CN117430711A - Bispecific fusion protein for treating liver cell type liver cancer and application thereof - Google Patents

Bispecific fusion protein for treating liver cell type liver cancer and application thereof Download PDF

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CN117430711A
CN117430711A CN202210864815.9A CN202210864815A CN117430711A CN 117430711 A CN117430711 A CN 117430711A CN 202210864815 A CN202210864815 A CN 202210864815A CN 117430711 A CN117430711 A CN 117430711A
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fusion protein
bispecific fusion
cells
bispecific
antibody
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陈琳
孙倍成
刘思远
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Abstract

The invention relates to a bispecific fusion protein for treating liver cancer and application thereof. Specifically, the invention provides a bispecific fusion protein targeting GPC3 antigen and NKG2D receptor, which can mediate NK cells to recognize tumor cells, and simultaneously improve the killing efficiency of NK cells on the tumor cells. The invention also provides application of the bispecific fusion protein in preparing medicines for treating HCC.

Description

Bispecific fusion protein for treating liver cell type liver cancer and application thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a bispecific fusion protein for treating liver cancer and application thereof.
Background
Primary liver cancer is one of the most common malignant tumors in our country, of which about 90% are Hepatocellular liver cancers (Hepatocellular carcinoma, HCC) (Villanueva, a., hepatocelularis carpinoma. N Engl J Med,2019.380 (15): p.1450-1462.), which not only localizes in the sixth place of incidence worldwide, but also cancer cause 2 in the third place worldwide (Forner, a., m.reig, and J. Bruix, hepatocelularis carpinoma. Lancet,2018.391 (10127): p.1301-1314). Currently, only 10-25% of early HCC patients can be treated with surgical resection, ablation and liver transplantation, and most HCC patients have progressed to mid-to-late stages when diagnosed, lacking effective treatment (Yang, j.d., et al A global view of hepatocellular carcinoma: treatments, risk, precursors and management. Nat Rev Gastroenterol Hepatol,2019.16 (10): p.589-604).
30-50% of lymphocytes in the Liver are natural killer cells (natural killer cells, NK cells) while the proportion of NK cells in the Liver is 5 times higher than that in spleen or peripheral blood, suggesting that NK cells play an important role in the Liver (Gao, B., W.I. Jeong, and Z.Tian, lever: an organ with predominant innate immunity.hepatology,2008.47 (2): p.729-36.). NK cells have a number of receptors, largely divided into two major classes, the activating and the inhibitory receptors. Tumor cells tend to evade immune surveillance by down-regulating their own major histocompatibility complex (major histocompatibility complex, MHC), but when NK cells are encountered, NK cells are caused to recognize killer tumor cells due to loss of inhibitory signals (Peng, H., E.Wisse, and Z.Tian, liver natural killer cells: subsets and roles in liver Immunity.Cell Mol Immunol,2016.13 (3): p.328-36.Koch, J., et al, activating natural cytotoxicity receptors of natural killer cells in cancer and Infection.trends Immunol,2013.34 (4): p.182-91.). Meanwhile, besides the strong capability of killing tumor cells, NK cells have no MHC restriction, which indicates that NK cells can be used as a general cell for treating patients. Thus, the use of NK cells for the treatment of HCC patients is currently a very valuable immunotherapy.
GPC3 is a very specific tumor antigen that is not only specifically overexpressed on about 80% of HCC, but is also critical for cell proliferation, closely related to poor prognosis (Capurro, M., et al, glypican-3:a novel serum and histochemical marker for hepatocellular carcinoma.Gastroenterology,2003.125 (1): p.89-97.Filmus,J.and M.Capurro,Glypican-3:a marker and a therapeutic target in hepatocellular carcinoma.FEBS J,2013.280 (10): p.2471-6.). There are many clinical trials currently performed for this target of GPC3, including classical chimeric antigen receptor (chimeric antigen receptor) -T cells, bispecific antibodies (Shi, D., et al, chimeric Antigen Receptor-Glypican-3T-Cell Therapy for Advanced Hepatocellular Carcinoma: results of Phase I Trials.Clin Cancer Res,2020.26 (15): p.3979-3989.Ishiguro, T., et al, an anti-Glypican 3/CD3 bispecific T cell-redirecting antibody for treatment of solid tuners. Sci Transl Med,2017.9 (410). However, although GPC3 CAR-T cells exhibit considerable clinical therapeutic effects, they cannot be widely used due to not only high cost, but also side effects such as graft versus host reaction (graft versus host disease, GVHD). However, bispecific antibodies are artificial antibodies comprising two specific antigen binding sites, which are capable of linking tumor cells to effector cells, and have great potential in tumor immunotherapy. Meanwhile, bispecific antibodies targeting CD19 and CD3 have been marketed for the treatment of acute lymphoblastic leukemia, suggesting that bispecific antibodies are of great value in the future immunotherapeutic market. Thus, the best solution is to design a bispecific fusion protein against this target of GPC3 to mediate NK cells for treatment of HCC patients.
The natural killer cell receptor G2D (natural killer cell receptor G2D) is an important activating receptor on the surface of NK cells and possesses a variety of ligands, including UL16-binding proteins (ULBPs) family members (Cosman, D., et al, ULBPs, novel MHC class I-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor.immunity,2001.14 (2): p.123-33). The expression level of NKG2D ligand on tumor cells directly affects the intensity of NK cell killing function (Pende, D., et al Major histocompatibility complex class I-related chain A and UL-binding protein expression on tumor cell lines of different histotypes: analysis of tumor susceptibility to NKG2D-dependent natural killer cell cytotoxity. Cancer Res,2002.62 (21): p.6178-86.). Of the ULBPs family, ULBP2 protein and NKG2D have the highest affinity (Kubin, m., et al, ULBP1,2,3:novel MHC class I-related molecules that bind to human cytomegalovirus glycoprotein UL, active NK cells, eur J Immunol,2001.31 (5): p.1428-37.). However, tumor cells release Soluble ULBP2 to inhibit NK cell function (Kegasawa, T., et al, solution UL16-binding protein 2is associated with a poor prognosis in pancreatic cancer patients.Biochem Biophys Res Commun,2019.517 (1): p.84-88.).
Thus, there is an urgent need in the art to develop an excellent bispecific fusion protein that simultaneously targets GPC3 antigen and NKG2D receptor, thereby mediating NK cell de-recognition of killer tumor cells.
Disclosure of Invention
The invention aims to provide a bispecific fusion protein which can mediate NK cells to recognize tumor cells and improve the killing efficiency of NK cells on the tumor cells.
It is another object of the present invention to provide the use of the bispecific fusion protein of the present invention for the preparation of a medicament for the treatment of HCC.
In a first aspect of the present invention, there is provided a bispecific fusion protein having the structure from N-terminus to C-terminus of formula (I):
Z1-L1-Z2 (I)
wherein,
z1 is a ligand or element of NKG 2D;
l1 is a bond or a linker element;
z2 is an antibody or element against GPC 3;
"-" represents a peptide bond.
In another preferred embodiment, the bispecific fusion protein comprises from N-terminus to C-terminus: ULBP2-linker 1-VH-hYP-linker 2-hYP7-VL;
wherein, the VH-hYP7 is the heavy chain variable region of the anti-GPC 3 antibody,
the VL-hYP7 is the light chain variable region of an anti-GPC 3 antibody,
the linker1 and the linker2 are each independently flexible peptide linkers,
said VH-hYP7 forms an antigen binding site with said VL-hYP7 that specifically binds GPC 3;
the ULBP2 specifically binds to the NKG2D receptor.
In another preferred embodiment, the bispecific fusion protein has the activity of simultaneously binding to GPC3 antigen and binding to NKG2D receptor.
In another preferred embodiment, the flexible peptide linker comprises 0-30 amino acids, preferably 1-10 amino acids.
In another preferred embodiment, the flexible peptide linker is 1-4 GGGGS and/or GGGS.
In another preferred example, the linker1 is GGGGS.
In another preferred embodiment, the amino acid sequence of the bispecific fusion protein is shown in SEQ ID NO. 2.
In a second aspect of the invention, there is provided an isolated nucleotide encoding a bispecific fusion protein according to the first aspect of the invention.
In another preferred embodiment, the nucleotide sequence of the bispecific fusion protein is shown in SEQ ID NO. 1.
In a third aspect of the invention there is provided an expression vector comprising a nucleotide according to the second aspect of the invention.
In another preferred embodiment, the expression vector is a eukaryotic expression vector.
In another preferred embodiment, the eukaryotic expression vector is pcDNA3.1.
In a fourth aspect of the invention there is provided a host cell comprising an expression vector according to the third aspect of the invention.
In a fifth aspect of the present invention, there is provided a method of preparing a bispecific fusion protein, the method comprising the steps of:
(a) Culturing the host cell according to the fourth aspect of the invention under expression, thereby expressing the bispecific fusion protein according to the first aspect of the invention;
(b) Isolating and purifying the bispecific fusion protein of (a).
In a sixth aspect of the invention, there is provided a pharmaceutical composition comprising a bispecific fusion protein according to the first aspect of the invention and a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition further comprises an anti-tumor agent.
In another preferred embodiment, the pharmaceutical composition is in unit dosage form.
In another preferred embodiment, the anti-tumor agent may be present in a separate package from the bispecific fusion protein, or the anti-tumor agent may be used in combination with the bispecific fusion protein.
In another preferred embodiment, the pharmaceutical composition is administered in a form that includes a gastrointestinal administration form or a parenteral administration form.
In another preferred embodiment, the parenteral administration comprises intravenous injection, intravenous drip, subcutaneous injection, topical injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.
In a seventh aspect of the invention, there is provided an immunoconjugate comprising:
(a) The bispecific fusion protein of the first aspect of the invention; and
(b) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
In another preferred embodiment, the conjugate moiety is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (e.g., NK cells, etc.) capable of producing detectable products.
In another preferred embodiment, the immunoconjugate comprises an antibody-drug conjugate (ADC).
In another preferred embodiment, the immunoconjugate is used for preparing a pharmaceutical composition for treating a tumor.
In an eighth aspect of the invention, there is provided a method of treating cancer comprising administering to a subject in need thereof a bispecific fusion protein according to the first aspect of the invention, or a pharmaceutical composition according to the sixth aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention.
In another preferred embodiment, the cancer is primary liver cancer.
In a ninth aspect of the invention there is provided the use of a bispecific fusion protein according to the first aspect of the invention, or a pharmaceutical composition according to the sixth aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention, for the manufacture of a medicament, reagent, assay plate or kit for the treatment of cancer.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is a schematic diagram of the construction of a plasmid of the bispecific fusion protein ULBP2-hYP 7.
FIG. 2is a schematic representation of the expression of anti-GPC 3/NKG2D double anti-ULBP 2-hYP fusion proteins in 293T and 293F cells.
FIG. 3 is a schematic representation of the quality control of ULBP2-hYP7 fusion proteins after purification.
FIG. 4 is a schematic representation of the effect of ULBP2-hYP7 fusion proteins on the binding of the respective antigen.
FIG. 5 is a schematic representation of the enhancement of NK cell activity by ULBP2-hYP7 fusion protein.
FIG. 6 is a schematic representation of ULBP2-hYP7 fusion proteins enhancing NK cell cytokine release.
FIG. 7 is a schematic representation of ULBP2-hYP7 fusion proteins to enhance NK cell killing of tumor cells.
Detailed Description
The present inventors have conducted extensive and intensive studies and have conducted extensive screening to obtain a bispecific fusion protein composed of an anti-GPC 3 antibody hYP7 and an NKG2D ligand ULBP2. The bispecific fusion protein of the invention can recognize not only GPC3 on the surface of tumor cells, but also NKG2D of NK cells. Experimental results show that the bispecific fusion protein of the invention obviously enhances the activation of NK cells, enhances the release capacity of NK cell factors and mediates and enhances the killing capacity of NK cells. On this basis, the present inventors have completed the present invention.
Terminology
In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meanings given below, unless expressly specified otherwise herein. Other definitions are set forth throughout the application.
As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
In the present invention, the terms "Antibody (abbreviated Ab)" and "Immunoglobulin G (abbreviated IgG)" are isotetralin proteins having the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes (isotype). Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a constant region, the heavy chain constant region consisting of three domains CH1, CH2, and CH 3. One end of each light chain has a variable region (VL) and the other end has a constant region, the light chain constant region comprising a domain CL; the constant region of the light chain is paired with the CH1 domain of the constant region of the heavy chain and the variable region of the light chain is paired with the variable region of the heavy chain. The constant regions are not directly involved in binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC, anti-independent cell-mediated cytotoxicity), and the like. Heavy chain constant regions include the IgG1, igG2, igG3, igG4 subtypes; the light chain constant region includes Kappa (Kappa) or Lambda (Lambda). The heavy and light chains of an antibody are covalently linked together by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of an antibody are covalently linked together by inter-polypeptide disulfide bonds formed between the hinge regions.
In the present invention, the term "bispecific antibody (or diabody)" refers to an antibody molecule capable of specifically binding to two antigens (targets) or two epitopes simultaneously. Bispecific antibodies can be classified into structurally symmetrical and asymmetrical molecules according to symmetry. Bispecific antibodies can be classified into bivalent, trivalent, tetravalent, and multivalent molecules depending on the number of binding sites.
In the present invention, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically a mixture of different antibodies with different epitopes) each monoclonal antibody is directed against a single epitope on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.
In the present invention, the terms "Fab" and "Fc" refer to papain that cleaves antibodies into two identical Fab fragments and one Fc fragment. The Fab fragment consists of VH and CH1 of the heavy chain and VL and CL domains of the light chain of the antibody. The Fc fragment, i.e., the crystallisable fragment (fragment crystallizable, fc), consists of the CH2 and CH3 domains of the antibody. The Fc segment has no antigen binding activity and is the site where an antibody interacts with an effector molecule or cell.
In the present invention, the term "scFv" is a single chain antibody (single chain antibody fragment, scFv) comprising an antibody heavy chain variable region and a light chain variable region, which are usually linked by a linking short peptide (linker) of 15 to 25 amino acids.
In the present invention, the term "variable" means that some portion of the variable region in an antibody differs in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the heavy and light chain variable regions, known as complementarity-determining region (CDR) or hypervariable regions. The more conserved parts of the variable region are called the Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)).
As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for the domains of the light and heavy chains to fold into an exchanged double variable region immunoglobulin. In the present invention, preferred Linker means Linker1, linker1 is used to link scFv to ligand ULBP2 of NKG2D, while Linker2 links VH and VL of single chain antibody (scFv).
Examples of suitable linkers include mono glycine (Gly), or serine (Ser) residues, the identity and sequence of the amino acid residues in the linker may vary with the type of secondary structural element that needs to be achieved in the linker.
Bispecific fusion proteins of the invention
As used herein, the terms "bispecific antibody-ligand fusion protein", "fusion protein of the invention", "bispecific protein of the invention", "protein of the invention" are used interchangeably to refer to the bispecific antibody-ligand fusion protein of the first aspect of the invention.
The bispecific fusion protein of the present invention is a bispecific fusion protein against GPC3×nkg2d, including anti-GPC 3 antibody hYP7 and the ligand ULBP2 of NKG2D.
Preferably, the anti-hYP antibody of the invention is a single-chain antibody, and the amino acid sequence of the antibody is shown as SEQ ID No. 2.
Wherein, the underlined "___" is the VH chain of GPC3 antibody hYP7, underlinedIs the VL chain of GPC3 antibody hYP.
It will be appreciated that the hYP antibodies of the invention may also be modified or engineered by techniques well known in the art, such as by adding, deleting and/or substituting one or more amino acid residues, to further increase the affinity or structural stability of the anti-hYP 7 and to obtain modified or engineered results by conventional assay methods.
In the present invention, a "conservative variant of a bispecific antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of a bispecific antibody of the present invention. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.
Table A
In the present invention, the terms "anti", "binding", "specific binding" refer to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. Typically, the antibody is present at less than about 10 -7 M, e.g. less than about 10 -8 M、10 -9 M、10 -10 M、10 -11 An equilibrium dissociation constant (KD) of M or less binds to the antigen. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. For example, the binding affinity of an antibody to an antigen is determined in a BIACORE instrument using surface plasmon resonance (Surface Plasmon Resonance, abbreviated SPR) or the relative affinity of an antibody to antigen binding is determined using ELISA.
In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. An epitope of the invention is a region of an antigen to which an antibody binds.
The bispecific fusion proteins of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, or a combination of any of the above.
Coding nucleic acids and expression vectors
The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
In the present invention, the term "expression vector" refers to a vector, such as a plasmid, viral vector (e.g., adenovirus, retrovirus), phage, yeast plasmid, or other vector, carrying an expression cassette for expression of a particular protein of interest or other substance. Representative examples include, but are not limited to: pTT5, pSECtag series, pCGS3 series, pcDNA series vectors and the like, as well as other vectors for use in mammalian expression systems and the like. Included in the expression vector are fusion DNA sequences linked to appropriate transcriptional and translational regulatory sequences.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. 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.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
In the present invention, the term "host cell" refers to a cell suitable for expressing the above expression vector, and may be eukaryotic, such as mammalian or insect host cell culture systems, all of which can be used for expression of the fusion protein of the present invention, CHO (chinese hamster ovary ), HEK293, COS, BHK and derivatives of the above cells are suitable for use in the present invention.
Pharmaceutical composition and application
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavity injection.
In the present invention, the term "pharmaceutical composition" means that the bispecific antibodies of the present invention can be combined with a pharmaceutically acceptable carrier to form pharmaceutical formulation compositions that provide more stable therapeutic effects, such formulations ensuring the conformational integrity of the amino acid core sequences of the disclosed bispecific antibodies while also protecting the multifunctional groups of the proteins from degradation (including, but not limited to, aggregation, deamination or oxidation).
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the bispecific antibody (or conjugate thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, bispecific antibodies of the invention can also be used with other therapeutic agents.
When a pharmaceutical composition is used, a safe and effective amount of the bispecific antibody or immunoconjugate thereof is administered to a mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
The main advantages of the invention include:
1) The invention greatly enriches the treatment means of the advanced refractory HCC patient for the research and development of the bispecific fusion protein of the HCC patient, is extremely likely to reduce the toxic and side effects of the drug, and simultaneously controls the treatment accuracy.
2) The bispecific fusion protein can mediate NK cells to the surface of tumor cells, greatly improves the killing efficiency of the NK cells on the tumor cells, and fills the gap of treatment means of HCC patients.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
The experimental materials used in the following examples are illustrated below:
pcDNA3.1 eukaryotic expression vector: purchased from gold sri;
293F cells: purchased from university of science and technology;
mag-beams His-Tag protein purification magnetic Beads: purchased from a manufacturer, cat No.: c650033;
NK cells: laboratory induction;
HepG2 cells: purchased from ATCC;
anti-CD56: biolegend, cat: 318310;
anti-CD107a: biolegend, cat: 328606;
anti-NKG2D antibody: biolegend, cat: 320808.
the experimental reagents used in the following examples are illustrated below:
transfection reagent PEI: purchased from YESEN, cat No.: 40816ES02;
NK cell sorting kit: purchased from meitian gentle, cat No.: 130092657;
NK MACS Medium+5% human serum+IL-2+IL-15 Medium: purchased from meitian gentle, cat No.: 130114429;
LDH kit: purchased from Promega, cat: G1780.
EXAMPLE 1 construction and expression purification of bispecific fusion proteins
1.1 Construction of ULBP2-hYP7 fusion protein expression vector
The amino acids of ULBP2-linker-hYP heavy chain variable region-hYP light chain variable region were codon optimized and ligated to eukaryotic expression vector pcDNA3.1 (see FIG. 1). The recombinant plasmid was then transfected into 293T or 293F cells using the transfection reagent PEI. After 4 days of culture, the culture supernatants were collected and the expression of the bispecific fusion proteins was detected by polypropylene gel electrophoresis (SDS-PAGE).
Results
As shown in FIG. 2, the expression level of the fusion protein of ULBP2-hYP7 in 293T is weaker than that in 293F cells, and the fusion protein has a hetero-band. And in 293F cells, the expression is specific and the expression exists stably.
1.2 Expression purification of ULBP2-hYP7 fusion proteins
50mL of 293F cells were transfected with PEI, the cell culture supernatant was collected after 4 days, and the target protein was eluted by using Mag-beams His-Tag protein purification Beads, followed by two overnight dialysis with PBS buffer. And concentrating by using a ultrafiltration tube, and finally analyzing the expression and purification effects of the protein by using SDS-PAGE and western blotting.
Results
The expression and purification effects of the protein are shown in figure 3, and the expression of the ULBP2-hYP7 fusion protein in 50mL 293F cells still keeps better effect and better purity. Meanwhile, the size and the expected size of the purified protein are consistent through western blotting verification, which shows that the protein is successfully purified. M represents a protein marker,1 represents ULBP2-hYP7 fusion protein, and 2 represents hYP protein.
Example 2 validation of bispecific fusion protein biological Activity
10. Mu.g of antigen GPC3 or NKG2D was coated in 96-well plates, 1. Mu.g of the fusion protein hYP and ULBP2-hYP was added to 96-well plates on the next day, and after 2 hours anti-His-HRP antibody was added for incubation, and finally TMB was added for color development, and the OD at 370nm was detected with an ELISA.
Results
As shown in FIG. 4, both hYP7 and ULBP2-hYP7 fusion proteins recognized GPC3 antigen, but hYP7 did not recognize NKG2D, only ULBP2-hYP7 fusion protein recognized NKG2D. This demonstrates that expression of the purified ULBP2-hYP fusion protein not only recognizes GPC3 on the surface of tumor cells, but also NKG2D on NK cells, which can be used to mediate NK cells to exert good reactivity.
EXAMPLE 3 Effect of bispecific fusion proteins on NK cells
3.1 preparation of NK cells
Cord blood was collected and counted by sorting using NK cell sorting kit. Then NK MACS Medium+5% human serum+IL-2+IL-15 Medium was used according to NK cell density of 1×10 6 Culture was performed in mL. After 7 days, NK cells were stained with anti-NKG2D antibody and the expression of NKG2D on the NK cell surface was examined by flow cytometry.
Results
As shown in FIG. 5A, NK cells after culture highly expressed NKG2D, and selected target cells HepG2 also highly expressed GPC3. This shows that the prepared NK has higher activation capacity and is expected to be mediated by ULBP2-hYP7 fusion protein to identify HepG2 cells.
3.2 Effect of ULBP2-hYP7 fusion proteins on NK cell Activity
NK cells and HepG2 cells were used at an effective target ratio of 1:1 were plated in 96-well plates with the addition of hYP and ULBP2-hYP fusion proteins, respectively. After 4 hours of co-culture, cells were collected, NK cells were stained with anti-CD56 and anti-CD107a, and finally stained with flow cytometry.
Results
As shown in FIG. 5B, the NK cells prepared had been activated in the presence of the target cells, but hYP7 did not increase NK cell activation in this co-culture system, but ULBP2-hYP7 fusion protein significantly enhanced NK cell activation, which was shown by increased CD107a expression.
3.3 Effect of ULBP2-hYP7 fusion proteins on NK cytokine Release
Will be 1X 10 5 NK cells and 1X 10 5 HepG2 cells were plated in 48-well plates with the addition of hYP7 and ULBP2-hYP fusion proteins, respectively. Each well was then added with a Golgi blocking agent (BFA, brefeldin A) and incubated for 4 hours after mixing. Cells were collected and NK cells were stained with anti-CD56 and anti-TNFα antibodies. Analysis of NK cell TNFα with flow cytometry on CD56 Positive cellsAnd thus exhibits the ability of NK cells to release factors.
Results
As shown in FIG. 6, the prepared NK cells release TNF alpha by 24.9% of NK cells in the presence of target cells, but the addition of hYP7 does not increase the NK cells releasing TNF alpha, but the fusion protein ULBP2-hYP7 obviously increases the NK cell proportion releasing TNF alpha, which indicates that the fusion protein ULBP2-hYP7 can better enhance the ability of NK cells to release TNF alpha.
3.4 Effect of ULBP2-hYP7 fusion proteins on NK cell killing
Will be 2X 10 4 NK cells and 2X 10 4 HepG2 cells were plated in 96-well plates with the addition of hYP and ULBP2-hYP fusion protein (0.1 mM), respectively, and incubated for 4 hours. Collecting supernatant, detecting OD value at 490nm wavelength by using an enzyme-linked detector according to LDH kit instruction, and calculating the killing function of the T cells according to a calculation formula. Repeated three times.
The calculation formula is as follows: killing rate = (experimental well-effector cell spontaneous well-target cell spontaneous well)/(target cell maximum lysis well-target cell spontaneous well).
Results
The experimental results are shown in FIG. 7.
In the blank, NK cells can kill about 60% of HepG2 cells in the presence of target cells and in the absence of hYP7 and ULBP2-hYP7 fusion proteins.
In the hYP7 addition group, the addition of hYP did not increase the killing effect of NK cells, and the killing rate of HepG2 cells was still about 60%.
In the ULBP 2-added group, use of ULBP2 protein alone not only does not increase the killing rate of NK cells against HepG2 cells, but rather also inhibits the killing function of NK cells against tumor cells, resulting in a killing rate of less than 60% against HepG2 cells (not shown).
In the ULBP2-hYP7 addition group, unexpectedly, the addition of ULBP2-hYP7 fusion protein significantly increased the NK cell killing effect, with a killing rate of HepG2 cells up to about 80%. This suggests that ULBP2-hYP7 fusion proteins may surprisingly and synergistically mediate and enhance NK cell killing.
Discussion of the invention
ULBP 2is the highest affinity member of the ULBPs family for NKG2D, and its expression on tumor cells is positively correlated with NK cell killing ability. In the tumor microenvironment, however, tumor cells will inhibit NK cell function by releasing soluble ULBP2, monitored by the escape immune system. Thus, ULBP2 protein alone does not only increase NK cell function, but rather inhibits NK cell function on tumor cells.
In summary, the bispecific fusion protein of the present invention can specifically bind to both GPC3 and NKG2D targets at the same time, which not only maintains the activity of the anti-GPC 3 antibody, but also improves the killing function of NK cells. Therefore, the bispecific fusion protein can be prepared into a medicament with excellent curative effect for treating primary liver cancer, and has a certain application prospect.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (10)

1. A bispecific fusion protein, characterized in that the bispecific fusion protein has the structure of formula (I) from N-terminus to C-terminus:
Z1-L1-Z2 (I)
wherein,
z1 is a ligand or element of NKG 2D;
l1 is a bond or a linker element;
z2 is an antibody or element against GPC 3;
"-" represents a peptide bond.
2. The bispecific fusion protein of claim 1, wherein the bispecific fusion protein has the activity of simultaneously binding to GPC3 antigen and binding to NKG2D receptor.
3. The bispecific fusion protein of claim 1, wherein the amino acid sequence of said bispecific fusion protein is shown in SEQ ID No. 2.
4. An isolated nucleotide encoding the bispecific fusion protein of claim 1.
5. An expression vector comprising the nucleotide sequence of claim 4.
6. A host cell comprising the expression vector of claim 5.
7. A method for preparing a bispecific fusion protein, comprising the steps of:
(a) Culturing the host cell of claim 6 under expression, thereby expressing the bispecific fusion protein of claim 1;
(b) Isolating and purifying the bispecific fusion protein of (a).
8. A pharmaceutical composition comprising the bispecific fusion protein of claim 1 and a pharmaceutically acceptable carrier.
9. An immunoconjugate, the immunoconjugate comprising:
(a) The bispecific fusion protein of claim 1; and
(b) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
10. Use of a bispecific fusion protein according to claim 1, or a pharmaceutical composition according to claim 8, or an immunoconjugate according to claim 9, for the preparation of a medicament, reagent, assay plate or kit for the treatment of cancer.
CN202210864815.9A 2022-07-21 2022-07-21 Bispecific fusion protein for treating liver cell type liver cancer and application thereof Pending CN117430711A (en)

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