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WO2023028914A1 - 抗Her3抗体及其应用 - Google Patents

抗Her3抗体及其应用 Download PDF

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Publication number
WO2023028914A1
WO2023028914A1 PCT/CN2021/115952 CN2021115952W WO2023028914A1 WO 2023028914 A1 WO2023028914 A1 WO 2023028914A1 CN 2021115952 W CN2021115952 W CN 2021115952W WO 2023028914 A1 WO2023028914 A1 WO 2023028914A1
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Prior art keywords
antibody
her3
light chain
heavy chain
seq
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PCT/CN2021/115952
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English (en)
French (fr)
Inventor
郭青松
沈毅珺
杨彤
陈珍
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上海复旦张江生物医药股份有限公司
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Priority to PCT/CN2021/115952 priority Critical patent/WO2023028914A1/zh
Priority to CN202180100378.7A priority patent/CN117616044A/zh
Publication of WO2023028914A1 publication Critical patent/WO2023028914A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to an anti-Her3 antibody and its application.
  • Her Human epidermal growth factor receptor, human epidermal receptor
  • 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.
  • 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.
  • NSG Neuregulin
  • 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.
  • 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.
  • NRG1 the main ligand of Her3
  • 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.
  • 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.
  • Her3 therapeutic drugs mainly target the extracellular region of Her3 to develop 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.
  • 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.
  • 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.
  • 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.
  • 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 the light chain constant region of human antibody or mouse antibody; more preferably the constant region of ⁇ or ⁇ chain of human antibody.
  • 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.
  • amino acid sequence of the light chain is shown in SEQ ID NO: 13.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:3.
  • 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.
  • the nucleotide sequence encoding the light chain variable region is shown in positions 1-339 of SEQ ID NO:14.
  • nucleotide sequence of the heavy chain variable region is shown in positions 1-351 of SEQ ID NO:6.
  • nucleotide sequence encoding the light chain is shown in SEQ ID NO: 14.
  • 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.
  • 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;
  • 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.
  • 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 disease is a tumor
  • the tumor is a Her3-positive tumor.
  • 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.
  • 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.
  • the colorectal cancer may be colorectal cancer generally understood in the art, and colorectal cancer cells include SW620 cells, for example.
  • 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.
  • the ovarian cancer may be ovarian cancer commonly understood in the art, and ovarian cancer cells contain OVCAR-8 cells, for example.
  • the breast cancer may be breast cancer commonly understood in the art, and breast cancer cells include, for example, SK-BR-3 cells.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • the compounds of the present invention 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.
  • base 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).
  • 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.
  • treatment 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 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.
  • 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.
  • prevention refers to a reduction in the risk of acquiring or developing a disease or disorder.
  • 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 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.
  • 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;
  • 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;
  • 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;
  • A is 3 days in an instant
  • 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;
  • A is 2F8 light chain mutant transient
  • B is 2F8Lm1 repeated transient.
  • FIG. 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.
  • FIG. 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.
  • CHO Chinese hamster ovary cells
  • HTRF Homogeneous Time-Resolved Fluorescence.
  • 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-Resolv
  • 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.
  • 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 ⁇ g/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.
  • 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.
  • 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
  • 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 combination of 2F8 light chain mutant 2F8-Lm1 and heavy chain 2F8-H-new was renamed antibody 3F5.
  • 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.
  • 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.
  • 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 100 ⁇ L of 3F5 with corresponding concentration gradient to the cells Mutants (10 ⁇ g/mL, 3.33 ⁇ g/mL, 1.11 ⁇ g/mL, 0.37 ⁇ g/mL, 0.12 ⁇ g/mL), incubated in ice bath for 1h, centrifuged to remove supernatant, added 0.5mL/tube of FACS staining buffer and centrifuged at 1000rpm Treat
  • 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 ⁇ g/mL and combined with the balanced sensor, and the combined sensor was rebalanced with Loading Buffer again to obtain Baseline 2.

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Abstract

本发明公开了一种抗Her3抗体及其应用。所述抗Her3抗体包含轻链和重链，所述轻链中轻链可变区的氨基酸序列如SEQ ID NO:13中的第1-113位所示，所述重链中重链可变区的氨基酸序列如SEQ ID NO:3中的第1-117位所示。本发明的抗Her3抗体的表达量达到现有抗体的3～5倍，同时保持了与靶点Her3的高亲和力。

Description

抗Her3抗体及其应用 技术领域
本发明属于生物医药领域，尤其涉及一种抗Her3抗体及其应用。
背景技术
Her(Human epidermal growth factor receptor，人表皮受体)家族由4个结构相近、功能相关的RTK组成，分别为Her1(EGFR)、Her2、Her3和Her4，受体间的二聚化是Her家族发挥其功能及信号转导活性的基本条件。受体二聚化后，诱导胞内高度保守的激酶残端发生交联磷酸化，进而招募并激活下游蛋白，引起信号级联反应，调节细胞增殖、生存、迁移、发生、转移等过程。Her3虽可与神经调节蛋白(Neuregulin，NRG)结合，但由于其缺乏内在的酪氨酸激酶活性，只能通过与其他受体形成异二聚体发挥功能。相反，Her2虽具有酪氨酸激酶活性，但并无相应的配体与之结合。Her2胞外区结构为天然的开放构象，此构象使Her2无需配体激活即可与其他受体发生二聚化，因此Her2是Her3首选的二聚化伴侣。
Her2与Her3形成异二聚体，可直接激活PI3K/AKT通路，PI3K/AKT通路是促进肿瘤细胞增殖的最重要的信号通路，参与调节基因表达、细胞代谢、细胞骨架重排等过程，因此Her2-Her3异二聚体被认为是Her家族中信号传导能力最强的二聚体。研究表明，直接或间接抑制PI3K/AKT通路的药物可导致Her3转录增加，通过PI3K/AKT负反馈调节使Her3表达上调及活化，重新激活下游通路而产生耐药，该现象与抗Her2及抗EGFR靶向治疗的耐药相关。此外，Her3的主要配体NRG1可通过旁分泌和自分泌途径参与Her3的信号转导等进而诱导Her3通路活化，也可能参与Her家族靶向治疗的耐药。此外，Her3还可与其他Her家族成员如EGFR，以及非Her家族成员如MET、IGF-1R形成二聚体，参与肿瘤的发生、发展。鉴于Her3可与其他靶点形成异二聚体传递最强的PI3K/AKT，并且由于缺乏酪氨酸激酶活性，因此Her3的治疗药物主要以Her3的胞外区为靶点开发抗体类药物。
抗体类药物的开发，其抗体的表达对于生产成本的控制至关重要，影响抗体基因在哺乳动物细胞高效表达的因素主要包括抗体基因本身的序列、抗体基因在宿主染色体上的整合位点、抗体基因的拷贝数、转录翻译水平、宿主细胞的选择和修饰以及轻、重链基因的均衡表达等。其中，抗体序列中除重链CDR3以外的五个CDR，在序列和结构上具 有固定的特点，表现为特定的CDR长度，并且抗体序列在CDR或Framework区存在一些保守的位点，这些位点对于维持CDR/loop区的构象和功能至关重要。
发明内容
本发明所要解决的技术问题是为了克服现有技术缺少高表达量的抗Her3抗体的缺陷，提供一种抗Her3抗体及其应用。本发明的抗Her3抗体的表达量达到现有抗体的3～5倍，同时保持了与靶点Her3的高亲和力。
为了提高抗体的表达量，节约生产成本，发明人尝试对抗体的各个方面进行改造。例如，发明人对现有的单克隆抗体进行密码子优化、更换信号肽、更换宿主细胞、调整稳定转染条件等，试图改善抗体表达。但是，经过前述改造的抗体在宿主细胞中的表达量仍然达不到预期标准。在大量试验中，发明人经过层层筛选，意外在轻重链组合交叉转染中发现抗体的轻链氨基酸序列会对抗体的表达量具有关键性影响。基于该研究结果，发明人对抗体的轻链进行了改造，在轻链的突变体中筛选得到具有高表达量、同时维持与Her3结合活性的抗体，其表达量得到显著改善，相对改造前的抗体提高了3～5倍。
本发明通过以下技术方案解决上述技术问题。
本发明的第一方面提供一种抗Her3抗体，所述抗Her3抗体包含轻链和重链；
所述轻链中轻链可变区的氨基酸序列如SEQ ID NO:13中的第1-113位所示，所述重链中重链可变区的氨基酸序列如SEQ ID NO:3中的第1-117位所示。
在本发明一些实施方案中，所述轻链还包含轻链恒定区，所述重链还包含重链恒定区。
本发明中，所述轻链恒定区较佳地为人源抗体或小鼠抗体的轻链恒定区；更佳地为人源抗体的κ或λ链的恒定区。
本发明中，所述重链恒定区较佳地为人源抗体或小鼠抗体的重链恒定区；更佳地为人源抗体IgG1、IgG2、IgG3或者IgG4的重链恒定区。
在本发明一些实施方案中，所述轻链的氨基酸序列如SEQ ID NO:13所示。
在本发明一些实施方案中，所述重链的氨基酸序列如SEQ ID NO:3所示。
本发明中，所述的抗Her3抗体的重链可为如SEQ ID NO:3所示的氨基酸序列的重链可变区发生了一个或多个氨基酸残基的突变(缺失、取代或添加)，且与如序列表SEQ ID NO:3所示的氨基酸序列具有至少70％、75％、80％、85％、90％、95％、98％或99％以上的同源性；优选为至少85％的同源性，更优选为至少90％的同源性，再优选为至少95％、96％、97％或98％的同源性，最优选为至少99％的同源性。
本发明的第二方面提供一种融合蛋白，所述融合蛋白包含如第一方面所述的抗Her3抗体。
本发明的第三方面提供一种分离的核酸，所述核酸编码如第一方面所述的抗Her3抗体或第二方面所述的融合蛋白。
在本发明一些实施方案中，编码所述轻链可变区的核苷酸序列如SEQ ID NO:14中的第1-339位所示。
在本发明一些实施方案中，所述重链可变区的核苷酸序列如SEQ ID NO:6中的第1-351位所示。
在本发明一些实施方案中，编码所述轻链的核苷酸序列如SEQ ID NO:14所示。
在本发明一些实施方案中，编码所述重链的核苷酸序列如SEQ ID NO:6所示。
本发明的第四方面提供一种重组表达载体，所述重组表达载体包含如第三方面所述的核酸。
本发明的第五方面提供一种抗体偶联药物，所述抗体偶联药物包含如第一方面所述的抗Her3抗体或者如第二方面所述的融合蛋白。
本发明的第六方面提供一种双特异性抗体分子，所述双特异性抗体分子包含如第一方面所述的抗Her3抗体或者如第二方面所述的融合蛋白。
本发明的第七方面提供一种嵌合抗原受体T细胞，所述嵌合抗原受体T细胞包含如第一方面所述的抗Her3抗体或者如第二方面所述的融合蛋白。
本发明的第八方面提供一种药物组合物，所述药物组合物包含如第一方面所述的抗Her3抗体、如第二方面所述的融合蛋白、如第五方面所述的抗体偶联药物、如第六方面所述的双特异性抗体分子、或者如第七方面所述的嵌合抗原受体T细胞及其药学上可接受的盐、溶剂合物、或者其药学上可接受的盐的溶剂合物。
在本发明一些实施方案中，所述药物组合物还包括药用辅料。
本发明的第九方面提供一种试剂盒，所述试剂盒包含如第一方面所述的抗Her3抗体或者如第二方面所述的融合蛋白。
本发明的第十方面提供一种套装药盒，所述套装药盒包括药盒A和药盒B；
其中，所述药盒A包括如第一方面所述的抗Her3抗体或者如第八方面所述的药物组合物；所述药盒B包括其他治疗剂。
在本发明一些实施方案中，所述药盒A与药盒B的施用时间不分先后或者先施用所述药盒A。
本发明的第十一方面提供一种给药装置，所述给药装置包含：(1)用于对有需要的 受试者施用如第八方面所述的药物组合物的输注模块，以及(2)任选的药效监控模块。
本发明的第十二方面提供一种治疗/预防疾病的方法，所述方法包括向有需要的患者施用有效剂量的如第一方面所述的抗Her3抗体、如第二方面所述的融合蛋白、如第五方面所述的抗体偶联药物、如第六方面所述的双特异性抗体分子、如第七方面所述的嵌合抗原受体T细胞、如第八方面所述的药物组合物、或施用如第十一方面所述的给药装置。
在本发明一些实施方案中，所述疾病为肿瘤；
在本发明一些较佳的实施方案中，所述肿瘤为Her3阳性肿瘤。
在本发明一些更佳的实施方案中，所述Her3阳性肿瘤选自Her3阳性肺癌、卵巢癌、结直肠癌、乳腺癌、前列腺癌和胃癌中的一种或多种。
本发明的第十三方面提供一种如第一方面所述的抗Her3抗体、如第二方面所述的融合蛋白、如第五方面所述的抗体偶联药物、如第六方面所述的双特异性抗体分子、如第七方面所述的嵌合抗原受体T细胞、或者如第八方面所述的药物组合物在制备Her3蛋白抑制剂、或者制备治疗和/或预防肿瘤的药物中的应用。
所述肿瘤的优选限定如第十二方面所述。
本发明中，所述前列腺癌可为本领域通常理解的前列腺癌，前列腺癌细胞例如含有22Rv1细胞和/或LNCaP细胞。
本发明中，所述结直肠癌可为本领域通常理解的结直肠癌，结直肠癌细胞例如含有SW620细胞。
本发明中，所述肺癌可为本领域通常理解的肺癌，肺癌细胞例如含有NCI-H820细胞或HCC827细胞。
本发明中，所述卵巢癌可为本领域通常理解的卵巢癌，卵巢癌细胞例如含有OVCAR-8细胞。
本发明中，所述乳腺癌可为本领域通常理解的乳腺癌，乳腺癌细胞例如含有SK-BR-3细胞。
除非另有说明，在本发明说明书中出现的以下术语具有下述含义：
所述药用辅料可为药物生产领域中广泛采用的辅料。辅料主要用于提供一个安全、稳定和功能性的药物组合物，还可以提供方法，使受试者接受给药后活性成分以所期望速率溶出，或促进受试者接受组合物给药后活性成分得到有效吸收。所述的药用辅料可以是惰性填充剂，或者提供某种功能，例如稳定该组合物的整体pH值或防止组合物活性成分的降解。所述的药用辅料可包括下列辅料中的一种或多种：缓冲剂、螯合剂、防腐剂、助溶剂、稳定剂、赋形剂和表面活性剂着色剂、矫味剂和甜味剂。
术语“药学上可接受的”是指盐、溶剂、辅料等一般无毒、安全，并且适合于患者使用。所述的“患者”优选哺乳动物，更优选为人类。
术语“药学上可接受的盐”是指本发明化合物与相对无毒的、药学上可接受的酸或碱制备得到的盐。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的药学上可接受的碱与这类化合物的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括但不限于：锂盐、钠盐、钾盐、钙盐、铝盐、镁盐、锌盐、铋盐、铵盐、二乙醇胺盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的药学上可接受的酸与这类化合物的中性形式接触的方式获得酸加成盐。所述的药学上可接受的酸包括无机酸，所述无机酸包括但不限于：盐酸、氢溴酸、氢碘酸、硝酸、碳酸、磷酸、亚磷酸、硫酸等。所述的药学上可接受的酸包括有机酸，所述有机酸包括但不限于：乙酸、丙酸、草酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、水杨酸、酒石酸、甲磺酸、异烟酸、酸式柠檬酸、油酸、单宁酸、泛酸、酒石酸氢、抗坏血酸、龙胆酸、富马酸、葡糖酸、糖酸、甲酸、乙磺酸、双羟萘酸(即4,4’-亚甲基-双(3-羟基-2-萘甲酸))、氨基酸(例如谷氨酸、精氨酸)等。当本发明的化合物中含有相对酸性和相对碱性的官能团时，可以被转换成碱加成盐或酸加成盐。具体可参见Berge et al.,"Pharmaceutical Salts",Journal of Pharmaceutical Science 66:1-19(1977)、或、Handbook of Pharmaceutical Salts:Properties,Selection,and Use(P.Heinrich Stahl and Camille G.Wermuth,ed.,Wiley-VCH,2002)。
术语“溶剂合物”是指本发明化合物与化学计量或非化学计量的溶剂结合形成的物质。溶剂合物中的溶剂分子可以有序或非有序排列的形式存在。所述的溶剂包括但不限于：水、甲醇、乙醇等。
术语“治疗”或它的同等表达当用于例如癌症时，指用来减少或消除患者体内癌细胞数目或减轻癌症的症状的程序或过程。癌症或另外的增生性障碍的“治疗”不一定指癌症细胞或其它障碍会实际上被消除，细胞或障碍的数目会实际上被减少或者癌症或其它障碍的症状会实际上被减轻。通常，即使只具有低的成功可能性也会进行治疗癌症的方法，但是考虑到患者的病史和估计的生存预期，其仍然被认为诱导总体有益的作用过程。
术语“预防”是指获得或发生疾病或障碍的风险降低。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
如无特殊说明，本发明中的室温是指20-30℃。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明的抗Her3抗体具有高表达量，可达到现有抗体的3～5倍，同时保持了与靶点Her3的高亲和力，对表达Her3等的多种肿瘤细胞具有很好的抑制效果。
附图说明
图1为实施例2中2F8瞬转表达上清(2天)的SDS-PAGE检测纯化产物的表达量检测结果示意图。
图2为实施例2中2F8瞬转纯化产物的SDS-PAGE检测结果；
其中：A为6％非还原性SDS-PAGE，B为12％还原性SDS-PAGE。
图3为实施例2中调整轻链转染比例后2F8瞬转表达上清的12％还原性SDS-PAGE检测结果；
其中：A为转染2天，B为转染4天。
图4为实施例3中2F8与1A9轻、重链组合瞬转表达上清的12％还原性SDS-PAGE检测结果。
图5为实施例3中更换轻链信号肽的2F8瞬转纯化产物的6％非还原性SDS-PAGE检测结果。
图6为实施例3中更换轻链信号肽的2F8瞬转纯化产物的12％还原性SDS-PAGE检测结果。
图7为实施例4中2F8轻链突变体瞬转后表达上清的12％还原性SDS-PAGE检测结果示意图；
其中：A为瞬转3天，B为瞬转5天。
图8为实施例4中2F8轻链突变体瞬转5天后的纯化产物的12％还原性SDS-PAGE检测结果示意图；
其中：A为2F8轻链突变体瞬转，B为2F8Lm1重复瞬转。
图9为实施例5中3F5(上)与2F8-L(下)氨基酸序列Blast图。
图10为实施例6中FACS检测2F8及3F5在T-47D细胞上的结合分析结果示意图。
图11为实施例6中FACS检测2F8及3F5在MDA-MB-453细胞上的结合分析结果示意图。
图12为2F8与Her3-His重组蛋白的反应曲线图。
图13为3F5与Her3-His重组蛋白的反应曲线图。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
缩写说明：
PCR：聚合酶链式反应；
CHO：中国仓鼠卵巢细胞；
HTRF：均相时间分辨荧光。
实施例1 抗Her3抗体稳定表达细胞株的制备
如表1所示，本实施例选取高亲和力并特异性靶向Her3的单克隆抗体2F8，其轻链的氨基酸序列如SEQ ID NO:1所示，其重链的氨基酸序列如SEQ ID NO:3所示。2F8的轻、重链核苷酸序列通过全基因合成(苏州金唯智生物科技有限公司)，其轻链的核苷酸序列如SEQ ID NO:2所示，其重链的核苷酸序列如SEQ ID NO:4所示。通过EcoR I和Hind III(TAKARA，R3104S、R3101S)双酶切将轻、重链分别单独构建至表达载体pV81中(上海翊圣生物，pEE14.2)，通过连接转化至Trans 1-T1感受态细胞(北京全式金生物，CD501-03)中，从中挑取克隆进行PCR鉴定并送检、测序确认，培养扩增阳性克隆进行质粒中抽，从而获得抗体轻链真核表达质粒2F8-L/pV81和抗体重链真核表达质粒2F8-H/pV81，轻、重链真核表达质粒比例1.5/1，通过电击转化至已适应悬浮生长的CHO细胞中(ATCC，CCL-61 TM)，电转48小时后采用HTRF法(Homogeneous Time-Resolved Fluorescence，均相时间分辨荧光)测得平均表达量为1.8μg/mL，表达量较低。经过稳定转染筛选及亚克隆，从中挑选表达量TOP10的克隆进行培养评估，在发酵罐中经过14天fed-batch培养(基础培养基：Dynamis AGT Medium，Gibco；补料培养基：EfficientFeedC+，Gibco)，最高表达量仅为1.2g/。综合以上，抗体2F8在宿主细胞CHO中表达量未达到预期目标。
表1. 2F8轻重链氨基酸和核苷酸序列
Figure PCTCN2021115952-appb-000001
Figure PCTCN2021115952-appb-000002
Figure PCTCN2021115952-appb-000003
实施例2.抗Her3抗体表达优化
对于抗体表达问题，研究中尝试通过更换信号肽加密码子优化、更换宿主细胞以及调整稳定转染条件等技术方法，以期改善抗体表达。
向抗体2F8的氨基酸序列中加上不同的信号肽，利用三联体密码子编码原则，进行密码子优化，得到氨基酸序列相同、核苷酸不同的2种2F8轻、重链分子(2F8-L-new，其核苷酸序列如SEQ ID NO:5所示；2F8-H-new，其核苷酸序列如SEQ ID NO:6所示；2F8-L-2，其核苷酸序列如SEQ ID NO:7所示；2F8-H-2，其核苷酸序列如序列SEQ ID NO:8所示，具体如表2所示)。重新进行基因合成，并构建至表达载体pV81，将轻、重链进行组合(组合方式如表2所示)，通过转染试剂293fectin(Invitrogen，12347019)转入HEK293细胞(中科院细胞库，GNHu43)中，转染体积20mL；转染2天后，取少量细胞培养上清，检测表达量；转染4天后收集细胞培养上清，上清体积为25mL，经一步Protein-A亲和纯化后，检测纯化产物表达量。检测结果如图1所示，结果显示HEK293瞬时转染2天，2F8的野生型和2种优化序列的表达量无明显差异。
表2. 2F8轻链核苷酸序列、重链核苷序列及组合方式
Figure PCTCN2021115952-appb-000004
Figure PCTCN2021115952-appb-000005
Figure PCTCN2021115952-appb-000006
Figure PCTCN2021115952-appb-000007
转染4天后，对2F8-new、2F8-1和2F8-2进行纯化、定量；其中2F8-new、2F8-1表达无明显差异，2F8-2表达稍高于2F8-1；抗体的基础表达量均偏低，约20μg/mL；蛋白纯化信息如表3所示；纯化产物6％非还原性SDS-PAGE检测结果如图2的A所示，12％还原性SDS-PAGE检测结果如图2的B所示，可见2F8分子轻链表达比例偏低。
表3. 2F8及FDA029瞬转表达上清纯化信息
Figure PCTCN2021115952-appb-000008
调整2F8分子轻链转染比例整，将2F8-new的轻、重链质粒按正常转染比例以及轻链多加一倍的比例，通过转染试剂293fectin，共转入HEK293细胞中，转染体积20mL；转染2天和4天时，取少量细胞培养上清，检测表达量。瞬转细胞培养上清的检测结果如图3的A和B所示，增加轻链转染比例后，2F8-new的基础表达量没有改善。
将2F8-new的轻重链片段构建到新的表达载体中，命名为2F8-3，并电击转染至宿主细胞CHOK1SV(载体和细胞均源于Lonza)，具有良好表达的抗体1A9(其轻链氨基酸序列如SEQ ID NO:9所示；其重链氨基酸序列如SEQ ID NO:10所示)作为系统对照，培养基为CD CHO Medium(Gibco，10743029)，表达结果如表5所示，实验组2F8-3在CHOK1SV中的表达量较原实验体系的2F8-new有所提升，但远低于系统对照，表明抗体2F8属于难表达抗体。
表4. 1A9轻重链氨基酸序列
Figure PCTCN2021115952-appb-000009
Figure PCTCN2021115952-appb-000010
表5. 2F8稳定转染48小时表达量
抗体 培养基 48小时表达量(mg/L) 备注
2F8-new CD CHO Medium 0.158 /
2F8-3 CD CHO Medium 0.327 实验组
1A9 CD CHO Medium 0.778 系统对照
实施例3.交叉转染组合评价
将2F8分子轻、重链组合交叉转染，将2F8-new的轻、重链质粒及抗体1A9的轻、重链质粒进行组合(组合方式如表6所示)，通过转染试剂293fectin，转入HEK293细胞中，转染体积20mL；转染2天后，取细胞培养上清检测表达量。瞬转细胞培养上清的检测结果如图4所示，与2F8-new相比，2F8的重链与1A9轻链组合表达明显增强，2F8的轻链与1A9重链组合表达没有改善，2F8的轻链对2F8的表达有关键性的影响。
表6. 2F8轻、重链与1A9轻、重链组合方式
Figure PCTCN2021115952-appb-000011
Figure PCTCN2021115952-appb-000012
更换2F8轻链信号肽；将2F8-new的轻链信号肽MGWSCIILFLVATATGVHS(SEQ ID NO:11)更换为新的信号肽MSVPTQVLGLLLLWLTDARC(SEQ ID NO:12)，通过转染试剂293fectin，转入HEK293细胞中，转染体积20mL；转染4天后收集细胞培养上清，上清体积为25mL，经一步Protein-A亲和纯化后，检测纯化产物表达量。更换轻链信号肽的2F8分子瞬转纯化产物的6％非还原性SDS-PAGE检测结果如图5所示，12％还原性SDS-PAGE检测结果如图6所示。更换轻链信号肽后，2F8的基础表达有轻微提高，但未得到明显改善。
实施例4.抗体序列分析
根据2F8分子轻链的序列特征，对其进行了3种轻链突变体的设计(Lm1、Lm2和Lm3)，其氨基酸序列与核苷酸序列如表7所示。按实施例1相同方式进行载体构建，获得轻链突变体2F8-Lm1、2F8-Lm2和2F8-Lm3。
表7. 2F8分子突变体序列
Figure PCTCN2021115952-appb-000013
Figure PCTCN2021115952-appb-000014
Figure PCTCN2021115952-appb-000015
2F8分子轻链突变体的转染
将2F8分子的轻链突变体、重链进行组合(组合方式如表8所示)通过转染试剂293fectin，转入HEK293细胞中，转染体积20mL；转染3天后，取少量细胞培养上清，检测表达量；转染5天后收集细胞培养上清，上清体积为25mL，经一步Protein-A亲和纯化后，检测纯化产物表达量。
表8 2F8轻链突变体组合方式
名称 重链 轻链
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
2F8轻链突变体转染后3天表达上清的12％还原性SDS-PAGE检测结果如图7的A所示，转染后5天表达上清的12％还原性SDS-PAGE检测结果如图7的B所示，上清纯化产物的12％还原性SDS-PAGE检测结果如图8的A所示，其中2F8-m1的表达量有明显提高，对其进行重复转染确认，转染后5天表达上清的12％还原性SDS-PAGE检测结果如图8的B所示，2F8-m1的表达明显得到改善，以SDS-PAGE的灰度分析及纯化后蛋白量计算，表达量提高约3-5倍；轻链突变体2F8-Lm1与2F8-L氨基酸差异如图9所示。
实施例5.位点改造抗体的表达制备
将2F8轻链突变体2F8-Lm1与重链2F8-H-new的组合，重命名为抗体3F5。使用ExpiCHO细胞瞬转2F8、3F5两个抗体，按照如下表9进行转染操作，转染体积每个摇瓶100mL，培养8天经HTRF检测表达量；结果显示3F5的瞬转表达量为2F8的2.9倍。
表9. 2F8和3F5瞬时转染
Figure PCTCN2021115952-appb-000016
抗体3F5进行稳定细胞株筛选、亚克隆，从中挑选表达量高的细胞进行培养评估，发酵罐中经过14天fed-batch培养，3F5的表达量最高为3.3g/L；而2F8的表达量仅为1.2g/L，实现了在大规模培养下的表达提升。综合以上结果，抗体3F5表达量较抗体2F8有明显提高，满足项目抗体表达量预期。
实施例6.抗Her3抗体突变序列的功能分析
3F5突变体在T-47D细胞上的结合分析
利用FACS检测结合能力，首先按3×10 5～1×10 6密度收集T-47D细胞(中科院细胞库，TCHu 87)，去上清；加入FACS染色缓冲液(Moregate，3827104)洗涤细胞一次， 再次离心细胞去上清，并使用2ml FACS重悬均分至2个1.5mL离心管中，冰浴封闭0.5小时，1000rpm离心处理5分钟，去上清；向细胞中加入100μL相应浓度梯度的3F5突变体(10μg/mL、3.33μg/mL、1.11μg/mL、0.37μg/mL、0.12μg/mL)，冰浴孵育1h后，离心去上清，加入0.5mL/管FACS染色缓冲液1000rpm离心处理5min(4℃)，去上清，重复两次。加入100μL荧光标记二抗(Thermo，A11013)，冰浴孵育30min后，离心去上清，加入0.5mL/管FACS染色缓冲液1000rpm离心处理5min(4℃)，去上清，重复两次；加入0.5mL PBS稀释液(上海双螺旋生物科技有限公司，P10033)重悬细胞，冰上避光放置；流式细胞仪检测荧光值。FACS检测结果如图10所示。结果显示，3F5与T-47D细胞有结合，且具有量效关系。在不同抗体浓度下，与亲本分子2F8结合能力无明显差异。
按照上述方法进行FACS检测的结果如图11所示，2F8Lm1与MDA-MB-453细胞(中科院细胞库，TCHu233)有结合，且具有良好的量效关系。在不同抗体浓度下，与亲本分子2F8结合能力无明显差异。
实施例7. 3F5突变体的亲和力分析
检测抗体2F8与3F5对Her3-His重组蛋白结合的亲和力，通过使用BLI的方法检测固化的抗体与Her3的结合动力学曲线，检测的方法参照仪器(Fortebio，Octet 96e)的使用说明进行，简言之，先用Loading Buffer/Sample dilution buffer(1×PBS，pH7.4，0.1％BSA+0.02％Tween-20)对AMC传感器平衡60s，得到Baseline 1。将待测抗体用Loading Buffer稀释成10μg/mL的浓度与平衡后的传感器进行结合，结合后的传感器再次用Loading Buffer进行再平衡，得到Baseline 2。然后把载有抗体的传感器至于用样品稀释液稀释为100～3.13nM的人Her3-His中结合90s，得到抗体与蛋白的结合曲线，如图12和图13所示。然后将结合有抗原的传感器再次置于Sample Dilution Buffer中解离180s，得到解离曲线。通过结合与解离曲线分别计算抗体与蛋白结合的k-on、k-off值，计算KD值，所得结果显示2F8与3F5对Her3-His重组蛋白的亲和力无差异，2F8亲和力为2.43E-09mol/L(以下表10中简写为M)，3F5亲和力为2.53E-09mol/L，详细动力学参数如下表10所示。
表10. 2F8及2F8Lm1亲和力测定结果
样品名称 批号 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
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。
Figure PCTCN2021115952-appb-000017
Figure PCTCN2021115952-appb-000018
Figure PCTCN2021115952-appb-000019
Figure PCTCN2021115952-appb-000020
Figure PCTCN2021115952-appb-000021
Figure PCTCN2021115952-appb-000022
Figure PCTCN2021115952-appb-000023
Figure PCTCN2021115952-appb-000024
Figure PCTCN2021115952-appb-000025
Figure PCTCN2021115952-appb-000026
Figure PCTCN2021115952-appb-000027
Figure PCTCN2021115952-appb-000028
Figure PCTCN2021115952-appb-000029
Figure PCTCN2021115952-appb-000030
Figure PCTCN2021115952-appb-000031
Figure PCTCN2021115952-appb-000032
Figure PCTCN2021115952-appb-000033
Figure PCTCN2021115952-appb-000034

Claims (15)

  1. 一种抗Her3抗体，其特征在于，所述抗Her3抗体包含轻链和重链，
    所述轻链中的轻链可变区的氨基酸序列如SEQ ID NO:13中的第1-113位所示，所述重链中的重链可变区的氨基酸序列如SEQ ID NO:3中的第1-117位所示。
  2. 如权利要求1所述的抗Her3抗体，其特征在于，所述轻链还包含轻链恒定区，所述重链还包含重链恒定区；
    所述轻链恒定区较佳地为人源抗体或小鼠抗体的轻链恒定区；更佳地为人源抗体的κ或λ链的恒定区；和/或，
    所述重链恒定区较佳地为人源抗体或小鼠抗体的重链恒定区；更佳地为人源抗体IgG1、IgG2、IgG3或者IgG4的重链恒定区。
  3. 如权利要求2所述的抗Her3抗体，其特征在于，所述轻链的氨基酸序列如SEQ ID NO:13所示；和/或，所述重链的氨基酸序列如SEQ ID NO:3所示。
  4. 一种融合蛋白，其特征在于，所述融合蛋白包含如权利要求1～3任一项所述的抗Her3抗体。
  5. 一种分离的核酸，其特征在于，所述核酸编码如权利要求1～3任一项所述的抗Her3抗体或如权利要求4所述的融合蛋白；
    较佳地，编码所述轻链可变区的核苷酸序列如SEQ ID NO:14中的第1-339位所示；和/或，编码所述重链可变区的核苷酸序列如SEQ ID NO:6中的第1-351位所示；
    更佳地，编码所述轻链的核苷酸序列如SEQ ID NO:14所示；和/或，编码所述重链的核苷酸序列如SEQ ID NO:6所示。
  6. 一种重组表达载体，其特征在于，所述重组表达载体包含如权利要求5所述的核酸。
  7. 一种抗体偶联药物，其特征在于，所述抗体偶联药物包含如权利要求1～3任一项所述的抗Her3抗体或如权利要求4所述的融合蛋白。
  8. 一种双特异性抗体分子，其特征在于，所述双特异性抗体分子包含如权利要求1～3任一项所述的抗Her3抗体或如权利要求4所述的融合蛋白。
  9. 一种嵌合抗原受体T细胞，其特征在于，所述嵌合抗原受体T细胞包含如权利要求1～3任一项所述的抗Her3抗体或如权利要求4所述的融合蛋白。
  10. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1～3任一项所述的抗Her3抗体、如权利要求4所述的融合蛋白、如权利要求7所述的抗体偶联药物、如 权利要求8所述的双特异性抗体分子、或者如权利要求9所述的嵌合抗原受体T细胞及其药学上可接受的盐、溶剂合物、或者其药学上可接受的盐的溶剂合物；
    较佳地，所述药物组合物还包括药用辅料。
  11. 一种试剂盒，其特征在于，所述试剂盒包含如权利要求1～3任一项所述的抗Her3抗体或者如权利要求4所述的融合蛋白。
  12. 一种套装药盒，其特征在于，所述套装药盒包括药盒A和药盒B；
    其中，所述药盒A包括如权利要求1～3任一项所述的抗Her3抗体或者如权利要求10所述的药物组合物；所述药盒B包括其他治疗剂；
    较佳地，所述药盒A与药盒B的施用时间不分先后或者先施用所述药盒A。
  13. 一种给药装置，其特征在于，所述给药装置包含：(1)用于对有需要的受试者施用如权利要求10所述的药物组合物的输注模块，以及(2)任选的药效监控模块。
  14. 一种治疗/预防疾病的方法，其特征在于，所述方法包括向有需要的患者施用有效剂量的如权利要求1～3任一项所述的抗Her3抗体、如权利要求4所述的融合蛋白、如权利要求7所述的抗体偶联药物、如权利要求8所述的双特异性抗体分子、或者如权利要求9所述的嵌合抗原受体T细胞、如权利要求10所述的药物组合物、或施用如权利要求13所述的给药装置；
    较佳地，所述疾病为肿瘤；
    更佳地，所述肿瘤为Her3阳性肿瘤；
    进一步更佳地，所述Her3阳性肿瘤选自Her3阳性肺癌、卵巢癌、结直肠癌、乳腺癌、前列腺癌和胃癌中的一种或多种。
  15. 一种如权利要求1～3任一项所述的抗Her3抗体、如权利要求4所述的融合蛋白、如权利要求7所述的抗体偶联药物、如权利要求8所述的双特异性抗体分子、如权利要求9所述的嵌合抗原受体T细胞、或者如权利要求10所述的药物组合物在制备Her3蛋白抑制剂、或者制备治疗和/或预防肿瘤的药物中的应用；
    较佳地，所述肿瘤为Her3阳性肿瘤；
    更佳地，所述Her3阳性肿瘤选自Her3阳性肺癌、卵巢癌、结直肠癌、乳腺癌、前列腺癌和胃癌中的一种或多种。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101808680B (zh) * 2007-08-01 2014-07-02 F·霍夫曼-拉罗氏股份公司 带有用于监测和控制流体输送的器件的便携式输注设备
CN110724194A (zh) * 2018-07-17 2020-01-24 上海生物制品研究所有限责任公司 抗her3人源化单克隆抗体及其制剂
CN113135995A (zh) * 2020-01-17 2021-07-20 上海生物制品研究所有限责任公司 抗her3单克隆抗体及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101808680B (zh) * 2007-08-01 2014-07-02 F·霍夫曼-拉罗氏股份公司 带有用于监测和控制流体输送的器件的便携式输注设备
CN110724194A (zh) * 2018-07-17 2020-01-24 上海生物制品研究所有限责任公司 抗her3人源化单克隆抗体及其制剂
CN113135995A (zh) * 2020-01-17 2021-07-20 上海生物制品研究所有限责任公司 抗her3单克隆抗体及其应用

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