CN117603916B - Human B7-H3 antibody based on fully humanized antibody mouse, and preparation method and application thereof - Google Patents
Human B7-H3 antibody based on fully humanized antibody mouse, and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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Abstract
The invention belongs to the field of fully humanized antibody development, and particularly discloses a fully humanized antibody mouse-based human B7-H3 antibody, and a preparation method and application thereof; the preparation method comprises the following steps: 1) Animal immunization: the antibody mouse is a 6-8 week female fully human antibody mouse, the immunogen cells are B7-H3-CHO-K1 living cells, and the immune mode is intraperitoneal injection; 2) Antibody titer detection: collecting serum, incubating antibodies, analyzing results by an up-flow cytometer, and selecting the higher antibody titer for cell fusion; 3) Preparing hybridoma cells and screening to obtain the human B7-H3 antibody based on the fully humanized antibody mouse; the novel humanized B7-H3 antibody screened by the invention has better affinity, lower immunogenicity and more similar to the natural antibody in a human body, can be used for targeted treatment of cancers or combined medication, and improves the efficacy and safety of the medicament.
Description
Technical Field
The invention belongs to the field of development of fully humanized antibodies, and particularly discloses a fully humanized antibody mouse-based human B7-H3 antibody, and a preparation method and application thereof.
Background
Since the approval of the first monoclonal antibody drug in 1986, the development of more than 30 years has led to the development of antibody drugs, which have become not only the leading drugs for the treatment of tumor diseases, but also increasingly used for the treatment of autoimmune diseases, metabolic diseases and infectious diseases. It is counted that the ratio of antibody drugs in the drugs newly approved to be marketed every year has reached 1/5, and the antibody drugs have become the most popular drugs.
The conventional antibody drug development method mainly comprises the traditional hybridoma cell fusion technology and phage library display technology. In 1975, the establishment of hybridoma cell fusion technology opened the preorder of monoclonal antibody drug development. In 1986, the FDA approved the first monoclonal antibody, moromonas (muromonab), for the treatment of immune rejection at the time of organ transplantation. However, as the antibody is a mouse monoclonal antibody developed based on hybridoma technology after mice are immunized, researchers find that the toxic and side effects of the antibody for human body treatment are more and more obvious along with the increase of treatment cases. This is because murine antibodies are highly immunogenic when used in humans, and can elicit an immune response in humans, thereby producing human anti-mouse antibodies (HMAM).
For the above reasons, researchers have begun to search for and improve antibody development techniques to reduce the immunogenicity of antibody drugs and to improve the therapeutic effects of antibody drugs. Summarizing, antibody drug development generally goes through four stages of development: first generation murine monoclonal antibodies, second generation human mouse chimeric antibodies, third generation humanized antibodies and fourth generation fully human antibodies. The human mouse chimeric antibody is obtained by fusing the variable region of a mouse antibody with the constant region of a human antibody, thereby reducing the immunogenicity of the antibody. The humanized antibody is prepared by grafting the CDR region of a murine antibody onto the human antibody framework, so that the immunogenicity of the antibody to a human body is weakened to a greater extent, and the therapeutic effect of the antibody is improved. However, although the components of mice can be significantly reduced by humanization, humanized antibodies still do not reach a satisfactory level, and the effect of immunogenicity of the remaining mouse components on the therapeutic effects of the anti-drug is still not negligible. Therefore, the development of fully human antibody drugs has become a necessary trend.
At present, the development of fully human antibodies mainly comprises two technical approaches: one is based on phage display of a human antibody library; one is to construct transgenic animals, such as humanized antibody mice. The phage library-based screening technology has the advantages of rapidness and simplicity, and is one of the important means for developing fully human antibodies. However, since the B cells used to construct phage libraries are typically derived from healthy donors, the constructed antibody libraries are natural antibody libraries, and thus the developed antibodies typically have drawbacks in terms of affinity, and complex affinity maturation steps are typically required after obtaining the antibodies. The antibody development technology based on transgenic animals can effectively overcome the defect of phage library screening technology. Humanized antibody mice are immunized by normal immunization procedures, and B cells develop and differentiate normally after antigen immunostimulation, usually to obtain high affinity antibodies.
B7-H3 (CD 276) is an important immune checkpoint molecule in the B7 family, playing a dual role of co-stimulation/co-suppression in the immune system. In addition, B7-H3 is under-expressed in most normal tissues, but is widely expressed in various human malignant tumors such as prostate cancer, pancreatic cancer, breast cancer, colorectal cancer, lung cancer, ovarian cancer, and the like. The series of characteristics make B7-H3 a potential target for developing various anticancer drugs.
In the invention, B7-H3-CHO-K1 cells are used as immunogens, a fully human antibody mouse developed by the company is immunized, a plurality of antibodies with high binding capacity to human B7-H3 antigen on the cell surface are developed and obtained based on a hybridoma cell fusion technology, and the sequences of the antibodies are extracted from two of the antibodies, so that the heavy chain variable region sequences and the light chain variable region sequences of the antibodies are all fully human antibody sequences. Therefore, the invention provides a fully human antibody development method based on an immune fully human antibody mouse, which is hopeful to greatly assist and accelerate the development process of fully human antibody medicines.
Disclosure of Invention
In order to solve the problems, the invention discloses a human B7-H3 antibody based on a fully humanized antibody mouse, and a preparation method and application thereof.
The invention comprises the following technical scheme:
a method for preparing hybridoma capable of generating humanized antibody capable of specifically recognizing human B7-H3 immune checkpoint, comprising the following steps,
1) Animal immunization: the antibody mouse is a 6-8 week female fully human antibody mouse, the immunogen cells are B7-H3-CHO-K1 living cells, and the immune mode is intraperitoneal injection;
2) Antibody titer detection: collecting serum, incubating antibodies, analyzing results by an up-flow cytometer, and selecting the higher antibody titer for cell fusion;
3) Preparing and obtaining hybridoma cells.
Furthermore, the invention discloses a preparation method of a human B7-H3 antibody based on a fully humanized antibody mouse, which comprises the following steps:
1) Animal immunization: the antibody mouse is a 6-8 week female fully human antibody mouse, the immunogen cells are B7-H3-CHO-K1 living cells, and the immune mode is intraperitoneal injection;
2) Antibody titer detection: collecting serum, incubating antibodies, analyzing results by an up-flow cytometer, and selecting the higher antibody titer for cell fusion;
3) Preparing and obtaining hybridoma cells;
4) And 3) screening and preparing the human B7-H3 antibody based on the fully humanized antibody mouse in the hybridoma cells prepared in the step 3).
Furthermore, the invention discloses a human B7-H3 antibody based on a fully humanized antibody mouse, which is prepared by the method.
Furthermore, the invention discloses a human B7-H3 antibody based on a fully humanized antibody mouse, wherein the amino acid sequence of a heavy chain variable region of the B7-H3 antibody is shown as SEQ ID No.2 or SEQ ID No. 10; the amino acid sequence of the light chain variable region is shown as SEQ ID No.4 or SEQ ID No. 12.
Further, the amino acid sequence of the heavy chain of the B7-H3 antibody is shown as SEQ ID No.1 or SEQ ID No. 9; the amino acid sequence of the light chain is shown as SEQ ID No.3 or SEQ ID No. 11.
Further, the invention discloses a biological material, which comprises any one of a) -d),
A) A nucleic acid molecule comprising an amino acid sequence as set forth in any one of SEQ ID nos. 1,2, 3, 4, 9, 10, 11, 12;
b) A recombinant vector comprising the nucleic acid molecule of a);
c) A recombinant microorganism comprising the nucleic acid molecule as described in a) or comprising the recombinant vector as described in b);
d) A recombinant cell comprising the nucleic acid molecule as set forth in a) or comprising the recombinant vector as set forth in b).
Further, the biological material is a nucleotide molecule containing a nucleic acid sequence shown in any one of SEQ ID No.5-8 and 13-16.
Furthermore, the invention discloses application of the antibody or the biological material in preparing medicines for treating cancers.
Further, in the above application, the drug comprises one or more of blocking antibodies, fc-enhanced ADCC-inducing antibodies, ADCs, bispecific antibodies, CAR-T.
Further, in the above application, the cancer includes one or more of prostate cancer, pancreatic cancer, breast cancer, colorectal cancer, lung cancer and ovarian cancer.
Compared with the prior art, the invention has the following beneficial effects:
According to the invention, by immunizing a fully humanized antibody mouse, based on a hybridoma technology, multiple hybridoma cells are obtained, and antibody sequences of two strains are prepared. As a result of sequence analysis, the variable region sequences of the two antibodies are all human sequences. Therefore, the invention provides a fully human antibody development method based on fully human antibody murine immunity, which can theoretically obtain high affinity antibodies without further affinity maturation compared with the traditional phage library display method; compared with the development method of the antibody immunized by the common mice, the antibody sequence obtained by the mouse immunization based on the fully-humanized antibody has affinity comparable to that of the human antibody, but no further humanized transformation is needed. Therefore, the development of the antibody based on the fully humanized antibody mouse greatly accelerates the development process of antibody drugs, and omits complicated steps of affinity maturation and humanized transformation.
Drawings
FIG. 1 is a mouse trisomy serum titer assay wherein Ab mouse represents a fully humanized antibody mouse and WT mouse represents a C57BL/6N mouse;
FIG. 2 is a representative example of the detection of the binding capacity of hybridoma cell supernatants to antigen-positive cells;
FIG. 3 is a graph showing the statistics of average fluorescence intensity of binding of hybridoma cell supernatants to antigen-positive cells;
FIG. 4 is a representative example of the detection of the binding capacity of hybridoma cell supernatants to antigen-positive cells;
FIG. 5 shows the results of hybridoma cell antibody sequence modulation experiments.
Detailed Description
The basic flow of the invention is as follows:
1. Immunization of animals
The antibody mice are 3 female fully human antibody mice of 6-8 weeks, the immunogen cells are B7-H3-CHO-K1 living cells, and the immunization mode is intraperitoneal injection. The invention selects the immunogen cell B7-H3-CHO-K1 (product number: ASC 003) and the fully humanized antibody mouse (HUGO-AB TM) which are independently researched and commercially prepared by the company. Immunization procedures were as follows: the first immunogen is 100 mul Freund's complete adjuvant and 2X 10 7 cells emulsified; the immunization of the 2 nd and the 3 rd adopts 100 mul Freund incomplete adjuvant and 2X 10 7 living cells after 1:1 emulsification, and the immunization time interval is two weeks; mouse serum was used to detect antibody titers 7 days after 3 rd immunization, boost was performed 10 days later, the immunization procedure was the same as 3 rd immunization, and cell fusion was performed 4 days of boost.
2. Antibody titer detection
Mouse serum hB7-H3 antibody titers were determined by FACS with 200 μl of mouse peripheral blood collected from the inner canthus. Mouse serum PBS was diluted 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 100. Mu.l each was incubated for 293T cells, 1X 10 5 cells per sample, 30: 30min at 37 ℃; after incubation, 3min was centrifuged at 4500 rpm, the supernatant was discarded, washed twice with 1ml of 1 XPBS, the supernatant was discarded, resuspended in 100. Mu.l of 1 XPBS, each of which was added with 0.5. Mu. l Goat anti Mouse IgG PE secondary antibody and incubated at 37℃in the incubator in the absence of light for 30min; after the secondary antibody incubation, 3min was centrifuged at 4500 rpm, the supernatant was discarded, washed twice with 1ml of 1 XPBS, the supernatant was discarded, resuspended in 100. Mu.l of 1 XPBS, and the results were analyzed by an up-flow cytometer, and the higher antibody titer was selected for cell fusion.
3. Preparation of hybridoma cells
3.1 Preparation of myeloma cell SP2/0
The SP2/0 cells were resuscitated one week prior to cell fusion, cultured by cell expansion, and passaged 1 day prior to hybridoma cell fusion, so that SP2/0 cells were in the logarithmic growth phase when SP2/0 cells and mouse B cells were fused the next day.
3.2 Preparation of mouse B cells
1 Immunized mouse was sacrificed by CO 2, and the mice were immersed in a box containing 75% alcohol; fixing the sterilized mice on an dissecting plate, dissecting, taking out spleen, grinding with a filter screen, washing the filter screen with 10ml of 1 XPBS, and collecting spleen cells; centrifuging spleen cell suspension at 25deg.C, 400×g, and 10 min to remove supernatant, and sucking with gun head; mixing three times volume of erythrocyte lysate, precipitating, standing at room temperature for 10 min, centrifuging at 25deg.C, 300 g, and 10 min, removing supernatant, and sucking with gun head; repeating for about 3 times. SP2/0 was removed at the 3 rd lysis of erythrocytes, at which time both spleen cells and SP2/0 were resuspended in HD complete medium, centrifuged, washed 3 times, and the cell ratio ready for fusion was SP2/0: spleen cells = 1:5-1:10. Finally, uniformly mixing the two cells in a 50ml centrifuge tube by using a preheated HD complete culture medium, and discarding the supernatant after centrifugation at 25 ℃, 400: 400 g and 7: 7 min; the bottom of the centrifuge tube was gently scraped on the centrifuge tube rack to loosen the cells slightly.
3.3 Cell fusion
Ensuring that the fusion process is carried out at 37 ℃, firstly adding 1ml 50% PEG4000 solution into spleen cells and myeloma cell sediment in 1min, adding 1ml PEG4000 while rotating a centrifuge tube, and slightly blowing and resuspending cells in 1min after the addition; then 4 min is added with 4 ml HD complete culture medium, the centrifuge tube is rotated and the mixture is added, and after the addition, 1min is slightly rotated and mixed uniformly; then 10 ml HD complete culture medium is added to the wall, the mixture is gently mixed, and the mixture is kept stand for 15 min in a 37 ℃ incubator; adding 30ml HD complete culture medium containing 20% FBS, slightly blowing, centrifuging at 25deg.C and 400 g for 7 and min, and discarding supernatant; re-suspending with 40 ml HD complete culture medium, centrifuging at 25deg.C and 400 g to 7 and min, and discarding supernatant; the fused cells were resuspended in 5ml of 1 XHAT medium and counted; cell suspensions were resuspended in1 XHAT medium and 96-well plates were plated at 8X 10 6 cells/plate, 100. Mu.L/well and cell plates were added;
4. Culture of hybridoma cells
Observing the growth condition of hybridomas in cell plates on the 4 th day after fusion, marking hybridoma cell holes, supplementing 100 mu l of 1 XHT culture medium for each hole, and continuously placing the cells in an incubator for culture; observing the growth condition of the hybridomas in the cell plates on the 7 th day after fusion, gently sucking 100 μl/well by a row gun, then supplementing 100 μl of 1×HT medium into each well, and continuously placing in an incubator for culture; at about 10 days post-fusion, hybridoma cell supernatants were harvested for FACS detection, positive hybridomas were picked, and hybridoma cells subcloned.
5. Hybridoma cell subcloning
The invention selects a plurality of hybridoma cell holes with strong positive flow result analysis to subclone, adopts a limiting dilution method to count the hybridoma cells, carries out 96-hole cell plate plating according to 1 cell per hole, and each hybridoma cell uses 1 XHT culture medium to respectively plate 3 cell plates; on days 3-4 of subcloning plating, observing the number of cell stacks in each well under a microscope, marking the cell wells containing single cell stacks, supplementing the single cell stack wells with 100 μl of 1×HT, and culturing in a CO 2 incubator at 37deg.C; detecting supernatant of single cell stacking holes with marks on day 7 of subcloning and plating, detecting cell positive rate by a FACS method, slightly blowing off cells when strong positive cell stacking grows to more than 80% of cell holes, transferring to a plate with larger volume, and expanding and culturing the cells; finally, 1ml of serum-free cell cryopreservation solution (special for hybridoma cells) is added to every 5×10 6 cells for cryopreservation, and cell sequencing is performed.
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The reagents or instruments used in the examples of the present invention were not manufacturer-identified and were conventional reagent products commercially available.
The sequences in the invention are as follows: (SEQ ID No. 1-16)
Sequence 1
325-5H9-3G11 heavy chain full-length amino acid sequence
HRKPPHISLNSGSSSHGKYFLRLMDLLHKNMKHLWFFLLLVAAPGWVLSQVQLQESGPGLVKPSGTLSLTCALSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLTSRVTISVDKSRNQFSLKLNSVTAADTAVYYCARGFNWFDPWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.
Sequence 2
325-5H9-3G11 heavy chain variable region amino acid sequence
QVQLQESGPGLVKPSGTLSLTCALSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLTSRVTISVDKSRNQFSLKLNSVTAADTAVYYCARGFNWFDPWGQGTLVTVSS.
Sequence 3
325-5H9-3G11 light chain full-length amino acid sequence
PLFQLSEMETDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLTISGLEPEDFALYYCQQYGRSPLTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.
Sequence 4
325-5H9-3G11 light chain variable region amino acid sequence
DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLTISGLEPEDFALYYCQQYGRSPLTFGGGTKVEIK.
Sequence 5
325-5H9-3G11 heavy chain full-length DNA sequence
CACAGGAAACCACCACACATTTCCTTAAATTCAGGGTCCAGCTCACATGGGAAATACTTTCTGAGACTCATGGACCTCCTGCACAAGAACATGAAACACCTGTGGTTCTTCCTCCTCCTGGTGGCAGCTCCCGGATGGGTCCTGTCTCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCTGCGCTCTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTATCATAGTGGGAGCACCAACTACAACCCGTCCCTCACGAGTCGAGTCACCATTTCAGTAGACAAGTCCAGGAACCAGTTCTCCCTGAAGCTGAACTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGGCTTCAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCCAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGAAACCCCGGGAGGAGCAGATCAACAGCACTTTCCGTTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAAACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGATCCCAGTGTCCTTGGAGCCCTCTGGTCCTACAGGACTCTGACACCTACCTCCACCCCTCCCTGTGTAAATAAAGCACCCAGCACTGCCTTGGGACCCTGCAAAAAAAAAAAAAAAAAAAAAAA.
Sequence 6
325-5H9-3G11 heavy chain variable region DNA sequence
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCTGCGCTCTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTATCATAGTGGGAGCACCAACTACAACCCGTCCCTCACGAGTCGAGTCACCATTTCAGTAGACAAGTCCAGGAACCAGTTCTCCCTGAAGCTGAACTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGGCTTCAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA.
Sequence 7
325-5H9-3G11 light chain full-length DNA sequence
CCTCTCTTCCAGCTCTCAGAGATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCGGACTGGAGCCTGAAGATTTTGCACTGTATTACTGTCAGCAGTATGGTAGGTCACCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAGAGACAAAGGTCCTGAGACGCCACCACCAGCTCCCCAGCTCCATCCTATCTTCCCTTCTAAGGTCTTGGAGGCTTCCCCACAAGCGACCTACCACTGTTGCGGTGCTCCAAACCTCCTCCCCACCTCCTTCTCCTCCTCCTCCCTTTCCTTGGCTTTTATCATGCTAATATTTGCAGAAAATATTCAATAAAGTGAGTCTTTGCACTTGAAAAAAAAAAAAAAAAAAAAAAA.
Sequence 8
325-5H9-3G11 light chain variable region DNA sequence
GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCGGACTGGAGCCTGAAGATTTTGCACTGTATTACTGTCAGCAGTATGGTAGGTCACCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA.
Sequence 9
329-6F12-2C6 heavy chain full-length amino acid sequence
LMDLLHKNMKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSGTLSLTCAVSGGSISSGNWWTWVRQSPGKGLEWIGEIHQSGSTNYNPSLKSRVTMSVDRSKNHFSLKVSSVTAADTATYYCAKMARGAPGDFWGQGILVTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK.
Sequence 10
329-6F12-2C6 heavy chain variable region amino acid sequence
QVQLQESGPGLVKPSGTLSLTCAVSGGSISSGNWWTWVRQSPGKGLEWIGEIHQSGSTNYNPSLKSRVTMSVDRSKNHFSLKVSSVTAADTATYYCAKMARGAPGDFWGQGILVTVSS.
Sequence 11
329-6F12-2C6 light chain full-length amino acid sequence
MDMRVPAQLLGLLLLWLPGARCDIQLTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLETGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDGYSRTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.
Sequence 12
329-6F12-2C6 light chain variable region amino acid sequence
DIQLTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLETGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDGYSRTFGQGTKVEIK.
Sequence 13
329-6F12-2C6 heavy chain full-length DNA sequence
GACTCATGGACCTCCTGCACAAGAACATGAAACACCTGTGGTTCTTCCTCCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCTCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTGGTAACTGGTGGACTTGGGTCCGCCAGTCCCCAGGAAAGGGGCTGGAATGGATTGGGGAAATACATCAGAGTGGGAGCACCAACTATAATCCGTCCCTCAAGAGTCGAGTCACCATGTCAGTAGACAGGTCCAAGAACCACTTCTCCCTGAAGGTGAGCTCTGTGACCGCCGCGGACACGGCCACATATTACTGTGCGAAAATGGCTCGGGGAGCCCCCGGTGACTTCTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAGCCAAAACAACACCCCCATCAGTCTATCCACTGGCCCCTGGGTGTGGAGATACAACTGGTTCCTCTGTGACTCTGGGATGCCTGGTCAAGGGCTACTTCCCTGAGTCAGTGACTGTGACTTGGAACTCTGGATCCCTGTCCAGCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGACTCTACACTATGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCAAGTCAGACCGTCACCTGCAGCGTTGCTCACCCAGCCAGCAGCACCACGGTGGACAAAAAACTTGAGCCCAGCGGGCCCATTTCAACAATCAACCCCTGTCCTCCATGCAAGGAGTGTCACAAATGCCCAGCTCCTAACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAATATCAAGGATGTACTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCGGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTATCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCATCACCCATCGAGAGAACCATCTCAAAAATTAAAGGGCTAGTCAGAGCTCCACAAGTATACATCTTGCCGCCACCAGCAGAGCAGTTGTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAGACATCAGTGTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACCGCACCAGTCCTGGACTCTGACGGTTCTTACTTCATATACAGCAAGCTCGATATAAAAACAAGCAAGTGGGAGAAAACAGATTCCTTCTCATGCAACGTGAGACACGAGGGTCTGAAAAATTACTACCTGAAGAAGACCATCTCCCGGTCTCCGGGTAAATGAGCTCAGCACCCACAAAGCTCTCAGGTCCTAAGAGACACTGGCACCCATATCCATGCATCCCTTGTATAAATAAAGCACCCAGCAAAGCCTGGGACCAT.
Sequence 14
329-6F12-2C6 heavy chain variable region DNA sequence
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTGGTAACTGGTGGACTTGGGTCCGCCAGTCCCCAGGAAAGGGGCTGGAATGGATTGGGGAAATACATCAGAGTGGGAGCACCAACTATAATCCGTCCCTCAAGAGTCGAGTCACCATGTCAGTAGACAGGTCCAAGAACCACTTCTCCCTGAAGGTGAGCTCTGTGACCGCCGCGGACACGGCCACATATTACTGTGCGAAAATGGCTCGGGGAGCCCCCGGTGACTTCTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCA.
Sequence 15
329-6F12-2C6 light chain full-length DNA sequence
TCTGCAGACAAATTTGTGCTACCCTGGTCTTACCTGGGACACCTGGGGACACTGAGCTGGTGCTGAGTTACTGAGATGAGCCAGCCCTGCAGCTGCGCCCAGCCTGCCCCATCCCCTGCTCATTTGCATGTTCCCAGAGCACAGTCTCCTGACCTGAAGACTTATTAACAGGCTGATCACACCCTGTGCAGGAGTCAGACCCAGTCAGGACACAGCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAACTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATTAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATGATGGTTATTCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAGAGACAAAGGTCCTGAGACGCCACCACCAGCTCCCCAGCTCCATCCTATCTTCCCTTCTAAGGTCTTGGAGGCTTCCCCACAAGCGACCTACCACTGTTGCGGTGCTCCAAACCTCCTCCCCACCTCCTTCTCCTCCTCCTCCCTTTCCTTGGCTTTTATCATGCTAATATTTGCAGAAAATATTCAATAAAGTGAGTCTTTGCAC.
Sequence 16
329-6F12-2C6 light chain variable region DNA sequence
GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAACTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATTAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATGATGGTTATTCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA.
Example 1
Detection of mouse serum antibody titers (FACS method)
1) Dilution of serum: the serum of the mice is diluted by PBS respectively with the proportions of 1:4000, 1:8000, 1:16000, 1:32000, 1:64000 and 1:128000 in advance, and is put in a centrifuge tube with the concentration of 1.5 ml for temporary storage at the temperature of 4 ℃ for standby;
2) Treating cells: taking a10 cm-dish WT-293T cell, discarding the supernatant, washing the cell once with 5ml 1 XPBS, adding 1ml pancreatin to digest the cell, adding 5ml culture medium to stop digestion after the cell is shed, centrifuging the cell by 1100 rpm for 4 min, discarding the supernatant, and adding 5ml 1 XPBS to re-suspend the cell;
3) Cell count: after mixing cells uniformly, taking 20 mu l of cell liquid and 20 mu l of trypan blue, mixing uniformly, sucking 20 mu l of the mixture, and dripping the mixture into an automatic cell counter, wherein the number of the displayed cells is 1ml of cell liquid;
4) Incubation of serum: taking 2×105 cells from each detection sample, centrifuging cells 4500 rpm for 4: 4 min, discarding supernatant, adding 100 μl diluted serum into each well, setting control well as1×PBS (phosphate buffered saline) without serum, and culturing at 37deg.C for 30min;
5) Incubating a secondary antibody: after incubation, 3min was centrifuged at 4500 rpm, the supernatant was discarded, washed twice with 1ml of 1 XPBS, the supernatant was discarded, resuspended in 100. Mu.l of 1 XPBS, each of which was added with 0.5. Mu. l Goat anti Mouse IgG PE secondary antibody and incubated at 37℃for 30min in the absence of light;
6) And (3) flow detection: after the secondary antibody incubation was completed, 3 min was centrifuged at 4500 rpm, the supernatant was discarded, washed twice with 1ml of 1 XPBS, the supernatant was discarded, 100. Mu.l of 1 XPBS was added to resuspend the mixture, and the results of the analysis performed by an up-flow cytometer were as shown in FIG. 1, wherein Ab mouse represents the fully humanized antibody mouse and WT mouse represents the C57BL/6N mouse. The results show that the fully humanized antibody mice and wild type C57BL/6N mice have comparable or even better immune performance, and the serum antibody titer of the fully humanized antibody mice and the wild type C57BL/6N mice can basically reach 1:256000.
Example 2
Preparation of hybridoma cells
Resuscitates SP2/0 cells (4 d before fusion):
taking 1 SP2/0 cell from a liquid nitrogen tank, quickly thawing in a water bath at 37 ℃, adding the cell into 1 centrifuge tube filled with 5 ml of 10% HD complete medium, centrifuging 4 min in a centrifuge at 1100 rpm, discarding the supernatant, re-suspending the supernatant with 10 ml of 10% HD complete medium, transferring the suspension to a 10 cm cell culture dish by a pipette, and culturing in a carbon dioxide incubator at 37 ℃.
SP2/0 cell passage (Pre-fusion 1 d)
The cell culture dish was removed from the incubator, the cell supernatant was gently aspirated, 5ml was gently blown against the bottom of the cell culture dish, the blown cells were collected in a 158 ml centrifuge tube, centrifuged at 1100 rpm in a centrifuge at 4 min, the supernatant was discarded, resuspended in 30ml of 10% hd complete medium, and transferred to a 10 cm cell culture dish with a pipette, and incubated in a 37 ℃ carbon dioxide incubator. And (3) injection: passaging was performed the day before fusion, and for the next day when SP2/0 cells and mouse B cells were fused, SP2/0 cells were in the logarithmic growth phase.
Hybridoma cell fusion (day 1)
3.1 Preparation of spleen cells
1) When the mice are subjected to booster immunization for 4 days, 1 booster immunized mouse is taken, eyeballs are taken out for bloodletting and sacrifice, and the mice are soaked in a box filled with 75% alcohol;
2) The sterilized mice were fixed to the dissecting plate, the lower abdominal skin was held with forceps, a small incision was made, the peritoneum was exposed by tearing the skin, the peritoneum was opened, the spleen was removed, connective tissue was removed in a 10 cm dish, and the fresh dish was filled with 1 XPBS.
3) Grinding spleen: grinding spleen in a filter screen, washing the filter screen with 10 ml of 1 XPBS, and collecting spleen cells;
4) Lysing erythrocytes: centrifuging spleen cell suspension at 25deg.C, 400×g, and 10min to remove supernatant, and sucking with gun head; mixing three times volume of erythrocyte lysate, homogenizing cell precipitate, standing at room temperature for 10min at 25deg.C, centrifuging at 300g and 10min, removing supernatant, and sucking with gun head; repeating for about 3 times.
5) Treatment of SP2/0 at the 3 rd lysis of erythrocytes: SP2/0 cells were purged with 10ml HD complete medium, and the supernatant was discarded by centrifugation at 25 ℃,300 g, 10 min; at this time, splenocytes and SP2/0 were resuspended in HD complete medium, centrifuged, and washed 3 times (PEG and 50 ml HD complete medium were preheated in 37 ℃ water bath at 1 st centrifugation) to prepare a fused cell ratio of SP2/0: splenocytes = 1:5-1:10. Finally, uniformly mixing the two cells in a 50 ml centrifuge tube by using a preheated HD complete culture medium, and discarding the supernatant after centrifugation at 25 ℃, 400: 400 g and 7: 7 min; gently scraping the bottom of the centrifuge tube on a centrifuge tube rack to loosen the cells slightly;
3.2 cell fusion
1) Fusion (Whole process operation in a large beaker with 37 ℃ distilled water, pre-heating PEG and complete medium at 37 ℃ in advance)
2) Adding 1 ml PEG solution into spleen cells and myeloma cell sediment in 1 min, adding 1 ml PEG while rotating a centrifuge tube, and lightly blowing and resuspending cells in 1 min after the addition;
3) HD complete medium was added: adding 4 ml into 4 min, adding while rotating the centrifuge tube, and mixing after adding 1 min;
4) HD complete medium was added: 10 The ml is lightly attached, a 50 ml centrifuge tube can be lightly rotated and evenly mixed, and the incubator is kept stand for 15 min at 37 ℃;
5) HD complete medium with 20% FBS was added: 30 Wall-attaching ml, lightly blowing, centrifuging: 25. c, 400 g, 7 and min, and discarding the supernatant;
6) HD complete medium: 40 ml was resuspended, centrifuged: 25. directly pouring out the supernatant at the temperature of 400 g and 7 min;
7) Cell count: the fused cells were resuspended in 5ml of 1 XHAT medium, 20. Mu.l of the cell fluid was aspirated, and 20. Mu.l of the cell fluid was mixed with 20. Mu.l of trypan blue and counted;
8) And (3) paving: cell suspensions were resuspended in 1 XHAT medium and 96-well plates were plated at 5X 106 cells/plate, 100. Mu.l/well and cell plates were added;
3.3 Hybridoma cell culture
1) Hybridoma cell replacement fluid (day 4): observing the growth condition of hybridomas in a cell plate, marking the holes with the hybridomas, supplementing liquid by a discharge gun, supplementing 100 μl of 1×HT culture medium in each hole, and continuously placing in an incubator for culture;
2) Hybridoma cell half-change (day 7): observing the growth condition of the hybridomas in the cell plates, gently sucking 100 μl/well by a row gun, supplementing 100 μl of 1×HT medium for each well, and continuously placing in an incubator for culturing; subcloning when the hybridoma cells grow to more than 1/4 of the 96-hole cell plate;
3.4 Positive hybridoma cell screening
1) Taking a 10 cm dish of 293T cells, discarding the supernatant, washing the cells with 5 ml of 1 XPBS once, adding 1ml pancreatin to digest the cells, adding 5 ml culture medium to stop digestion after the cells fall off, centrifuging at 110 rpm for 4min, discarding the supernatant, and adding 5 ml of 1 XPBS to resuspend the cells;
2) Cell count: after mixing cells uniformly, taking 20 mu l of cell liquid and 20 mu l of trypan blue, mixing uniformly, sucking 20 mu l of the mixture, and dripping the mixture into an automatic cell counter, wherein the number of the displayed cells is 1ml of cell liquid;
3) Taking 2X 105 293T cells from each detection sample, centrifuging the cells 4500 rpm to 4X min, discarding the supernatant, adding 100 μl of the supernatant of the subclone hybridoma cells to be detected into each well, setting a control well to be 100 μl of 1 XPBS/well without adding the supernatant of the cells, and culturing in a 37 ℃ incubator for 30min;
4) After incubation, 3 min was centrifuged at 4500 rpm, the supernatant was discarded, washed twice with 1 ml of 1 XPBS, the supernatant was discarded, resuspended in 100. Mu.l of 1 XPBS, each of which was added with 0.5-mu l Goat anti Mouse IgG APC secondary antibody and incubated at 37℃in an incubator in the absence of light for 30 min;
5) After the secondary antibody incubation was completed, the supernatant was centrifuged at 450 rpm for 3 min, washed twice with 1 ml of 1 XPBS, the supernatant was discarded, resuspended in 100. Mu.l of 1 XPBS, and the results were analyzed by an upflow cytometer.
The detection results show that 70 hybridoma cells which secrete antibodies and can be combined with hB7-H3 antigen-positive 293T cells are obtained through screening, a part of representative cases are shown in FIG. 2, average fluorescence intensity statistics results of the combination of hybridoma supernatants and the hB7-H3 antigen-positive 293T cells are shown in FIG. 3, and four hybridoma cells with higher positive rates are selected for further subcloning to prepare cloned cells.
3.5 Positive hybridoma subcloning
1) Firstly, 90 μl of 1×HT medium is aspirated and added to 4-5 wells of a 96-well plate, respectively;
2) Blowing off and mixing hybridoma cells to be subcloned, sucking 10 μl, adding into the 1 st hole, then sucking 10 μl, adding into the 2 nd hole after mixing, and sequentially carrying out 10-time dilution;
3) Observing the cell holes, and performing cell counting on the cell holes with the cell number not less than 20 and the visually-counted cell holes;
4) The 1 st well corresponds to the number of cells contained in 10. Mu.l of the stock solution, the 2 nd well corresponds to the number of cells contained in 1. Mu.l of the stock solution, and so on, and the 3 rd well corresponds to 0.1. Mu.l;
in general, 4 subclones of 96 well plates are selected and the number of cells per well is about 1, namely, cell stock solution containing 96 cells is sucked, 40 ml of 1 XHT culture medium is added and mixed uniformly, 100 μl/well of 96 well plates are paved, and the mixture is placed in a CO2 incubator at 37 ℃ for culture;
5) On days 3-4 of subcloning, the number of cell stacks per well was observed under a microscope, labeling cell wells containing single cell stacks;
6) Supplementing the single cell stack hole with 100 μl of 1×HT, and culturing in a CO2 incubator at 37deg.C;
3.6 Positive subclone hybridoma cell selection
1) On day 7 of subclone plating, single cell stack supernatant with label was assayed for cell positive rate by FACS method
2) Taking a 10 cm dish of 293T cells, discarding the supernatant, washing the cells with 5ml of 1 XPBS once, adding 1 ml pancreatin to digest the cells, adding 5ml culture medium to stop digestion after the cells fall off, centrifuging the cells at 1100 rpm for 4 min, discarding the supernatant, and adding 5ml of 1 XPBS to re-suspend the cells;
3) Cell count: after mixing cells uniformly, taking 20 mu l of cell fluid and 20 mu l of trypan blue, mixing uniformly, sucking 20 mu l of the mixture, and dripping the mixture into an automatic cell counter, wherein the number of the displayed cells is 1 ml;
4) Taking 2X 10 5 293T cells from each detection sample, centrifuging the cells 4500. 4500 rpm for 4. 4 min, discarding the supernatant, adding 100 μl of the supernatant of the subclone hybridoma cells to be detected into each well, setting a control well as 100 μl of 1 XPBS/well without adding the supernatant of the cells, and culturing in a incubator at 37deg.C for 30min;
5) After incubation, 3 min was centrifuged at 4500 rpm, the supernatant was discarded, washed twice with 1 ml of 1 XPBS, the supernatant was discarded, resuspended in 100. Mu.l of 1 XPBS, each of which was added with 0.5. Mu. l Goat anti Mouse IgG APC secondary antibody and incubated at 37℃in the incubator in the absence of light for 30 min;
6) After the secondary antibody incubation was completed, 3: 3 min was centrifuged at 4500 rpm, the supernatant was discarded, washed twice with 1 ml of 1 XPBS, the supernatant was discarded, resuspended in 100. Mu.l of 1 XPBS, and the results were analyzed by an upflow cytometer.
The experimental results show that four subcloned hybridoma cells are positive cells, three long subcloned cells are positive cells, only one positive cell is obtained by screening one long subcloned cell, the result is shown in fig. 4, and two long subcloned hybridoma cells are randomly selected for sequencing.
Example 3
Hybridoma cell sequencing
The subcloned positive hybridoma cells were frozen at 5X 10 6 cells/cell line and the hybrid antibody sequences were called by Seu Hongxun Biotech Co. The sequencing result is shown in FIG. 5, and it can be seen that the heavy chain variable region sequence and the light chain variable region sequence of the two extracted antibody sequences are both human antibody sequences, and the specific sequences are shown in SEQ ID No. 1-16.
As can be seen from the above examples: according to the invention, by immunizing a fully humanized antibody mouse, based on a hybridoma technology, multiple hybridoma cells are obtained, and antibody sequences of two strains are prepared. As a result of sequence analysis, the variable region sequences of the two antibodies are all human sequences. Therefore, the invention provides a fully human antibody development method based on fully human antibody murine immunity, which can theoretically obtain high affinity antibodies without further affinity maturation compared with the traditional phage library display method; compared with the development method of the antibody immunized by the common mice, the antibody sequence obtained by the mouse immunization based on the fully-humanized antibody has affinity comparable to that of the human antibody, but no further humanized transformation is needed. Therefore, the development of the antibody based on the fully humanized antibody mouse greatly accelerates the development process of antibody drugs, and omits complicated steps of affinity maturation and humanized transformation.
The foregoing is a description of only a limited number of preferred embodiments of the invention, which are described in greater detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (3)
1. A human B7-H3 antibody based on a fully humanized antibody mouse is characterized in that,
The amino acid sequence of the heavy chain variable region of the B7-H3 antibody is shown as SEQ ID No.2, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 4;
Or:
The amino acid sequence of the heavy chain variable region of the B7-H3 antibody is shown as SEQ ID No.10, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 12.
2. The human B7-H3 antibody based on the fully humanized antibody mouse according to claim 1, wherein the amino acid sequence of the heavy chain of the B7-H3 antibody is shown in SEQ ID No.1, and the amino acid sequence of the light chain is shown in SEQ ID No. 3;
Or:
The amino acid sequence of the heavy chain of the B7-H3 antibody is shown as SEQ ID No.9, and the amino acid sequence of the light chain is shown as SEQ ID No. 11.
3. A biomaterial comprising any one of a) to d),
A) Nucleic acid molecules encoding an amino acid sequence as set forth in any one of SEQ ID NO.2, 4 or 10, 12 or 1,3 or 9 or 11;
b) A recombinant vector comprising the nucleic acid molecule of a);
c) A recombinant microorganism comprising the nucleic acid molecule as described in a) or comprising the recombinant vector as described in b);
d) A recombinant cell comprising the nucleic acid molecule as set forth in a) or comprising the recombinant vector as set forth in b).
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