WO2023028914A1 - Anti-her3 antibody and application thereof - Google Patents

Abstract

Disclosed in the present invention are an anti-Her3 antibody and an application thereof. The anti-Her3 antibody comprises a light chain and a heavy chain, the amino acid sequence of a light chain variable region in the light chain is as shown in positions 1-113 in SEQ ID NO: 13, and the amino acid sequence of a heavy chain variable region in the heavy chain is as shown in positions 1-117 in SEQ ID NO: 3. The expression amount of the anti-Her3 antibody of the present invention is 3-5 times that of an existing antibody, while retaining a high affinity with the target Her3.

Description

Anti-Her3 antibody and its application technical field

The invention belongs to the field of biomedicine, and in particular relates to an anti-Her3 antibody and its application.

Background technique

The Her (Human epidermal growth factor receptor, human epidermal receptor) family consists of four RTKs with similar structures and functions, namely Her1 (EGFR), Her2, Her3 and Her4. The dimerization between receptors is the function of the Her family. Essential conditions for its function and signal transduction activity. After receptor dimerization, it induces cross-linking and phosphorylation of highly conserved kinase residues in the cell, and then recruits and activates downstream proteins, causing signal cascade reactions, and regulating cell proliferation, survival, migration, occurrence, and metastasis. Although Her3 can bind to Neuregulin (NRG), it can only function by forming heterodimers with other receptors due to its lack of intrinsic tyrosine kinase activity. On the contrary, although Her2 has tyrosine kinase activity, there is no corresponding ligand to bind to it. The structure of the extracellular region of Her2 is in a natural open conformation, which allows Her2 to dimerize with other receptors without ligand activation, so Her2 is the preferred dimerization partner of Her3.

Her2 and Her3 form heterodimers, which can directly activate the PI3K/AKT pathway. The PI3K/AKT pathway is the most important signaling pathway to promote tumor cell proliferation and participate in the regulation of gene expression, cell metabolism, and cytoskeleton rearrangement. Therefore, Her2 -Her3 heterodimer is considered to be the dimer with the strongest signaling ability in the Her family. Studies have shown that drugs that directly or indirectly inhibit the PI3K/AKT pathway can increase Her3 transcription, upregulate and activate Her3 expression through PI3K/AKT negative feedback regulation, and reactivate downstream pathways to produce drug resistance. This phenomenon is similar to anti-Her2 and anti-EGFR. Resistance to targeted therapy. In addition, NRG1, the main ligand of Her3, can participate in Her3 signal transduction through paracrine and autocrine pathways, thereby inducing the activation of Her3 pathway, 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 participate in the occurrence and development of tumors. In view of the fact that Her3 can form a heterodimer with other targets to deliver the strongest PI3K/AKT, and because of the lack of tyrosine kinase activity, Her3 therapeutic drugs mainly target the extracellular region of Her3 to develop antibody drugs.

In the development of antibody drugs, the expression of antibodies is crucial to the control of production costs. Factors affecting the efficient expression of antibody genes in mammalian cells mainly include the sequence of the antibody gene itself, the integration site of the antibody gene on the host chromosome, the antibody Gene copy number, transcription and translation level, host cell selection and modification, and balanced expression of light and heavy chain genes, etc. Among them, the five CDRs in the antibody sequence except the heavy chain CDR3 have fixed characteristics in sequence and structure, showing a specific CDR length, and there are some conserved positions in the CDR or Framework region of the antibody sequence. It is essential to maintain the conformation and function of the CDR/loop region.

Contents of the invention

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

In order to increase the expression level of the antibody and save production costs, the inventors tried to modify various aspects of the antibody. For example, the inventors optimized the codons of existing monoclonal antibodies, replaced signal peptides, replaced host cells, adjusted stable transfection conditions, etc., in an attempt to improve antibody expression. However, the expression level of the modified antibody in the host cell is still not up to the expected standard. In a large number of experiments, the inventors went through layers of screening and unexpectedly found that the amino acid sequence of the light chain of the antibody has a key impact on the expression level of the antibody in the combined cross-transfection of the light and heavy chains. Based on the results of this study, the inventors modified the light chain of the antibody, and screened the light chain mutants to obtain an antibody with high expression while maintaining Her3-binding activity. Antibodies increased 3 to 5 times.

The present invention solves the above-mentioned technical problems through the following technical solutions.

A first aspect of the present invention provides an anti-Her3 antibody, the anti-Her3 antibody comprising 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 of 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 Positions 1-117 are shown.

In some embodiments of the invention, the light chain further comprises a light chain constant region and the heavy chain further comprises a heavy chain constant region.

In the present invention, the light chain constant region is preferably the light chain constant region of human antibody or mouse antibody; more preferably the constant region of κ or λ chain of human antibody.

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

In some embodiments of the present invention, the amino acid sequence of the light chain is shown in SEQ ID NO: 13.

In some embodiments of the present invention, the amino acid sequence of the heavy chain is shown in SEQ ID NO:3.

In the present invention, the heavy chain of the anti-Her3 antibody can be mutated (deleted, substituted or added) by one or more amino acid residues in the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:3 , and have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more than 99% homology with the amino acid sequence shown in the sequence table 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, most preferably at least 99% homology.

The second aspect of the present invention provides a fusion protein comprising the anti-Her3 antibody as described in the first aspect.

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

In some embodiments of the present invention, the nucleotide sequence encoding the light chain variable region is shown in positions 1-339 of SEQ ID NO:14.

In some embodiments of the present invention, the nucleotide sequence of the heavy chain variable region is shown in positions 1-351 of SEQ ID NO:6.

In some embodiments of the present invention, the nucleotide sequence encoding the light chain is shown in SEQ ID NO: 14.

In some embodiments of the present invention, the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO:6.

The fourth aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the third aspect.

The fifth aspect of the present invention provides an antibody-drug conjugate, which comprises the anti-Her3 antibody as described in the first aspect or the fusion protein as described in the second aspect.

The sixth aspect of the present invention provides a bispecific antibody molecule comprising the anti-Her3 antibody as described in the first aspect or the fusion protein as described in the second aspect.

The seventh aspect of the present invention provides a chimeric antigen receptor T cell comprising the anti-Her3 antibody as described in the first aspect or the fusion protein as described in the second aspect.

The eighth aspect of the present invention provides a pharmaceutical composition, which comprises the anti-Her3 antibody as described in the first aspect, the fusion protein as described in the second aspect, and the antibody-conjugated antibody as described in the fifth aspect. Drugs, bispecific antibody molecules as described in the sixth aspect, or chimeric antigen receptor T cells as described in the seventh aspect, and pharmaceutically acceptable salts, solvates, or pharmaceutically acceptable salts thereof Salt solvates.

In some embodiments of the present invention, the pharmaceutical composition further includes pharmaceutical excipients.

The ninth aspect of the present invention provides a kit comprising the anti-Her3 antibody as described in the first aspect or the fusion protein as described in the second aspect.

The tenth aspect of the present invention provides a kit of medicines, the kit of medicines includes a medicine box A and a medicine box B;

Wherein, the kit A includes the anti-Her3 antibody as described in the first aspect or the pharmaceutical composition as described in the eighth aspect; the kit B includes other therapeutic agents.

In some embodiments of the present invention, the administration time of the medicine box A and the medicine box B is not in any order, or the medicine box A is administered first.

The eleventh aspect of the present invention provides a drug delivery device, comprising: (1) an infusion module for administering the pharmaceutical composition as described in the eighth aspect to a subject in need, and (2) Optional drug efficacy monitoring module.

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

In some embodiments of the invention, the disease is a tumor;

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

In some better 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.

The thirteenth aspect of the present invention provides 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, and an anti-Her3 antibody as described in the sixth aspect. Bispecific antibody molecules, chimeric antigen receptor T cells as described in the seventh aspect, or pharmaceutical compositions as described in the eighth aspect are used in the preparation of Her3 protein inhibitors, or the preparation of drugs for treating and/or preventing tumors Applications.

The preferred definition of the tumor is as described in the twelfth aspect.

In the present invention, the prostate cancer may be prostate cancer commonly understood in the art, and the prostate cancer cells include, for example, 22Rv1 cells and/or LNCaP cells.

In the present invention, the colorectal cancer may be colorectal cancer generally understood in the art, and colorectal cancer cells include SW620 cells, for example.

In the present invention, the lung cancer can be lung cancer commonly understood in the art, and lung cancer cells include NCI-H820 cells or HCC827 cells, for example.

In the present invention, the ovarian cancer may be ovarian cancer commonly understood in the art, and ovarian cancer cells contain OVCAR-8 cells, for example.

In the present invention, the breast cancer may be breast cancer commonly understood in the art, and breast cancer cells include, for example, SK-BR-3 cells.

Unless otherwise stated, the following terms appearing in the description of the present invention have the following meanings:

The pharmaceutical adjuvant can be an adjuvant widely used in the field of pharmaceutical production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method for the subject to dissolve the active ingredient at a desired rate after administration, or to promote the activity of the subject after administration of the composition. The ingredients are effectively absorbed. The pharmaceutical excipients can be inert fillers, or provide certain functions, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredients of the composition. The pharmaceutical adjuvant can include one or more of the following adjuvants: buffering agent, chelating agent, preservative, cosolvent, stabilizer, excipient and surfactant colorant, corrective agent and sweetener .

The term "pharmaceutically acceptable" means that salts, solvents, auxiliary materials, etc. are generally non-toxic, safe and suitable for use by patients. The "patient" is preferably a mammal, more preferably a human.

The term "pharmaceutically acceptable salt" refers to a salt prepared from a compound 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, the base addition can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable base in pure solution or in a suitable inert solvent. A salt. 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, diethanolamine salts. When the compounds of the present invention contain relatively basic functional groups, acid addition can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in neat solution or in a suitable inert solvent. A salt. The pharmaceutically acceptable acid includes inorganic acids, including but not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid and the like. 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, suberic acid , fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid , tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid (ie 4,4'-methylene-bis( 3-hydroxy-2-naphthoic acid)), amino acids (eg glutamic acid, arginine) and the like. 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 of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).

The term "solvate" refers to a compound of the present invention in combination with a stoichiometric or non-stoichiometric amount of solvent. The solvent molecules in a solvate may exist in an ordered or non-ordered arrangement. The solvent includes but not limited to: water, methanol, ethanol and the like.

The term "treatment" or its equivalents when applied to, eg, cancer, refers to a procedure or process to reduce or eliminate the number of cancer cells in a patient or to alleviate the symptoms of cancer. "Treatment" of cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will actually be eliminated, that the number of cells or disorder will actually be reduced, or that the symptoms of the cancer or other disorder will actually be alleviated. Often, methods of treating cancer are pursued even with a low probability of success, but which are still considered to induce an overall beneficial course of action, taking into account the patient's medical history and estimated life expectancy.

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

On the basis of not violating common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

Unless otherwise specified, room temperature in the present invention refers to 20-30°C.

The reagents and raw materials used in the present invention are all commercially available.

The positive progress effect of the present invention is:

The anti-Her3 antibody of the present invention has a high expression level, which can reach 3 to 5 times that of the existing antibody, while maintaining a high affinity with the target Her3, and has a good inhibitory effect on various tumor cells expressing Her3 and the like.

Description of drawings

Figure 1 is a schematic diagram of the expression level detection results of the purified product detected by SDS-PAGE of the 2F8 transiently expressed supernatant (2 days) in Example 2.

Fig. 2 is the SDS-PAGE detection result of 2F8 transient purification product in embodiment 2;

Among them: A is 6% non-reducing SDS-PAGE, B is 12% reducing SDS-PAGE.

Figure 3 is the 12% reducing SDS-PAGE detection result of the 2F8 transient expression supernatant after adjusting the light chain transfection ratio in Example 2;

Among them: A is transfection 2 days, B is transfection 4 days.

Fig. 4 is the 12% reducing SDS-PAGE detection result of the transient expression supernatant of the combination of 2F8 and 1A9 light and heavy chains in Example 3.

Fig. 5 is the 6% non-reducing SDS-PAGE detection result of the 2F8 transiently purified product replaced with the light chain signal peptide in Example 3.

Fig. 6 is the 12% reducing SDS-PAGE detection result of the 2F8 transiently purified product replaced with the light chain signal peptide in Example 3.

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

Among them: A is 3 days in an instant, and B is 5 days in an instant.

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

Among them: A is 2F8 light chain mutant transient, B is 2F8Lm1 repeated transient.

9 is a Blast map of the amino acid sequences of 3F5 (upper) and 2F8-L (lower) in Example 5.

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

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

Figure 12 is a graph showing the reaction between 2F8 and Her3-His recombinant protein.

Figure 13 is a graph showing the reaction between 3F5 and Her3-His recombinant protein.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

Description of abbreviations:

PCR: polymerase chain reaction;

CHO: Chinese hamster ovary cells;

HTRF: Homogeneous Time-Resolved Fluorescence.

Example 1 Preparation of Anti-Her3 Antibody Stable Expression Cell Line

As shown in Table 1, this embodiment selects the monoclonal antibody 2F8 with high affinity and specificity targeting 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 shown. The light and heavy chain nucleotide sequences of 2F8 are synthesized through the whole gene (Suzhou Jinweizhi 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 Shown in SEQ ID NO:4. The light and heavy chains were separately constructed into the expression vector pV81 (Shanghai Yisheng Biology, pEE14.2) by EcoR I and Hind III (TAKARA, R3104S, R3101S) double enzyme digestion, and transformed into Trans 1-T1 competent by ligation Cells (Beijing Quanshijin Biology, CD501-03), from which clones were selected for PCR identification, sent for inspection, and sequenced for confirmation, and positive clones were cultured and amplified for plasmid extraction to obtain the antibody light chain eukaryotic expression plasmid 2F8-L /pV81 and antibody heavy chain eukaryotic expression plasmid 2F8-H/pV81, light and heavy chain eukaryotic expression plasmid ratio 1.5/1, transformed into CHO cells (ATCC, CCL-61 ^TM ) adapted to suspension growth by electric shock, After 48 hours of electroporation, the average expression level measured by HTRF method (Homogeneous Time-Resolved Fluorescence, homogeneous time-resolved fluorescence) was 1.8 μg/mL, which was relatively low. After stable transfection screening and subcloning, clones expressing TOP10 were selected for culture evaluation, fed-batch cultured in a fermenter for 14 days (basic medium: Dynamis AGT Medium, Gibco; feed medium: EfficientFeedC+, Gibco ), the highest expression level was only 1.2g/. Based on the above, the expression level of the antibody 2F8 in the host cell CHO did not reach the expected target.

Table 1. Amino acid and nucleotide sequences of 2F8 light and heavy chains

Example 2. Optimization of Anti-Her3 Antibody Expression

For the problem of antibody expression, the research tried to improve antibody expression by replacing signal peptides and optimizing codons, changing host cells, and adjusting stable transfection conditions.

Add different signal peptides to the amino acid sequence of antibody 2F8, use the principle of triplet codon coding, and optimize the codon to obtain two kinds of 2F8 light and heavy chain molecules with the same amino acid sequence and different nucleotides (2F8-L- new, its nucleotide sequence is shown in SEQ ID NO:5; 2F8-H-new, its nucleotide sequence is shown in SEQ ID NO:6; 2F8-L-2, its nucleotide sequence is shown in SEQ ID NO: shown in 7; 2F8-H-2, its nucleotide sequence is shown in sequence SEQ ID NO: 8, specifically as shown in table 2). Gene synthesis was re-constructed into the expression vector pV81, the light and heavy chains were combined (the combination method is shown in Table 2), and the transfection reagent 293fectin (Invitrogen, 12347019) was transferred into HEK293 cells (Cell Bank of Chinese Academy of Sciences, GNHu43) Medium, the transfection volume is 20mL; 2 days after transfection, take a small amount of cell culture supernatant to detect the expression level; Purified product expression. The detection results are shown in Figure 1. The results showed that HEK293 was transiently transfected for 2 days, and there was no significant difference in the expression levels of the wild type 2F8 and the two optimized sequences.

Table 2. 2F8 light chain nucleotide sequence, heavy chain nucleotide sequence and combination

4 days after transfection, 2F8-new, 2F8-1 and 2F8-2 were purified and quantified; there was no significant difference in the expression of 2F8-new and 2F8-1, and the expression of 2F8-2 was slightly higher than that of 2F8-1; the basic expression of the antibody The amounts were all low, about 20 μg/mL; the protein purification information is shown in Table 3; the 6% non-reducing SDS-PAGE test results of the purified products are shown in Figure 2 A, and the 12% reducing SDS-PAGE test results are shown in Figure 2 As shown in B of 2, it can be seen that the expression ratio of the light chain of the 2F8 molecule is low.

Table 3. Purification information of 2F8 and FDA029 transiently expressed supernatants

Adjust the transfection ratio of the light chain of 2F8 molecules, and transfer the light and heavy chain plasmids of 2F8-new into HEK293 cells with the transfection reagent 293fectin according to the normal transfection ratio and double the ratio of the light chain, and the transfection volume 20mL; 2 days and 4 days after transfection, take a small amount of cell culture supernatant to detect the expression level. The detection results of the transient cell culture supernatant are shown in Figure 3 A and B, after increasing the light chain transfection ratio, the basal expression level of 2F8-new did not improve.

The light and heavy chain fragments of 2F8-new were constructed into a new expression vector, named 2F8-3, and electroporated and transfected into the host cell CHOK1SV (both the vector and the cell were derived from Lonza), with well-expressed antibody 1A9 (its light chain The amino acid sequence is shown in SEQ ID NO: 9; its heavy chain amino acid sequence is shown in SEQ ID NO: 10) as a system control, the medium is CD CHO Medium (Gibco, 10743029), and the expression results are shown in Table 5. Experimental The expression level of group 2F8-3 in CHOK1SV was higher than that of 2F8-new in the original experimental system, but far lower than that of the system control, indicating that antibody 2F8 is a difficult-to-express antibody.

Table 4. 1A9 light and heavy chain amino acid sequences

Table 5. 2F8 stable transfection 48 hours expression level

Antibody	culture medium	48 hours expression level (mg/L)	Remark
2F8-new	CD CHO Medium	0.158	/
2F8-3	CD CHO Medium	0.327	test group
1A9	CD CHO Medium	0.778	System comparison

Example 3. Evaluation of cross-transfection combinations

Cross-transfect the combination of light and heavy chains of 2F8 molecules, combine the light and heavy chain plasmids of 2F8-new and the light and heavy chain plasmids of antibody 1A9 (combination methods are shown in Table 6), and transfect with the transfection reagent 293fectin. Into HEK293 cells, the transfection volume was 20mL; 2 days after transfection, the cell culture supernatant was taken to detect the expression level. The detection results of the transient cell culture supernatant are shown in Figure 4. Compared with 2F8-new, the expression of the combination of 2F8 heavy chain and 1A9 light chain was significantly enhanced, and the expression of the combination of 2F8 light chain and 1A9 heavy chain was not improved. The light chain has a critical effect on the expression of 2F8.

Table 6. Combination of 2F8 light and heavy chains and 1A9 light and heavy chains

Replace the 2F8 light chain signal peptide; replace the 2F8-new light chain signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO: 11) with a new signal peptide MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 12), and transfer it into HEK293 cells through the transfection reagent 293fectin , the transfection volume was 20mL; the cell culture supernatant was collected 4 days after transfection, and the volume of the supernatant was 25mL. After one-step Protein-A affinity purification, the expression level of the purified product was detected. The 6% non-reducing SDS-PAGE detection result of the 2F8 molecular transient purification product replaced with the light chain signal peptide is shown in Figure 5, and the 12% reducing SDS-PAGE detection result is shown in Figure 6. After replacing the light chain signal peptide, the basal expression of 2F8 was slightly increased, but not significantly improved.

Example 4. Antibody Sequence Analysis

According to the sequence characteristics of the molecular light chain of 2F8, three light chain mutants (Lm1, Lm2 and Lm3) were designed, the amino acid and nucleotide sequences of which are shown in Table 7. Vector construction was carried out in the same manner as in Example 1 to obtain light chain mutants 2F8-Lm1, 2F8-Lm2 and 2F8-Lm3.

Table 7. 2F8 molecular mutant sequences

Transfection of 2F8 Molecular Light Chain Mutants

Combine the light chain mutants and heavy chains of 2F8 molecules (the combination method is shown in Table 8) and transfer them into HEK293 cells through the transfection reagent 293fectin, with a transfection volume of 20 mL; 3 days after transfection, take a small amount of cell culture supernatant , to detect the expression level; 5 days after transfection, the cell culture supernatant was collected, the volume of the supernatant was 25 mL, and after one-step Protein-A affinity purification, the expression level of the purified product was detected.

Table 8 Combination of 2F8 light chain mutants

name	heavy chain	light chain
2F8-new	2F8-H-new	2F8-L-new

2F8-m1	2F8-H-new	2F8-Lm1
2F8-m2	2F8-H-new	2F8-Lm2
2F8-m3	2F8-H-new	2F8-Lm3

The 12% reducing SDS-PAGE detection result of the expression supernatant 3 days after the transfection of the 2F8 light chain mutant is shown in A of Figure 7, and the 12% reducing SDS-PAGE detection result of the expression supernatant 5 days after the transfection is shown in As shown in B of Figure 7, the 12% reducing SDS-PAGE detection results of the supernatant purified product are shown in A of Figure 8, in which the expression of 2F8-m1 was significantly increased, and it was confirmed by repeated transfection. The 12% reducing SDS-PAGE test results of the expression supernatant after 5 days are shown in Figure 8B, the expression of 2F8-m1 has been significantly improved, and the expression level is calculated based on the grayscale analysis of SDS-PAGE and the amount of protein after purification. The increase is about 3-5 times; the amino acid differences between the light chain mutants 2F8-Lm1 and 2F8-L are shown in Figure 9 .

Example 5. Expression and Preparation of Site Modified Antibody

The combination of 2F8 light chain mutant 2F8-Lm1 and heavy chain 2F8-H-new was renamed antibody 3F5. Use ExpiCHO cells to transiently transfect the two antibodies 2F8 and 3F5, and perform the transfection operation according to the following table 9. The transfection volume is 100mL per shake flask, and the expression level is detected by HTRF after 8 days of culture; the results show that the transient expression level of 3F5 is that of 2F8 2.9 times.

Table 9. Transient transfection of 2F8 and 3F5

Antibody 3F5 was screened and subcloned for stable cell lines, and cells with high expression levels were selected for culture evaluation. After 14 days of fed-batch culture in the fermenter, the highest expression level of 3F5 was 3.3g/L; while the expression level of 2F8 was only It was 1.2g/L, realizing the expression improvement under large-scale culture. Based on the above results, the expression level of antibody 3F5 is significantly higher than that of antibody 2F8, which meets the expectation of the antibody expression level of the project.

Example 6. Functional Analysis of Anti-Her3 Antibody Mutant Sequences

Binding assay of 3F5 mutants on T-47D cells

FACS was used to detect the binding ability. First, T-47D cells (Cell Bank of Chinese Academy of Sciences, TCHu 87) were collected at a density of 3×10 ⁵ to 1×10 ⁶ , and the supernatant was removed; FACS staining buffer (Moregate, 3827104) was added to wash the cells once, Centrifuge the cells again to remove the supernatant, and use 2ml FACS to resuspend and divide evenly into two 1.5mL centrifuge tubes, block in ice bath for 0.5 hours, centrifuge at 1000rpm for 5 minutes, remove the supernatant; add 100μL of 3F5 with corresponding concentration gradient to the cells Mutants (10μg/mL, 3.33μg/mL, 1.11μg/mL, 0.37μg/mL, 0.12μg/mL), incubated in ice bath for 1h, centrifuged to remove supernatant, added 0.5mL/tube of FACS staining buffer and centrifuged at 1000rpm Treat for 5 minutes (4°C), remove the supernatant, and repeat twice. Add 100 μL fluorescently labeled secondary antibody (Thermo, A11013), incubate on ice for 30 min, centrifuge to remove the supernatant, add 0.5 mL/tube of FACS staining buffer, centrifuge at 1000 rpm for 5 min (4°C), remove the supernatant, repeat twice; add The cells were resuspended in 0.5mL PBS diluent (Shanghai Double Helix Biotechnology Co., Ltd., P10033), and placed on ice in the dark; the fluorescence value was detected by flow cytometry. The results of FACS detection are shown in Figure 10. The results showed that 3F5 was combined with T-47D cells and had a dose-effect relationship. At different antibody concentrations, there was no significant difference in the binding ability to the parental molecule 2F8.

The results of FACS detection according to the above method are shown in Figure 11, 2F8Lm1 binds to MDA-MB-453 cells (Cell Bank of Chinese Academy of Sciences, TCHu233), and has a good dose-effect relationship. At different antibody concentrations, there was no significant difference in the binding ability to the parental molecule 2F8.

Example 7. Affinity analysis of 3F5 mutants

Detect the binding affinity of antibodies 2F8 and 3F5 to the Her3-His recombinant protein, and use the BLI method to detect the binding kinetic curve between the immobilized antibody and Her3. The detection method is carried out according to the instructions of the instrument (Fortebio, Octet 96e). Briefly First, use Loading Buffer/Sample dilution buffer (1×PBS, pH7.4, 0.1% BSA+0.02% Tween-20) to equilibrate the AMC sensor for 60s to obtain Baseline 1. The antibody to be tested was diluted with Loading Buffer to a concentration of 10 μg/mL and combined with the balanced sensor, and the combined sensor was rebalanced with Loading Buffer again to obtain Baseline 2. Then bind the antibody-loaded sensor to human Her3-His diluted to 100-3.13nM with sample diluent for 90s to obtain the binding curve of antibody and protein, as shown in Figure 12 and Figure 13 . Then put the antigen-bound sensor in the Sample Dilution Buffer again for 180s to dissociate to obtain the dissociation curve. The k-on and k-off values of the antibody-protein binding were calculated through the binding and dissociation curves, and the KD value was calculated. The results showed that there was no difference in the affinity of 2F8 and 3F5 to the Her3-His recombinant protein, and the affinity of 2F8 was 2.43E-09mol /L (abbreviated as M in Table 10 below), the affinity of 3F5 is 2.53E-09mol/L, and the detailed kinetic parameters are shown in Table 10 below.

Table 10. 2F8 and 2F8Lm1 affinity determination results

sample name	batch number	KD(M)	kon(1/Ms)	koff(1/s)
2F8	Elu-20200911	2.43E-09	1.47E+05	3.57E-04
3F5	20201214G	2.53E-09	1.56E+05	3.93E-04

Although the specific implementations of the present invention have been described above, those skilled in the art should understand that these are only examples, and various changes or changes can be made to these implementations without departing from the principle and essence of the present invention. Revise. Accordingly, the protection scope of the present invention is defined by the appended claims.

Claims (15)

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 the 1-113 positions in SEQ ID NO:13, and the amino acid sequence of the heavy chain variable region in the heavy chain is as SEQ ID NO:3 Shown in bits 1-117 of .
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;

   The light chain constant region is preferably a light chain constant region of a human antibody or a mouse antibody; more preferably a constant region of a κ or λ chain of a human antibody; and/or,

   The heavy chain constant region is preferably a heavy chain constant region of a human antibody or a mouse antibody; more preferably a heavy chain constant region of a human antibody IgG1, IgG2, IgG3 or IgG4.
3. The anti-Her3 antibody according to claim 2, wherein the amino acid sequence of the light chain is as shown in SEQ ID NO:13; and/or, the amino acid sequence of the heavy chain is as shown in SEQ ID NO:3 .
4. A fusion protein, characterized in that the fusion protein comprises the anti-Her3 antibody according to any one of claims 1-3.
5. An isolated nucleic acid, characterized in that the nucleic acid encodes the anti-Her3 antibody according to any one of claims 1 to 3 or the fusion protein according to claim 4;

   Preferably, the nucleotide sequence encoding the variable region of the light chain is shown in positions 1-339 of SEQ ID NO: 14; and/or, the nucleotide sequence encoding the variable region of the heavy chain As shown in the first 1-351 in SEQ ID NO:6;

   More preferably, 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.
6. A recombinant expression vector, characterized in that the recombinant expression vector comprises the nucleic acid according to claim 5.
7. An antibody-coupled drug, characterized in that the antibody-coupled drug comprises the anti-Her3 antibody according to any one of claims 1-3 or the fusion protein according to claim 4.
8. A bispecific antibody molecule, characterized in that the bispecific antibody molecule comprises the anti-Her3 antibody according to any one of claims 1-3 or the fusion protein according to claim 4.
9. 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 1-3 or the fusion protein according to claim 4.
10. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the anti-Her3 antibody according to any one of claims 1 to 3, the fusion protein according to claim 4, and the antibody according to claim 7 The conjugated drug, the bispecific antibody molecule as claimed in claim 8, or the chimeric antigen receptor T cell as claimed in claim 9 and its pharmaceutically acceptable salt, solvate, or its pharmaceutically acceptable Solvates of accepted salts;

    Preferably, the pharmaceutical composition also includes pharmaceutical excipients.
11. A kit, characterized in that the kit comprises the anti-Her3 antibody according to any one of claims 1-3 or the fusion protein according to claim 4.
12. A set of medicine boxes, characterized in that the set of medicine boxes includes a medicine box A and a medicine box B;

    Wherein, the kit A includes the anti-Her3 antibody according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 10; the kit B includes other therapeutic agents;

    Preferably, the administration time of the medicine box A and the medicine box B is not in any order, or the medicine box A is administered first.
13. A drug delivery device, characterized in that the drug delivery device comprises: (1) an infusion module for administering the pharmaceutical composition according to claim 10 to a subject in need, and (2) any The selected efficacy monitoring module.
14. A method for treating/preventing a disease, characterized in that the method comprises administering an effective dose of the anti-Her3 antibody as claimed in any one of claims 1 to 3, the fusion protein as described in claim 4, to a patient in need protein, the antibody-drug conjugate as claimed in claim 7, the bispecific antibody molecule as claimed in claim 8, or the chimeric antigen receptor T cell as claimed in claim 9, or the chimeric antigen receptor T cell as claimed in claim 10 A pharmaceutical composition, or administering a drug delivery device as claimed in claim 13;

    Preferably, the disease is a tumor;

    More preferably, the tumor is a Her3-positive tumor;

    Further more preferably, 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.
15. An anti-Her3 antibody as claimed in any one of claims 1 to 3, a fusion protein as claimed in claim 4, an antibody-drug conjugate as claimed in claim 7, and a bispecific drug as claimed in claim 8 Application of the antibody molecule, the chimeric antigen receptor T cell as claimed in claim 9, or the pharmaceutical composition as claimed in claim 10 in the preparation of Her3 protein inhibitors, or in the preparation of drugs for treating and/or preventing tumors;

    Preferably, the tumor is a Her3-positive tumor;

    More preferably, 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.

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