Anti-Her3 antibodies and their applications

Abstract

This invention discloses an anti-Her3 antibody and its applications. The anti-Her3 antibody comprises a light chain and a heavy chain. The amino acid sequence of the variable region of the light chain is shown as positions 1-113 in SEQ ID NO:13, and the amino acid sequence of the variable region of the heavy chain is shown as positions 1-117 in SEQ ID NO:3. The expression level of the anti-Her3 antibody of this invention reaches 3-5 times that of existing antibodies, while maintaining a high affinity for the target Her3.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to an anti-Her3 antibody and application thereof. BACKGROUND

[0002] The Her (Human epidermal growth factor receptor) family consists of four structurally similar and functionally related RTKs, namely Her1 (EGFR), Her2, Her3 and Her4, and the dimerization between receptors is the basic condition for the Her family to exert its function and signal transduction activity. After the dimerization of the receptors, the highly conserved kinase tail in the cell is induced to cross-link and phosphorylate, and then the downstream proteins are recruited and activated, causing a cascade of signal reactions, regulating cell proliferation, survival, migration, occurrence, metastasis and other processes. Her3 can bind to neuregulin (NRG), but due to its lack of intrinsic tyrosine kinase activity, it can only function by forming a heterodimer with other receptors. On the contrary, Her2 has tyrosine kinase activity, but there is no corresponding ligand to bind to it. The extracellular region structure of Her2 is a natural open conformation, which makes Her2 dimerize with other receptors without ligand activation, so Her2 is the preferred dimerization partner of Her3.

[0003] Her2 and Her3 form a heterodimer, which can directly activate the PI3K / AKT pathway, which is the most important signal pathway for promoting tumor cell proliferation, and is involved in regulating gene expression, cell metabolism, cytoskeleton rearrangement and other processes, so the Her2-Her3 heterodimer is considered to be the dimer with the strongest signal transduction ability in the Her family. Studies have shown that drugs that directly or indirectly inhibit the PI3K / AKT pathway can lead to an increase in Her3 transcription, up-regulate Her3 expression and activation through PI3K / AKT negative feedback regulation, re-activate the downstream pathway and produce drug resistance. This phenomenon is related to the drug resistance of anti-Her2 and anti-EGFR targeted therapy. In addition, the main ligand of Her3, NRG1, can participate in Her3 signal transduction through paracrine and autocrine pathways, thereby inducing Her3 pathway activation, and may also be involved in the drug resistance of Her family targeted therapy. In addition, Her3 can also form dimers with other Her family members such as EGFR, and non-Her family members such as MET and IGF-1R, and is involved in the occurrence and development of tumors. Given that Her3 can form heterodimers with other targets to transmit the strongest PI3K / AKT, and due to the lack of tyrosine kinase activity, the treatment drugs for Her3 mainly target the extracellular region of Her3 to develop antibody drugs.

[0004] The development of antibody drugs, the expression of antibodies is crucial to the control of production cost, and the factors affecting the efficient expression of antibody genes in mammalian cells mainly include the sequence of antibody genes, the integration site of antibody genes on the host chromosome, the copy number of antibody genes, the transcription and translation level, the selection and modification of host cells, and the balanced expression of light and heavy chain genes. Among them, the five CDRs in the antibody sequence except the heavy chain CDR3 have fixed characteristics in sequence and structure, which show specific CDR length, and there are some conservative sites in the CDR or framework region of the antibody sequence, which are crucial to maintaining the conformation and function of the CDR / loop region. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the lack of high expression of anti-Her3 antibody in the prior art, and to provide an anti-Her3 antibody and its application. The expression of the anti-Her3 antibody of the present application reaches 3-5 times that of the existing antibody, while maintaining high affinity to the target Her3.

[0006] In order to improve the expression of antibodies and save production cost, the inventors tried to modify various aspects of antibodies. For example, the inventors optimized the codons of existing monoclonal antibodies, replaced the signal peptide, replaced the host cells, adjusted the stable transfection conditions, etc., in an attempt to improve antibody expression. However, the expression of the antibodies modified by the foregoing methods in host cells still did not meet the expected standard. In a large number of experiments, the inventors found by layer-by-layer screening that the amino acid sequence of the light chain of the antibody had a key influence on the expression of the antibody in the cross-transfection of light and heavy chain combinations. Based on this research result, the inventors modified the light chain of the antibody, and screened the light chain mutants to obtain an antibody with high expression and high binding activity to Her3. The expression of the antibody was significantly improved, and the expression was increased by 3-5 times compared with the antibody before modification.

[0007] The present application solves the above technical problems by the following technical solutions.

[0008] The first aspect of the present application provides an anti-Her3 antibody, which comprises a light chain and a heavy chain.

[0009] The amino acid sequence of the light chain variable region in the light chain is shown in SEQ ID NO: 13, and the amino acid sequence of the heavy chain variable region in the heavy chain is shown in SEQ ID NO: 3.

[0010] In some embodiments of the present application, the light chain further comprises a light chain constant region, and the heavy chain further comprises a heavy chain constant region.

[0011] In the present application, the light chain constant region is preferably the light chain constant region of a human antibody or a mouse antibody; more preferably the constant region of the kappa or lambda chain of a human antibody.

[0012] In the present application, the heavy chain constant region is preferably the heavy chain constant region of a human antibody or a mouse antibody; more preferably the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4 of a human antibody.

[0013] In some embodiments of the present application, the amino acid sequence of the light chain is as shown in SEQ ID NO: 13.

[0014] In some embodiments of the present application, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 3.

[0015] In the present application, the heavy chain of the anti-Her3 antibody can be a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 3 with one or more mutations (deletion, substitution or addition) of amino acid residues, and having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% or more homology with the amino acid sequence as shown in SEQ ID NO: 3; preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95%, 96%, 97% or 98% homology, and most preferably at least 99% homology.

[0016] The second aspect of the present application provides a fusion protein comprising the anti-Her3 antibody of the first aspect.

[0017] The third aspect of the present application provides an isolated nucleic acid encoding the anti-Her3 antibody of the first aspect or the fusion protein of the second aspect.

[0018] In some embodiments of the present application, the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO: 14.

[0019] In some embodiments of the present application, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID NO: 6.

[0020] In some embodiments of the present application, the nucleotide sequence encoding the light chain is as shown in SEQ ID NO: 14.

[0021] In some embodiments of the present application, the nucleotide sequence encoding the heavy chain is as shown in SEQ ID NO: 6.

[0022] The fourth aspect of the present application provides a recombinant expression vector comprising the nucleic acid according to the third aspect.

[0023] The fifth aspect of the present application provides an antibody conjugate comprising the anti-Her3 antibody according to the first aspect or the fusion protein according to the second aspect.

[0024] The sixth aspect of the present application provides a bispecific antibody molecule comprising the anti-Her3 antibody according to the first aspect or the fusion protein according to the second aspect.

[0025] The seventh aspect of the present application provides a chimeric antigen receptor T cell comprising the anti-Her3 antibody according to the first aspect or the fusion protein according to the second aspect.

[0026] The eighth aspect of the present application provides a pharmaceutical composition comprising the anti-Her3 antibody according to the first aspect, the fusion protein according to the second aspect, the antibody conjugate according to the fifth aspect, the bispecific antibody molecule according to the sixth aspect, or the chimeric antigen receptor T cell according to the seventh aspect, and pharmaceutically acceptable salts, solvates, or solvates of the pharmaceutically acceptable salts thereof.

[0027] In some embodiments of the present application, the pharmaceutical composition further comprises a pharmaceutical excipient.

[0028] The ninth aspect of the present application provides a kit comprising the anti-Her3 antibody according to the first aspect or the fusion protein according to the second aspect.

[0029] The tenth aspect of the present application provides a pharmaceutical kit comprising a kit A and a kit B;

[0030] wherein the kit A comprises the anti-Her3 antibody according to the first aspect or the pharmaceutical composition according to the eighth aspect; and the kit B comprises another therapeutic agent.

[0031] In some embodiments of the present application, the kit A and the kit B are administered simultaneously or the kit A is administered first.

[0032] The eleventh aspect of the present application provides a drug delivery device comprising: (1) an infusion module for administering the pharmaceutical composition according to the eighth aspect to a subject in need thereof, and (2) an optional pharmacodynamic monitoring module.

[0033] The twelfth aspect of the present invention provides a method for treating / preventing a disease, the method comprising administering to a patient in need an effective dose of an anti-Her3 antibody as described in the first aspect, a fusion protein as described in the second aspect, an antibody-drug conjugate as described in the fifth aspect, a bispecific antibody molecule as described in the sixth aspect, a chimeric antigen receptor T cell as described in the seventh aspect, a pharmaceutical composition as described in the eighth aspect, or administering a drug delivery device as described in the eleventh aspect.

[0034] In some embodiments of the present invention, the disease is a tumor;

[0035] In some preferred embodiments of the present invention, the tumor is a Her3-positive tumor.

[0036] In some preferred embodiments of the present invention, the Her3-positive tumor is selected from one or more of Her3-positive lung cancer, ovarian cancer, colorectal cancer, breast cancer, prostate cancer, and gastric cancer.

[0037] The thirteenth aspect of the present invention provides the use of an anti-Her3 antibody as described in the first aspect, a fusion protein as described in the second aspect, an antibody-drug conjugate as described in the fifth aspect, a bispecific antibody molecule as described in the sixth aspect, a chimeric antigen receptor T cell as described in the seventh aspect, or a pharmaceutical composition as described in the eighth aspect in the preparation of a Her3 protein inhibitor or in the preparation of a medicament for the treatment and / or prevention of tumors.

[0038] The preferred definition of the tumor is as described in aspect 12.

[0039] In this invention, the prostate cancer may be prostate cancer as commonly understood in the art, and prostate cancer cells may include, for example, 22Rv1 cells and / or LNCaP cells.

[0040] In this invention, the colorectal cancer may be colorectal cancer as commonly understood in the art, and colorectal cancer cells may include, for example, SW620 cells.

[0041] In this invention, the lung cancer can be lung cancer as commonly understood in the art, and the lung cancer cells may include, for example, NCI-H820 cells or HCC827 cells.

[0042] In this invention, the ovarian cancer may be ovarian cancer as commonly understood in the art, and ovarian cancer cells may include, for example, OVCAR-8 cells.

[0043] In this invention, the breast cancer may be breast cancer as commonly understood in the art, and breast cancer cells may contain, for example, SK-BR-3 cells.

[0044] Unless otherwise stated, the following terms appearing in this specification have the following meanings:

[0045] The pharmaceutical excipients mentioned are those widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods to ensure that the active ingredient dissolves at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipients may be inert fillers, or provide a function such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The pharmaceutical excipients may include one or more of the following: buffers, chelating agents, preservatives, solubilizers, stabilizers, excipients and surfactants, colorants, flavoring agents, and sweeteners.

[0046] The term "pharmaceutically acceptable" means that the salts, solvents, excipients, etc., are generally non-toxic, safe, and suitable for patient use. The term "patient" preferably refers to a mammal, and more preferably a human.

[0047] The term "pharmaceutically acceptable salt" refers to a salt prepared from the compounds of the present invention with a relatively non-toxic, pharmaceutically acceptable acid or base. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting a sufficient amount of a pharmaceutically acceptable base with the neutral form of such compounds in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, lithium salts, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, zinc salts, bismuth salts, ammonium salts, and diethanolamine salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting a sufficient amount of a pharmaceutically acceptable acid with the neutral form of such compounds in a pure solution or a suitable inert solvent. Pharmaceutically acceptable acids include inorganic acids, including but not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, and sulfuric acid. The pharmaceutically acceptable acids include organic acids, including but not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acidic citric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentic acid, fumaric acid, gluconic acid, glycolic acid, formic acid, ethanesulfonic acid, dihydroxynaphthyl acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthoic acid)), amino acids (e.g., glutamic acid, arginine), etc. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts. For details, see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977), or Handbook ofPharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl andCamille G. Wermuth, ed., Wiley-VCH, 2002).

[0048] The term "solvate" refers to a substance formed by the combination of the compound of this invention with a stoichiometric or non-stoichiometric solvent. Solvent molecules in a solvate can exist in an ordered or disordered arrangement. The solvents include, but are not limited to, water, methanol, and ethanol.

[0049] The term "treatment" or its equivalents, when used, for example, in the context of cancer, refers to a procedure or process used to reduce or eliminate the number of cancer cells in a patient's body or to alleviate the symptoms of cancer. "Treatment" for cancer or other proliferative disorders does not necessarily mean that cancer cells or other disorders will actually be eliminated, that the number of cells or disorders will actually be reduced, or that the symptoms of cancer or other disorders will actually be alleviated. Often, methods for treating cancer are undertaken even with only a low probability of success, but are still considered to induce an overall beneficial effect, taking into account the patient's medical history and estimated survival expectations.

[0050] The term "prevention" refers to the reduction of the risk of acquiring or developing a disease or disorder.

[0051] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0052] Unless otherwise specified, room temperature in this invention refers to 20-30°C.

[0053] The reagents and raw materials used in this invention are all commercially available.

[0054] The positive and progressive effects of this invention are as follows:

[0055] The anti-Her3 antibody of the present invention has a high expression level, which can reach 3 to 5 times that of existing antibodies, while maintaining a high affinity for the target Her3, and has a good inhibitory effect on various tumor cells expressing Her3 and other substances. Attached Figure Description

[0056] Figure 1 This is a schematic diagram showing the SDS-PAGE detection results of the purified product expression level in the 2F8 transient expression supernatant (2 days) in Example 2.

[0057] Figure 2 The results of SDS-PAGE analysis of the purified product from the 2F8 transient conversion in Example 2 are shown.

[0058] Where: A is 6% non-reducing SDS-PAGE, and B is 12% reducing SDS-PAGE.

[0059] Figure 3 The results of 12% reducing SDS-PAGE analysis of the supernatant of 2F8 transient transfection after adjusting the light chain transfection ratio in Example 2;

[0060] Where: A represents 2 days after transfection, and B represents 4 days after transfection.

[0061] Figure 4 The results are obtained by 12% reducing SDS-PAGE analysis of the supernatant of the transient expression of the 2F8 and 1A9 light and heavy chain combination in Example 3.

[0062] Figure 5 The result is a 6% non-reducing SDS-PAGE assay of the purified product of 2F8 transient conversion with the light chain signal peptide replaced in Example 3.

[0063] Figure 6 The results are obtained by 12% reducing SDS-PAGE analysis of the purified product of 2F8 transient conversion with the light chain signal peptide replaced in Example 3.

[0064] Figure 7 This is a schematic diagram of the 12% reducing SDS-PAGE detection results of the expression supernatant after transient transduction of the 2F8 light chain mutant in Example 4;

[0065] Where: A is a 3-day instantaneous cycle, and B is a 5-day instantaneous cycle.

[0066] Figure 8 This is a schematic diagram of the 12% reducing SDS-PAGE detection results of the purified product of the 2F8 light chain mutant after 5 days of transient transformation in Example 4.

[0067] Where: A is the transient transformation of the 2F8 light chain mutant, and B is the transient transformation of the 2F8Lm1 repeat.

[0068] Figure 9 This is a Blast diagram of the amino acid sequences of 3F5 (top) and 2F8-L (bottom) in Example 5.

[0069] Figure 10 This is a schematic diagram of the FACS detection results of 2F8 and 3F5 binding on T-47D cells in Example 6.

[0070] Figure 11 This is a schematic diagram of the FACS detection results of 2F8 and 3F5 binding on MDA-MB-453 cells in Example 6.

[0071] Figure 12 The reaction curve of 2F8 with Her3-His recombinant protein is shown.

[0072] Figure 13 The reaction curve of 3F5 with Her3-His recombinant protein is shown. Detailed Implementation

[0073] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0074] Abbreviation Explanation:

[0075] PCR: Polymerase chain reaction;

[0076] CHO: Chinese hamster ovary cells;

[0077] HTRF: Homogeneous time-resolved fluorescence.

[0078] Example 1: Preparation of a cell line stably expressing anti-Her3 antibody

[0079] As shown in Table 1, this embodiment selected the monoclonal antibody 2F8, which has high affinity and specifically targets Her3. The amino acid sequence of its light chain is shown in SEQ ID NO: 1, and the amino acid sequence of its heavy chain is shown in SEQ ID NO: 3. The light and heavy chain nucleotide sequences of 2F8 were synthesized through whole-genome synthesis (Suzhou Genewiz Biotechnology Co., Ltd.). The nucleotide sequence of its light chain is shown in SEQ ID NO: 2, and the nucleotide sequence of its heavy chain is shown in SEQ ID NO: 4. The light and heavy chains were separately constructed into expression vector pV81 (Shanghai Yisheng Biotechnology, pEE14.2) by double digestion with EcoRI and Hind III (TAKARA, R3104S, R3101S). These vectors were then ligated and transformed into Trans 1-T1 competent cells (Beijing Quanshijin Biotechnology, CD501-03). Clones were selected for PCR identification and sequencing confirmation. Positive clones were cultured and amplified, and plasmids were extracted to obtain the antibody light chain eukaryotic expression plasmid 2F8-L / pV81 and the antibody heavy chain eukaryotic expression plasmid 2F8-H / pV81, with a light / heavy chain eukaryotic expression plasmid ratio of 1.5 / 1. These plasmids were then electroporated into CHO cells adapted for suspension growth (ATCC, CCL-61). TM After electroporation for 48 hours, the average expression level measured by HTRF (Homogeneous Time-Resolved Fluorescence) was 1.8 μg / mL, which is relatively low. Following stable transfection screening and subcloning, the top 10 clones with the highest expression levels were selected for further evaluation. After 14 days of fed-batch culture in a fermenter (basal medium: Dynamis AGTMedium, Gibco; supplemental feeding medium: EfficientFeed C+, Gibco), the highest expression level was only 1.2 g / mL. In conclusion, the expression level of antibody 2F8 in host cells CHO did not reach the expected target.

[0080] Table 1. Amino acid and nucleotide sequences of the 2F8 light and heavy chains

[0081]

[0082]

[0083] Example 2. Optimization of anti-Her3 antibody expression

[0084] To address the issue of antibody expression, the study explored various techniques, including optimizing the signal peptide codon, changing the host cell, and adjusting stable transfection conditions, in an attempt to improve antibody expression.

[0085] Different signal peptides were added to the amino acid sequence of antibody 2F8, and codon optimization was performed using the triplet codon encoding principle to obtain two 2F8 light and heavy chain molecules with the same amino acid sequence but different nucleotides (2F8-L-new, whose nucleotide sequence is shown in SEQ ID NO: 5; 2F8-H-new, whose nucleotide sequence is shown in SEQ ID NO: 6; 2F8-L-2, whose nucleotide sequence is shown in SEQ ID NO: 7; 2F8-H-2, whose nucleotide sequence is shown in SEQ ID NO: 8, as detailed in Table 2). Genes were resynthesized and constructed into the expression vector pV81. The light and heavy chains were combined (as shown in Table 2). The cells were then transfected into HEK293 cells (Chinese Academy of Sciences Cell Bank, GNHu43) using the 293fectin transfection reagent (Invitrogen, 12347019), with a transfection volume of 20 mL. Two days after transfection, a small amount of cell culture supernatant was collected to detect the expression level. Four days after transfection, 25 mL of cell culture supernatant was collected, purified using Protein-A affinity in one step, and the expression level of the purified product was detected. The results are as follows: Figure 1 As shown, the results indicate that after transient transfection of HEK293 for 2 days, there was no significant difference in expression levels between wild-type 2F8 and the two optimized sequences.

[0086] Table 2. 2F8 light chain nucleotide sequence, heavy chain nucleoside sequence and combination.

[0087]

[0088]

[0089]

[0090] Four days after transfection, 2F8-new, 2F8-1, and 2F8-2 were purified and quantified. There was no significant difference in expression between 2F8-new and 2F8-1, while 2F8-2 expression was slightly higher than 2F8-1. The baseline expression levels of the antibodies were all low, approximately 20 μg / mL. Protein purification information is shown in Table 3. The results of 6% non-reducing SDS-PAGE analysis of the purified products are shown below. Figure 2 As shown in A, the 12% reducing SDS-PAGE detection results are as follows: Figure 2 As shown in B, the expression ratio of the light chain of the 2F8 molecule is relatively low.

[0091] Table 3. Purification information of supernatant for 2F8 and FDA029 transient expression

[0092]

[0093] The transfection ratio of the 2F8 light chain was adjusted. The light and heavy chain plasmids of 2F8-new were transfected into HEK293 cells at the normal transfection ratio, with the light chain increased by twice the amount. The transfection volume was 20 mL using the 293fectin transfection reagent. On days 2 and 4 after transfection, a small amount of cell culture supernatant was collected to detect expression levels. The detection results of the transiently transfected cell culture supernatant are as follows: Figure 3 As shown in A and B, increasing the light chain transfection ratio did not improve the basic expression level of 2F8-new.

[0094] The light and heavy chain fragments of 2F8-new were constructed into a new expression vector named 2F8-3 and transfected into host cells CHOK1SV (both the vector and cells were derived from Lonza). Antibody 1A9, which showed good expression (its light chain amino acid sequence is shown in SEQ ID NO: 9; its heavy chain amino acid sequence is shown in SEQ ID NO: 10), was used as a system control. The culture medium was CDCHO Medium (Gibco, 10743029). The expression results are shown in Table 5. The expression level of 2F8-3 in CHOK1SV was higher than that of 2F8-new in the original experimental system, but much lower than that of the system control, indicating that antibody 2F8 is a difficult antibody to express.

[0095] Table 4. Amino acid sequences of the 1A9 light and heavy chains

[0096]

[0097] Table 5. Expression levels of 2F8 stably transfected 48 hours later

[0098]

[0099] Example 3. Evaluation of cross-transfection combinations

[0100] The light and heavy chains of the 2F8 molecule were cross-transfected. The light and heavy chain plasmids of 2F8-new and antibody 1A9 were combined (the combination methods are shown in Table 6). These were then transfected into HEK293 cells using the 293fectin transfection reagent, with a transfection volume of 20 mL. Two days after transfection, the expression level was detected using the cell culture supernatant. The detection results of the transiently transfected cell culture supernatant are shown below. Figure 4 As shown, compared with 2F8-new, the expression of 2F8 heavy chain combined with 1A9 light chain is significantly enhanced, while the expression of 2F8 light chain combined with 1A9 heavy chain is not improved. The light chain of 2F8 has a key influence on the expression of 2F8.

[0101] Table 6. Combination methods of 2F8 light and heavy chains with 1A9 light and heavy chains

[0102]

[0103] The 2F8 light chain signal peptide was replaced; the 2F8-new light chain signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO:11) was replaced with a new signal peptide MSVPTQVLGLLLLWLTDARC (SEQ ID NO:12), and transfected into HEK293 cells using the 293fectin transfection reagent, with a transfection volume of 20 mL. Four days after transfection, the cell culture supernatant was collected, with a supernatant volume of 25 mL. After one-step Protein-A affinity purification, the expression level of the purified product was detected. The 6% non-reducing SDS-PAGE results of the purified product of the 2F8 molecule with the replaced light chain signal peptide are as follows. Figure 5 As shown, the results of 12% reducing SDS-PAGE detection are as follows: Figure 6 As shown, after changing the light chain signal peptide, the basal expression of 2F8 was slightly increased, but not significantly improved.

[0104] Example 4. Antibody Sequence Analysis

[0105] Based on the sequence characteristics of the 2F8 light chain, three light chain mutants (Lm1, Lm2, and Lm3) were designed, and their amino acid and nucleotide sequences are shown in Table 7. Vectors were constructed in the same manner as in Example 1 to obtain the light chain mutants 2F8-Lm1, 2F8-Lm2, and 2F8-Lm3.

[0106] Table 7. Sequences of 2F8 molecular mutants

[0107]

[0108]

[0109]

[0110] Transfection with the 2F8 light chain mutant

[0111] The light chain mutant and heavy chain mutant of 2F8 molecule were combined (the combination method is shown in Table 8) and transfected into HEK293 cells using the 293fectin transfection reagent, with a transfection volume of 20 mL. Three days after transfection, a small amount of cell culture supernatant was collected to detect the expression level. Five days after transfection, the cell culture supernatant was collected, with a supernatant volume of 25 mL. After one-step Protein-A affinity purification, the expression level of the purified product was detected.

[0112] Table 8 Combinations of 2F8 light chain mutants

[0113]

[0114] The results of 12% reducing SDS-PAGE analysis of the expression supernatant 3 days after transfection with the 2F8 light chain mutant are as follows: Figure 7 As shown in A, the 12% reducing SDS-PAGE results of the expression supernatant 5 days after transfection are as follows: Figure 7 As shown in B, the 12% reducing SDS-PAGE results of the purified supernatant are as follows: Figure 8 As shown in Figure A, the expression level of 2F8-m1 was significantly increased. Repeat transfection confirmed this, and the 12% reducing SDS-PAGE results of the expression supernatant 5 days post-transfection are shown below. Figure 8 As shown in B, the expression of 2F8-m1 was significantly improved, with an increase of approximately 3-5 times based on SDS-PAGE grayscale analysis and the amount of purified protein. The amino acid differences between the light chain mutant 2F8-Lm1 and 2F8-L are as follows: Figure 9 As shown.

[0115] Example 5. Expression and preparation of site-modified antibodies

[0116] The combination of the 2F8 light chain mutant 2F8-Lm1 and the heavy chain mutant 2F8-H-new was renamed antibody 3F5. ExpiCHO cells were transiently transfected with both antibodies 2F8 and 3F5 according to Table 9 below. The transfection volume was 100 mL per shake flask. After 8 days of culture, the expression level was detected by HTRF. The results showed that the transient transfection expression level of 3F5 was 2.9 times that of 2F8.

[0117] Table 9. Transient transfection of 2F8 and 3F5

[0118]

[0119] Stable cell lines for antibody 3F5 were screened and subcloned. Cells with high expression levels were selected for culture and evaluation. After 14 days of fed-batch culture in a fermenter, the highest expression level of 3F5 was 3.3 g / L, while the expression level of 2F8 was only 1.2 g / L, achieving enhanced expression under large-scale culture. In summary, the expression level of antibody 3F5 is significantly higher than that of antibody 2F8, meeting the expected antibody expression level for the project.

[0120] Example 6. Functional analysis of the mutant sequence of anti-Her3 antibody

[0121] Binding analysis of 3F5 mutant on T-47D cells

[0122] Utilizing the FACS detection binding capability, firstly, according to 3×10 5 ~1×10 6T-47D cells (Chinese Academy of Sciences Cell Bank, TCHU 87) were collected at density and the supernatant was discarded. Cells were washed once with FACS staining buffer (Moregate, 3827104), centrifuged again to discard the supernatant, and resuspended in 2 ml of FACS into two 1.5 mL centrifuge tubes. The cells were incubated on ice for 0.5 h, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. 100 μL of the corresponding concentration gradient of 3F5 mutant (10 μg / mL, 3.33 μg / mL, 1.11 μg / mL, 0.37 μg / mL, 0.12 μg / mL) was added to the cells. After incubation on ice for 1 h, the cells were centrifuged to discard the supernatant. 0.5 mL of FACS staining buffer was added to each tube, and the cells were centrifuged at 1000 rpm for 5 min (4℃). The supernatant was discarded. This process was repeated twice. Add 100 μL of fluorescently labeled secondary antibody (Thermo, A11013), incubate on ice for 30 min, centrifuge to remove supernatant, add 0.5 mL / tube of FACS staining buffer, centrifuge at 1000 rpm for 5 min (4℃), remove supernatant, repeat twice; add 0.5 mL of PBS dilution buffer (Shanghai Double Helix Biotechnology Co., Ltd., P10033) to resuspend cells, and incubate on ice in the dark; detect fluorescence values ​​by flow cytometry. FACS detection results are as follows. Figure 10 As shown in the figure. The results showed that 3F5 bound to T-47D cells in a dose-dependent manner. There was no significant difference in binding ability to the parental molecule 2F8 at different antibody concentrations.

[0123] The results of FACS testing using the above method are as follows: Figure 11 As shown, 2F8Lm1 binds to MDA-MB-453 cells (Chinese Academy of Sciences Cell Bank, TCHU233) with a good dose-response relationship. There is no significant difference in binding ability to the parental molecule 2F8 at different antibody concentrations.

[0124] Example 7.3 Affinity Analysis of F5 Mutant

[0125] The affinity of antibodies 2F8 and 3F5 for the recombinant Her3-His protein was detected using the BLI method to measure the binding kinetics of the immobilized antibody to Her3. The method followed the instrument's (Fortebio, Octet 96e) instructions. In short, the AMC sensor was first equilibrated for 60 s with Loading Buffer / Sample Dilution Buffer (1×PBS, pH 7.4, 0.1% BSA + 0.02% Tween-20) to obtain Baseline 1. The antibody was then diluted to a concentration of 10 μg / mL with Loading Buffer and bound to the equilibrated sensor. The bound sensor was then reequilibrated with Loading Buffer to obtain Baseline 2. Finally, the antibody-loaded sensor was placed in human Her3-His diluted to 100–3.13 nM with sample dilution buffer and bound for 90 s to obtain the antibody-protein binding curve, as shown below. Figure 12 and Figure 13 As shown in the figure. The antigen-bound sensor was then placed in Sample Dilution Buffer for 180 s to dissociate, yielding the dissociation curve. The k-on and k-off values ​​of antibody-protein binding were calculated from the binding and dissociation curves, and the KD value was calculated. The results showed that there was no difference in affinity between 2F8 and 3F5 for the Her3-His recombinant protein. The affinity of 2F8 was 2.43E-09 mol / L (abbreviated as M in Table 10 below), and the affinity of 3F5 was 2.53E-09 mol / L. Detailed kinetic parameters are shown in Table 10 below.

[0126] Table 10. Affinity test results of 2F8 and 2F8Lm1

[0127]

[0128] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims. SEQUENCE LISTING <110> Shanghai Fudan Zhangjiang Bio-Pharmaceutical Co., Ltd. <120> Anti-Her3 antibodies and their applications <130> P23118220CP <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 220 <212> PRT <213> artificial sequence <220> <223> 2F8‑L <400> 1 Asp Ile Glu Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Ser Asn Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Asn Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 2 <211> 663 <212> DNA <213> Synthetic sequence <220> <223> 2F8-L <400> 2 gacatcgaga tgacacagag ccctgacagc ctggccgtga gcctgggcga gagagccacc 60 atcaactgca gaagctctca gagcgtgctg tacagcagca gcaacagaaa ctacctggcc 120 tggtatcagc agaaccctgg acagcctcct aagctgctga tctactgggc tagcacaaga 180 gagagcggcg tgcctgacag attcagcggc agcggcagcg gcaccgactt caccctgacc 240 atcagcagcc tgcaagccga ggacgtggcc gtgtactact gtcagcagta ctacagcacc 300 cctagaacct tcggccaagg caccaaggtg gagatcaaga gaaccgtggc cgcccctagc 360 gtgttcatct tccctcctag cgacgagcag ctgaagagcg gcaccgctag cgtggtgtgc 420 ctgctgaaca acttctaccc tagagaggcc aaggtgcagt ggaaggtgga caacgccctg 480 cagagcggca acagccaaga gagcgtgacc gagcaagaca gcaaggacag cacctacagc 540 ctgagcagca ccctgaccct gagcaaggcc gactacgaga agcacaaggt gtacgcctgc 600 gaggtgaccc accaaggcct gagcagccct gtgaccaaga gcttcaacag aggcgagtgc 660 tga 663 <210> 3 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> 2F8-H <400> 3 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 上标10 上标15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Notes: 1. For the superscript numbers in line [[ID=]]32 , they are presented as "上标" in the translation to show the original format. In a more proper scientific or technical context, they should be formatted correctly as superscripts. 2. The tags - are preserved as they are as per the requirement. 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Glu Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Lys Trp Thr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 4 <211> 1341 <212> DNA <213> Artificial sequence <220> <223> 2F8-H <400> 4 caagtgcagc tgcagcagtg gggtgctggt ctgctgaagc ctagcgagac cctgagcctg 60 acctgcgccg tgtacggcgg cagcttcagc ggctactact ggagctggat cagacagcct 120 cctggcaagg gcctggagtg gatcggcgag atcaaccaca gcggcagcac caactacaac 180 cctagcctga agagcagagt gaccatcagc gtggagacaa gcaagaatca gttcagcctg 240 aagctgtcgt ccgtgaccgc cgccgacacc gccgtgtact actgcgctag agaaagtgg 300 acctggtact tcgacctgtg gggcagaggc accctggtga cggtaagctc cgctagcacc 360 aagggtccta gcgtttttcc gctggcccct agcagcaaga gcacaagcgg cggcaccgcc 420 gccctgggct gtttggtgaa ggactacttc cctgagcctg tcacagtgtc ctggaatagc 480 ggcgccctga caagcggcgt gcacaccttc cctgccgtgc tgcagagcag cggcctgtac 540 agcctgagca gcgtggttac cgtgcctagc agcagcctgg gcacacagac ctacatctgc 600 aacgtgaacc acaagcctag caacaccaag gtggacaaga aggtggagcc taagagctgc 660 gacaagaccc acacctgccc tccttgccct gcccctcctg tggccggcgg ccctagcgtg 720 ttcctgttcc ctcctaagcc taggcacc ctgatgatca gcagaacccc tgaggtgacc 780 tgcgtggtgg tggacgtgag ccacgaggac cctgaggtga agttcaactg gtacgtggac 840 ggcgtggagg tgcacaacgc caagaccaag cctagagagg agcagtacaa cagcacctac 900 agagtggtga gcgtgctgac cgtgctgcac caagactggc tgaacggcaa ggagtacaag 960 tgcaaggtga gcaacaaggc cctgcctgcc cctatcgaga agaccatcag caaggccaag 1020 ggacagccta gagagcctca agtgtacacc ctgcctccta gcagagagga gatgaccaag 1080 aaccaagtga gcctgacttg tcttgtgaag ggcttttacc ctagcgacat cgccgtggag 1140 tgggagagca acggacagcc tgagaacaac tacaagacca cccctcctgt gctggacagc 1200 gacggcagct tcttcctgta cagcaagctg accgtggaca agagcagatg gcagcaaggc 1260 aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac acagaagagc 1320 ctgagcctga gccctggcaa g 1341 <210> 5 <211> 660 <212> DNA <213> Artificial Sequence <220> <223> 2F8-L-new <400> 5 gacatcgaga tgacccagtc tcccgactcc ctggccgtgt ccctgggcga gagagccacc 60 atcaactgcc ggtcctccca gtccgtgctg tactcctcct ccaaccggaa ctacctggcc 120 tggtaccagc agaaccctgg ccagcctccc aagctgctga tctactgggc ctccaccagg 180 gagtctggcg tgcccgaccg gttctctggc tccggatctg gcaccgactt caccctgacc 240 atctcctccc tgcaggccga ggacgtggcc gtgtactact gccagcagta ctactccaca 300 cctcggacct tcggccaggg caccaaggtg gagatcaagc ggaccgtggc ggcgccatct 360 gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gtaccgctag cgttgtgtgc 420 ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480 caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540 ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600 gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660 <210> 6 <211> 1341 <212> DNA <213> Artificial Sequence <220> <223> 2F8‑H‑new <400> 6 caggtgcagc tgcagcagtg gggagctggc ctgctgaagc cctccgagac cctgtccctg 60 acctgcgccg tgtacggagg ctccttctcc ggctactact ggtcctggat cagacagcct 120 cctggcaagg gcctggagtg gatcggcgag atcaaccact ccggctccac caactacaac 180 ccctccctga agtccagggt gaccatctcc gtggagacct ccaagaacca gttctccctg 240 aagctgtcct ccgtgacagc tgccgacaca gccgtgtact actgcgctcg ggacaagtgg 300 acctggtact tcgacctgtg gggcagaggc accctggtga ccgtgtcctc cgctagcacc 360 aagggcccat cggtcttcc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660 gacaaaactc acacatgccc accgtgccca gcacctcctg tggccgggg accgtcagtc 720 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1260 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320 ctctccctgt ctccgggtaa a 1341 <210> 7 <211> 660 <212> DNA <213> Artificial sequence <220> <223> 2F8-L-2 <400> 7 gacattgaga tgacacagag ccctgacagc ctggctgtga gcctgggaga gagagccacc atcaactgca gaagctctca gtctgtgctg gcaacagaaa ctacctggcc tggtatcagc agaaccctgg acagcctcct aagctgctga tctactgggc tagcacaaga gagtctggag tgcctgacag attctctggc tctggctctg gcacagactt caccctgacc 240 atcagcagcc tgcaagctga ggatgtggct gtgtactact gtcagcagta ctacagcacc cctagaacct ttggccaagg caccaaggtg gagatcaaga gaacagtggc tgccccttct gtgttcatct tccctccttc tgatgagcag ctgaagtctg gcacagcctc tgtggtgtgc 420 ctgctgaaca acttctaccc tagagaggcc aaggtgcagt ggaaggtgga caatgccctg cagtctggca acagccaaga gtctgtgaca cagcaagaca gcaaggacag cacctacagc ctgagcagca ccctgaccct gagcaaggct gactatgaga agcacaaggt gtatgcctgt 660. gaggtgaccc accaaggcct gagcagccct gtgaccaga gcttcaacag aggagagtgc <210> 8 <211> 1341 <212> DNA <213> The snowstorm <220> <223> 2F8‑H‑2 <400> 8 caagtgcagc tgcagcagtg gggtgctggt ctgctgaagc cttctgagac cctgagcctg 60 acctgtgctg tgtatggagg cagcttctct ggctactact ggagctggat cagacagcct 120 cctggcaagg gcctggagtg gattggagag atcaaccact ctggcagcac caactacaac 180 cctagcctga agagcagagt gaccatctct gtggagacaa gcaagaatca gttcagcctg 240 aagctgagct ctgtgacagc tgctgacaca gctgtgtact actgtgctag agacaagtgg 300 acctggtact ttgacctgtg gggcagaggc accctggtga cattaagctc tgctagcacc 360 aagggtcctt ctgtgtttcc tctggcccct agcagcaaga gcacctctgg aggcacagct 420 gccctgggct gtttggtgaa ggactacttc cctgagcctg tcacagtgtc ctggaactct 480 ggagccctga cctctggagt gcacaccttc cctgctgtgc tgcagagctc tggcctgtac 540 agcctgagct ctgtggttac agtgcctagc agcagcctgg gcacacagac ctacatctgc 600 aatgtgaacc aaagcctag caacaccaag gtggagaaga aggtggagcc taagagctgt 660 gacaagaccc acacctgccc tccttgccct gcccctcctg tggctggagg cccttctgtg 720 ttcctgttcc ctcctaagcc taaggacacc ctgatgatca gcagaacccc tgaggtgacc 780 tgtgtggtgg tggatgtgag ccatgaggac cctgaggtga agttcaactg gtatgtggat 840 ggagtggagg tgcacaatgc caagaccaag cctagagagg agcagtacaa cagcacctac 900 agagtggtgt ctgtgctgac agtgctgcac caagactggc tgaatggcaa ggagtacaag 960 tgcaaggtga gcaacaaggc cctgcctgcc cctattgaga agaccatcag caaggccaag 1020 ggacagccta gagagcctca agtgtacacc ctgcctccta gcagagagga gatgaccaag 1080 aaccaagtga gcctgacttg tcttgtgaag ggcttttacc cttctgacat tgctgtggag 1140 tgggagagca atggacagcc tgagaacaac tacaagacca cccctcctgt gctggactct 1200 gatggcagct tcttcctgta cagcaagctg acagtggaca agagcagatg gcagcaaggc 1260 aatgtgttca gctgctctgt gatgcatgag gccctgcaca accactacac acagaagagc 1320 ctgagcctga gccctggcaa g 1341 <210> 9 <211> 215 <212> PRT <213> artificial sequence <220> <223> 1A9‑L <400> 9 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 10 <211> 456 <212> PRT <213> Synthetic Sequence <220> <223> 1A9-H <400> 10 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr 20 25 30[[ID=…]] Ala Met His Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Val 35 40 45 Ala Phe Met Ser Tyr Asp Gly Ser Asn Lys Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Ile Ala Ala Gly Gly Asn Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser 115 120 125 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145 150 155 160 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 165 170 175 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 180 185 190 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 195 200 205 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 210 215 220 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 225 230 235 240 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 245 250 255 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 260 265 270 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 275 280 285 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 290 295 300 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310 315 320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 325 330 335 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 340 345 350 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 355 360 365 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 370 375 380 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390 395 400 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 405 410 415 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 420 425 430 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 435 440 445 Ser Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 11 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Original signal peptide <400> 11 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser <210> 12 <211> 20 <212> PRT <213> Artificial sequence <220> <223> New signal peptide <400> 12 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys 20 <210> 13 <211> 220 <212> PRT <213> artificial sequence <220> <223> 2F8‑Lm1 <400> 13 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Ser Asn Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 14 <211> 660 <212> DNA <213> Artificial sequence <220> <223> 2F8-Lm1 <400> 14 gacatcgtga tgacccagtc tcccgactcc ctggccgtgt ccctgggcga gagagccacc 60 atcaactgcc ggtcctccca gtccgtgctg tactcctcct ccaaccggaa ctacctggcc 120 tggtaccagc agaagcctgg ccagcctccc aagctgctga tctactgggc ctccaccagg 180 gagtctggcg tgcccgaccg gttctctggc tccggatctg gcaccgactt caccctgacc 240 atctcctccc tgcaggccga ggacgtggcc gtgtactact gccagcagta ctactccaca 300 cctcggacct tcggccaggg caccaaggtg gagatcaagc ggaccgtggc ggcgccatct 360 gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gtaccgctag cgttgtgtgc 420 ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480 caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540 ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600 gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660 <210> 15 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> 2F8-Lm2 <400> 15 Asp Ile Glu Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Ser Asn Arg Asn 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Asn Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45 Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro 85 90 95 Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 16 <211> 645 <212> DNA <213> Artificial sequence <220> <223> 2F8-Lm2 <400> 16 gacatcgaga tgacccagtc tcccgactcc ctggccgtgt ccctgggcga gagagccacc 60 atcaactgcc ggtcctccca gtcctccaac cggaactacc tggcctggta ccagcagaac 120 cctggccagc ctcccaagct gctgatctac tgggcctcca ccagggagtc tggcgtgccc 180 gaccggttct ctggctccgg atctggcacc gacttcaccc tgaccatctc ctccctgcag 240 gccgaggacg tggccgtgta ctactgccag cagtactact ccacacctcg gaccttcggc 300 cagggcacca aggtggagat caagcggacc gtggcggcgc catctgtctt catcttcccg 360 ccatctgatg agcagttgaa atctggtacc gctagcgttg tgtgcctgct gaataacttc 420 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt 645 <210> 17 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> 2F8-Lm3 <400> 17 Asp Ile Glu Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Val Leu Ser Ser 20 25 30 Asn Tyr Leu Ala Trp Tyr Gln Gln Asn Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr 85 90 95 Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 18 <211> 648 <212> DNA <213> Synthetic sequence <220> <223> 2F8-Lm3 <400> 18 gacatcgaga tgacccagtc tcccgactcc ctggccgtgt ccctgggcga gagagccacc 60 atcaactgcc ggtcctccca gtccgtgctg tcctccaact acctggcctg gtaccagcag 120 aaccctggcc agcctcccaa gctgctgatc tactgggcct ccaccaggga gtctggcgtg 180 cccgaccggt tctctggctc cggatctggc accgacttca ccctgaccat ctcctccctg 240 caggccgagg acgtggccgt gtactactgc cagcagtact actccacacc tcggaccttc 300 ggccagggca ccaaggtgga gatcaagcgg accgtggcgg cgccatctgt cttcatcttc 360 ccgccatctg atgagcagtt gaaatctggt accgctagcg ttgtgtgcct gctgaataac 420 ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 480 tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 540 ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 600 cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgt 648

Claims

1. An anti-Her3 antibody, characterized in that, The anti-Her3 antibody comprises a light chain and a heavy chain, the amino acid sequence of the light chain variable region in the light chain is shown in positions 1-113 in SEQ ID NO: 13, and the amino acid sequence of the heavy chain variable region in the heavy chain is shown in positions 1-117 in SEQ ID NO:

3.

2. The anti-Her3 antibody of claim 1, wherein The light chain further comprises a light chain constant region, and the heavy chain further comprises a heavy chain constant region.

3. The anti-Her3 antibody of claim 2, wherein The light chain constant region is a light chain constant region of a human antibody or a mouse antibody; or, The heavy chain constant region is a heavy chain constant region of a human antibody or a mouse antibody.

4. The anti-Her3 antibody of claim 2, wherein The light chain constant region is a light chain constant region of a human antibody or a mouse antibody; and, The heavy chain constant region is a heavy chain constant region of a human antibody or a mouse antibody.

5. The anti-Her3 antibody of claim 3, wherein The light chain constant region is a constant region of a kappa or lambda chain of a human antibody; or, The heavy chain constant region is a heavy chain constant region of a human antibody IgG1, IgG2, IgG3 or IgG4.

6. The anti-Her3 antibody of claim 4, wherein The light chain constant region is a constant region of a kappa or lambda chain of a human antibody; and, The heavy chain constant region is a heavy chain constant region of a human antibody IgG1, IgG2, IgG3 or IgG4.

7. The anti-Her3 antibody according to any one of claims 2 to 6, wherein The amino acid sequence of the light chain is shown in SEQ ID NO: 13; and / or, the amino acid sequence of the heavy chain is shown in SEQ ID NO:

3.

8. An isolated nucleic acid, comprising, The nucleic acid encodes the anti-Her3 antibody according to any one of claims 1-7.

9. The isolated nucleic acid of claim 8, wherein, The nucleotide sequence encoding the light chain variable region is shown in positions 1-339 in SEQ ID NO: 14; and / or, the nucleotide sequence encoding the heavy chain variable region is shown in positions 1-351 in SEQ ID NO:

6.

10. The isolated nucleic acid of claim 9, wherein, The nucleotide sequence encoding the light chain is shown in SEQ ID NO: 14; and / or, the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO:

6.

11. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid according to any one of claims 8-10.

12. A bispecific antibody molecule, characterized in that, The bispecific antibody molecule comprises the anti-Her3 antibody according to any one of claims 1-7.

13. A chimeric antigen receptor T cell, characterized in that, The chimeric antigen receptor T cell comprises the anti-Her3 antibody according to any one of claims 1-7.

14. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the anti-Her3 antibody according to any one of claims 1-7, the bispecific antibody molecule according to claim 12, or the chimeric antigen receptor T cell according to claim 13, and pharmaceutically acceptable salts, solvates, or solvates of the pharmaceutically acceptable salts thereof.

15. The pharmaceutical composition of claim 14, wherein, The pharmaceutical composition further comprises a pharmaceutical excipient.

16. A kit comprising, The kit comprises the anti-Her3 antibody according to any one of claims 1-7.

17. A kit, characterized in that The kit comprises kit A and kit B; The kit A comprises the anti-Her3 antibody according to any one of claims 1-7 or the pharmaceutical composition according to claim 14 or 15; and the kit B comprises other therapeutic agents.

18. The kit of claim 17, wherein The kit A and the kit B are administered in no particular order, or the kit A is administered first.

19. Use of an anti-Her3 antibody according to any one of claims 1 to 7, a bispecific antibody molecule according to claim 12, a chimeric antigen receptor T cell according to claim 13, or a pharmaceutical composition according to claim 14 or 15 for the manufacture of a medicament for the treatment and / or prevention of a tumor; said tumor is selected from one or more of Her3-positive lung cancer, ovarian cancer, colorectal cancer, breast cancer, prostate cancer and gastric cancer.

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