)以人工合成的抗体重链DNA为底物扩增重链基因：
5'端引物M13-F(SEQ ID NO:1)：5'-TGTAAAACGACGGCCAGT-3'，位于pUC57载体上。
3'端引物KDP004(SEQ ID NO:2)：5'-TCCTGGGGACAGTGACAGTG-3'， 是抗体重链基因专一引物。
同样，用PCR方法扩增成熟CCL11蛋白部分(Gly24-Pro97)的基因：
5'端引物KDP008(SEQ ID NO:3)：
5'-CACTGTCACTGTCCCCAGGAGGGCCAGCTTCTGTCCCAACC-3'；
3'端引物KDP007(SEQ ID NO:4)：
5'-TGGTGGTGTCTAGAGACTTATGGCTTTGGAGTTGG-3'，是CCL11基因专一引物。
其中引物KDP008的头20个核苷酸序列与引物KDP004的核苷酸序列互补，这样在第二步的重叠延伸PCR过程中，可以把这2个PCR片段连接起来。
上面2个PCR片段经DNA胶纯化后(天根生化科技有限公司，北京)，进行第二步重叠PCR。5'端引物M13-F(SEQ ID NO:1)，3'端引物KDP007(SEQ ID NO:4)含有用于克隆的Xba I酶切序列。
曲妥珠单抗重链基因转录起始位点前有Not I的酶切位点，在胶纯化重叠PCR得到的片段后，进行Not I/Xba I双酶切(Takara)。然后把酶切的PCR片段克隆到同样酶切的哺乳动物细胞表达载体上。此哺乳动物细胞表达载体是改进的pcDNA3.1(Invitrogen)，pcDNA3.1里的抗neomycin(新霉素)基因被DHFR(二氢叶酸还原酶)基因取代，改进后的载体适用于筛选稳定转染蛋白高表达的哺乳动物细胞。将重组质粒转染进DH5a感受态细菌，用菌落PCR方法鉴定含有正确重组质粒的阳性菌落，提纯重组质粒。经酶切和测序鉴定，曲妥珠单抗重链-CCL11重组基因具有正确的序列。
用亚克隆方法把曲妥珠单抗轻链cDNA克隆到另一个改进的pcDNA3.1质粒，克隆酶是Not I和Xba I。
2.曲妥珠单抗LC-CCL11融合蛋白表达质粒的构建
以曲妥珠单抗作为Her-2抗体的示例。CCL11直接连接到曲妥珠单抗轻链的C末端(LC-CCL11)，或通过连接肽连接到曲妥珠单抗轻链的C末端(LC-linker-CCL11)。结构如图1B所示。
(i)编码曲妥珠单抗单链LC-CCL11的基因用两步聚合酶链式反应技术(PCR)方法连接起来。第一步，用PCR方法(高保真聚合酶Pfx，Invitrogen)以人工合成的抗体轻链链DNA为底物扩增轻链基因：
5'端引物KDP068(SEQ ID NO:5)：5'-CTTTGGCAAAGAATTGGG-3'，位 于载体上。
3'端引物KDP206(SEQ ID NO:6)：5'-ACATTCGCCACGATTAAAGGAT-3'，是抗体轻链基因专一引物。
同样，用PCR方法扩增成熟CCL11蛋白部分(Gly24-Pro97)的基因：
5'端引物KDP603(SEQ ID NO:7)：
5'-CTTTAATCGTGGCGAATGTGGGCCAGCTTCTGTC-3'；
3'端引物BGHR(SEQ ID NO:8)：
5'-AACTAGAAGGCACAGTCGAGGC-3'，位于载体上。
其中引物KDP603的头19个核苷酸序列与引物KDP206的核苷酸序列互补，这样在第二步的重叠延伸PCR过程中，可以把这2个PCR片段连接起来。
上面2个PCR片段经DNA胶纯化后(天根生化科技有限公司，北京)，进行第二步重叠延伸PCR反应。
5’端引物KDP066(SEQ ID NO:9)：5'-CGAACATCGATTGAATTCC-3'；
3’端引物KDP605(SEQ ID NO:10)：
5'-GAATAGGGCCCTCTAGAGACTTATGGCTTTGGAGTTG-3'
用与实施例1中构建HC-CCL11表达基因相同的方法，把LC-CCL11重组基因克隆到哺乳动物细胞表达载体中，然后制备质粒。
(ii)编码曲妥珠单抗LC-linker-CCL11的基因用两步聚合酶链式反应技术(PCR)方法连接起来。方法与构建LC-CCL11基因的方法相同，只是轻链PCR的3’端引物和CCL11PCR的5’端引物不同。
轻链PCR的3’端引物KDP602(SEQ ID NO:11)：
5'-GATCCGCCACCGCCGCTGCCACATTCGCCACGATTAAAG-3'
CCL11PCR的5’端引物KDP601(SEQ ID NO:12)：
5'-GGCAGCGGCGGTGGCGGATCCGGGCCAGCTTCTGTC-3'
其中引物KDP601的头20个核苷酸序列与引物KDP602的头20个核苷酸序列互补，这样在第二步的重叠延伸PCR过程中，可以把这2个PCR片段连接起来。
上面2个PCR片段经DNA胶纯化后(天根生化科技有限公司，北京)，进行第二步重叠延伸PCR反应。
用与实施例1中构建HC-CCL11表达基因相同的方法，把LC-linker-CCL11重组基因克隆到哺乳动物细胞表达载体中，然后制备质粒。
肽接头Linker核苷酸序列(SEQ ID NO:13)：
GGCAGCGGCGGTGGCGGATCC
(iii)用亚克隆方法把曲妥珠单抗重链cDNA克隆到哺乳动物细胞表达载体中，比如pcDNA3.1质粒，克隆酶是Not I和Xba I。
实施例3.建立曲妥珠单抗-CCL11融合蛋白稳定表达细胞株
用于稳定表达曲妥珠单抗-CCL11融合蛋白的宿主细胞为中国仓鼠卵巢细胞CHO-KS。CHO-KS是生长在含胎牛血清(FBS)培养基里的CHO-K1细胞经过逐渐降低培养基中FBS含量的培养直至无FBS培养基培养，最终驯化成在不含FBS的OptiCHO培养基(Invitrogen)中悬浮生长的细胞。含有融合蛋白基因的pcDNA3.1载体中的抗新霉素基因用大鼠谷氨酰胺合成酶基因取代，采用电转染(Bio-Rad，Gene Pulser Xcell)的方法把重链表达质粒和轻链表达质粒共转染进CHO-KS细胞，转染的细胞在培养24-48个小时后，用有限稀释法在96孔培养板上对转染的细胞进行筛选培养。筛选培养基是OptiCHO，5μg/ml重组人胰岛素和10μM氨基亚砜蛋氨酸(MSX)。在37℃，8％CO 2的培养箱里培养细胞。3个星期后，用ELISA方法(碱性磷酸酶偶联的羊抗人IgG Fc抗体，Jackson ImmunoResearch Lab)对每个长有细胞群的孔的细胞培养液进行分析，把融合蛋白表达阳性的细胞群进一步扩增，再ELISA检测，再扩增，最后得到融合蛋白表达稳定细胞群。
用于表达曲妥珠单抗HC-CCL融合蛋白的质粒为HC-CCL表达质粒和LC表达质粒，用于表达曲妥珠单抗LC-linker-CCL11融合蛋白的质粒为HC表达质粒和LC-linker-CCL11表达质粒。
实施例4.曲妥珠单抗-CCL11融合蛋白的制备、纯化和鉴定
将实施例3所得曲妥珠单抗HC-CCL11融合蛋白和曲妥珠单抗HC-linker-CCL11融合蛋白高表达的细胞系，分别培养扩增至2升。培养液上清用来纯化制备抗体。Protein-A亲和层析(POROS MabCapture A，Life Tech)，接着用阴离子柱(流穿)纯化。
结果与分析
图2的4-12％非还原SDS-PAGE电泳胶(泳道1、3和5)显示完整曲妥珠单抗HC-CCL11融合蛋白和曲妥珠单抗LC-linker-CCL11融合蛋白的分子量均略大于曲妥珠单抗，接近其理论值162kDa。4-12％还原SDS-PAGE电泳胶(泳道2、4 和6)显示：(i)曲妥珠单抗HC-CCL11融合蛋白的重链(泳道4，星号标记)略大于曲妥珠单抗重链(泳道2)，与其理论分子量一致(58kDa)，曲妥珠单抗HC-CCL11融合蛋白的轻链与曲妥珠单抗的轻链相同；(ii)曲妥珠单抗LC-linker-CCL11融合蛋白的轻链(泳道6，星号标记)略大于曲妥珠单抗轻链(泳道2)，与其理论分子量一致(32kDa)，曲妥珠单抗LC-CCL11融合蛋白的重链与曲妥珠单抗的重链相同。
实施例5.曲妥珠单抗-CCL11融合蛋白与Her-2的结合研究
用流式细胞仪方法(flow cytometry)检测以上制备的曲妥珠单抗-CCL11融合蛋白与细胞膜上Her-2受体的结合。人乳腺癌细胞BT-474(购自中国科学院细胞库)是高表达Her-2的肿瘤细胞。各取适量的BT-474细胞，用预冷的FACS工作液(PBS含0.1％FBS)调整其细胞密度为2×10 6个/ml，分装100μL/管，冰上封闭1小时。然后用FACS工作液系列稀释曲妥珠单抗、曲妥珠单抗HC-CCL11融合蛋白和曲妥珠单抗LC-linker-CCL11融合蛋白到50μg/mL、10μg/mL和2μg/mL，取系列稀释的10μL加到100μL的细胞悬液中，使抗体终浓度分别为5、1和0.2μg/mL。用同型IgG1作阴性对照。冰上孵育30分钟后，向每管细胞悬液加1mL FACS工作液，涡旋混匀细胞，离心5分钟，1200rpm/min，弃去上清，重复洗涤一遍。用FACS工作液稀释FITC标记的羊抗人IgG Fc抗体(Jackson ImmunoResearch Lab)，每管细胞悬液加10μL抗体，使其终浓度为1μg/mL，避光，冰上孵育30分钟。孵育完成后，向每管细胞悬液加1mL FACS工作液，涡旋混匀细胞，离心5分钟，1200rpm/min，弃去上清，重复洗涤一遍。用流式细胞仪C6(BD Biosciences)检测细胞。
结果与分析
流式细胞术试验结果显示：曲妥珠单抗、曲妥珠单抗HC-CCL11融合蛋白和曲妥珠单抗LC-linker-CCL11融合蛋白都能够结合细胞膜上的Her-2，其中曲妥珠单抗LC-linker-CCL11融合蛋白结合能力和曲妥珠单抗相当，曲妥珠单抗HC-CCL11融合蛋白的细胞膜Her-2结合能力稍微弱一些(见图3)。
因此，本发明不同结构的曲妥珠单抗-CCL11融合蛋白基本完好保留了与Her-2的结合特性。
实施例6.曲妥珠单抗-CCL11融合蛋白与表达CCR3的MRC-5细胞体外结 合
人胚肺细胞MRC-5表达CCL11的受体膜蛋白CCR3。用流式细胞仪方法研究各曲妥珠单抗-CCL11融合蛋白与细胞MRC-5的结合(MRC-5细胞购自中国科学院细胞库)。实验方法参照实施例4中的方法。各曲妥珠单抗-CCL11融合蛋白的浓度分别为0.2、1和5μg/mL。用曲妥珠单抗作为阴性对照。
结果与分析
结果如图4所示，曲妥珠单抗HC-CCL11融合蛋白和曲妥珠单抗LC-linker-CCL11融合蛋白均能与MRC-5结合，而曲妥珠单抗不与MRC-5结合，证明本发明的曲妥珠单抗HC-CCL11融合蛋白和曲妥珠单抗LC-linker-CCL11融合蛋白完整的保留了特异性结合细胞膜上CCL11受体CCR3的功能。此外，本发明的曲妥珠单抗HC-CCL11融合蛋白在5μg/mL浓度时，与表达CCR3的MRC-5细胞体外结合的能力明显优于曲妥珠单抗LC-linker-CCL11融合蛋白和曲妥珠单抗。
实施例7.人Her-2稳定表达小鼠肿瘤细胞株的构建
小鼠黑色素瘤细胞B16来自中国科学院典型培养物保藏委员会细胞库，小鼠卵巢癌细胞ID-8购自上海弘顺生物科技有限公司，肿瘤细胞均培养在RPMI1640/10％FBS(Gibco)培养基里。
人Her-2表达基因克隆在表达载体pcDNA3.1中(Invitrogen)，重组质粒用Lipofectmaine 3000(Invitrogen)分别转染进小鼠黑色素瘤细胞B16和小鼠卵巢癌细胞ID-8里，转染的细胞在含G418(Sigma)的RPMI/10％FBS培养基里培养，得到稳转细胞池。用流式细胞分选仪(Influx，BD Biosciences)从稳转细胞池中筛选出Her-2高表达的单克隆稳定细胞株B16/Her-2和ID8/Her-2。
实施例8.曲妥珠单抗-CCL11融合蛋白抑制表达人Her-2的小鼠黑色素瘤B16在小鼠体内生长的研究
C57BL/6小鼠来自上海斯莱克有限公司，饲养在SPF级别环境里。
将6-7周龄的C57/B6小鼠，分组，每组8-10只，用皮下接种的方法，在小鼠腋下注射B16/Her-2细胞3×10 6个/只。细胞接种2天后，各组小鼠尾静脉分别给予小鼠PBS(对照组)、曲妥珠单抗4mg/kg、CCL11 0.4mg/kg(与曲妥珠单抗同 等摩尔质量)、曲妥珠单抗+CCL11 4mg/kg(3.6mg/kg+0.4mg/kg，曲妥珠单抗-CCL11融合蛋白中CCL11的重量百分比是10％)、曲妥珠单抗HC-CCL11融合蛋白4mg/kg、曲妥珠单抗LC-CCL11融合蛋白4mg/kg和曲妥珠单抗HC-linker-CCL11融合蛋白4mg/kg。然后每周给药1次，共4次。每次给药时测量瘤体积，称小鼠重量。肿瘤体积测量按(长×宽×宽/2)计算。在接种细胞第27天结束实验，引颈脱臼处死小鼠，摘眼球取血，收集小鼠血液，并解剖小鼠，记录肿瘤重量、脾脏重量大小，同时，记录各组肿瘤照片。实验结果如图5和表1所示。其中，表1显示在不同时间点曲妥珠单抗HC-CCL11融合蛋白和曲妥珠单抗对B16/Her-2肿瘤生长的抑制率。数据表明曲妥珠单抗HC-CCL11融合蛋白对肿瘤生长的抑制效果要优于曲妥珠单抗的效果。
表1.不同时间点曲妥珠单抗HC-CCL11融合蛋白和曲妥珠单抗对B16/Her-2肿瘤生长的抑制率
Figure PCTCN2021129639-appb-000007
结果与分析
图5显示，与对照组小鼠肿瘤平均体积相比，曲妥珠单抗HC-CCL11融合蛋白能显著抑制B16/Her-2肿瘤在小鼠体内的生长，具有统计学意义(p值<0.01)。曲妥珠单抗对B16/Her-2肿瘤生长有些影响，但与对照组相比，没有统计学差异。此外，本发明的曲妥珠单抗-CCL11融合蛋白中的各部分具有协同作用，不仅优于CCL11或曲妥珠单抗单独施用的效果，也优于曲妥珠单抗和CCL11的联合使用效果。
讨论：
近期研究显示，嗜酸性粒细胞在抑制肿瘤生长的过程中发挥了显著的作用， 其可以通过直接和间接机制介导抗肿瘤反应，嗜酸性粒细胞能够分泌细胞毒蛋白(碱性蛋白MBP，嗜酸性粒细胞阳离子蛋白ECP，嗜酸性粒细胞衍生神经毒素EDN)来诱导肿瘤细胞死亡。另外嗜酸性粒细胞可通过IL-12、IL-10、NK细胞、IFNγ、CD8+T细胞等途径间接介导抗肿瘤作用。(Sharon Grisaru-Tal，A new dawn for eosinophils in the tumour microenvironment，Nat Rev Cancer.2020 Jul 16)研究显示，LMP胰腺癌肿瘤和BRAF黑色素瘤模型中发现了大量嗜酸性粒细胞浸润，这对后续肿瘤免疫将起到很大作用。(Allen B M,Systemic dysfunction and plasticity of the immune macroenvironment in cancer models.Nature Medicine,2020:1-10.)在大量黑色素瘤患者中已证实绝对嗜酸性粒细胞计数(AEC)与黑色素瘤患者的预后呈正相关。(Martens,A.et al.Baseline peripHeral blood biomarkers associated with clinical outcome of advanced melanoma patients treated with ipilimumab.Clin.Cancer Res.22,2908–2918(2016).)
在体内，CCL11对嗜酸性粒细胞的调控起到了重要的作用。主要可分为以下两大途径：一是诱导嗜酸性粒细胞向肿瘤微环境趋化(Lorena,S Eotaxin expression in oral squamous cell carcinomas with and without tumour associated tissue eosinophilia.Oral.Dis.9,279–283(2003))；二是增强嗜酸性细胞介导的肿瘤杀伤作用(Simson,L.Regulation of carcinogenesis by IL-5 and CCL11:a potential role for eosinophils in tumor immune surveillance.J.Immunol 178,4222–4229(2007))。在结直肠癌活检切片中观察到了CCL11的表达与肿瘤浸润嗜酸性粒细胞具有统计学的正相关性，在相应的动物模型中也得到了相同的结论。(Eosinophils in colorectal neoplasms associated with expression of CCL11 and CCL24.J.Pathol.Transl.Med.50,45–51(2016))肿瘤微环境中的CCL11将有助于嗜酸性粒细胞参与肿瘤微环境中的免疫识别与肿瘤杀伤。
本发明的相关序列
SEQ ID NO.1
5'端引物M13-F:TGTAAAACGACGGCCAGT
SEQ ID NO.2
3'端引物KDP004:TCCTGGGGACAGTGACAGTG
SEQ ID NO.3
5'端引物KDP008:CACTGTCACTGTCCCCAGGAGGGCCAGCTTCTGTCCCAACC
SEQ ID NO.4
3'端引物KDP007:TGGTGGTGTCTAGAGACTTATGGCTTTGGAGTTGG
SEQ ID NO.5
5'端引物KDP068:CTTTGGCAAAGAATTGGG
SEQ ID NO.6
3'端引物KDP206:ACATTCGCCACGATTAAAGGAT
SEQ ID NO.7
5'端引物KDP603:CTTTAATCGTGGCGAATGTGGGCCAGCTTCTGTC
SEQ ID NO.8
3'端引物BGHR:AACTAGAAGGCACAGTCGAGGC
SEQ ID NO.9
5’端引物KDP066:CGAACATCGATTGAATTCC
SEQ ID NO.10
3’端引物KDP605:GAATAGGGCCCTCTAGAGACTTATGGCTTTGGAGTTG
SEQ ID NO.11
轻链PCR的3’端引物KDP602:GATCCGCCACCGCCGCTGCCACATTCGCCACGATTAAAG
SEQ ID NO.12CCL11PCR的5’端引物KDP601:
GGCAGCGGCGGTGGCGGATCCGGGCCAGCTTCTGTC
SEQ ID NO.13肽接头Linker核苷酸序列:
GGCAGCGGCGGTGGCGGATCC
SEQ ID NO.14曲妥珠单抗HC-CCL11融合蛋白重链核苷酸序列:
Figure PCTCN2021129639-appb-000008
Figure PCTCN2021129639-appb-000009
SEQ ID NO.15
曲妥珠单抗HC-CCL11融合蛋白轻链核苷酸序列:
Figure PCTCN2021129639-appb-000010
SEQ ID NO.16
曲妥珠单抗LC-CCL11或LC-linker-CCL11融合蛋白重链核苷酸序列
Figure PCTCN2021129639-appb-000011
SEQ ID NO.17
曲妥珠单抗LC-CCL11融合蛋白轻链核苷酸序列
Figure PCTCN2021129639-appb-000012
Figure PCTCN2021129639-appb-000013
SEQ ID NO.18
曲妥珠单抗LC-linker-CCL11融合蛋白轻链核苷酸序列
Figure PCTCN2021129639-appb-000014
SEQ ID NO.19
曲妥珠单抗HC-CCL11融合蛋白重链氨基酸序列
Figure PCTCN2021129639-appb-000015
Figure PCTCN2021129639-appb-000016
SEQ ID NO.20
曲妥珠单抗HC-CCL11融合蛋白轻链氨基酸序列
Figure PCTCN2021129639-appb-000017
SEQ ID NO.21
曲妥珠单抗LC-CCL11或LC-linker-CCL11融合蛋白重链氨基酸序列
Figure PCTCN2021129639-appb-000018
SEQ ID NO.22
曲妥珠单抗LC-CCL11融合蛋白轻链氨基酸序列
Figure PCTCN2021129639-appb-000019
SEQ ID NO.23
曲妥珠单抗LC-linker-CCL11融合蛋白轻链氨基酸序列
Figure PCTCN2021129639-appb-000020
SEQ ID NO.24接头:GSGGGGS
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 一种融合蛋白单链，其特征在于，所述融合蛋白单链包括融合在一起的以下元件：
    (a)第一蛋白元件；
    (b)第二蛋白元件；以及
    (c)任选的位于第一蛋白元件和第二蛋白元件之间的接头元件；
    其中，所述第一蛋白元件为抗Her-2抗体或其活性片段的蛋白元件；
    第二蛋白元件为选自趋化因子CC家族的蛋白元件。
  2. 如权利要求1所述的融合蛋白单链，其特征在于，所述趋化因子选自CCL-11或CCL2。
  3. 如权利要求1所述的融合蛋白单链，其特征在于，所述抗Her-2抗体或其活性片段为含有F(ab)、scFv、VH、CH、VL或VHH的活性片段。
  4. 由权利要求1所述的融合蛋白单链组成的融合蛋白，其特征在于，所述融合蛋白具有下式I或II所示的二聚体结构：
    Figure PCTCN2021129639-appb-100001
    或
    Figure PCTCN2021129639-appb-100002
    式中，
    H-Chain--V-Chain为抗Her-2抗体或其活性片段的蛋白元件，其中，
    H-Chain为无、或抗Her-2抗体或其活性片段中的重链融合蛋白；
    V-Chain为无、或抗Her-2抗体或其活性片段中的轻链融合蛋白；
    CCL为选自趋化因子CC家族的蛋白元件；
    Figure PCTCN2021129639-appb-100003
    表示重链和轻链之间的二硫键；
    “-”代表肽键或肽接头。
  5. 如权利要求4所述的融合蛋白，其特征在于，所述融合蛋白的序列选自下组：
    (1)轻链氨基酸序列如SEQ ID NO:20所示；和重链氨基酸序列如SEQ ID NO:19所示；
    (2)轻链氨基酸序列如SEQ ID NO:22所示；和重链氨基酸序列如SEQ ID NO:21所示；或
    (3)轻链氨基酸序列如SEQ ID NO:23所示；和重链氨基酸序列如SEQ ID NO:21所示；
    (4)将(1)至(3)中的氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有同时结合Her-2蛋白以及结合CCL-11活性的由(1)至(3)衍生的多肽。6.一种分离的多核苷酸，其特征在于，所述的多核苷酸编码权利要求4-5任一所述的融合蛋白。7.一种载体，其特征在于，它含有权利要求6所述的多核苷酸。
  6. 一种宿主细胞，其特征在于，它含有权利要求7所述的载体或基因组中整合有权利要求6所述的多核苷酸。
  7. 一种产生权利要求2所述融合蛋白的方法，其特征在于，它包括步骤：
    (1)在适合表达的条件下，培养权利要求8所述的宿主细胞，从而表达出权利要求4-5任一所述的融合蛋白；和
    (2)任选地分离所述融合蛋白。
  8. 一种免疫偶联物，其特征在于，所述免疫偶联物包含：
    (a)如权利要求4-5任一所述的融合蛋白；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。11.一种免疫细胞，其特征在于，所述的免疫细胞携带权利要求4-5任一所述的融合蛋白。
  9. 一种药物组合物，其特征在于，所述的组合物包含：
    权利要求4-5任一所述的融合蛋白、或权利要求11所述的免疫细胞，以及
    药学上可接受的载体。
  10. 如权利要求12所述的药物组合物，其特征在于，所述的药物组合物还含有：额外的活性成分，较佳地所述的活性成分包括：小分子化合物、细胞因子、抗体(如抗PD-1抗体、抗OX40抗体、抗CD137抗体、抗CD47抗体、ADC、CAR-免疫细胞)。
  11. 如权利要求4-5任一所述的融合蛋白或其药物组合物、免疫偶联物或权 利要求11所述免疫细胞的用途，用于制备治疗肿瘤的药物。
  12. 如权利要求14所述的用途，其特征在于，所述的肿瘤为Her-2阳性的肿瘤。
  13. 如权利要求14所述的用途，其特征在于，所述的肿瘤包括：乳腺癌肿瘤、胃癌肿瘤、膀胱癌肿瘤、胰腺癌肿瘤、大肠癌肿瘤、肺癌肿瘤、肝癌肿瘤、黑色素肿瘤。
  14. 一种治疗肿瘤的方法，其特征在于，所述方法包括向有需要的受试者施用根据权利要求4-5任一项所述的融合蛋白、或其免疫偶联物、或根据权利要求12所述的药物组合物。
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