A single-domain antibody against vegfr2 and uses thereof

Abstract

The application belongs to the field of immunology and relates to an anti-VEGFR2 single-domain antibody and application thereof. The single-domain antibody is composed of a heavy chain, the heavy chain comprising heavy chain CDR1 shown in any one of SEQ ID NO: 29-SEQ ID NO: 33, heavy chain CDR2 shown in any one of SEQ ID NO: 34-SEQ ID NO: 37 and heavy chain CDR3 shown in any one of SEQ ID NO: 38-SEQ ID NO: 42. Compared with the prior art, the application has the beneficial effect that the single-domain antibody specific to VEGFR2 is screened by using biological genetic engineering technology, and the antibody has good affinity.

Description

Technical Field

[0001] This invention relates to a single-domain antibody capable of specifically binding to VEGFR2 (hereinafter abbreviated as "anti-VEGFR2 single-domain antibody"), a pharmaceutical composition containing the single-domain antibody as an active ingredient, and its use in pharmaceutical therapy. Background Technology

[0002] VEGF is a family of pro-angiogenic factors with a molecular weight of 40-45 kDa, short for Vascular Endothelial Growth Factors. VEGF regulates tumor angiogenesis, primarily by binding to its receptors (VEGFR1, VEGFR2, and VEGFR3), thereby activating intracellular signaling pathways. VEGF and its receptors (VEGFR) play a crucial role in tumor growth and metastasis. Blocking the VEGF-VEGFR signaling pathway can control tumor growth, thus achieving the goal of treating tumors. Anti-VEGF-VEGFR drugs broadly include the following categories: antibody drugs that directly target VEGF and VEGFR proteins, such as bevacizumab and ramucirumab; inhibitors of intracellular tyrosine kinase signaling pathways, such as sorafenib; and drugs such as fusion proteins and immunomodulators.

[0003] VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) is one of the most well-understood VEGF receptors, playing a crucial role in the regulation of angiogenesis, vascular development, vascular permeability, and embryonic hematopoiesis. The binding of VEGF to VEGFR2 triggers the formation of a receptor dimer and activates downstream signaling pathways. The extracellular region of VEGFR2 consists of seven Ig-like subunits. In addition, VEGFR2 includes a transmembrane domain and a catalytic tyrosine kinase domain. In recent years, significant progress has been made in the development of novel anti-tumor drugs targeting VEGFR2. For the VEGFR2 pathway, drugs that inhibit ligands include monoclonal antibodies (bevacizumab and ranibizumab), siRNA drugs (pilgatanib), and soluble receptor VEGF-Trap. There are also many small molecule drugs that inhibit intracellular tyrosine kinases of the receptor, such as sorafenib, sunitinib, antisense RNA, and siRNA.

[0004] Therefore, the development of antibodies that can specifically recognize and bind to VEGFR2 is of great significance in the diagnosis, treatment, and prognosis of diseases involving VEGFR2. Currently, there is still a lack of single-domain antibody products against VEGFR2 with strong affinity and pharmaceutical value in existing technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a single-domain antibody that can specifically bind to VEGFR2 and its uses.

[0006] A first aspect of the present invention provides a single-domain antibody against VEGFR2, said single-domain antibody being composed of heavy chains, the heavy chains including heavy chain CDR1 shown in any one of SEQ ID NO:29-SEQ ID NO:33, heavy chain CDR2 shown in any one of SEQ ID NO:34-SEQ ID NO:37, and heavy chain CDR3 shown in any one of SEQ ID NO:38-SEQ ID NO:42.

[0007] Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1)-(5):

[0008] (1) CDR1 shown in SEQ ID NO:29, CDR2 shown in SEQ ID NO:35, and CDR3 shown in SEQ ID NO:40;

[0009] (2) CDR1 shown in SEQ ID NO:30, CDR2 shown in SEQ ID NO:37, and CDR3 shown in SEQ ID NO:41;

[0010] (3) CDR1 shown in SEQ ID NO:31, CDR2 shown in SEQ ID NO:37, and CDR3 shown in SEQ ID NO:42;

[0011] (4) CDR1 shown in SEQ ID NO:32, CDR2 shown in SEQ ID NO:36, and CDR3 shown in SEQ ID NO:39;

[0012] (5) CDR1 shown in SEQ ID NO:33, CDR2 shown in SEQ ID NO:34, and CDR3 shown in SEQ ID NO:38.

[0013] All of the above sequences can be replaced with sequences that have "at least 80% homology" or sequences that replace only one or a few amino acids; preferably "at least 85% homology", more preferably "at least 90% homology", even more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0014] In one embodiment, in any one or more CDRs of the heavy chain CDR1, CDR2, and CDR3, one to five arbitrary amino acid residues may be substituted with their conserved amino acids. Specifically, in the heavy chain CDR1, one to five amino acid residues may be substituted with their conserved amino acids; in the heavy chain CDR2, one to five amino acid residues may be substituted with their conserved amino acids; and in the heavy chain CDR3, one to five amino acid residues may be substituted with their conserved amino acids.

[0015] As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% homology.

[0016] In some embodiments, the inventive objective can also be achieved by substituting only one or a few amino acids compared to the aforementioned sequence, for example, by including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. These variations include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, most preferably up to 5) at the C-terminus and / or N-terminus. In practice, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 in a single-domain antibody, those skilled in the art can consider so-called “conserved” amino acid substitutions, in which case the substitution would preferably be a conserved amino acid substitution. A conserved amino acid can generally be described as an amino acid residue substituted by another amino acid residue having a similar chemical structure, and this substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are common in the art. For example, a conserved amino acid substitution is the substitution of one or a few amino acids in the following groups (a)-(d) by another or a few amino acids in the same group: (a) polar negatively charged residues and their non-charged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. The particularly preferred conserved amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. Furthermore, those skilled in the art will understand that the frame region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conserved sequence variants of the sequences disclosed in this invention.

[0017] The term "anti-VEGFR2 single-domain antibody" in this invention includes not only complete single-domain antibodies but also fragments, derivatives, and analogs of said anti-VEGFR2 single-domain antibodies. As used herein, the terms "fragment," "derivative," and "analyte" have the same meaning and refer to polypeptides that substantially retain the same biological function or activity as the antibodies of this invention. The polypeptide fragments, derivatives, or analogs of this invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence, secretion sequence, or sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with an Fc tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0018] In a preferred embodiment, the heavy chain further includes a framework region FR; the framework region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the framework region FR are as follows:

[0019] The FR1 or a variant thereof shown in any one of SEQ ID NO:15-18, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR1;

[0020] FR2 or a variant thereof shown in any one of SEQ ID NO:19-21, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR2;

[0021] The FR3 or a variant thereof shown in any one of SEQ ID NO:22-26, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR3;

[0022] FR4 or a variant thereof shown in any one of SEQ ID NO:27-28, wherein the variant thereof contains substitutions of up to 5 amino acids.

[0023] A second aspect of the present invention is to provide an amino acid sequence of a single-domain antibody capable of binding VEGFR2, wherein the amino acid sequences of the single-domain antibody are as shown in SEQ ID NO: 1-7, or the single-domain antibody has at least 80% sequence homology with the amino acid sequences of SEQ ID NO: 1-7 and is capable of specifically binding to the VEGFR2 protein.

[0024] In one embodiment, the anti-VEGFR2 single-domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with an amino acid sequence selected from SEQ ID NO: 1-7, and is capable of specifically binding to the VEGFR2 protein.

[0025] A third aspect of the invention is to provide an Fc fusion antibody or humanized antibody that provides any of the aforementioned single-domain antibodies against VEGFR2.

[0026] A fourth aspect of the present invention is to provide nucleotide molecules encoding the aforementioned anti-VEGFR2 single-domain antibody, the aforementioned Fc fusion antibody, or the aforementioned humanized antibody, wherein the nucleotide sequences are as shown in SEQ ID NO: 8-14, or have at least 95% sequence homology with any one of SEQ ID NO: 8-14.

[0027] In one embodiment, the nucleic acid molecule encoding the single-domain antibody against VEGFR2 is selected from SEQ ID NO: 8-14 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a nucleotide sequence selected from SEQ ID NO: 8-14, and the single-domain antibody against VEGFR2 encoded therefrom is capable of specifically binding to the VEGFR2 protein.

[0028] A fifth aspect of the present invention is to provide an expression vector comprising a nucleotide molecule encoding a single-domain antibody, an Fc fusion antibody, or a humanized antibody against VEGFR2, wherein the nucleotide sequences are as shown in SEQ ID NO: 8-14 or have at least 80% sequence homology with any one of SEQ ID NO: 8-14.

[0029] In a preferred embodiment, the expression vector used is RJK-V4-hFC1 (a nucleotide molecule encoding a single-domain antibody or its Fc fusion antibody or humanized antibody encoding anti-VEGFR2 is integrated into RJK-V4-hFC1 by genetic engineering). Other universal expression vectors may also be selected as needed.

[0030] A sixth aspect of the invention is to provide a host cell capable of expressing the aforementioned anti-VEGFR2 single-domain antibody, Fc fusion antibody, or humanized antibody, or an expression vector comprising the aforementioned. Preferably, the host cell is a bacterial cell, fungal cell, or mammalian cell.

[0031] In another preferred embodiment, the host cell includes prokaryotic or eukaryotic cells, including bacteria and fungi.

[0032] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0033] In another preferred embodiment, the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0034] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomiae, Trichoderma, or combinations thereof.

[0035] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of insect cells such as armyworms, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

[0036] In another preferred embodiment, the host cell is a suspension ExpiCHO-S cell.

[0037] In another preferred embodiment, the host cell is a suspension 293F cell.

[0038] A seventh aspect of the present invention is to provide a recombinant protein comprising the aforementioned anti-VEGFR2 single-domain antibody. The recombinant protein may be the single-domain antibody shown in SEQ ID NO: 1-7, or a single-domain antibody having at least 80% homology with SEQ ID NO: 1-7, or a multi-epitope antibody, multi-specific antibody, or multivalent antibody; for example, the multi-epitope antibody may consist of more than one sequence from SEQ ID NO: 1-7; the multivalent antibody may consist of a sequence from SEQ ID NO: 1-7 repeated a certain number of times; the multi-specific antibody includes, but is not limited to, bispecific antibodies and trispecific antibodies; furthermore, the recombinant protein may be a fragment, derivative, or analog of the aforementioned antibody.

[0039] An eighth aspect of the invention is to provide a pharmaceutical composition comprising the aforementioned single-domain antibody binding VEGFR2 and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically determined based on the isoelectric point of the antibody (the pH of the aqueous carrier medium must deviate from the isoelectric point of the antibody by approximately 2).

[0040] The pharmaceutical composition of the present invention can be directly used to bind to VEGFR2 protein molecules.

[0041] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the aforementioned single-domain antibody and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under aseptic conditions.

[0042] A ninth aspect of the present invention is to provide a medicament for treating diseases, comprising the aforementioned single-domain antibody for binding the VEGFR2 protein as an active ingredient. The diseases may include liver cancer, colorectal cancer, gastrointestinal stromal tumors, non-small cell lung cancer, adenocarcinoma of the gastroesophageal junction, gastric cancer, esophageal cancer, adenocarcinoma, biliary tract tumors, bladder cancer, bile duct cancer, melanoma, ovarian cancer, kidney cancer, breast cancer, high microsatellite instability cancers, pancreatic cancer, carcinoid tumors, and solid tumors.

[0043] A tenth aspect of the present invention is to provide a kit for detecting VEGFR2 levels, comprising the aforementioned anti-VEGFR2 single-domain antibody. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc.

[0044] In a preferred embodiment, the kit includes an antibody that recognizes the VEGFR2 protein, a lysis medium for dissolving the sample, and universal reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0045] In a preferred embodiment, the kit further contains a second antibody and an enzyme or fluorescent or radiolabeled marker for detection, as well as a buffer solution.

[0046] In a preferred embodiment, the second antibody in the kit may be an antibody (as an anti-antibody) of the aforementioned single-domain antibody against VEGFR2, and may be a single-domain antibody, monoclonal antibody, polyclonal antibody, or any other form of antibody.

[0047] In an eleventh aspect of the present invention, a method for generating a single-domain antibody against VEGFR2 is provided, comprising the steps of:

[0048] (a) Under conditions suitable for the production of single-domain antibodies, the host cells described in the sixth aspect of the invention are cultured, thereby...

[0049] Obtain a culture containing the single-domain antibody against VEGFR2; and

[0050] (b) Isolating or recovering the single-domain antibody against VEGFR2 from the culture; and

[0051] (c) Optionally, purify and / or modify the anti-VEGFR2 single-domain antibody obtained in step (b).

[0052] The twelfth aspect of the invention is the use of the aforementioned anti-VEGFR2 single-domain antibody or the aforementioned pharmaceutical composition in the preparation of a medicament for treating a disease.

[0053] In a preferred embodiment, the disease can be liver cancer, colorectal cancer, gastrointestinal stromal tumor, non-small cell lung cancer, adenocarcinoma of the gastroesophageal junction, gastric cancer, esophageal cancer, adenocarcinoma, biliary tract tumor, bladder cancer, bile duct cancer, melanoma, ovarian cancer, kidney cancer, breast cancer, high microsatellite instability cancer, pancreatic cancer, carcinoid tumors, and solid tumors.

[0054] Beneficial effects

[0055] Compared with the prior art, the beneficial effects of the present invention are:

[0056] (1) The single-domain antibody of the present invention is specifically targeted at the VEGFR2 protein with the correct spatial structure.

[0057] (2) The single-domain antibody obtained by the present invention has a flexible expression system. It can be expressed in a prokaryotic system or in a eukaryotic system of yeast cells or mammalian cells. Moreover, its expression cost in the prokaryotic expression system is low, which can reduce the later production cost.

[0058] (3) The single-domain antibody obtained by the present invention has simple multi-combination form modification. It can be obtained by simple tandem through genetic engineering to obtain multivalent and multispecific antibodies. Moreover, its immune heterogeneity is very low and it will not produce a strong immune response without humanization modification.

[0059] (4) The single-domain antibody obtained by the present invention has a wider affinity range. Before affinity maturation, its affinity range can be from nM to pM, providing multiple options for antibodies for different purposes in the later stage. Attached Figure Description

[0060] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0061] Figure 1 SDS-PAGE gel image for identification of purified human VEGFR2 protein;

[0062] Figure 2 The enrichment status of the library for screening antibodies targeting VEGFR2 in Example 3;

[0063] Figure 3 The antibody-antigen binding dose-response curve assay chromatogram (1A8) is shown in Example 12.

[0064] Figure 4 The antibody-antigen binding dose-response curve assay chromatogram (2B7) in Example 12;

[0065] Figure 5 The antibody-antigen binding dose-response curve assay chromatogram (3B12) in Example 12;

[0066] Figure 6 The antibody-antigen binding dose-response curve (3C4) is shown in Example 12.

[0067] Figure 7 The antibody-antigen binding dose-response curve assay chromatogram (1F9) is shown in Example 12.

[0068] Figure 8 The antibody-antigen binding dose-response curves (3F12, 3F2) from Example 12 are shown.

[0069] Figure 9 Experimental results for neutralizing the VEGF165-VEGFR2 signaling pathway (1A8);

[0070] Figure 10 To neutralize the experimental results of the VEGF165-VEGFR2 signaling pathway (Tab1, hIgG);

[0071] Figure 11 Experimental results for neutralizing the VEGF165-VEGFR2 signaling pathway (2B7);

[0072] Figure 12 Experimental results for neutralizing the VEGF165-VEGFR2 signaling pathway (3B12, 3C4);

[0073] Figure 13 Experimental results for neutralizing the VEGF165-VEGFR2 signaling pathway (1F9);

[0074] Figure 14 Experimental results (3F12, 3F2) for neutralizing the VEGF165-VEGFR2 signaling pathway. Detailed Implementation

[0075] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0076] As used herein, “sdAb” (also referred to as nanobodies or VHH by the developer Ablynx) is well known to those skilled in the art. A single-domain antibody is an antibody whose complementarity-determining region is part of a single-domain polypeptide. Therefore, a single-domain antibody contains a single complementarity-determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single-domain antibodies include antibodies containing only heavy chains (which naturally do not contain light chains), single-domain antibodies derived from conventional antibodies, and engineered antibodies.

[0077] Single-domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species such as camels, dromedaries, llamas, and guanacos. Like complete antibodies, single-domain antibodies can selectively bind to specific antigens. Single-domain antibodies may contain only variable domains of the immunoglobulin chain, which have CDR1, CDR2, and CDR3, as well as a frame region.

[0078] As used in this article, the term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc fragment of a target antibody with a biologically active functional protein molecule using genetic engineering techniques.

[0079] The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (such as an animal antibody) with the constant region of a human antibody, or by transplanting the complementarity-determining region (CDR1-3 sequence) of a target antibody into the variable region of a human antibody, or by mutating the target antibody according to the characteristics of the human antibody backbone region (FR1-4). Humanized antibodies can be produced using synthetic methods or site-directed mutagenesis methods.

[0080] In this invention, sequences with high homology to the CDR1-3 sequences disclosed herein can also yield single-domain antibodies against VEGFR2. In some embodiments, sequences having "at least 80% homology," or "at least 85% homology," "at least 90% homology," "at least 95% homology," or "at least 98% homology" with the sequences in SEQ ID NO: 1-7 can achieve the purpose of the invention.

[0081] In some embodiments, the inventive objective can also be achieved by replacing only one or a few amino acids compared to the sequences in SEQ ID NO: 1-7, for example, by including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. In practice, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 in a single-domain antibody, those skilled in the art can consider so-called “conserved” amino acid substitutions. In the case of substitution, the substitution will preferably be a conserved amino acid substitution, which can generally be described as an amino acid residue replaced by another amino acid residue having a similar chemical structure, and this substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are common in the art. For example, a conserved amino acid substitution is the substitution of one or a few amino acids in the following groups (a)-(d) by another or a few amino acids in the same group: (a) polar negatively charged residues and their non-charged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conserved amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. The preferred host cell of this invention is a bacterial cell, a fungal cell, or a mammalian cell.

[0082] This patent involves preparing a target protein and its truncated form using genetic engineering technology. The obtained antigen protein is then used to immunize Bactrian camels in Alashan, Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells from the camels are obtained. Through genetic engineering, the variable region coding sequence of the camel-derived antibody is recombined into a phage display vector. Specific antibodies against the antigen protein are then screened using phage display technology, and their ability to bind to the antigen and their application in inhibiting tumors are further tested.

[0083] The above technical solution will now be broken down and explained in detail, and described with specific embodiments:

[0084] Example 1: Preparation of human VEGFR2 protein:

[0085] The human recombinant extracellular domain protein used in this patent was expressed and purified by the company itself. The specific design scheme of the expression vector for the human recombinant VEGFR2 protein is as follows:

[0086] (1) The coding sequence of VEGFR2 was obtained by searching in NCBI. Its accession number is NM_002253.2, and the accession number of the amino acid sequence encoded by this sequence is NP_002244.1.

[0087] (2) The nucleotide sequence encoding amino acids 20 to 764 of the VEGFR2 protein was cloned into the vector pcDNA3.4 using gene synthesis. The constructed vector was subjected to Sanger sequencing and compared with the original sequence. After confirming that there were no errors, the recombinant plasmid was extracted in large quantities, endotoxin was removed, and it was transfected into suspension 293F cells for expression and purification of the target protein. The purity reached more than 90%, which met the requirements for animal immunization.

[0088] SDS-PAGE gel image of purified human VEGFR2 protein as shown below Figure 1 As shown, M is a protein marker.

[0089] Example 2: Construction of a single-domain antibody library targeting the VEGFR2 protein:

[0090] One mg of the purified human recombinant VEGFR2 protein obtained in Example 1 was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alashan, Inner Mongolia. The camel was immunized once a week for a total of 7 weeks. Except for the first immunization, the remaining six immunizations were performed by mixing one mg of VEGFR2 protein with an equal volume of Freund's incomplete adjuvant. This immunization process was intended to concentrate the stimulation of the camel to produce antibodies against VEGFR2 protein.

[0091] After animal immunization, 150 mL of peripheral blood lymphocytes were collected from camels, and RNA was extracted from the cells. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified using nested PCR with the cDNA as a template.

[0092] Then, the pMECS vector and VHH fragment were digested with restriction endonucleases, and the digested fragments were ligated to the vector. The ligated fragments were electroporated into competent TG1 cells to construct a phage display library of VEGFR2 protein, and the library size was determined to be approximately 1 × 10⁻⁶. 9 Meanwhile, the correct insertion rate of the target fragment in the library was detected by colony PCR.

[0093] The results showed that after PCR amplification of 30 colonies randomly selected from the library, 28 clones were able to amplify bands of the predicted size, while 2 clones amplified bands incorrectly. Therefore, the correct insertion rate was 28÷30×100%≈93%.

[0094] Example 3: Screening for single-domain antibodies against VEGFR2 protein:

[0095] Take 200 μL of the recombinant TG1 cells from Example 2 and culture them in 2×TY medium. During the culture, add 40 μL of helper phage VCSM13 to infect the TG1 cells and culture them overnight to amplify the phage. The next day, precipitate the phage with PEG / NaCl and collect the amplified phage by centrifugation.

[0096] 500 μg of VEGFR2 protein diluted in 100 mM pH 8.3 NaHCO3 was coupled onto an ELISA plate and incubated overnight at 4°C. A negative control well (culture medium control) was also included. The next day, 200 μL of 3% skim milk was added, and the plate was blocked at room temperature for 2 hours. After blocking, 100 μL of the amplified phage library (approximately 2 × 10⁻⁶) was added. 11 (1 phage particle), incubate at room temperature for 1 hour; after 1 hour, wash 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0097] Phages that specifically bind to VEGFR2 protein were dissociated using trypsin at a final concentration of 25 mg / mL and then used to infect E. coli TG1 cells in the logarithmic growth phase. The cells were cultured at 37°C for 1 h to produce and collect phages for the next round of screening. The same screening process was repeated once to gradually enrich the cells.

[0098] When the enrichment factor reaches 10 times or more, the enrichment effect is as follows: Figure 2 As shown.

[0099] Figure 2 In this context, P / N = the number of monoclonal bacteria grown from phages eluted from positive wells in the biopanning process after infecting TG1 bacteria / the number of monoclonal bacteria grown from phages eluted from negative wells after infecting TG1 bacteria. This parameter gradually increases after enrichment occurs. I / E = the total number of phages added to positive wells in each round of the biopanning process / the total number of phages eluted from positive wells in each round of the biopanning process. This parameter gradually approaches 1 after enrichment occurs.

[0100] Example 4: Screening for VEGFR2-specific positive clones using phage enzyme-linked immunosorbent assay (ELISA):

[0101] Two rounds of screening were conducted on single-domain antibodies against the anti-VEGFR2 protein according to the screening method in Example 3 above. The phage enrichment factor against the anti-VEGFR2 protein reached more than 10. After the screening, 384 single colonies were selected from the positive clones and inoculated into 96-well plates of 2×TY medium containing 100 μg / mL ampicillin. A blank control was set up. After incubation at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM and incubated overnight at 28°C.

[0102] Crude antibody was obtained using the osmotic burst method. VEGFR2 recombinant protein was released into 100 mM NaHCO3 (pH 8.3), and 100 μg of protein was coated overnight at 4°C in an ELISA plate. 100 μL of the obtained crude antibody extract was transferred to an ELISA plate containing the antigen and incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and 100 μL of Mouse Anti-HA tag Antibody (HRP) (Mouse anti-HA horseradish peroxidase labeled antibody, ThermoFisher) diluted 1:2000 was added. The plate was incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and horseradish peroxidase chromogenic solution was added. The reaction was carried out at 37°C for 15 min, and then stop solution was added. The absorbance was read at 450 nm using an ELISA reader.

[0103] When the OD value of the sample well is more than 5 times that of the control well, it is determined to be a positive clone well. The bacteria in the positive clone well are transferred to LB medium containing 100 μg / mL ampicillin for plasmid extraction and sequencing.

[0104] Gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while clones with different sequences were considered different clones. Ultimately, single-domain antibodies specifically targeting the VEGFR2 protein were obtained (including single-domain antibodies 1A8, 1F9, 2B7, 3B12, 3C4, 3F12, 3F2, and single-domain antibodies with sequences not shown: 1A11, 1A3, 1A4, 1A9, 1C3, 2C11, 2C12, 2C3, 2C4, 2D1, 3A7, 3B2, 3B3, 3C1, 3C10, 3C12, 3C2, 3C3, 3C5, 3C6, 1C9, 2A10, 2A9, 2B1, 2B5, 3C7, 3D6, 3E6, 3F3).

[0105] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, constituting the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system to ultimately obtain the single-domain antibody protein.

[0106] The amino acid sequences of single-domain antibodies 1A8, 1F9, 2B7, 3B12, 3C4, 3F12, and 3F2 are shown in SEQ ID NO:1-7, and their nucleotide sequences are shown in SEQ ID NO:8-14.

[0107] The CDR sequences of the seven single-domain antibodies are shown in Tables 1-3. The FR1 sequences of the seven single-domain antibodies are shown in Table 4, the FR2 sequences of the seven single-domain antibodies are shown in Table 5, the FR3 sequences of the seven single-domain antibodies are shown in Table 6, and the FR4 sequences of the seven single-domain antibodies are shown in Table 7.

[0108] Table 1. CDR1 sequences of 7 single-domain antibodies

[0109]

[0110] Table 2. CDR2 sequences of 7 single-domain antibodies

[0111]

[0112] Table 3. CDR3 sequences of 7 single-domain antibodies

[0113]

[0114] Table 4. FR1 sequences of 7 single-domain antibodies

[0115]

[0116] Table 5. FR2 sequences of 7 single-domain antibodies

[0117]

[0118] Table 6. FR3 sequences of 7 single-domain antibodies

[0119]

[0120] Table 7. FR4 sequences of seven single-domain antibodies

[0121]

[0122] Example 5: Purification and expression of a specific single-domain antibody against VEGFR2 protein in the host bacterium *Escherichia coli*.

[0123] The plasmids (pMECS-VHH) of different clones obtained from sequencing analysis in Example 4 were electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose culture plates containing ampicillin and glucose, and incubated overnight at 37°C. Single colonies were selected and inoculated into 5 mL of LB culture medium containing ampicillin and incubated overnight on a shaker at 37°C.

[0124] Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium and incubate at 37°C in a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and incubate overnight at 28°C in a shaker. Centrifuge to collect E. coli and obtain crude antibody extract using the osmotic rupture method.

[0125] The antibody was purified by nickel column affinity chromatography.

[0126] Example 6: Construction of a eukaryotic expression vector for an Fc fusion antibody against a single domain antibody of anti-VEGFR2

[0127] (1) Subcloning the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibody screened in Example 4 was obtained by Sanger sequencing to obtain its nucleotide sequence;

[0128] (2) The above nucleotide sequences (SEQ ID NO:8-14 and nucleotide sequences of other single-domain antibody clones not shown) were synthesized into the vector RJK-V4-hFC1 designed and modified by our company by sequence synthesis to obtain a recombinant eukaryotic expression vector. The modification method of the vector is as described in Example 10.

[0129] (3) Transform the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culture it for plasmid extraction, and remove endotoxins;

[0130] (4) The extracted plasmids were then sequenced and identified.

[0131] (5) After confirming the recombinant vector, prepare it for subsequent eukaryotic cell transfection and expression. After expressing the Fc protein of VHH by the method of Example 7 or 8, purify the above antibody by the method of Example 9.

[0132] Example 7: Expression of single-domain antibody against VEGFR2 protein in suspension ExpiCHO-S cells

[0133] (1) Three days before transfection, use 2.5×10 5 / mL cell passage and expansion culture ExpiCHO-S TM Cells, the calculated desired cell volume, were transferred to 120 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium, the cell concentration was increased to approximately 4 × 10⁻⁶ cells / mL. 6 -6×10 6 live cells / mL;

[0134] (2) One day before transfection, ExpiCHO-S TM Cells were diluted to a concentration of 3.5 × 10⁻⁶.6 Live cells / mL, incubate cells overnight;

[0135] (3) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 7 × 10⁻⁶ cells / day before transfection. 6 -10×10 6 live cells / mL;

[0136] (4) Use fresh ExpiCHO preheated to 37°C TM The expression medium was used to dilute the cells to 6 × 10⁶. 6 viable cells / mL. The calculated desired cell volume was transferred to 100 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium;

[0137] (5) Gently invert to mix ExpiFectamine TM CHO reagent, using 3.7 mL OptiPRO TM Culture medium for diluting ExpiFectamine TM CHO reagent, vortex or mix well;

[0138] (6) Use 4 mL of refrigerated OptiPRO TM Dilute the plasmid DNA in the culture medium and vortex to mix.

[0139] (7) Incubate the ExpiFectamine CHO / plasmid DNA (the plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the anti-VEGFR2 single-domain antibody prepared in Example 6) complex at room temperature for 1-5 minutes, and then gently add it to the prepared cell suspension while gently shaking the flask during the addition process.

[0140] (8) The cells were cultured with shaking in humidified air at 37°C and 8% CO2.

[0141] (9) Add 600 μl of ExpiFectamine on the first day after transfection (18-22 hours later). TM CHO Enhancer and 24mLExpiCHO feed;

[0142] (10) Collect the supernatant about 8 days after transfection (when cell viability is less than 70%).

[0143] Example 8: Expression of single-domain antibody against VEGFR2 protein in suspension 293F cells

[0144] Recombinant single-domain antibody expression experimental procedure (taking a 500mL shake flask as an example):

[0145] (1) Three days before transfection, use 2.5×10 5 After passage and expansion of 293F cells at / mL, the calculated desired cell volume was transferred to a 500mL shake flask containing 120mL (final volume) of fresh, preheated OPM-293CD05 Medium to achieve a cell concentration of approximately 2×10⁻⁶. 6 -3×10 6 Live cells / mL.

[0146] (2) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 2 × 10⁻⁶ cells / day before transfection. 6 -3×10 6 Live cells / mL.

[0147] (3) Dilute the cells to 1×10⁻⁵ using preheated OPM-293CD05 Medium. 6 1 live cells / mL. Calculate the required cell volume and transfer it to a 500 mL shake flask containing 100 mL (final volume) of fresh, preheated culture medium.

[0148] (4) Dilute PEI (1 mg / mL) reagent with 4 mL Opti-MEM medium, and mix by swirling or pipetting; dilute plasmid DNA (the plasmid DNA is the eukaryotic expression vector of the Fc fusion antibody of the anti-VEGFR2 single-domain antibody prepared in Example 6) with 4 mL Opt-MEM medium, mix by swirling, and filter with a 0.22 μm filter. Incubate at room temperature for 5 min.

[0149] (5) Add the diluted PEI reagent to the diluted DNA and mix by inverting. Incubate the PEI / plasmid DNA complex at room temperature for 15-20 minutes, then gently add it to the prepared cell suspension while gently shaking the flask during the addition process.

[0150] (6) The cells were cultured at 37°C, 5% CO2, and 120 rpm with shaking.

[0151] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0152] (8) Collect the supernatant about 7 days after transfection (when cell viability is less than 70%).

[0153] Example 9: Purification of single-domain antibody against VEGFR2 protein

[0154] (1) The protein expression supernatant obtained in Example 7 or 8 was filtered with a 0.45 μm disposable filter to remove insoluble impurities;

[0155] (2) The above filtrate was purified by affinity chromatography using a protein purifier. The agarose packing material coupled with Protein A was used to purify the filtrate by utilizing the ability of human Fc to bind to Protein A.

[0156] (3) Pass the filtrate through a pre-packed Protein A column at a flow rate of 1 mL / min. In this step, the target protein in the filtrate will bind to the packing material.

[0157] (4) Wash the impurity proteins bound to the column with low-salt and high-salt buffers;

[0158] (5) Elute the target protein bound to the column with a low pH buffer;

[0159] (6) Quickly add the eluent to a Tris-HCl solution with a pH of 9.0 to neutralize it;

[0160] (7) After dialysis of the above-neutralized protein solution, perform SDS-PAGE analysis to confirm that the protein purity is above 95% and the concentration is above 0.5 mg / mL, and then store it at low temperature for later use.

[0161] Example 10: Construction of a single-domain antibody eukaryotic expression vector

[0162] The universal target vector RJK-V4-hFC1 for the nanobody mentioned is a modified version of Invitrogen's commercial vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf), which incorporates the Fc region of the human IgG1 heavy chain coding sequence. Specifically, this vector contains the CH2 and CH3 hinge regions of the IgG1 heavy chain. The specific modification scheme is as follows:

[0163] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0164] (2) Multiple cloning sites (MCS) and 6×His tags were introduced at the 5' and 3' ends of the Fc fragment coding sequence, respectively, by overlapping PCR.

[0165] (3) The above fragment was amplified by PCR using a pair of primers with XbaI and AgeI restriction sites respectively;

[0166] (4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction endonucleases XbaI and AgeI, respectively;

[0167] (5) The digested vector and the insert fragment were ligated with T4 ligase, and then the ligation product was transformed into E. coli, amplified, sequenced and verified to obtain the recombinant plasmid.

[0168] The name with hFc refers to: the Fc fusion antibody obtained by cloning the corresponding single-domain antibody sequence into RJK-V4-hFC1 and then expressing it in eukaryotes (e.g., 1A8-hFc is the Fc fusion antibody obtained by cloning the nucleotide sequence corresponding to the 1A8 single-domain antibody into RJK-V4-hFC1 and then expressing it in eukaryotes).

[0169] Example 11: Expression and purification of a tool antibody (Tab1) targeting human VEGFR2

[0170] In this article, Tab1 refers to ramucirumab (CYRAMZA). TM )).

[0171] The searched sequences were commissioned to General Biosystems (Anhui) Co., Ltd. for codon optimization in mammalian cell expression systems and cloned into the pcDNA3.1 vector. After antibiotic selection, plasmid-positive bacteria were selected for amplification, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat#740412.50). 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) was added per 100 mL of cells, and PEI was used to transiently express the plasmid in 293F cells (medium: FreeStyle 293Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032). 6–24 h after transfection, 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added, and the cells were cultured in 8% CO2 at 130 rpm for approximately 7–8 days. When cell viability decreased to 50%, the expression supernatant was collected and purified using a gravity column with Protein A (GE, Cat#17-5438-02). After dialysis with PBS, the concentration was determined using Nanodrop, purity was identified by SEC, and binding capacity was verified by indirect ELISA.

[0172] The Tab1 obtained by this method has a concentration of not less than 2 mg / ml and a purity greater than 95%.

[0173] Example 12: Determination of Antibody-Antigen Binding Dose-Reaction Curve

[0174] This embodiment uses the standard enzyme-linked immunosorbent assay (ELISA) procedure.

[0175] (1) Coat 50 μL of 1 μg / mL VEGFR2 protein and incubate overnight at 4°C.

[0176] (2) Wash the plate; add 200 μL of 5% milk and seal at 37°C for 2 hours.

[0177] (3) VHH-hFc was diluted to 2 μg / mL, and then the antibody was serially diluted 5-fold to a total of 8 concentration gradients. VHH-hFc was obtained from the Fc fusion antibody of the single-domain antibody against VEGFR2 protein prepared in Example 8 (expressed in 293F cells) purified in Example 9. In addition, hIgG and Tab1 controls were set up respectively; Tab1 was prepared in Example 11; hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target and was obtained through commercial purchase.

[0178] (4) Wash the plate; add 50 μL of the single-domain antibody obtained by dilution in step (3), double replicates, and incubate at 37°C for 1 h.

[0179] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37°C for 30 min.

[0180] (6) Wash the plate (wash several times); add 50 μL of TMB that has been brought back to room temperature, and react at room temperature in the dark for 15 min.

[0181] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0182] (8) Plot the curve and calculate EC50, as follows: Figures 3-8 As shown.

[0183] Example 13: Experiment on neutralizing the VEGF165-VEGFR2 signaling pathway.

[0184] The following operations shall be performed in accordance with methods commonly used by those skilled in the art:

[0185] HEK293-VEGFR2-NFκB-Luciferase reporter system cells were seeded at 30,000 cells per well in a 96-well plate. 4 ng / mL VEGF165 protein solution was added, followed by mixing with antibody at an initial concentration of 75 μg / mL. After incubation for 4.5 hours, cell fluorescence intensity was detected using One-Glo. The volume of VEGF165 protein solution added was half the volume of the HEK293-VEGFR2-NFκB-Luciferase reporter system cells, and the volume of antibody added was equal to the volume of the VEGF165 protein solution. The reporter system cells were purchased from Suzhou Ruian Biotechnology Co., Ltd. (catalog number RA-CK24). VEGF165 protein was purchased from Novoprotein (catalog number C083). The antibody was obtained by purifying the Fc fusion antibody of the anti-VEGFR2 single-domain antibody prepared in Example 8 using the antibody obtained in Example 9.

[0186] hIgG and Tab1 controls were set up separately; Tab1 was prepared in Example 11; hIgG refers to the isotype control, which is an immunoglobulin molecule that does not bind to any target and is obtained through commercial purchase.

[0187] The results are as follows Figures 9-14 As shown.

[0188] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An anti-VEGFR2 single-domain antibody, characterized in that: The single-domain antibody is composed of a heavy chain, which comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3; the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are: a CDR1 as represented by SEQ ID NO: 31, a CDR2 as represented by SEQ ID NO: 37, and a CDR3 as represented by SEQ ID NO:

42.

2. The single-domain antibody against VEGFR2 according to claim 1, characterized in that: The heavy chain further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR4; the amino acid sequences of the framework region FR are: FR1 or a variant of FR1 as represented by SEQ ID NO: 15 or 16, the variant of FR1 comprising up to 5 substitutions of amino acids in the FR1; FR2 or a variant of FR2 as represented by SEQ ID NO: 21, the variant of FR2 comprising up to 5 substitutions of amino acids in the FR2; FR3 or a variant of FR3 as represented by SEQ ID NO: 24, the variant of FR3 comprising up to 5 substitutions of amino acids in the FR3; and FR4 or a variant of FR4 as represented by SEQ ID NO: 28, the variant of FR4 comprising up to 5 substitutions of amino acids in the FR4.

3. The single-domain antibody against VEGFR2 according to claim 1, characterized in that: The amino acid sequence of the single-domain antibody is represented by SEQ ID NO: 1 or SEQ ID NO:

4.

4. An Fc fusion antibody or a humanized antibody of the single-domain antibody against VEGFR2 according to any one of claims 1 to 3.

5. A nucleotide molecule encoding the anti-VEGFR2 single-domain antibody of any one of claims 1 to 3, characterized in that: The nucleotide sequence thereof is represented by SEQ ID NO: 8 or SEQ ID NO:

11.

6. An expression vector, characterized by: The nucleotide molecule encoding the single-domain antibody against VEGFR2 according to any one of claims 1 to 3 or the Fc fusion antibody or the humanized antibody according to claim 4, or the nucleotide molecule according to claim 5.

7. A host cell, characterized in that: The single-domain antibody against VEGFR2 according to any one of claims 1 to 3 or the Fc fusion antibody or the humanized antibody according to claim 4, which can be expressed, or the expression vector according to claim 6, which comprises the same.

8. A pharmaceutical composition, characterized by: The pharmaceutical composition comprises the single-domain antibody against VEGFR2 according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

9. Use of the anti-VEGFR2 single-domain antibody according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 8 for the manufacture of a medicament for the treatment of a disease, characterized in that, The disease is liver cancer, colorectal cancer, non-small cell lung cancer, gastroesophageal junction adenocarcinoma, gastric cancer, esophageal cancer, bladder cancer, cholangiocarcinoma, melanoma, ovarian cancer, renal cancer, breast cancer or pancreatic cancer.

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