Immunotoxin molecule of anti-HER2 single-domain antibody as well as preparation method and application of immunotoxin molecule

Abstract

The invention provides an immunotoxin molecule of an anti-HER2 single-domain antibody as well as a preparation method and application of the immunotoxin molecule. The invention provides an anti-HER2 immunotoxin molecule. The anti-HER2 immunotoxin molecule comprises three parts, namely an anti-HER2 single-domain antibody, PE25 and a connexon. The immunotoxin molecule of the anti-HER2 single-domain antibody disclosed by the invention can target a specific antigen HER2 on the surface of a tumor cell with high specificity, and can efficiently kill cancer cells and inhibit tumor growth.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology, and specifically, the present invention relates to an immunotoxin molecule of an anti-HER2 single domain antibody, and a preparation method and application thereof. Background Art

[0002] Human epidermal growth factor receptor 2 (HER2) is one of the four members of the epidermal growth factor receptor family and is expressed in many tumors such as breast cancer, ovarian cancer, gastric cancer, bladder cancer, and non-small cell lung cancer.

[0003] According to the latest global cancer burden data for 2020 recently released by the World Health Organization's International Agency for Research on Cancer (IARC), there are 2.26 million new cases of breast cancer worldwide, exceeding the 2.2 million cases of lung cancer, becoming the world's largest cancer. 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 rates. Trastuzumab, pertuzumab, and fixed-dose combination preparations of the two drugs have significantly improved the clinical treatment benefits and survival of HER2-positive breast cancer, but the second-line and subsequent treatment of such patients still faces the dilemma of insufficient effective treatment methods.

[0004] Antibody-drug conjugates (ADCs) deliver cytotoxic drugs to tumor lesions by binding monoclonal antibodies to specific antigens on the surface of tumor cells. As small molecule toxins are released, they can induce apoptosis of cancer cells through various mechanisms such as binding to the minor groove of DNA or tubulin, exerting the cytotoxicity of drugs, thus providing new treatment options 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 targeted tumor therapy) in the next decade. According to forecasts by 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.

[0005] Currently, the most successful ADC drug commercialized globally is Kadcyla (enmetazudzumab, T-DM1) for the treatment of HER2-positive breast cancer. The drug has become the international standard second-line treatment for HER2-positive breast cancer, with global sales reaching 1.393 billion Swiss francs in 2019. In May 2019, Kadcyla was also approved by the FDA as an adjuvant treatment for patients with HER2-positive early breast cancer (who still have residual invasive disease after receiving taxanes and trastuzumab treatment), further expanding the market space. Following closely behind is Enhertu (DS8201a), 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 and has excellent efficacy.

[0006] In the past few years, immunoconjugates have been developed as an alternative treatment for malignant tumors. Immunoconjugates were originally composed of antibodies and plant or bacterial toxins chemically coupled, which are in a form called immunotoxins. The antibody binds to the antigen expressed on the target cell, and the toxin is internalized, thereby causing cell death by preventing protein synthesis and inducing apoptosis (Brinkmann, U., Mol. Med. Today, 2: 439-446 (1996)). Recently, the genes encoding the antibody and toxin have been fused and the immunotoxin is expressed as a fusion protein.

[0007] Much research has been conducted on immunotoxins that utilize a bacterial toxin called Pseudomonas exotoxin A ("PE") as the toxic moiety. Typically, PE is truncated or mutated to reduce its nonspecific toxicity without destroying its toxicity to cells targeted by the targeting moiety of the immunotoxin. Currently, clinical trials are underway to test the use of PE-based immunotoxins as treatments for a variety of cancers.

[0008] Current PE-based immunotoxins are highly immunogenic. This has been shown to be not a problem in the treatment of hematological malignancies, where the immune system's ability to respond is often impaired. Immunotoxins can usually be given multiple times to patients with hematological malignancies. However, patients with solid tumors usually produce neutralizing antibodies to PE-based immunotoxins within a few weeks after the first dose. Because many regimens require a 3-week period between immunotoxin administrations, the production of antibodies during this period of time actually means that for solid tumors, there is usually only a 3-week window before the patient's antibodies render the PE-based immunotoxin ineffective. Despite this, a single dose of a PE-based immunotoxin can also be very helpful in reducing a patient's tumor burden, eliminating smaller metastases, and relieving symptoms. However, it is still desirable to have a PE-based immunotoxin in a less antigenic form that can reduce a patient's immunogenic response.

[0009] Currently, the second-line and later-stage treatments for HER2-positive breast cancer still face the dilemma of insufficient effective treatments, and new therapies are needed. Therefore, there is still a need to develop new immunotoxin molecules of anti-HER2 single-domain antibodies, and their preparation methods and applications. Summary of the invention

[0010] The purpose of the present invention is to provide an immunotoxin molecule of an anti-HER2 single domain antibody, and a preparation method and application thereof.

[0011] In the first aspect of the present invention, an anti-HER2 immunotoxin molecule is provided, which comprises a fusion polypeptide shown in the following molecular formula from N-terminus to C-terminus:

[0012] Ab-L-PE;

[0013] Wherein, the Ab is an anti-HER2 antibody or an antigen-binding fragment thereof;

[0014] L is a linker;

[0015] PE comprises the amino acid sequence shown in SEQ ID NO: 4 or a variant of SEQ ID NO: 4;

[0016] Wherein, the anti-HER2 antibody or antigen-binding fragment thereof comprises an anti-HER2 single-domain antibody, and the single-domain antibody comprises the following three complementary determining regions CDR:

[0017] CDR1 shown in SEQ ID NO.10,

[0018] CDR2 shown in SEQ ID NO.11, and

[0019] CDR3 shown in SEQ ID NO.12;

[0020] In another preferred example, the anti-HER2 single-domain antibody comprises the amino acid sequence shown in SEQ ID NO.2; or comprises an amino acid sequence that is at least 98% or 99% identical to SEQ ID NO.2.

[0021] In another preferred embodiment, the L comprises a cleavage site as shown in SEQ ID NO: 13.

[0022] In another preferred embodiment, the L comprises (G4S)m or GS(G4S)m, where m is a positive integer selected from 1 to 5.

[0023] In another preferred embodiment, the sequence of L is shown as SEQ ID NO.3.

[0024] In another preferred embodiment, the immunotoxin molecule further comprises

[0025] (i) optional functional domains that enhance the physicochemical properties or druggability of the protein; and / or

[0026] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0027] In another preferred embodiment, the improving the physicochemical properties or drugability of the protein includes prolonging the half-life of the anti-HER2 single domain antibody.

[0028] In another preferred embodiment, the tag sequence is selected from the following group: 6His tag, GGGS sequence, FLAG tag and / or SUMO tag.

[0029] In another preferred embodiment, the tag sequence is shown as SEQ ID NO.5.

[0030] In another preferred embodiment, the anti-HER2 immunotoxin molecule comprises an amino acid sequence as described in SEQ ID NO.1 or 6; or comprises an amino acid sequence that is at least 98% or 99% identical to SEQ ID NO.1 or 6.

[0031] In the second aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the anti-HER2 immunotoxin molecule described in the first aspect of the present invention.

[0032] In another preferred example, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:7.

[0033] In the third aspect of the present invention, an expression vector is provided, wherein the expression vector comprises the nucleic acid molecule as described in the second aspect of the present invention.

[0034] In the fourth aspect of the present invention, a host cell is provided, wherein the host cell comprises the expression vector as described in the third aspect of the present invention.

[0035] In a fifth aspect of the present invention, a method for preparing an anti-HER2 immunotoxin molecule as described in the first aspect of the present invention is provided, the preparation method comprising the following steps:

[0036] a) culturing the host cell according to the fourth aspect of the present invention under expression conditions, thereby expressing an anti-HER2 immunotoxin molecule;

[0037] b) separating and purifying the anti-HER2 immunotoxin molecules described in step a).

[0038] In another preferred embodiment, during the purification process, the ULP1His enzyme as shown in SEQ ID NO. 9 is used to enzymatically digest the immunotoxin molecule with the SUMO tag, thereby removing the SUMO tag.

[0039] In the sixth aspect of the present invention, a pharmaceutical composition is provided, comprising an effective amount of the anti-HER2 immunotoxin molecule as described in the first aspect of the present invention and one or more pharmaceutically acceptable carriers.

[0040] In the seventh aspect of the present invention, provided is the use of the anti-HER2 immunotoxin molecule as described in the first aspect of the present invention and the pharmaceutical composition as described in the sixth aspect in the preparation of a drug for treating HER2-positive cancer.

[0041] In the eighth aspect of the present invention, the use according to the seventh aspect of the present invention is provided, wherein the cancer is selected from breast cancer, gastric cancer, ovarian cancer, bladder cancer and non-small cell lung cancer.

[0042] In the ninth aspect of the present invention, a method for treating HER2-positive cancer is provided, the method comprising administering to a subject in need thereof the anti-HER2 immunotoxin molecule as described in the first aspect of the present invention or the pharmaceutical composition as described in the sixth aspect of the present invention.

[0043] In another preferred embodiment, the cancer is selected from any one or more of breast cancer, gastric cancer, ovarian cancer, bladder cancer or non-small cell lung cancer.

[0044] 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 below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Preparation flow chart.

[0046] Figure 2 Expression, purification and enzyme digestion SDS-PAGE of 5F7PE010 fusion protein, including expression, purification and enzyme digestion SDS-PAGE of 5F7PE010 fusion protein; M: protein marker; A: His-ULP1 enzyme; B: E.Coli expressing fusion protein Hissumo5F7PE010, Ni column purification; C: enzyme digestion solution; D: enzyme digestion solution Ni column flow-through, 5F7PE010.

[0047] Figure 3 5F7PE010 mass spectrometry detection.

[0048] Figure 4 ELISA test results of 5F7PE010 binding to antigen.

[0049] Figure 5 In vitro killing curve of 5F7PE010 on human breast ductal carcinoma cell HCC1954 cells.

[0050] Figure 6 Detection of the cytotoxic activity of 5F7PE010 against human breast cancer cells JIMT1.

[0051] Figure 7 Detection of the killing activity of 5F7PE010 on human gastric cancer cell NCI-N87 cells.

[0052] Figure 8 In vivo antitumor activity of 5F7PE010 in the human gastric cancer NCI-N87 xenograft mouse model.

[0053] Fig. 9 Low-dose efficacy testing of 5F7PE010 in the human gastric cancer NCI-N87 xenograft mouse model. DETAILED DESCRIPTION

[0054] After extensive and in-depth research, the inventors have constructed an anti-HER2 single-domain antibody immunotoxin molecule, which includes three parts: an anti-HER2 single-domain antibody, a mutant of the DIII domain of Pseudomonas exotoxin A (PE25), and a linker. Experimental results show that the anti-HER2 single-domain antibody 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. Subsequently, the cytotoxic drugs will be internalized, processed and release toxin PE25 by the cells, inhibiting cell protein translation and leading to cell apoptosis. The present invention was completed on this basis.

[0055] the term

[0056] 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 to which this invention belongs.

[0057] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0058] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0059] Anti-HER2 Immunotoxin Molecules

[0060] In the present invention, "immunotoxin molecule" refers to a fusion polypeptide (fusion protein) composed of an antibody or its antigen-binding fragment (antibody part) and a toxin (toxin part). The antibody part can specifically bind to the antigen and target the cell; the toxin part is endocytosed by the cell bound to the antibody part, thereby exerting the toxin's cell-killing or cell-promoting apoptosis effects in the cell.

[0061] As used herein, "anti-HER2 immunotoxin molecules", "anti-HER2 single domain antibody immunotoxin molecules", and "immunotoxin molecules of the present invention" can be used interchangeably and all refer to the HER2-targeting immunotoxin molecules of the first aspect of the present invention.

[0062] 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 "fusion" refers to direct connection by a peptide bond or effective connection via one or more linkers.

[0063] In the present invention, the terms "anti" and "binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed. -7 M, for example, less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, surface plasmon resonance (SPR) is used to measure the binding affinity of an antibody to an antigen in a BIACORE instrument or to measure the relative affinity of an antibody to an antigen using ELISA.

[0064] In the present invention, the term "linker" is used to connect two polypeptide chains. Suitable linkers can be flexible polypeptide sequences, examples of which include single glycine (Gly) or serine (Ser) residues, and the identity and sequence of amino acid residues in the linker can vary with the type of secondary structural elements that need to be implemented in the linker.

[0065] Preferably, the linker in the present invention may also contain an enzymatic cleavage site that is recognized and degraded in the tumor microenvironment, for example, a furin protease cleavage site (as shown in SEQ ID NO: 13). When HER2-positive cancer cells take up the immunotoxin of the anti-HER2 single domain antibody, the site can be recognized and degraded by proteolytic enzymes present in the lysosome, such as cathepsin B that is highly expressed in various malignant tumor tissues such as lung cancer, gastric cancer, prostate cancer, and breast cancer. The immunotoxin molecules of the present invention are cleaved by intracellular lysosomes to release PE25, which accurately kills cancer cells.

[0066] Those skilled in the art can modify or transform the antibody portion or toxin portion 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 (e.g., improve affinity), and obtain the modified or transformed results by conventional measurement methods.

[0067] In the present invention, the antibody portion of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 7, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody or antigen-binding fragment of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0068] Table A

[0069]

[0070]

[0071] In the present invention, the term "PE" refers to Pseudomonas exotoxin A (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 domains DI, DⅡ, and DⅢ. Among them, DI is the binding region, which is composed of amino acid residues at positions 1-252, and can guide PE to recognize and bind to receptors on the target cell membrane. DⅡ is the translocation region, which is composed of amino acid residues at positions 253-384, and is responsible for the transmembrane transport of PE, allowing it to enter the cell; DⅢ is the active region, which is composed of amino acid residues at positions 385-613, catalyzing the ADP ribosylation of elongation factor 2, resulting in the inactivation of Ef2, thereby inhibiting cellular protein synthesis and ultimately leading to cell death.

[0072] 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, wherein the variant refers to SEQ ID NO: 4 comprising at least one mutation (such as a substitution or insertion mutation), and the PE25 retains the activity of PE in inhibiting cellular protein synthesis and reduces its immunogenicity.

[0073] In the present invention, the terms "polypeptide" and "protein" are used interchangeably.

[0074] In the present invention, "-" represents a peptide bond.

[0075] Encoding nucleic acid and expression vector

[0076] The present invention also provides a nucleic acid molecule encoding the above-mentioned anti-HER2 immunotoxin molecule. The nucleic acid molecule of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a 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 substance, such as a plasmid, a viral vector (such as adenovirus, retrovirus), a phage, a yeast plasmid or other vector. For example, a conventional expression vector in the art containing appropriate regulatory sequences, such as promoters, terminators, enhancers, marker genes and / or sequences, etc., includes but is not limited to: viral vectors (such as adenovirus, retrovirus), plasmids, phages, yeast plasmids 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, for example, Sambrook et al., Molec μLar Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989. Many known techniques and protocols for nucleic acid manipulation can be found in Current Protocols in Molec μLar Biology, 2nd edition, Ausubel et al., eds.

[0077] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0078] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0079] In the present invention, the term "host cell" refers to various host cells conventional in the art, as long as the vector can stably replicate itself and the polynucleotide molecules carried can be effectively expressed. The host cells include prokaryotic expression cells and eukaryotic expression cells, and the host cells preferably include: COS, CHO, NS0, sf9, sf21, DH5α, BL21 (DE3), TG1, BL21 (DE3), 293F or 293E cells.

[0080] Pharmaceutical compositions and applications

[0081] 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, diluent or excipient. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 4-8, preferably about 5-7, although the pH value may vary with the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal), intracranial injection, or intracavitary injection.

[0082] 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, release agents or excipients to form a pharmaceutical preparation composition so as to more stably exert therapeutic effects. These preparations can ensure the conformational integrity of the amino acid core sequence of the anti-HER2 immunotoxin molecules disclosed in the present invention, while also protecting the multifunctional groups of the protein to prevent its degradation (including but not limited to aggregation, deamination or oxidation).

[0083] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, and more preferably 0.1-80wt%) of the above-mentioned anti-HER2 immunotoxin molecules of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. In addition, the anti-HER2 immunotoxin molecules of the present invention can also be used with other therapeutic agents.

[0084] In the present invention, the term "effective amount" refers to the amount or dosage of the anti-HER2 immunotoxin molecule of the present invention that produces the expected effect in the treated individual after being administered to the subject, and the expected effect includes the improvement of the individual's symptoms. The term "subject" includes but is not limited to mammals, such as humans, non-human primates, rats and mice, etc.

[0085] The main advantages of the present invention include

[0086] 1) The immunotoxin molecules of the anti-HER2 single domain antibody of the present invention can specifically bind to the HER2 antigen on the surface of tumor cells, can target HER2 high-expressing cells, and have good targeting properties.

[0087] 2) The immunotoxin molecule of the anti-HER2 single domain antibody of the present invention has strong HER-positive cancer cell killing activity, and its killing activity is better than DS8201 (ENHERTU, ADC containing HER2 antibody + deruxtecan), achieving unexpected technical effects.

[0088] 3) The immunotoxin molecules of the anti-HER2 single domain antibody of the present invention are soluble when expressed in Escherichia coli, do not require denaturation, have a simple purification process, good purity, high yield, and low production cost.

[0089] 4) The linker connecting the HER2-targeting single-domain antibody and PE25 contains a furin protease cleavage site. When HER2-positive cancer cells take up the immunotoxin of the anti-HER2 single-domain antibody, PE25 is released after enzymatic cleavage by intracellular lysosomes, thus accurately killing cancer cells.

[0090] The present invention is further described below in conjunction with specific examples. 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 methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0091] The experimental materials and reagents used in the following examples were all commercially available unless otherwise specified.

[0092] The detection methods used in the following examples are described as follows:

[0093] 1. SDS-PAGE detection:

[0094] Detection system: Mini protein TeTra system

[0095] Test conditions: 140V constant voltage 45-55min

[0096] 2. UV detection:

[0097] Instrument model: Nanodrop one (Thermo)

[0098] Extinction coefficient: 1.42

[0099] 3. Protein A affinity chromatography

[0100] Chromatography column: XK16 / 20 (GE)

[0101] Filler: Protein L (GenScript)

[0102] Chromatography system: AKTA Pure150 (GE)

[0103] Operating system: unicorn 7.0 (GE)

[0104] Flow rate: 1.0mL / min

[0105] 4. ELISA detection and processing

[0106] Microplate reader: SpecTraMax 190, wavelength 450nm

[0107] Processing software: GraphPad Prism 9

[0108] Example 1. Preparation of anti-HER2 single domain antibody immunotoxin

[0109] 1.1 Anti-HER2 single domain antibody immunotoxin amino acid design and coding nucleotide optimization

[0110] The anti-HER2 immunotoxin molecule 5F7PE010 (also known as 5F7-PE010) has a structure of 5F7-L-PE25, and its amino acid sequence is shown in SEQ ID NO: 1. The single-domain antibody immunogen is derived from 5F7 (SEQ ID NO: 2). The numbering is based on the Kabat system. The linker L is GSGGGGSGGGGSRHRQPRGWEQLGGS (SEQ ID NO: 3); PE25 (SEQ ID NO: 4) is a mutant of the DIII region (positions 395-613) of Pseudomonas exotoxin PE, which has complete catalytic activity and lower immunogenicity.

[0111] To facilitate separation and purification, Hissumo (SEQ ID NO: 5) was added to the N-terminus of 5F7PE010 to obtain Hissumo5F7PE010 (amino acid SEQ ID NO: 6). To increase the expression level, the nucleotide sequence encoding the amino acid sequence of Hissumo5F7PE010 (SEQ ID NO: 6) was optimized using the codons of Escherichia coli to obtain SEQ ID NO: 7.

[0112] Table B Sequence Listing (Kabat System Rules Numbering and Definitions)

[0113]

[0114]

[0115] 1.2 Construction of anti-HER2 single-domain antibody immunotoxin expression vector

[0116] The whole gene was synthesized and the nucleotide sequence of Hissumo5F7PE010 (SEQ ID NO: 7) was inserted into the expression frame of pET-28a to construct a plasmid and its plasmid bacteria.

[0117] 1.3 Construction of anti-HER2 immunotoxin expression strain

[0118] The plasmid Hissumo5F7PE010 PET28a constructed in Example 1.2 was transformed into BL21 (DE3) competent cells, coated with 2YT agar plates containing kanamycin resistance, and then single clones were selected for IPTG induction and SDS-PAGE detection and screening. The cell line with the highest expression level was selected for subsequent experiments.

[0119] 1.4 Expression and purification of anti-HER2 immunotoxin

[0120] 1.4.1 IPTG-induced expression of Hissumo5F7PE010

[0121] The glycerol strain was inoculated into a kanamycin-resistant culture medium at a volume ratio of 1:1000, and cultured at 37°C, 150rpm overnight; the seed liquid was inoculated into a kanamycin-resistant culture medium at a volume ratio of 1:1000, and cultured at 37°C, 150rpm until the OD600 reached 0.7; IPTG was added to a final concentration of 1mM, and the culture was continued at 25°C, 120rpm for 16-18 hours; centrifuged at 8000rpm×5min, the supernatant was removed, and the bacteria were collected.

[0122] Weigh the collected bacteria, resuspend them with PBS at a weight ratio of 1:20, and stir them thoroughly; use a disruption instrument: ultra-high pressure continuous flow cell disruptor, model: JN-MiniPro, to disrupt them thoroughly; disruption conditions: 4°C, pressure value 1000Ba, disrupt twice; centrifuge the disrupted bacterial solution at 4°C, 10000rpm, 30min, collect the supernatant; filter the supernatant with a 0.22-micron filter membrane.

[0123] 1.4.2 Separation and purification of Hissumo5F7PE010 using Ni

[0124] Affinity chromatography was performed using an AKTA Pure 150 (GE) chromatography system, with Ni as the column. The instrument was operated according to the operating instructions, with the A1 pump being Buffer A (PBS, pH 7.4) and the B1 pump being Buffer B (PBS, pH 7.4 250 mM imidazole).

[0125] 1) Equilibration: Buffer A was flushed at a flow rate of 1 mL / min for 10 CV (column volume) to maintain the Ni gel environment suitable for the binding of anti-HER2 immunotoxin to Ni.

[0126] 2) Sample loading: Pass the sample through the Ni gel at a rate of 1 mL / min to allow Hissumo5F7PE010 to bind specifically to Ni.

[0127] 3) Elute: flush with Buffer A at a flow rate of 1 mL / min until the absorbance at 280 nm is less than 0.01.

[0128] 4) Elution: Wash the Ni gel with Buffer B at a flow rate of 1 mL / min and collect the elution peak.

[0129] Neutralization: Collect the eluted peak and adjust the pH to 7.4 with 50 mM glycine buffer (pH 9.0). 10 mg of Hissumo5F7PE010 can be purified per liter of culture medium.

[0130] 5) SDS-PAGE analysis of the purified Hissumo5F7PE010.

[0131] 1.4.3Hissumo5F7PE010 digestion and 5F7PE010 purification

[0132] 1) Replace Hissumo5F7PE010 with PBS buffer and digest with ULP1His enzyme (SEQ ID NO: 9) at a ratio of 100:1 at 30°C for 4 hours;

[0133] 2) Pass the digestion solution from 1) through a pre-equilibrated Ni column and collect the flow-through solution, which is 5F7PE010.

[0134] Preparation flow chart Figure 1 5F7PE010 fusion protein expression, purification and restriction enzyme digestion SDS-PAGE Figure 2 shown.

[0135] Depend on Figure 2 It can be seen that BL21 (DE3) expresses the fusion protein Hissumo5F7PE010. After Ni column purification, the fusion protein is highly purified and the enzyme cleavage efficiency is good. The enzyme cleavage solution flows through the Ni column to obtain a protein with higher purity.

[0136] Example 2.5F7PE010 mass spectrometry detection

[0137] 1) Mass spectrometry conditions:

[0138] Waters UPLC-XEVO G2 Q-TOF LC / MS system. The system liquid phase part is configured as: BSM binary high pressure mixing pump, SM sample manager, TUV UV detector; the mass spectrometry part is configured as: ESI source, Q-TOF detector. Data processing and analysis are performed using Masslynx V4.1 and BiopharmaLynx analysis software (Version: 1.2).

[0139] The MS data were collected in continuum mode under Resolution mode; the LockSpray acquisition mode was real-time acquisition and no calibration was applied.

[0140] Calibration solution: Real-time calibration (LockSpray) solution: 2 ng / μL LE solution;

[0141] Calibration solution for mass axis: 2 μg / μL sodium iodide solution.

[0142] The mass spectrometry parameters are shown in Table 1.

[0143] 2) Liquid phase conditions:

[0144] Chromatographic column: Mass PREPTM Micro Desalting Column 2.1×5mm (intact protein molecular weight analysis), column temperature: 80°C;

[0145] Mobile phase A: 0.1% FA-H2O

[0146] Mobile phase B: 0.1% FA-CAN

[0147] Seal Wash Solution: 10% IPA

[0148] Mass spectrometer cleaning solution: 50% ACN

[0149] Mass Spectrometer IntelliStart Valve Cleaning Solution: 50% MeOH

[0150] Injection volume: 10 μL

[0151] Sample chamber temperature: 10°C

[0152] The gradient elution conditions are shown in Table 2.

[0153] Table 1. Mass spectrometry parameters

[0154]

[0155] Table 2. Gradient elution conditions

[0156]

[0157]

[0158] Table 3 Intact molecular weight of 5F7PE010 sample

[0159] Sample name Theoretical molecular weight (Da) Measured molecular weight (Da) 5F7PE010 38270.61 38272.04

[0160] The mass spectrometry results are as follows Figure 3 As shown in Table 3, it is shown that the 5F7PE010 of the present invention is relatively uniform and basically consistent with the design.

[0161] Example 3. ELISA detection of 5F7PE010 binding to antigen

[0162] 1. Coating antigen HER2-ECD-His (SEQ ID NO: 8), 100 μL per well, concentration of 1 μg / mL, incubate at 4°C overnight.

[0163] 2. Washing: Take out the Elisa plate coated with antigen, wash the plate 3 times with washing buffer PBST, pat the Elisa plate dry and wait for the next step of blocking.

[0164] 3. Blocking: Add 200 μL of blocking solution (PBST + 1% BSA) to each well and block at room temperature for 2 hours.

[0165] 4. Washing: Wash the plate three times with washing buffer PBST and pat dry for later use.

[0166] 5. Add primary antibody: 5F7PE010 (SEQ ID NO: 1)

[0167] (1) Antibody dilution: dilute the primary antibody in a 96-well cell culture plate (the diluent is the blocking solution): the initial concentration is 100 nM, and the dilution is 3-fold from left to right, with 11 dilution gradients. The 12th column is set as a blank control without adding the primary antibody. For each sample, set up 2 replicate wells for each gradient.

[0168] (2) Add 100 μL of diluted primary antibody to each well and block at room temperature for 2 hours.

[0169] 6. Washing: Wash the plate three times with washing buffer PBST and pat dry for later use.

[0170] 7. Add secondary antibody: dilute AntiPEA-HRP with blocking solution (PBST + 1% BSA) at a ratio of 1:4000, add 100 μL of diluted secondary antibody to each well, and incubate at room temperature in the dark for 1 hour.

[0171] 8. Washing: Wash the plate three times with washing buffer PBST, pat dry and set aside.

[0172] 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 10 minutes.

[0173] 10. Stop: Add 70 μL of stop solution (2M H2SO4) to each well, mix well, and immediately read the results on a microplate reader at a detection wavelength of 450 nm.

[0174] 11. Use GraphPad Prism 9 to draw graphs and fit curves to analyze the EC50 (nM) of 5F7PE010 binding to antigen.

[0175] The ELISA results of 5F7PE010 binding to antigen are as follows Figure 4 As shown, the EC50 value was 0.174 nM.

[0176] Example 4. Detection of the in vitro cytotoxicity of 5F7PE010 against HER2-positive cancer cells

[0177] 4.1 In vitro cytotoxicity of 5F7PE010 against human breast ductal carcinoma HCC1954 cells (HER2 overexpression) I. Experimental materials

[0178] Cells: HCC1954 purchased from ATCC

[0179] Culture medium: RPMI 1640 + 15% FBS + 1% Pen-Strep

[0180] 2. Experimental steps

[0181] 1. Cell digestion: Cultivate cells until the density reaches 80%-90%, discard the supernatant, wash with DPBS, add 0.25% Trypsin-EDTA to digest, add complete culture medium to terminate, and mix well by pipetting.

[0182] 2. Density determination and cell plating: The cell suspension was mixed with 0.2% Trypan Blue at a ratio of 1:1. After counting using a cell counter, the cells were diluted to the desired density. The cell suspension was then plated onto a 96-well plate (Corning, 3599) at 2400 cells / well, 150 μL / well, and cultured overnight at 37°C, 5% CO2 to allow the cells to adhere.

[0183] 3. Drug dilution and addition: Prepare the drug to the required concentration in complete medium, filter it, dilute it in 5-fold gradient, and add it to a 96-well plate, 50 μL / well.

[0184] 4. Drug incubation: 37°C, 5% CO2 for 4 days.

[0185] 5. CCK8 color development: discard the supernatant, turn the 96-well plate upside down on absorbent paper to dry, add 10% CCK-8 color development, 100 μL / well. After incubation at 37°C for 1-2 hours, use an ELISA reader to read the plate at 450nm.

[0186] 6. Data analysis: The wells without cells were used as blank controls, recorded as 0%, and the wells with cells but no drugs were used as negative controls, recorded as 100%. GraphPad Prism 9 was used to plot and fit the curves to compare the differences in IC50 (nM) between different drugs. DS8201 was used as the control positive drug.

[0187] In vitro killing curve of 5F7PE010 on human breast ductal carcinoma cell HCC1954 Figure 5 shown. Figure 5 The results show that the IC50 of 5F7PE010 is 0.007nM, and the IC50 of the control positive drug DS8201 is 0.717nM. It can be seen that 5F7PE010 has strong anti-tumor activity and can kill human breast ductal cancer cells HCC1954. Its killing activity against cancer cells is significantly higher than that of the positive control drug DS8201, which is about 100 times that of the control positive drug DS8201.

[0188] 4.2 Cytotoxicity of 5F7PE010 against human breast cancer JIMT1 cells (HER2 overexpression) in vitro

[0189] 1. Experimental Materials

[0190] Strain: JIMT1

[0191] Culture medium: DMEM + 10% FBS + 1% Pen-Strep

[0192] 2. Experimental steps

[0193] 1. Cell digestion: Cultivate cells until the density reaches 80%-90%, discard the supernatant, wash with DPBS, add 0.25% Trypsin-EDTA to digest, add complete culture medium to terminate, and mix well by pipetting.

[0194] 2. Density determination and cell plating: Cell suspension was mixed with 0.2% Trypan Blue at a ratio of 1:1. After counting using a cell counter, the cells were diluted to the desired density. The cell suspension was then plated onto a 96-well plate (Corning, 3599) at 4200 cells / well, 150 μL / well, and cultured overnight at 37°C, 5% CO2 to allow the cells to adhere.

[0195] 3. Drug dilution and addition: Prepare the drug to the required concentration in complete medium, filter it, dilute it in 5-fold gradient, and add it to a 96-well plate, 50 μL / well.

[0196] 4. Drug incubation: 37°C, 5% CO2 for 4 days.

[0197] 5. CCK8 color development: discard the supernatant, turn the 96-well plate upside down on absorbent paper to dry, add 10% CCK-8 color development, 100 μL / well. After incubation at 37°C for 1-2 hours, use an ELISA reader to read the plate at 450nm.

[0198] 6. Data analysis: The wells without cells were used as blank controls, recorded as 0%, and the wells with cells but no drugs were used as negative controls, recorded as 100%. GraphPad Prism 9 was used to plot and fit the curves to compare the differences in IC50 (nM) between different drugs.

[0199] Effect of 5F7PE010 on the killing of human breast cancer JIMT1 cells Figure 6 shown. Figure 6 The results showed that the IC50 of 5F7PE010 was 0.025 nM, and the control positive drug DS8201 had almost no killing effect. It can be seen that 5F7PE010 has strong anti-tumor activity and can kill human breast cancer cells JIMT1, and its killing activity against cancer cells is significantly higher than that of the positive control drug DS8201.

[0200] 4.3 Cytotoxicity of 5F7PE010 against human gastric cancer cells NCI-N87 (HER2 overexpression) in vitro

[0201] 1. Experimental Materials

[0202] Strain: NCI-N87

[0203] Culture medium: RPMI 1640 + 10% FBS + 1% Pen-Strep

[0204] 2. Experimental steps

[0205] 1. Cell digestion: Cultivate cells until the density reaches 80%-90%, discard the supernatant, wash with DPBS, add 0.25% Trypsin-EDTA to digest, add complete culture medium to terminate, and mix well by pipetting.

[0206] 2. Density determination and cell plating: The cell suspension was mixed with 0.2% Trypan Blue at a ratio of 1:1. After counting using a cell counter, the cells were diluted to the desired density. The cell suspension was then plated onto a 96-well plate (Corning, 3599) at 10,000 cells / well, 150 μL / well, and cultured overnight at 37°C, 5% CO2 to allow the cells to adhere.

[0207] 3. Drug dilution and addition: Prepare the drug to the required concentration in complete medium, filter it, dilute it in 5-fold gradient, and add it to a 96-well plate, 50 μL / well.

[0208] 4. Drug incubation: 37°C, 5% CO2 for 4 days.

[0209] 5. CCK8 color development: discard the supernatant, turn the 96-well plate upside down on absorbent paper to dry, add 10% CCK-8 color development, 100 μL / well. After incubation at 37°C for 1-2 hours, use an ELISA reader to read the plate at 450nm.

[0210] 6. Data analysis: The wells without cells were used as blank controls, recorded as 0%, and the wells with cells but no drugs were used as negative controls, recorded as 100%. GraphPad Prism 9 was used to plot and fit the curves to compare the differences in IC50 (nM) between different drugs.

[0211] 5F7PE010 kills human gastric cancer cell NCI-N87 cells Figure 7 shown. Figure 7 The results show that the IC50 of 5F7PE010 is 0.002nM, and the IC50 of the control positive drug DS8201 is 0.705nM. It can be seen that 5F7PE010 has strong anti-tumor activity and can kill human breast cancer cells NCI-N87, and its killing activity against cancer cells is significantly higher than that of the positive control drug DS8201.

[0212] Example 5. Antitumor activity of 5F7PE010 in the NCI-N87 gastric cancer xenograft mouse model

[0213] NCI-N87 tumor xenografts in BALB / c nude mice were used as a model. 5×10 6 NCI-N87 cells were injected subcutaneously into the flanks of tumor-bearing mice. 3When the volume of the tumor reached 1.37 μg / cm2, the mice were randomly divided into 5 groups and intravenously injected with different doses of 5F7PE010 or PBS as a control twice a week, or DS8201 as a positive drug was administered once and monitored for 28 days. The tumor volume and body weight were measured twice a week (the results are shown in Table 2). Figure 8 shown).

[0214] Depend on Figure 8 It can be seen that: DS8201 4mg / kg single administration showed strong in vivo anti-tumor activity in the human gastric cancer NCI-N87 transplant tumor model. 5F7PE010 twice a week also showed strong in vivo anti-tumor activity in the human gastric cancer NCI-N87 transplant tumor model and was dose-dependent. In addition, the tumor inhibition activity of 5F7PE010 in the medium-dose group (5mg / kg) and the high-dose group (5mg / kg) was significantly better than that of the positive control DS8201, and the final tumor inhibition effect of the low-dose group (2.5mg / kg) was also better than that of the positive control DS8201.

[0215] The animals in the 5F7PE010 10mg / kg, twice-weekly group experienced a large weight loss in the early stage of the experiment, but gradually stabilized in the later stage, and the animals were in a good mental state. The animals in the other groups had normal weights and good mental states, indicating that the animals had good tolerance to the 5F7PE010 treatment.

[0216] The above results indicate that the immunotoxin molecules of the present invention can effectively inhibit tumor volume within a time window of about 3 weeks before the PE immunotoxin loses its effectiveness.

[0217] Example 6. Low-dose efficacy test of 5F7PE010 in the NCI-N87 human gastric cancer transplanted tumor mouse model

[0218] NCI-N87 tumor xenografts in BALB / c nude mice were used as a model. 5×10 6 NCI-N87 cells were injected subcutaneously into the flanks of tumor-bearing mice. 3 When the volume of the tumor reached 1.57 μg / kg, the mice were randomly divided into 7 groups and intravenously injected with 5F7PE010 at different doses (0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 2.5 mg / kg) and different times (once a week, twice a week, and three times a week) every week, and injected with PBS as a control, and monitored for 28 days. The tumor volume and body weight were measured twice a week (the results are shown in Table 2). Fig. 9 shown).

[0219] Depend on Fig. 9It can be seen that: 5F7PE010 0.1mg / kg administered twice a week has no anti-tumor activity, 0.3, 1, 2.5mg / kg three doses administered twice a week all show anti-tumor activity, and the anti-tumor activity shows obvious dose dependence. 0.3mg / kg administered three times a week also shows anti-tumor activity. Compared with 0.3mg / kg administered twice a week, the anti-tumor activity of three doses a week is slightly stronger than that of twice a week. 1mg / kg administered once a week also shows anti-tumor activity, which is equivalent to the anti-tumor activity of 0.3mg / kg administered twice a week. Compared with 1mg / kg administered twice a week, the anti-tumor activity of 1mg / kg administered once a week is significantly weaker than that of twice a week. The body weight of animals in each group showed an overall increase, and their mental state was good. The animals were well tolerated at these dosages.

[0220] The above results indicate that the immunotoxin molecules of the present invention can effectively inhibit tumor volume at a lower dose (0.1-1 mg / kg) within a time window of about 3 weeks before the PE immunotoxin loses its effectiveness.

[0221] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings 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 claims attached to this application.

Claims

1. An anti-HER2 immunotoxin molecule, characterized in that From N-terminus to C-terminus, the fusion polypeptide includes the following molecular formula: Ab-L-PE; Wherein, the Ab is an anti-HER2 antibody or an antigen-binding fragment thereof; L is a linker; PE comprises the amino acid sequence shown in SEQ ID NO: 4 or a variant of SEQ ID NO: 4; Wherein, the anti-HER2 antibody or antigen-binding fragment thereof comprises an anti-HER2 single-domain antibody, and the single-domain antibody comprises the following three complementary determining regions CDR: CDR1 shown in SEQ ID NO.10, CDR2 shown in SEQ ID NO.11, and CDR3 shown in SEQ ID NO.

12.

2. The anti-HER2 immunotoxin molecule according to claim 1, characterized in that The anti-HER2 single-domain antibody comprises the amino acid sequence shown in SEQ ID NO.2; or comprises an amino acid sequence that is at least 98% or 99% identical to SEQ ID NO.

2.

3. The anti-HER2 immunotoxin molecule according to claim 1, characterized in that The L comprises a cleavage site as shown in SEQ ID NO: 13; preferably, the sequence of the L is shown in SEQ ID NO.

3.

4. The anti-HER2 immunotoxin molecule according to claim 1, characterized in that The anti-HER2 immunotoxin molecule comprises an amino acid sequence as described in SEQ ID NO.1 or 6; or comprises an amino acid sequence that is at least 98% or 99% identical to SEQ ID NO.1 or 6.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-HER2 immunotoxin molecule according to any one of claims 1-4.

6. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 5.

7. A host cell, characterized in that The host cell comprises the expression vector according to claim 6.

8. A method for preparing an anti-HER2 immunotoxin molecule as claimed in claim 1, characterized in that: The preparation method comprises the following steps: a) culturing the host cell according to claim 7 under expression conditions, thereby expressing the anti-HER2 immunotoxin molecule; b) separating and purifying the anti-HER2 immunotoxin molecules described in step a).

9. 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 to 4 and one or more pharmaceutically acceptable carriers.

10. Use of the anti-HER2 immunotoxin molecule according to any one of claims 1 to 4 and the pharmaceutical composition according to claim 9 in the preparation of a drug for treating HER2-positive cancers, wherein the cancers preferably include any one or more of breast cancer, gastric cancer, ovarian cancer, bladder cancer or non-small cell lung cancer.

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