WO2023071987A1 - Anti-her2 immunotoxin molecule and preparation method therefor and application thereof - Google Patents

Abstract

The present invention relates to the technical field of biological pharmacy, and in particular to an anti-HER2 immunotoxin molecule and a preparation method therefor and an application thereof. The anti-HER2 immunotoxin molecule comprises, from an amino end to a carboxyl end, a fusion polypeptide of an H-L1-PE structure or a PE-L1-H structure, wherein H is an anti-HER2 antibody or an antigen binding fragment thereof, L1 is a linker 1, and PE comprises part or all of a structural domain III of Pseudomonas exotoxin A. The immunotoxin molecule of the present invention can target cancer cells having HER2 positive expression in a high-specificity manner, thereby achieving efficient killing of cancer cells and inhibition of tumor growth.

Description

A kind of anti-HER2 immunotoxin molecule and its preparation method and application technical field

The invention belongs to the technical field of biopharmaceuticals. Specifically, the invention relates to an anti-HER2 immunotoxin molecule and its preparation method and application.

Background technique

Epidermal growth factor receptor 2 (Human epidermal growth factor receptor 2, HER2) is one of the four members of the epidermal growth factor receptor family. There is expression. According to the latest global cancer burden data for 2020 recently released by the International Agency for Research on Cancer (IARC) of the World Health Organization, there are 2.26 million new cases of breast cancer worldwide, surpassing the 2.2 million cases of lung cancer and becoming the largest cancer in the world. In breast cancer patients, the proportion of HER2-positive patients reaches 20% to 25%, and the biological characteristics of the tumor are high malignancy, metastasis and mortality. Trastuzumab, pertuzumab, and fixed-dose combination formulations of both drugs improved clinical benefit and survival in HER2-positive breast cancer.

Antibody-conjugated drugs (ADCs) combine monoclonal antibodies with specific antigens on the surface of tumor cells, and deliver cytotoxic drugs to tumor lesions in a targeted manner. As small molecule toxins are released, they can bind to DNA minor groove or tubulin A variety of mechanisms, such as induction of apoptosis of cancer cells, exert the cytotoxicity of drugs, thus providing a new treatment option for patients with HER2-positive breast cancer. ADC is also considered to be one of the important directions for the development of monoclonal antibody drugs (especially in the field of tumor targeted therapy) in the next decade. According to the forecasts of Evaluate Pharma and BCG, the global ADC market is expected to reach US$12.9 billion in 2024, with a compound annual growth rate of approximately 35% from 2018 to 2024. Currently, the most commercially successful ADC drug in the world is Kadcyla (trastuzumab emmettuzumab, T-DM1) for the treatment of HER2-positive breast cancer. This drug has become the second-line standard treatment for HER2-positive breast cancer in the world. Annual global sales of CHF 1.393 billion (CHF 1.295 billion in the first three quarters of 2020, a year-on-year increase of 37%). In May 2019, Kadcyla was also approved by the FDA as an adjuvant therapy for patients with HER2-positive early breast cancer (residual invasive disease after taxane and trastuzumab treatment), further expanding the market space. It was followed by Enhertu (DS-8201a), jointly developed by Daiichi Sankyo and AstraZeneca. This ADC was first approved by the FDA in December 2019 for the treatment of HER2-positive breast cancer.

Over the past few years, immunoconjugates have been developed as therapeutic alternatives for the treatment of malignancies. Immunoconjugates, originally composed of antibodies chemically coupled to plant or bacterial toxins, are a form of immunotoxin. Antibodies bind to antigens expressed on target cells, and the toxin is internalized, causing cell death by preventing protein synthesis and inducing apoptosis (Brinkmann, U., Mol. Med. Today, 2:439-446 (1996)).

At present, the second-line and late-stage treatment of HER2-positive breast cancer still faces the dilemma of insufficient effective treatment methods, so there is a clinical need to develop immunotoxin molecules targeting HER2.

Contents of the invention

The purpose of the present invention is to provide a novel anti-HER2 immunotoxin molecule, which can highly specifically target cancer cells with positive expression of HER2, realize efficient killing of cancer cells, and thereby inhibit tumor growth. The object of the present invention is also to provide a nucleic acid molecule encoding the immunotoxin molecule; provide an expression vector comprising the nucleic acid molecule; provide a host cell comprising the expression vector; provide a preparation method for the immunotoxin molecule; A pharmaceutical composition of the immunotoxin molecule; an application of the immunotoxin molecule or the pharmaceutical composition in the preparation of a drug for treating cancer; a method for treating cancer with the immunotoxin molecule or the pharmaceutical composition.

In order to achieve the above object, the present invention provides the following technical solutions:

The first aspect of the present invention provides an anti-HER2 immunotoxin molecule, which comprises a fusion polypeptide of H-L1-PE or PE-L1-H structure from the amino terminus to the carboxyl terminus, wherein H is an anti-HER2 antibody or its Antigen-binding fragment; L1 is linker 1; PE contains part or all of domain III of Pseudomonas exotoxin A.

In a preferred embodiment, the anti-HER2 immunotoxin molecule comprises a fusion polypeptide of H-L1-PE structure from the amino terminus to the carboxyl terminus.

In a preferred embodiment, the anti-HER2 antibody or antigen-binding fragment thereof comprises a single-chain antibody scFv.

In a preferred embodiment, the single-chain antibody scFv comprises a fusion polypeptide of VL-L2-VH or VH-L2-VL structure from the amino terminus to the carboxyl terminus, wherein VL is the light chain variable region, and VH is the heavy chain Variable region, L2 is linker 2.

In a more preferred embodiment, the single-chain antibody scFv comprises a fusion polypeptide of VL-L2-VH structure from the amino terminus to the carboxyl terminus.

In a preferred embodiment, the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 11, and the amino acid sequence of HCDR2 is shown in SEQ ID NO: 12, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 13; the VL includes light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 8, and the amino acid sequence of LCDR2 is shown in SEQ ID NO : Shown in 9, the amino acid sequence of LCDR3 is shown in SEQ ID NO: 10.

In a preferred embodiment, the scFv of the single-chain antibody comprises a VL or a variant thereof having an amino acid sequence such as SEQ ID NO: 16, and a VH or a variant thereof having an amino acid sequence such as SEQ ID NO: 17, The variants contain 1-5 amino acid mutations.

In a more preferred embodiment, said VL comprises a Q100C mutation and said VH comprises a K44C mutation.

In a preferred embodiment, the scFv of the single-chain antibody comprises a VL having an amino acid sequence as shown in SEQ ID NO: 14, and a VH having an amino acid sequence as shown in SEQ ID NO: 15.

In a preferred embodiment, the L2 comprises (G4S) _n , wherein n is any integer selected from 1-6; preferably, the L2 comprises (G4S) ₄ .

In a preferred embodiment, said single-chain antibody scFv comprises an amino acid sequence as described in SEQ ID NO: 2, or comprises an amino acid sequence having at least 98% or more than 99% identity with SEQ ID NO: 2 .

In a preferred embodiment, the PE is PE25, comprising the amino acid sequence as set forth in SEQ ID NO:4.

In a preferred embodiment, said PE comprises at least 1 amino acid mutation.

In a preferred embodiment, the L1 comprises a cathepsin B cleavage site; more preferably, the L1 comprises a cleavage site Gly-Phe-Leu-Gly.

In a more preferred embodiment, said L1 comprises the amino acid sequence as set forth in SEQ ID NO: 3.

In a preferred embodiment, the anti-HER2 immunotoxin molecule comprises the amino acid sequence as set forth in SEQ ID NO:1.

The second aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned anti-HER2 immunotoxin molecule.

In a preferred embodiment, the nucleic acid molecule further comprises gene regulatory elements such as promoters, terminators, enhancers and the like.

In a preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6.

The third aspect of the present invention provides an expression vector, which comprises the above-mentioned nucleic acid molecule.

In a preferred embodiment, the expression vector is pET-28a.

The fourth aspect of the present invention provides a host cell comprising the above expression vector.

In a preferred embodiment, the host cell is Escherichia coli; more preferably, the host cell is BL21(DE3).

A fifth aspect of the present invention provides a method for preparing an anti-HER2 immunotoxin molecule, the preparation method comprising the following steps: a) cultivating the above-mentioned host cells under expression conditions, thereby expressing an anti-HER2 immunotoxin molecule and b) isolating and purifying the anti-HER2 immunotoxin molecules described in step a).

In a preferred embodiment, said purification comprises purification using Protein L affinity chromatography.

The sixth aspect of the present invention provides a pharmaceutical composition, which comprises an effective amount of the above-mentioned anti-HER2 immunotoxin molecule and one or more pharmaceutically acceptable carriers or excipients.

The seventh aspect of the present invention provides the application of the above-mentioned anti-HER2 immunotoxin molecule or pharmaceutical composition in the preparation of a drug for treating cancer, and the cancer is a cancer with positive expression of HER2.

In a preferred embodiment, the cancer is a cancer with high expression of HER2.

In a preferred embodiment, the cancer is selected from breast cancer, gastric cancer, ovarian cancer, bladder cancer and non-small cell lung cancer.

The eighth aspect of the present invention provides a method for treating HER2-positive cancer, the method comprising administering the above-mentioned anti-HER2 immunotoxin molecule or pharmaceutical composition to a subject in need, the cancer being HER2 positively expressed cancers.

In a preferred embodiment, the cancer is a cancer with high expression of HER2.

In a preferred embodiment, the cancer is selected from breast cancer, gastric cancer, ovarian cancer, bladder cancer and non-small cell lung cancer.

The ninth aspect of the present invention provides the application of the above-mentioned anti-HER2 immunotoxin molecule or pharmaceutical composition in the preparation of a drug for killing cancer cells or inhibiting the growth of cancer cells, and the cancer cells are cancer cells positively expressing HER2.

In a preferred embodiment, the cancer cells are cancer cells with high expression of HER2.

In a preferred embodiment, the cancer cells are selected from breast cancer, gastric cancer, ovarian cancer, bladder cancer and non-small cell lung cancer cells.

In a more preferred embodiment, the cancer cells are HCC19549 cells or SKBR3 cells.

The tenth aspect of the present invention provides a method for killing cancer cells or inhibiting the growth of cancer cells, the method comprising administering the above-mentioned anti-HER2 immunotoxin molecule or pharmaceutical composition to a subject in need, the cancer cells The cells are cancer cells with positive expression of HER2.

In a preferred embodiment, the cancer cells are cancer cells with high expression of HER2.

In a preferred embodiment, the cancer cells are selected from breast cancer, gastric cancer, ovarian cancer, bladder cancer and non-small cell lung cancer cells.

In a more preferred embodiment, the cancer cells are HCC19549 cells or SKBR3 cells.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

The beneficial effects of the present invention include:

1) The anti-HER2 immunotoxin molecule of the present invention can specifically bind to the HER2 antigen on the surface of tumor cells, can target cells with high expression of HER2, and has better targeting.

2) The anti-HER2 immunotoxin molecule of the present invention has strong breast cancer cell killing activity, and its killing activity is better than T-DM1 (trastuzumab-matansine conjugate), and unexpected technical effect.

3) The anti-HER2 immunotoxin molecule of the present invention can be expressed and soluble in Escherichia coli without renaturation, the purification process is simple, the purity is good, and the yield is high.

4) The Linker1 linking the antibody molecule Ds4d5Fv targeting HER2 and PE25 contains the cathepsin B cleavage site Gly-Phe-Leu-Gly. The Linker1 is very stable in plasma. When HER2-positive cancer cells take up Tra-PE25 , PE25 is released after intracellular lysosome digestion, which can accurately kill cancer cells.

Description of drawings

Figure 1 is the SDS-PAGE detection of Tra-PE25

Figure 2 is Tra-PE25 immunotoxin ELISA detection

Figure 3 is the detection of the killing activity of Tra-PE25 in HCC1954 cells

Figure 4 is the detection of the killing activity of Tra-PE25 in BEAS-2B cells

Figure 5 is Tra-PE25 mass spectrometry detection

Detailed ways

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention; in the description and claims of the present invention, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural forms.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the embodiments of the present invention can also be used Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.

After extensive and in-depth research, the inventors have constructed an anti-HER2 immunotoxin molecule, which includes three parts: anti-HER2 antibody or its antigen-binding fragment, part or all of Pseudomonas exotoxin A, and a linker. Experimental results show that the anti-HER2 immunotoxin molecule of the present invention can bind to the specific antigen HER2 on the surface of tumor cells, and deliver cytotoxic drugs to tumor lesions in a targeted manner, and then the cytotoxic drugs will be internalized by cells, processed and released toxin PE25 , Inhibit cellular protein translation, leading to apoptosis. The present invention has been accomplished on this basis.

The following experimental examples are to further illustrate the present invention, and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional methods or methods routine in the art, such as methods for the preparation of nucleic acid molecules, methods for constructing vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or introducing plasmids into Methods for host cells, methods for culturing host cells, methods for purifying proteins, etc., such methods are well known to those skilled in the art, and have been described in many publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

the term

Anti-HER2 immunotoxin molecules

In the present invention, the term "immunotoxin (Immunotoxin) molecule" refers to a fusion polypeptide (fusion protein) composed of an antibody or its antigen-binding fragment (antibody part) and toxin (toxin part). Among them, the antibody part can specifically bind to the antigen and target cells; the toxin part is endocytosed by the cells bound to the antibody part, so that the toxin can kill cells or promote cell apoptosis in the cells.

In the present invention, the term "fusion polypeptide" refers to a new polypeptide sequence obtained by fusing two or more identical or different polypeptide sequences. The term "fused" refers to direct linkage by peptide bonds or operably linkage via one or more linkers.

In the present invention, the term "antibody" refers to a full-length antibody, and the term "antigen-binding fragment" refers to a fragment derived from an antibody and capable of binding antigenic epitopes, including but not limited to scFv, Fv, Fd, Fab, F(ab' )2 or F(ab').

In the present invention, the term "scFv" refers to a polypeptide single chain formed by connecting a VH region and a VL region through a linker.

In the present invention, the term "full-length antibody" refers to a heterotetrameric glycoprotein of about 150,000 Daltons with the same structural features, comprising variable regions and constant regions, which consist of two identical heavy chains (HC) and two The same light chain (LC) composition. Each heavy chain has a heavy chain variable region (VH) at one end followed by a heavy chain constant region consisting of three domains CH1, CH2, and CH3. Each light chain has a light chain variable region (VL) at one end and a light chain constant region at the other end. The light chain constant region includes a domain CL; the light chain constant region is paired with the CH1 domain of the heavy chain constant region. The chain variable region is paired with the heavy chain variable region. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity) and so on. The heavy chain constant region includes 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 a disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are linked together by an interpolypeptide disulfide formed between the hinge regions. bonded together covalently.

In the present invention, the term "variable" means that certain parts of the variable regions in antibodies differ in sequence, which contribute to the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout antibody variable domains. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy-chain variable region and the light-chain variable region. The more conserved part of the variable region is called the frame region (FR). The variable domains of native heavy and light chains each contain four FR regions that are roughly in a β-sheet configuration connected by three CDRs that form connecting loops, in some cases forming partial β-sheet structures. The CDRs in each chain are in close proximity through 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 Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The CDRs of the heavy chain variable region (VH) and light chain variable region (VL) are called HCDR and LCDR, respectively.

In the present invention, the term "framework region" (FR)" refers to a relatively small part of the amino acid composition and arrangement sequence outside the hypervariable region in the variable region of the antibody. The light chain and the heavy chain of the antibody each have four FRs, Respectively referred to as FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H. Preferably, the FR of the present invention is a human antibody FR or a derivative thereof , the derivative of the human antibody FR is substantially identical to the naturally occurring human antibody FR, that is, the sequence identity reaches at least 85%, 90%, 95%, 96%, 97%, 98% or 99%. Skills in the art After knowing the amino acid sequence of the CDR, the personnel can determine the sequence of the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR4-H.

In the present invention, the terms "anti" and "binding" refer to the non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. Typically, the antibody binds the antigen with an equilibrium dissociation constant (KD) of less than about 10" ⁷ M, eg, less than about 10 ^"8 M, 10 ^"9 M, 10 ^"10 M, 10" ¹¹ M or less. The term "KD" refers to the equilibrium dissociation constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, surface plasmon resonance (Surface Plasmon Resonance, abbreviated as SPR) is used to measure the binding affinity of the antibody to the antigen in a BIACORE instrument or ELISA is used to determine the relative affinity of the antibody to the antigen.

In the present invention, the term "Linker" is used to connect two polypeptide chains. A suitable linker can be a flexible polypeptide sequence, an example of which includes a single glycine (Gly), or a serine (Ser) residue, and the identification and sequence of the amino acid residues in the linker can follow the secondary structure that needs to be realized in the linker The type of element varies.

Those skilled in the art can modify or transform the antibody part or toxin part of the present invention by techniques well known in the art, such as adding, deleting and/or replacing one or several amino acid residues, thereby further improving or optimizing the immunotoxin molecule (such as Improve affinity), and obtain modified or engineered results by conventional assay methods.

In the present invention, the antibody portion of the present invention also includes its conservative variants, which refer to at most 10, preferably at most 7, and more preferably Up to 5, optimally up to 3 amino acids are replaced by amino acids of similar or closely related properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.

Table A

initial residue	representative replacement	preferred substitution
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
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;	Leu

In the present invention, the term "PE" refers to Pseudomonas exotoxin A (Pseudomonas exotoxin, PE), which is a single-chain toxin protein (GeneBank accession number 1IKQ_A) consisting of 613 amino acid residues, with a molecular weight of about 66kD. PE contains three structural domains DI, DII, and DIII. Among them, DI is the binding region, consisting of amino acid residues 1-252, which can guide PE to recognize and bind to receptors on the target cell membrane. DII is the translocation region, composed of amino acid residues 253-384, responsible for the transmembrane transport of PE, allowing it to enter cells; DIII is the active region, composed of amino acid residues 385-613, catalyzing the elongation of factor 2 ADP ribosylation leads to the inactivation of Ef2, which inhibits cellular protein synthesis and eventually leads to cell death.

In the present invention, the term "PE25" is a truncated PE, comprising a polypeptide having a sequence as shown in SEQ ID NO: 4, or a variant of SEQ ID NO: 4, which means comprising at least one mutation (such as a substitution , deletion or insertion mutation) of SEQ ID NO: 4, the PE25 retains the activity of PE to inhibit cell protein synthesis.

In the present invention, the term "cathepsin B" is a cysteine proteolytic enzyme present in lysosomes. In various malignant tumor tissues such as lung cancer, gastric cancer, prostate cancer, and breast cancer, the expression of cathepsin B Both are exponentially higher than even 3-9 times higher than adjacent normal tissues. Cathepsin B can selectively recognize and degrade some polypeptide fragments, such as Val-Cit, Phe-Lys, Gly-Phe-Leu-Gly and so on.

In the present invention, the terms "polypeptide" and "protein" are used interchangeably.

In the present invention, "-" represents a peptide bond.

Encoding Nucleic Acids and Expression Vectors

The present invention also provides nucleic acid molecules encoding the above-mentioned anti-HER2 immunotoxin molecules. A nucleic acid molecule of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. In the present invention, the term "expression vector" refers to a vector carrying an expression cassette for expressing a specific target protein or other substances, such as a plasmid, a viral vector (such as adenovirus, retrovirus), phage, yeast plasmid or other vectors. For example, conventional expression vectors in the field comprising appropriate regulatory sequences, such as promoters, terminators, enhancers, marker genes and/or sequences, etc., including but not limited to: viral vectors (such as adenovirus, retroviral recording virus), plasmid, phage, yeast plasmid or other vectors. The expression vector preferably includes pDR1, pcDNA3.4(+), pcDNA3.1/ZEO(+), pDHFR, pTT5, pET-28a, pET-21a, pCGS3. For more technical details see, eg, Sambrook et al., Molec μLar Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Many known techniques and protocols for nucleic acid manipulation are found in Current Protocols in Molecular Biology, 2nd Edition, edited by Ausubel et al. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.

In the present invention, the term "host cell" refers to various conventional host cells in the art, as long as the vector can stably replicate itself and the polynucleotide molecules carried can be effectively expressed. Wherein said host cells include prokaryotic expression cells and eukaryotic expression cells, said host cells preferably include: COS, CHO, NSO, sf9, sf21, DH5α, BL21 (DE3), TG1, BL21 (DE3), 293F or 293E cells.

Pharmaceutical compositions and applications

The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned anti-HER2 immunotoxin molecule, and a pharmaceutically acceptable carrier or auxiliary material. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 4-8, preferably about 5-7, although the pH value can vary Depending on the nature of the substance formulated and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavitary injection.

In the present invention, the term "pharmaceutical composition" means that the anti-HER2 immunotoxin molecules of the present invention can be combined with pharmaceutically acceptable carriers or excipients to form a pharmaceutical preparation composition so as to exert a more stable therapeutic effect. These preparations can guarantee the efficacy of the present invention. The disclosed conformational integrity of the amino acid core sequence of the immunotoxin molecule against HER2, while also protecting the protein's multifunctional groups from its degradation (including but not limited to aggregation, deamination or oxidation).

"Pharmaceutically acceptable"means that the drug does not produce adverse, allergic or other adverse reactions when properly administered to an animal or human.

The "pharmaceutically acceptable carrier or excipient" should be compatible with the active ingredient, that is, it can be blended with it without greatly reducing the effect of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, Ethyl cellulose and methyl cellulose; Gum tragacanth powder; Malt; Gelatin; Talc; Solid lubricants such as stearic acid and magnesium stearate; Calcium sulfate; Vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oils, corn oil, and cocoa butter; polyols, such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; Flavoring agent; tableting agent, stabilizer; diluent; excipient; antioxidant; preservative; pyrogen-free water; isotonic saline solution; buffer such as phosphate buffer, etc. These substances are used as needed to aid the stability of the formulation or to help enhance the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.

The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned anti-HER2 immunotoxin molecule of the present invention and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day. In addition, the anti-HER2 immunotoxin molecules of the invention can also be used with other therapeutic agents.

In the present invention, the term "effective amount" refers to the amount or dose of the anti-HER2 immunotoxin molecule of the present invention administered to a subject, which produces the expected effect in the treated individual, and the expected effect includes the improvement of the individual's disease. The term "subject" includes, but is not limited to, mammals such as humans, non-human primates, rats and mice, and the like.

treatment method

The present invention also provides a method for treating HER2-positive cancers, the method comprising administering the above-mentioned anti-HER2 immunotoxin molecule or pharmaceutical composition to a subject in need.

"Treatment" or "therapy" for a condition includes preventing or alleviating a condition, reducing the rate at which a condition occurs or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition , to reduce or terminate symptoms associated with a condition, to produce a complete or partial reversal of a condition, to cure a condition, or a combination of the above. With respect to cancer, "treating" or "therapy" can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumor or malignant cells, or some combination thereof. With respect to tumors, "treatment" or "therapy" includes eradicating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor progression, or some combination of the above.

Specifically, when administering to a subject, the dosage varies according to the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. The results of animal experiments and various situations can be referred to. The total dosage should not exceed a certain range.

In some embodiments, the methods described herein can further be administered in combination with other compounds or other cancer treatment regimens known in the art.

Other cancer treatment options may include, but are not limited to: surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, cancer vaccines (e.g., HPV vaccine, hepatitis B vaccine, Oncophage, Provenge) and gene therapy, and their any combination of . Immunotherapy, including but not limited to adoptive cell therapy, derivation of stem cells and/or dendritic cells, blood transfusion, lavage and/or other therapies including but not limited to freezing tumors.

Below in conjunction with specific embodiment, further state the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate detailed conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Example

The experimental materials and experimental reagents used in the following examples are all commercially available unless otherwise specified.

The detection method used in the following examples is described as follows:

1. SDS-PAGE detection:

Detection system: Mini protein TeTra system

Detection conditions: 140V constant voltage 45-55min

2. Ultraviolet detection:

Instrument model: Nanodrop one (Thermo)

Extinction coefficient: 1.42

3. Protein A affinity chromatography

Chromatographic column: XK16/20(GE)

Filler: Protein L (Gensray)

Chromatography system: AKTA Pure150(GE)

Operating system: unicorn 7.0 (GE)

Flow rate: 1.0mL/min

4. ELISA detection and processing

Microplate reader: SpecTraMax 190, wavelength 450nm

Processing software: GraphPad Prism 9

Example 1. Construction of an anti-HER2 immunotoxin expression vector

1.1. Anti-HER2 immunotoxin amino acid design and its gene and related element sequence design

Anti-HER2 immunotoxin molecule Tra-PE25, its structure is: Ds4d5Fv-Linker1(L1)-PE25, its amino acid sequence is shown in SEQ ID NO:1.

Among them, Ds4d5Fv is derived from the Fv of trastuzumab, which is constructed by connecting VL and VH through the linker Linker2 (L2), wherein VL contains the Q100C mutation, and VH contains the K44C mutation, named as Q100C VL 4(G4S)K44C VH (SEQ ID NO: 2). Numbered according to Kabat system rules.

The linker Linker1 (L1) is GGGSGGGGSGSSGFLGSSGSSGLGFGGSSGG (SEQ ID NO: 3); PE25 (SEQ ID NO: 4) is the DIII region (position 395-613) of Pseudomonas exotoxin PE, which has complete catalytic activity.

In order to increase the expression level, the nucleotide sequence of the amino acid sequence SEQ ID NO: 1 encoding Tra-PE25 was optimized with the codons of Escherichia coli to obtain SEQ ID NO: 5. In order to facilitate expression, the start codon ATG is added to the 5' of SEQ ID NO: 5, and the stop codon TAA is added to the 5' of SEQ ID NO: 5 to obtain the Tra-PE25 gene encoding the Anti-HER2 immunotoxin molecule and related elements Sequence (SEQ ID NO: 6).

Table B sequence list (Kabat system rule number and definition)

Note: lowercase letters: PE amino acid sequence; single underline mark: amino acid sequence of antibody CDR region; double underline mark: linker amino acid sequence; italics: antibody mutation site.

1.2. Preparation of anti-HER2 immunotoxin expression vector

The whole gene synthesis Anti-HER2 immunotoxin molecule Tra-PE25 gene and related element sequence (SEQ ID NO: 6) was inserted into the expression frame of pET-28a, and the Tra-PE25 PET28a plasmid and its plasmid bacteria were constructed. The plasmid TRA-PE25PET28a was extracted for use.

Example 2. Construction of an anti-HER2 immunotoxin expression strain

The plasmid Tra-PE25 PET28a constructed in Example 1.2 was transformed into BL21(DE3) competent cells, spread on 2YT agar plates containing kanamycin resistance, and then picked monoclonal IPTG to induce expression, and SDS PAGE detection and screening. The cell line with the highest expression level was selected for subsequent experiments.

Example 3. Expression and purification of anti-HER2 immunotoxin

3.1.IPTG induced expression of anti-HER2 immunotoxin

Inoculate the glycerol strain with a ratio of 1:1000 by volume to the culture medium containing Kanna resistance, and cultivate overnight at 37 degrees at 150 rpm; inoculate the seed solution with the ratio of 1:1000 by volume to the culture medium containing Kanna resistance, at 37 degrees , 150rpm to OD600 of 0.7; add IPTG to make the final concentration of 1mM, 25 degrees, 120rpm, continue to cultivate for 18 hours; centrifuge 8000rpm × 5min, remove the supernatant, and collect the bacteria.

Weigh the collected bacteria, resuspend the bacteria in PBS according to the weight ratio of 1:20, and stir thoroughly; use a crushing instrument: ultra-high pressure continuous flow cell disruptor, number: JN-MiniPro, fully crush; crushing conditions : 4 degrees, pressure value 1000Ba, crush twice; centrifuge the crushed bacterial liquid, 4 degrees, 10000rpm, 30min, collect the supernatant; filter the supernatant with a 0.22 micron filter membrane.

3.2.Protein L affinity chromatography separation and purification of anti-HER2 immunotoxin

AKTA Pure150 (GE) chromatography system was used for affinity chromatography, and the chromatographic column HiTraP Protein L 5ml pre-column (cytiva). The operation of the instrument was carried out according to the operating instructions, the A1 pump was Buffer A (PBS, pH7.4), and the B1 pump was Buffer B (50mM glycine buffer, pH2.5).

1) Balance: Wash Buffer A for 10CV (column volume) at a flow rate of 1mL/min to maintain the Protein L gel environment suitable for the combination of anti-HER2 immunotoxin and Protein L.

2) Sample loading: pass through the Protein L gel at a rate of 1 mL/min, so that the anti-HER2 immunotoxin can specifically bind to Protein L.

3) Flushing: Buffer A was flushed at a flow rate of 1mL/min until the absorbance at 280nm was lower than 0.01.

4) Elution: Rinse the Protein L gel with Buffer B at a flow rate of 1 mL/min, and collect the elution peaks.

5) Neutralization: The collected samples of the elution peak were adjusted to pH 7.4 with 50mM glycine buffer (pH 9.0). 5mg Tra-PE25 can be purified per liter of culture medium.

6) SDS-PAGE detection and analysis of the purified anti-HER2 immunotoxin Tra-PE25. SDS-PAGE is shown in Figure 1.

Example 4. Anti-HER2 immunotoxin ELISA detection

1. Coating antigen Her2-ECD-His (SEQ ID NO: 7), 100 μL per well, concentration 1 μg/mL, incubate overnight at 4°C.

2. Washing: Take out the Elisa plate coated with the antigen, wash the plate 3 times with the washing buffer PBST, pat dry the Elisa plate and wait for the next step of blocking.

3. Blocking: 200 μL of blocking solution (PBST+1% BSA) was added to each well, and blocked at room temperature for 2 hours.

4. Washing: wash the plate 3 times with washing buffer PBST, and pat dry for later use.

5. Add primary antibody Tra-PE25

1) Dilute the antibody: Dilute the primary antibody in a 96-well cell culture plate (the diluent is the blocking solution): the initial concentration is 300nM, and it is diluted 2.5 times from left to right, and the dilution is 11 gradients, and the 12th column is set to no A blank control with primary antibody was added. For each sample, 2 replicate wells were set for each gradient.

2) Add 100 μL of diluted primary antibody to each well, and block for 2 hours at room temperature.

6. Washing: wash the plate 3 times with washing buffer PBST, and pat dry for later use.

7. Add secondary antibody: Dilute Protein L HRP (SEQ ID NO: 18) with blocking solution (PBST+1% BSA) at a ratio of 1:1000, add 100 μL of diluted secondary antibody to each well, and incubate at room temperature for 1 hour in the dark .

8. Washing: wash the plate 3 times with washing buffer PBST, and pat dry for later use.

9. Color development: add 100 μL of freshly prepared color development solution (TMB) to each well, and incubate at room temperature in the dark for 5 minutes.

10. Termination: Add 70 μL of stop solution (2M H ₂ SO ₄ ) to each well, mix well, and immediately read on a microplate reader, and the detection wavelength is 450 nm. The processed data map is shown in Figure 2.

Figure 2 shows that the EC50 of Tra-PE25 is 4.220nM, indicating that Tra-PE25 has better binding activity to the antigen HER2.

Example 5. Detection of anti-HER2 immunotoxin cell killing activity

5.1 Detection of killing activity on human breast cancer cells (HER2 high expression)

5.1.1 Experimental materials

1) Cells: HCC19549 (human breast ductal carcinoma cells) were purchased from ATCC.

2) Complete medium: RPMI 1640+15%FBS+1%Pen-Strep.

3) 0.25% Trypsin-EDTA, DPBS, Trypan Blue.

4) CCK-8 reagent.

5.1.2 Experimental steps

1) Cell digestion: Cultivate HCC1954 cells until the density reaches 80%-90%, discard the supernatant, wash with DPBS, add 0.25% Trypsin-EDTA, digest at 37°C for 3 minutes, add complete medium to stop, pipette and mix well .

2) Density determination and adjustment: the cell suspension was mixed with 0.2% Trypan Blue 1:1, and after counting with a cell counter, the cell density was adjusted to 1.4×10 ⁴ cells/mL.

3) Cell plating: After adjusting the density, the cell suspension was plated to a 96-well plate (3599), 150 μL/well, that is, 2100 cells/well, and cultured overnight at 37° C., 5% CO ₂ to allow the cells to adhere to the wall.

4) Dilution and addition of drug: prepare drug in complete medium to a final concentration of 400 nM, and filter. After 4-fold serial dilution, add to a 96-well plate, 50 μL/well, the final concentration is 100 nM starting, and 4-fold serial dilution. Control T-DM1500nM start, 4 times serial dilution.

5) Drug incubation: 37°C, 5% CO ₂ for 4 days.

6) CCK8 color development: the supernatant was discarded, the 96-well plate was turned upside down on absorbent paper to dry, and 10% CCK-8 was added for color development, 100 μL/well. After incubation at 37°C for 1.5 h, the plate was read at 450 nm using a microplate reader.

7) Data analysis: the well without cells was used as a blank control, which was recorded as 0%, and the well with cells but no drug added was used as a negative control, which was recorded as 100%. Use GraphPad Prism 9 to plot and fit the curve to compare the differences in IC50 (nM) between different drugs.

The biological activity detection is shown in Figure 3. Figure 3 shows that the EC50 of Tra-PE25 is 0.120nM, and the EC50 of the control positive drug T-DM1 is 17.05nM. It can be seen that Tra-PE25 has strong biological activity and can kill Human breast ductal carcinoma cells HCC1954, and the killing activity on cancer cells was significantly higher than that of the positive control T-DM1.

5.2 Detection of BEAS-2B killing activity on normal human lung cells (HER2 not expressed)

5.2.1 Experimental materials

1) Cells: BEAS-2B (human normal lung epithelial cells).

2) Complete medium: RPMI 1640+10%FBS+1%Sodium Pyruvate+1%GlutaMax+1%Pen-Strep.

3) 0.05% Trypsin-EDTA, DPBS, Trypan Blue, human serum albumin (HSA).

4) CCK-8 reagent.

5.2.2 Experimental steps

1) Cell digestion: Culture BEAS-2B cells until the density reaches 80%-90%, discard the supernatant, wash with DPBS, add 0.05% Trypsin-EDTA, digest at 37°C for 2 minutes, add complete medium to stop, pipette Mix well.

2) Density determination and adjustment: the cell suspension was mixed with 0.2% Trypan Blue 1:1, and after counting with a cell counter, the cell density was adjusted to 1×10 ⁴ cells/mL.

3) Cell plating: After adjusting the density, plate the cell suspension into a 96-well plate (3599), 150 μL/well, that is, 1500 cells/well, and culture overnight at 37° C., 5% CO ₂ to allow the cells to adhere to the wall.

4) Drug preparation and filtration: the complete medium was used to prepare the drug to a final concentration of 400 nM, and then sterilized by filtration. Prepare HSA in the complete medium to a final concentration of 8 μM, and filter to sterilize.

5) Drug dilution and addition: The drug and HSA solution (or complete medium) were mixed according to the volume ratio of 4:1, and after 4-fold gradient dilution, they were added to a 96-well plate, 50 μL/well, and the final concentration was 80 nM of the drug starting, HAS400nM start, 4-fold serial dilution.

6) Drug incubation: culture at 37°C, 5% CO ₂ for 6 days.

7) CCK8 color development: the supernatant was discarded, the 96-well plate was turned upside down on absorbent paper to dry, and 10% CCK-8 was added for color development, 100 μL/well. After incubation at 37°C for 1.5 h, the plate was read at 450 nm using a microplate reader.

8) Data analysis: the well without cells was used as a blank control, which was recorded as 0%, and the well with cells but no drug added was used as a negative control, which was recorded as 100%. Use GraphPad Prism 9 to plot and fit the curve to compare the differences in IC50 (nM) between different drugs.

The biological activity test is shown in Figure 4, which shows that Tra-PE25 has almost no killing activity on normal cells.

Example 6. Anti-HER2 immunotoxin mass spectrometry detection

1) Mass spectrometry conditions:

Waters company UPLC-XEVO G2 Q-TOF liquid mass spectrometry system. The liquid phase part of the system is configured as: BSM binary high-pressure mixing pump, SM sample manager, TUV ultraviolet detector; the mass spectrometry part is configured as: ESI source, Q-TOF detector. Masslynx V4.1 and BiopharmaLynx analysis software (Version: 1.2) were used for data processing and analysis.

The MS data are collected in the Resolution mode in the continuum mode; the LockSpray acquisition mode is: real-time acquisition without calibration.

Calibration solution: real-time calibration (LockSpray) solution: 2ng/μL LE solution.

Calibration solution for mass axis: 2 μg/μL sodium iodide solution.

The mass spectrometry parameters are shown in Table 1.

2) Liquid phase conditions:

Chromatographic column: Mass PREPTM Micro Desalting Column 2.1×5mm (intact protein molecular weight analysis), column temperature: 80°C.

Mobile phase A: 0.1% FA- _H2O .

Mobile phase B: 0.1% FA-CAN.

Seal Wash solution: 10% IPA.

Mass spectrometry cleaning solution: 50% ACN.

Mass Spec IntelliStart Valve Cleaning Solution: 50% MeOH.

Injection volume: 10 μL.

Sample chamber temperature: 10°C.

See Table 2 for gradient elution conditions.

Table 1. Mass Spectrometry Parameters

Table 2. Gradient elution conditions

Table 3 Complete molecular weight of Tra-PE25 samples

sample name	Theoretical molecular weight (Da)	Measured Molecular Weight (Da)
Tra-PE25	52364.15	52363.34

The results of mass spectrometry are shown in Figure 5 and Table 3, indicating that the Tra-PE25 of the present invention is more uniform and consistent with the design.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

The above examples are intended to illustrate the disclosed embodiments of the present invention, and should not be construed as limiting the present invention. In addition, various modifications set forth herein, as well as changes in the method of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in connection with various specific preferred embodiments of the invention, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as mentioned above which are obvious to those skilled in the art to obtain the invention should be included in the scope of the present invention.

Claims (20)

1. An anti-HER2 immunotoxin molecule, characterized in that the fusion polypeptide comprising H-L1-PE or PE-L1-H structure from the amino terminal to the carboxyl terminal, wherein H is an anti-HER2 antibody or an antigen-binding fragment thereof; L1 is linker 1; PE contains part or all of domain III of Pseudomonas exotoxin A.
2. The anti-HER2 immunotoxin molecule according to claim 1, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises a single-chain antibody scFv.
3. The immunotoxin molecule against HER2 according to claim 2, wherein said single-chain antibody scFv comprises a fusion polypeptide of VL-L2-VH or VH-L2-VL structure from the amino terminal to the carboxyl terminal, wherein VL is The variable region of the light chain, VH is the variable region of the heavy chain, and L2 is the linker 2; preferably, the single-chain antibody scFv comprises a fusion polypeptide of VL-L2-VH structure from the amino terminal to the carboxyl terminal.
4. The anti-HER2 immunotoxin molecule according to claim 3, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 11, and the amino acid sequence of HCDR2 The sequence is shown in SEQ ID NO: 12, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 13; the VL includes light chain complementarity determining regions LCDR1, LCDR2, LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 8 The amino acid sequence of LCDR2 is shown in SEQ ID NO: 9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 10.
5. The anti-HER2 immunotoxin molecule according to claim 4, wherein the single-chain antibody scFv comprises an amino acid sequence such as SEQ ID NO: 16 VL or its variants, and an amino acid sequence such as SEQ ID NO: The VH or its variant described in 17, the variant comprises 1-5 amino acid mutations; preferably, the VL comprises the Q100C mutation, and the VH comprises the K44C mutation.
6. The anti-HER2 immunotoxin molecule according to claim 3, wherein said L2 comprises (G4S) _n , wherein n is any integer selected from 1-6; preferably, said L2 comprises (G4S) ₄ .
7. The anti-HER2 immunotoxin molecule according to claim 3, wherein said single-chain antibody scFv comprises an amino acid sequence as described in SEQ ID NO: 2, or comprises an amino acid sequence having at least 98 Amino acid sequences with % or more than 99% identity.
8. The anti-HER2 immunotoxin molecule of claim 1, wherein the PE is PE25, comprising the amino acid sequence as set forth in SEQ ID NO:4.
9. The anti-HER2 immunotoxin molecule according to claim 1, wherein said L1 comprises a cathepsin B cleavage site; preferably, said L1 comprises an amino acid sequence as set forth in SEQ ID NO:3.
10. The anti-HER2 immunotoxin molecule of claim 1, wherein the anti-HER2 immunotoxin molecule comprises the amino acid sequence as described in SEQ ID NO:1.
11. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the anti-HER2 immunotoxin molecule according to any one of claims 1-10.
12. The nucleic acid molecule of claim 11, wherein the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6.
13. An expression vector, characterized in that the expression vector comprises the nucleic acid molecule according to claim 11 or 12.
14. A host cell, characterized in that the host cell comprises the expression vector according to claim 13.
15. A preparation method of the anti-HER2 immunotoxin molecule according to any one of claims 1-10, characterized in that the preparation method comprises the following steps: a) under expression conditions, culturing the host cells, thereby expressing the immunotoxin molecule against HER2; b) isolating and purifying the immunotoxin molecule against HER2 described in step a).
16. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises an effective amount of the anti-HER2 immunotoxin molecule according to any one of claims 1-10 and one or more pharmaceutically acceptable carriers or adjuvants .
17. The application of the anti-HER2 immunotoxin molecule according to any one of claims 1-10 and the pharmaceutical composition according to claim 16 in the preparation of medicines for treating HER2-positive cancers.
18. The use according to claim 17, wherein the cancer is selected from breast cancer, gastric cancer, ovarian cancer, bladder cancer and non-small cell lung cancer.
19. A method for treating cancers with positive expression of HER2, characterized in that the method comprises administering the anti-HER2 immunotoxin molecule according to any one of claims 1-10 or the immunotoxin molecule according to claim 16 to a subject in need pharmaceutical composition.
20. The method according to claim 19, wherein the cancer is selected from any one or more of breast cancer, gastric cancer, ovarian cancer, bladder cancer or non-small cell lung cancer.

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