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WO2023028914A1 - Anticorps anti-her3 et son utilisation - Google Patents

Anticorps anti-her3 et son utilisation 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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Ceased
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PCT/CN2021/115952
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English (en)
Chinese (zh)
Inventor
郭青松
沈毅珺
杨彤
陈珍
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL Co Ltd
Shanghai Fudan Zhangjiang Bio pharmaceutical Co Ltd
Original Assignee
SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL Co Ltd
Shanghai Fudan Zhangjiang Bio pharmaceutical Co Ltd
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Application filed by SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL Co Ltd, Shanghai Fudan Zhangjiang Bio pharmaceutical Co Ltd filed Critical SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL Co Ltd
Priority to PCT/CN2021/115952 priority Critical patent/WO2023028914A1/fr
Priority to CN202180100378.7A priority patent/CN117616044A/zh
Publication of WO2023028914A1 publication Critical patent/WO2023028914A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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

La présente invention concerne un anticorps anti-Her3 et son utilisation. L'anticorps anti-Her3 comprend une chaîne légère et une chaîne lourde, la séquence d'acides aminés d'une région variable de chaîne légère dans la chaîne légère est telle que représentée dans les positions 1 à 113 dans SEQ ID NO : 13, et la séquence d'acides aminés d'une région variable de chaîne lourde dans la chaîne lourde est telle que représentée dans les positions 1 à 117 dans SEQ ID NO : 3. La quantité d'expression de l'anticorps anti-Her3 selon la présente invention est de 3 à 5 fois celle d'un anticorps existant, tout en conservant une affinité élevée avec la cible Her3.
PCT/CN2021/115952 2021-09-01 2021-09-01 Anticorps anti-her3 et son utilisation Ceased WO2023028914A1 (fr)

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CN202180100378.7A CN117616044A (zh) 2021-09-01 2021-09-01 抗Her3抗体及其应用

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Citations (3)

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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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