TW202325737A - Anti-her2 immunotoxin molecule, preparation method and application thereof - Google Patents

Abstract

An anti-HER2 immunotoxin molecule, preparation method and application thereof, the anti-HER2 immunotoxin molecule comprises a fusion polypeptide of H-L1-PE or PE-L1-H structure from amino terminus to carboxyl terminus, wherein H is an anti-HER2 antibody or an antigen-binding fragment thereof; L1 is linker 1; PE comprises part or all of domain III of Pseudomonas exotoxin A. The immunotoxin molecule of the invention can target HER2-positive cancer cells with high specificity, so as to efficiently kill cancer cells and inhibit tumor growth.

Description

Anti-HER2 immunotoxin molecule and its preparation method and application

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.

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, which is found in various tumors such as breast cancer, ovarian cancer, gastric cancer, bladder cancer, and non-small cell lung cancer. Express. 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, exceeding the 2.2 million cases of lung cancer and becoming the largest cancer in the world. Among breast cancer patients, the proportion of HER2-positive patients reaches 20% to 25%, and their tumor biological characteristics are highly malignant, with higher metastasis and mortality. He Aiping (Trastuzumab), Pertuzumab (Pertuzumab), and fixed-dose combination preparations of the two drugs have improved clinical treatment benefits and survival in HER2-positive breast cancer.

Antibody-drug complexes (ADCs) combine monoclonal antibodies with specific antigens on the surface of tumor cells, and deliver cytotoxic drugs to tumor lesions in a targeted manner. With the release of small-molecule toxins, they can bind to DNA minor grooves or tubulin, etc. Various mechanisms induce the apoptosis of cancer cells and exert the cytotoxicity of drugs, thus providing new treatment options for HER2-positive breast cancer patients. 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 (ado-trastuzumab emtansine, T-DM1) for the treatment of HER2-positive breast cancer. This drug has become the second-line standard treatment for HER2-positive breast cancer internationally. According to the plan, global sales in 2019 will reach 1.393 billion Swiss francs (sales in the first three quarters of 2020 will be 1.295 billion Swiss francs, 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 He Aiping treatment), further expanding the market space. It was followed by Enhertu (DS-8201a), which was 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 are originally composed of antibodies and plant or bacterial toxins through chemical coupling. It should be noted that this immunoconjugate is 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.

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; to provide an expression vector comprising the nucleic acid molecule; to provide a host cell comprising the expression vector; to 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, comprising 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 variable region of the light chain, and VH is the variable region of 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 : 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 with an amino acid sequence as shown in SEQ ID NO: 16 or a variant thereof, and a VH with an amino acid sequence as shown in SEQ ID NO: 17 or a variant thereof, 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, the scFv of the single-chain antibody comprises an amino acid sequence as described in SEQ ID NO: 2, or comprises an amino acid sequence having at least 98% or more identity to 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 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 comprising 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-mentioned expression vector.

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

The 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; b) isolating and purifying the anti-HER2 immunotoxin molecule 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-positive expressed cancer.

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 HER2-positive cancer cells.

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 good 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 that of T-DM1 (ado-trastuzumab emtansine), which has obtained unexpected technical effects .

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

4) The Linker1 that connects 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.

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. Except the specific method, equipment, material used in the embodiment, according to those skilled in the art to the grasp of prior art and the record of the present invention, can also use the method, equipment, material similar to described in the embodiment of the present invention or any equivalent prior art methods, apparatus and materials to carry out 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 traditional 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 Methods or methods for introducing plastids into 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 molecule

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 fusion of 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 characteristics, including variable regions and constant regions, which consist of two identical heavy chains (HC) and two 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 framework region (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 brought into close proximity 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 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, antibodies are produced in less than approximately M, such as less than about M. M. M. An equilibrium dissociation constant (KD) of M or less binds the antigen. 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 an antibody to an antigen in a BIACORE instrument or ELISA is used to determine the relative affinity of an antibody to an antigen.

In the present invention, the term "linker" is used to connect two polypeptide chains. A suitable linker can be a flexible polypeptide sequence, examples of which include monoglycine (Gly) or serine (Ser) residues, and the identification and sequence of amino acid residues in the linker can be realized as needed in the linker varies with the type of secondary structural elements.

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, so as to further improve or optimize 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 composed of 613 amino acid residues (GeneBank accession number 1IKQ_A), 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, consisting of amino acid residues 253-384, responsible for the transmembrane transport of PE, allowing it to enter cells; DIII is the active region, consisting of amino acid residues 385-613, which catalyzes 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, said 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 is stable times higher than even 3-9 times higher than adjacent normal tissue. 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.

Coding 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 protein or other substance, such as a plastid, a viral vector (such as an adenovirus, a retrovirus), a phage, a yeast plastid 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., said expression vectors include but are not limited to: viral vectors (such as adenovirus, retroviral transcription virus), plastids, phages, yeast plastids 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, Second 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 the host cells include prokaryotic expression cells and eukaryotic expression cells, the 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 be formulated according to the Depending on the nature of the substance and the condition being 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, cellulose and methylcellulose; 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 molecules of the present invention and 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 cancer, 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 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 the development of a tumor, 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 45-55 min

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 (GentScript)

Chromatography system: AKTA Pure150 (GE)

OS: unicorn 7.0 (GE)

Flow rate: 1.0 mL/min

4. ELISA detection and processing

Microplate reader: SpecTraMax 190, wavelength 450 nm

Processing software: GraphPad Prism 9

Example 1. anti- HER2 Construction of immunotoxin expression vector

1.1. Anti-HER2 Design of Immunotoxin Amino Acids and Their Genes and Related Element Sequences

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

Among them, Ds4d5Fv is derived from He Aiping's Fv, 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 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) serial number sequence SEQ ID NO: 1 Tra-PE25（Ds4d5Fv-Linker-PE25）氨基酸序列DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FG C GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFNIK DTYIH WVRQAPGK C LEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDY WGQGTLVTVSS GGGSGGGGSGSSGFLGSSGSSGLGFGGSSGG ptgaeflgdggdvsfstrgtqnwtverllqahrqleergyvfvgyhgtfleaaqsivfggvrarsqdldaiwrgfyiagdpalaygyaqdqepdargrirngallrvyvprsslpgfyrtsltlaapeaageverlighplplrldaitgpeeeggrletilgwplaertvvipsaiptdprnvggdldpssipdkeqaisalpdyasqpgkppredlk SEQ ID NO: 2 Tra-PE25 Heaiping Fv (Ds4d5Fv) amino acid sequence DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FG C GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQ PGGSLRLSCAASGFNIK DTYIH WVRQAPGK C LEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDY WGQGTLVTVSS SEQ ID NO: 3 Tra-PE25 linker (Linker1) amino acid sequence GGGSGGGGSGSSGFLGSSGSSGLGFGGSSGG SEQ ID NO: 4 Tra-PE25 Pseudomonas exotoxin exotoxin PE25 amino acid sequence letilgwplaertvvipsaiptdprnvggdldpssipdkeqaisalpdyasqpgkppredlk SEQ ID NO: 5 Tra-PE25 nucleotide sequence (E. coli codon optimized) GATATACAAATGACACAGTCTCCCTCAAGTCTGAGCGCCTCCGTGGGTGATCGTGTGACCATTACCTGTCGTGCTTCCCAAGATGTTAATACCGCTGTGGCATGGTATCAGCAGAAACCGGGTAAGGCTCCGAAACTGCTGATCTACTCCGCGAGCTTTCTGTATAGCGGTGTTCCGAGCCGTTTCAGCG GCTCTCGCTCTGGCACTGACTTTTACCCTGACCATCAGCTCCTTACAACCGGAGGATTTTGCAACCTATTACTGCCAGCAACATTATACCACCCCCGCCAACCTTCGGGTGCGGTACGAAAGTCGAGATCAAGGGTGGCGGCGGCTCGGGCGGCGGTGGTTCCGGTGGCGGCGGTAGTGGCGGTGGTGGCTCTGAAGTGCAGCTGGTCGAGA GCGGCGGCGGTCTGGTGCAGCCGGGCGGCTCCCTGCGCCTGTCTTGTGCAGCTAGTGGTTTAACATTAAGGATACGTACATCCACTGGGTTCGTCAAGCTCCGGGTAAGTGCCTGGAATGGGTCGCACGTATTTACCCGACGAACGGTTACACCCGTTATGCGGATAGCGTTAAAGGTCGCTTTACCATCTCTGCGGATACCAGCAAAAACACCGCGC ATACCTGCAAATGAACAGCTTGCGCGCAGAGGACACCGCGGTTTACTACTGCAGCCGTTGGGGCGGTGACGGCTTCTACGCCATGGATTATTGGGGTCAGGGTACTCTGGTAACTGTGAGCTCAGGTGGTGGTAGCGGCGGTGGCGCTCCGGCTCTAGCGGTTTCCTGGGTTCTAGCGGCAGTTCCGGCCTGGGTTTCGGCGGG TCGTCGGGCGGACCGACTGGTGCAGAATTTTTGGGTGACGGCGGTGACGTTAGCTTTTCTACACGTGGTACGCAGAATTGGACCGTCGAGCGCCTTTTGCAGGCGCATCGTCAGCTGGAGGAGCGCGGTTACGTGTTCGTGGGCTACCACGGCACCTTCCTAGAGGCCGCGCAAAGCATTGTTTTCGGCGGGGTGCGTGCGCGTAGCCAAGATCT GGACGCGATTTGGCGTGGCTTCTATATTGCTGGAGACCCGGCATTGGCCTATGGTTATGCGCAGGATCAGGAGCCGGATGCGCGTGGACGTATTCGTAATGGCGCGCTGTTGCGCGTGTATGTTCCGAGAAGCTCCCTGCCGGGTTTCTACCGCACCAGTCTGACCCTGGCCGCGCCAGAAGCGGCGGGTGAAGTAGAGCGTTTAATTGG TCACCCGTTGCCTCTCCGTTTGGACGCCATCACCGGTCCGGAAGAGGAAGGTGGTCGCTTGGAAACCATCCTGGGCTGGCCGTTAGCAGAGCGCACGGTTGTTATTCCCGAGCGCGATTCCGACGGACCCACGCAACGTTGGTGGAGATCTGGACCCGAGCTCGATCCCGGATAAGGAACAGGCGATCAGCGCGCTGCCGGACTACGCGAGCCA ACCGGGTAAGCCGCCTCGTGAAGACCTGAAA SEQ ID NO: 6 Nucleotide sequence encoding Anti-HER2 immunotoxin molecule Tra-PE25 gene and related elements SEQ ID NO: 7 Coating antigen Her2-ECD-His amino acid sequence SEQ ID NO: 8 Amino acid sequence of Tra-PE25 He Aiping Fv (Ds4d5Fv) LCDR1 region RASQDVNTAVA SEQ ID NO: 9 Amino acid sequence of Tra-PE25 He Aiping Fv (Ds4d5Fv) LCDR2 region SASFLYS SEQ ID NO: 10 Amino acid sequence of Tra-PE25 He Aiping Fv (Ds4d5Fv) LCDR3 region QQHYTTPPT SEQ ID NO: 11 Amino acid sequence of Tra-PE25 He Aiping Fv (Ds4d5Fv) HCDR1 region DTYIH SEQ ID NO: 12 Amino acid sequence of Tra-PE25 He Aiping Fv (Ds4d5Fv) HCDR2 region RIYPTNGYTRYADSVKG SEQ ID NO: 13 Amino acid sequence of Tra-PE25 He Aiping Fv (Ds4d5Fv) HCDR3 region WGGDGFYAMDY SEQ ID NO: 14 Amino acid sequence of Tra-PE25 He Aiping Fv (Ds4d5Fv) VL region DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKP GKAPKLLIY SASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FG C GTKVEIK SEQ ID NO: 15 Tra-PE25 He Aiping Fv (Ds4d5Fv) VH region amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFNIK DTYIH WVRQAPGK C LEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDY WGQGTLVTVSS SEQ ID NO: 16 He Aiping VL region amino acid sequence DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKP GKAPKLLIY SASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FG Q GTKVEIK SEQ ID NO: 17 He Aiping VH region amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFNIK DTYIH WVRQAPGK K LEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDY WGQGTLVTVSS SEQ ID NO: 18 Protein L HRP amino acid sequenceGSSHHHHHHSSGKEETPETPETDSEEEVTIKANLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDNGEYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGKEKTPEEPKEE VTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEAEATAEAYRYADLLAKENGKYTADLEDGGYTINIRFAGKKVDEKPEEKEQVTIKENIYFEDGTVQTATFKGTF AEATAEAYRYADLL SKEHGKYTADLEDGGYTINIRFAGGGGGSGGGGSGGGSQLTPTFYDNSCPNVSNVRDTIVNELRSDPRIAASILRLHFHDCFVNGCDASILLDNTTSFRTEKDAFGNANSARGFPVIDRMKAAVESACPRTVSCADLLTIAAQSVTLAGGPSWRVPLGRRDSLQAFLDLANANLPAPFFTLPQLKDSFRNVGLNRSSDLVAL SGGHTFGKNQCRFIMDRLYNFSNTGLPDPTLNTTYLQTLRGLCPLNGNLSALVDFDLRTPTIFDNKYYVNLEEQKGLIQSDQELFSSPNATDTIPLVRSFANSTQTFFNAFVEAMDRMGNITPLTGTQGQIRLNCRVVNSNS SEQ ID NO: 19 Linker Linker2 amino acid sequence GGGGSGGGGSGGGGSGGGGS

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. anti- HER2 Preparation of Immunotoxin Expression Vectors

Whole-gene synthesis Anti-HER2 immunotoxin molecule Tra-PE25 gene and related element sequence (SEQ ID NO: 6) were inserted into the expression cassette of pET-28a to construct Tra-PE25 PET28a plastid and its plastid bacteria. The plastid TRA-PE25 PET28a was extracted for use.

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

The plasmid Tra-PE25 PET28a constructed in Example 1.2 was transformed into BL21 (DE3)"competent cells (competent cells), coated with 2YT agar plates containing kamycin resistance, and then picked monoclonal IPTG induced expression, SDS PAGE detection and screening .Choose the cell line with the highest expression level for follow-up experiments.

Example 3. anti- HER2 Expression and purification of immunotoxin

3.1. IPTG induced expression of anti HER2 immunotoxin

Glycerol strains were inoculated with anti-kanamycin-containing medium at a volume ratio of 1:1000, and cultured overnight at 37 degrees at 150 rpm; the seed liquid was inoculated with kanamycin-containing medium at a volume ratio of 1:1000. culture medium, 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 column HiTraP Protein L 5ml pre-column (cytiva). The instrument was operated according to the operating instructions, the A1 pump was Buffer A (PBS, pH7.4), and the B1 pump was Buffer B (50 mM glycine buffer, pH2.5).

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

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.

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

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

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

The purified anti-HER2 immunotoxin Tra-PE25 was detected and analyzed by SDS-PAGE. 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, incubated 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: add 200 μL of blocking solution (PBST+1%BSA) to each well, and block 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

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

Add 100 μL of diluted primary antibody to each well and block for 2 h 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 keep it in the dark at room temperature Culture for 1h.

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 for 5 minutes in the dark.

10. Termination: Add 70 μL of stop solution (2M H2SO4) to each well, mix well, and immediately read on a microplate reader, and the detection wavelength is 450nm. The processed data map is shown in Figure 2.

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

Example 5. anti- HER2 Immunotoxin Cell Killing Activity Assay

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

5.1.1 Experimental Materials

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

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

0.25% Trypsin-EDTA, DPBS, Trypan Blue.

CCK-8 reagent.

5.1.2 Experimental procedure

Cell digestion: HCC1954 cells were cultured until the density reached 80%-90%, discarded the supernatant, washed with DPBS, added 0.25% Trypsin-EDTA, digested at 37°C for 3 minutes, then added complete medium to terminate, and pipet and mix well.

Density determination and adjustment: Mix the cell suspension with 0.2% Trypan Blue 1:1, count with a cell counter, and adjust the cell density to 1.4×104 cells/mL.

Cell plating: After adjusting the density, plate the cell suspension into a 96-well plate (3599), 150 μL/well, that is, 2100 cells/well, and culture overnight at 37°C with 5% CO2 to allow the cells to adhere to the wall.

Drug dilution and addition: Prepare the drug in the complete medium to a final concentration of 400 nM, and filter. After 4-fold serial dilution, add to 96-well plate, 50 μL/well, the final concentration is 100 nM starting, 4-fold serial dilution. Control T-DM1 500nM starting, 4 times serial dilution.

Drug culture: 37°C, 5% CO2 culture for 4 days.

CCK8 color development: Discard the supernatant, turn the 96-well plate upside down on absorbent paper to dry, add 10% CCK-8 for color development, 100 μL/well. After incubating at 37° C. for 1.5 h, the plate was read at 450 nm using a microplate reader.

Data analysis: the well without cells was used as the blank control, which was recorded as 0%, and the well with cells but no drug added was used as the 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 0.120 nM, and the EC50 of the control positive drug T-DM1 is 17.05 nM. 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 normal human lung cells BEAS-2B Killing activity assay ( HER2 not expressed)

5.2.1 Experimental Materials

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

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

0.05% Trypsin-EDTA, DPBS, Trypan Blue, Human Serum Albumin (HSA).

CCK-8 reagent.

5.2.2 Experimental procedure

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 and mix well .

Density determination and adjustment: mix the cell suspension with 0.2% Trypan Blue 1:1, count with a cell counter, and adjust the cell density to 1×104 cells/mL.

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 with 5% CO2 to allow the cells to adhere to the wall.

Drug preparation and filtration: Prepare the drug in the complete medium to a final concentration of 400 nM, and filter to sterilize. Prepare HSA in the complete medium to a final concentration of 8 μM, and filter to sterilize.

Drug dilution and addition: The drug and HSA solution (or complete medium) were mixed at a volume ratio of 4:1, diluted sequentially by 4 times, added to a 96-well plate, 50 μL/well, and the final concentration was 80 nM of the drug starting, HAS 400nM starting, 4-fold serial dilution.

Drug culture: 37°C, 5% CO2 for 6 days.

CCK8 color development: Discard the supernatant, turn the 96-well plate upside down on absorbent paper to dry, add 10% CCK-8 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.

Data analysis: the well without cells was used as the blank control, which was recorded as 0%, and the well with cells but no drug added was used as the 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 Mass Spectrometry Detection of Immunotoxins

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. Data processing and analysis using Masslynx V4.1 and BiopharmaLynx analysis software (Version: 1.2).

The MS data are collected in the Resolution mode in the continuum mode; the LockSpray acquisition mode is: real-time acquisition and no calibration is applied.

Calibration solution: real-time calibration (LockSpray) solution: 2 ng/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 5 mm (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.

MS 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 Intact protein molecular weight analysis Capillary voltage (V) 3000 Collection time of online desalting column (min) 0.4-4 Primary cone voltage (V) 35 SEC column acquisition time (min) 0-8 Secondary cone voltage (V) 4/0.1 (ADC samples) Mass calibration range (m/z) 100-3500 or more Desolvation temperature (°C) 350 Acquisition mass range (m/z) 500-3500 or above Desolvation gas flow rate (L/h) 500 Sampling time (sec) 0.500 Cone gas flow rate (L/h) 50 LockSpray scan time (sec) 0.500 ESI source temperature ( ) 130 LockSpray scan interval (sec) 30

Table 2. Gradient elution conditions Intact protein molecular weight analysis Online desalting column time (min) Flow rate (mL/min) %A %B curve initial 0.5 95 5 initial 0.5 0.5 95 5 6 0.51 0.2 95 5 6 2 0.2 10 90 6 2.1 0.5 95 5 6 2.7 0.5 10 90 6 2.8 0.5 95 5 6 3.4 0.5 10 90 6 3.5 0.5 95 5 6 4.0 0.5 95 5 6

Table 3 The 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.

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.

Claims (18)

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. 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. The immunotoxin molecule against HER2 according to claim 2, wherein the 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. 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 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. The anti-HER2 immunotoxin molecule according to claim 4, wherein the single-chain antibody scFv comprises an amino acid sequence such as VL shown in SEQ ID NO: 16 or a variant thereof, 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. The immunotoxin molecule against HER2 as claimed in claim 3, wherein said L2 comprises , wherein n is selected from any integer of 1-6; preferably, said L2 comprises . The anti-HER2 immunotoxin molecule according to claim 3, wherein the 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. The anti-HER2 immunotoxin molecule according to claim 1, characterized in that the PE is PE25, comprising the amino acid sequence as described in SEQ ID NO:4. 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 described in SEQ ID NO:3. The anti-HER2 immunotoxin molecule according to claim 1, characterized in that the anti-HER2 immunotoxin molecule comprises the amino acid sequence as described in SEQ ID NO:1. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the anti-HER2 immunotoxin molecule described in any one of claims 1-10. The nucleic acid molecule according to claim 11, wherein the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6. An expression vector, characterized in that the expression vector comprises the nucleic acid molecule as claimed in claim 11 or 12. A host cell, characterized in that the host cell comprises the expression vector as claimed in item 13. A method for preparing an anti-HER2 immunotoxin molecule according to any one of claim items 1-10, characterized in that the preparation method comprises the following steps: a) under expression conditions, cultivating the host according to claim item 14 cells, thereby expressing the immunotoxin molecule against HER2; b) isolating and purifying the immunotoxin molecule against HER2 described in step a). A pharmaceutical composition, characterized in that the pharmaceutical composition comprises an effective amount of the anti-HER2 immunotoxin molecule as described in any one of Claims 1-10 and one or more pharmaceutically acceptable carriers or adjuvants. Application of the anti-HER2 immunotoxin molecule as described in any one of claim items 1-10 and the pharmaceutical composition described in claim item 16 in the preparation of drugs for treating HER2-positive cancers. The application according to claim 17, wherein the cancer is selected from breast cancer, gastric cancer, ovarian cancer, bladder cancer and non-small cell lung cancer.