A rabbit-derived Fab antibody against CD30, its preparation method and application

Abstract

The present invention provides a rabbit-derived Fab antibody against CD30, its preparation method and application. In the present invention, human CD30 protein is recombinantly expressed in vitro, and rabbits are immunized three times with the CD30 protein. Then, peripheral blood lymphocytes are isolated and their total RNA is extracted, which is reverse transcribed into cDNA. Using this cDNA as a template, the Fab sequence is amplified with specific primers. Furthermore, through yeast display technology, a Fab antibody capable of binding to the CD30 protein is screened, and the Fab sequence is combined with the rabbit Fc domain to obtain a fusion protein with high purity. The strong activity of this Fab antibody with CD30 and its specific binding ability to tumor cells are verified at the cellular level.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and particularly relates to a rabbit-derived Fab antibody against CD30, its preparation method and application. Background Art

[0002] Lymphoma is a malignant tumor originating from lymphoid tissue, including two major types: Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). The number of newly diagnosed lymphoma cases globally is increasing. Data shows that there were 589,600 newly diagnosed lymphoma cases globally in 2018. Although many patients have the possibility of cure, approximately 30% of patients will ultimately face the challenges of disease recurrence or non-responsiveness to existing treatments. In order to improve the treatment effect and survival rate of lymphoma patients, the medical community is actively seeking safer and more effective treatment strategies.

[0003] Brentuximab vedotin (BV) is an innovative antibody-drug conjugate that was approved for the Chinese market in May 2020. Currently, this drug has been officially approved for adult patients, especially those with CD30-positive lymphoma. BV is applicable to various types of lymphoma, including but not limited to relapsed or refractory classical Hodgkin lymphoma, systemic anaplastic large cell lymphoma, and patients with primary cutaneous anaplastic large cell lymphoma or mycosis fungoides who have received systemic treatment. The introduction of this drug provides new treatment options for lymphoma patients and is expected to improve their treatment effect and quality of life. Therefore, it is of great significance to develop more similar drugs. Summary of the Invention

[0004] In view of this, the present invention provides a rabbit-derived Fab antibody against CD30, its preparation method and application. The present invention uses yeast display technology to screen a Fab antibody that can specifically bind to CD30 protein, and verifies the activity of this Fab antibody binding to CD30 at the cellular level.

[0005] The technical solution of the present invention is realized as follows:

[0006] The present invention provides a rabbit-derived Fab antibody against CD30, which includes a heavy chain and a light chain; the heavy chain contains at least one antigen complementary determining region shown by the amino acid sequences such as SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; the light chain contains at least one antigen complementary determining region shown by the amino acid sequences such as SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0007] On the basis of the above technical solution, preferably, the amino acid sequence of the heavy chain is as shown in SEQ ID NO:1; the amino acid sequence of the light chain is as shown in SEQ ID NO:2.

[0008] On the basis of the above technical solutions, preferably, the rabbit-derived Fab antibody further includes a rabbit Fc domain having an amino acid sequence as shown in SEQ ID NO: 9.

[0009] The present invention also provides a coding gene encoding the rabbit-derived Fab antibody.

[0010] The present invention also provides an engineering strain containing the coding gene.

[0011] The present invention also provides a method for preparing the rabbit-derived Fab antibody, which includes the following steps:

[0012] (1) Immunize rabbits by expressing CD30 protein.

[0013] (2) Extract rabbit ear marginal vein blood and isolate lymphocytes, extract the total RNA of lymphocytes, use the cDNA obtained by reverse transcription of RNA as a template, amplify the Fab antibody sequence, and then clone the Fab antibody sequence into a yeast vector to obtain a recombinant product, which is the initial Fab antibody yeast library.

[0014] (3) Transform the recombinant product into Saccharomyces cerevisiae competent cells to obtain an electrotransformed product, and then culture the electrotransformed product to screen out yeast cells that can bind to CD30 protein.

[0015] (4) Perform single-cell cloning on the screened yeast cells, further screen clones with strong positivity to obtain Fab recombinant protein, and sequence to obtain the coding sequence of the Fab antibody.

[0016] (5) Connect the coding sequence of the Fab antibody with the coding sequence of the rabbit Fc domain, clone it into a mammalian expression vector, transfect HEK293F cells, culture and then purify to obtain the Fab-Fc fusion protein.

[0017] On the basis of the above technical solutions, preferably, in the step (4), flow cytometry is used to screen positive clones. It is to add tumor lymphocytes to yeast cells labeled with fluorescently labeled antibodies and incubate them before detection, and sequence positive clones with a double-positive signal of specific binding between yeast and tumor cells greater than 30%.

[0018] On the basis of the above technical solutions, preferably, in the step (5), the coding sequence of the Fab antibody is connected to the coding sequence of the rabbit Fc domain through a linker having an amino acid sequence as shown in SEQ ID NO: 10.

[0019] On the basis of the above technical solutions, preferably, the heavy chain of the Fab antibody is directly connected to the rabbit Fc domain.

[0020] The present invention also provides the use of the rabbit-derived Fab antibody, or the coding gene thereof, or the engineered strain in the preparation of a drug, preparation or kit for preventing, treating and / or diagnosing lymphoma.

[0021] Based on the above technical solutions, preferably, the lymphoma is non-Hodgkin lymphoma.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The antibody provided by the present invention has a Fab sequence derived from a rabbit antibody, so it has the advantages of high expression level, high affinity and high stability.

[0024] (2) In the present invention, the anti-human CD30 antibody is expressed and secreted using mammalian cells (293F). The Fab is fused with rabbit Fc, cloned into the mammalian expression vector pTT5, the vector is transfected into mammalian cells 293F, and the supernatant is collected after culture. The fusion protein in the supernatant is purified using a protein A column, and the obtained 0115-1-FC fusion protein has a high purity.

[0025] (3) ELISA binding experiments show that the antibody provided by the present invention can bind to human CD30 protein with high affinity, even reaching the nM level.

[0026] (4) Flow cytometry binding experiments show that the antibody of the present invention can specifically bind to tumor cell lines expressing human CD30, has specific binding ability, and the binding force is stronger than that of known antibodies. Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 It is the flow cytometry result of monoclonal yeast; among them, Figure A is the flow cytometry result of Hut78 cells, and Figure B is the flow cytometry result of L540 cells.

[0029] Figure 2 It is the antibody purification result; among them, lane 1 is Marker, and lane 2 is the fusion protein of Fab antibody 0115-1-Fc.

[0030] Figure 3 It is the result of ELISA identification of the binding force between 0115-1-Fc antibody and human CD30 protein.

[0031] Figure 4 Results of specific binding of 0115-1-Fc antibody to lymphoid tumor cell lines; where Figure A shows the results of Hut78 cells, Figure B shows the results of L540 cells, and Figure C shows the results of CHO cells.

[0032] Figure 5 Specific IP identification of 0115-1-Fc antibody with lymphoid tumor cell lines; where Lane M: Marker, Lane 1: Hut78 lysate, Lane 2: Negative control 0115-1, Lane 3: Experimental group.

[0033] Figure 6 Results of flow cytometry detection for comparison of binding activity between 0115-1-FC and known antibodies. Specific implementation mode

[0034] The following will describe the technical solutions in the embodiments of the present invention clearly and completely in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] In the present invention, the following definitions are included:

[0036] EC50: Full name is half maximal effective concentration, which refers to the concentration of a drug, antibody, or toxin, etc. that can achieve 50% of the maximum biological effect after a specific exposure time. It is usually used to measure the potency of a drug. The lower the EC50 value, the stronger the potency of the drug, that is, the same biological effect can be produced at a lower concentration. In a quantal response, EC50 refers to the drug dose that can cause 50% of the maximum response intensity; in a qualitative response, it refers to the drug dose that can only cause 50% of the experimental animals to show a positive response.

[0037] Example 1 Screening of Fab antibody

[0038] 1) Animal immunization

[0039] Use recombinant expressed CD30 protein (Company: Aimeijie Technology, Product number: PRO-1369) to immunize rabbits. Freund's complete adjuvant (Company: sigma, Product number: F5881) is used for the primary immunization, and Freund's incomplete adjuvant (Company: sigma, Product number: F5506) is used for the latter two times. Mix the antigen and adjuvant at a volume ratio of 1:1, and immunize the rabbits subcutaneously 3 times at intervals of 2 weeks each.

[0040] 2) Construction of Fab Antibody Yeast Display Library

[0041] After immunization three times, blood was drawn from the marginal ear vein of rabbits and lymphocytes were isolated. Total RNA was extracted using the RNA extraction kit from Novoprotein. Then, the RNA was reverse transcribed into cDNA using the Novoprotein RNA reverse transcription kit (HiScript II Q Select RT SuperMix for qPCR R232). Using the cDNA as a template, Fab heavy and light chains were amplified using rabbit-specific primers (synthesized by Tsingke Biological Company). The amplified Fab sequences were cloned into the yeast vector Pct302 by homologous recombination to obtain the recombinant product, which was the initial Fab yeast library (refer to Baek DS, Park SW, Adams C, et al. Yeast Mating as a Tool for Highly Effective Discovery and Engineering of Antibodies via Display Methodologies. Methods Mol Biol. 2022;2491:313 - 333. doi:10.1007 / 978 - 1 - 0716 - 2285 - 8 - 17).

[0042] Among them, the rabbit-specific primers for amplifying the heavy chain are as follows:

[0043] b-fabvh1: ccgccggaattccaggagcagctgaaggag (SEQ ID NO:11);

[0044] rb-fabvh2: ccgccggaattccaggagcagctgrtggag (SEQ ID NO:12);

[0045] rb-fabvh3: ccgccggaattccaggagcagctggaggagtcc (SEQ ID NO:13);

[0046] rb-ch1-r: cgccgcggatcccgtgggcttgctgcatgtcg (SEQ ID NO:14);

[0047] The rabbit-specific primers for amplifying the light chain are as follows:

[0048] rb-fabvk1: cggcggggtaccgaccctatgctgacccag (SEQ ID NO:15);

[0049] rb-fabvk2: cggcggggtaccgatgtcgtgatgacccag (SEQ ID NO:16);

[0050] rb-fabvk3: cggcggggtaccgcagccgtgctgacccag (SEQ ID NO:17);

[0051] rb-cl-r: gccggctctagattarcagtcacccctrttgaagc (SEQ ID NO:18).

[0052] The Saccharomyces cerevisiae EBY100 was streaked on YPD solid medium and cultured at 30 °C for about 48 h. Single colonies were picked and inoculated into 10 mL of YPD medium (formulation: 20 g of glucose, 20 g of peptone, 10 g of yeast extract, dissolved in 1000 mL of ddH2O, mixed and autoclaved), and cultured with shaking overnight. Then all were inoculated into 200 mL of fresh YPD medium and cultured overnight until the OD 600 was approximately 1.6. The cells were centrifuged at 4000 g for 3 min, and the supernatant was discarded. The cells were washed twice with 50 mL of ice-cold water, and then washed once with 50 mL of ice-cold electroporation buffer (1 M sorbitol, 1 mM CaCl2). The cells were resuspended in 20 mL of LiAc buffer (0.1 M LiAc, 10 mM DTT) and incubated at 30 °C and 180 rpm for 30 min. The cells were centrifuged at 4000 g for 3 min and washed once with 50 mL of pre-cooled electroporation buffer. The cells were resuspended in 200 μL of ice-cold electroporation buffer, and the final volume was approximately 1 mL, which could be used for two electroporation reactions.

[0053] Transformation of Saccharomyces cerevisiae EBY100 competent cells (prepared by oneself) using Gene Pulser Xcell electroporator (BioRad): Transform the yeast plasmid Fab antibody gene (the above homologous recombination product) into Saccharomyces cerevisiae EBY100 competent cells in a 0.1 cm electroporation cuvette. Resuspend the electroporated product (500 μL) in 20 mL of SD-Trp medium and incubate at 30 °C and 220 rpm for 1 h. Spread Saccharomyces cerevisiae EBY100 on 4 SD-Trp plates (formula: 10 g agar powder, 26.7 g mini SD-Base, 8.56 g NaH2PO4, 5.4 g Na2HPO4, and 0.64 g dropout mix Trp dissolved in 1000 mL of ddH2O, autoclave after mixing) to amplify the yeast library and culture at 30 °C for 48 h. Scrape the yeast from the plate, vortex it thoroughly with glycerol at a final concentration of 25%, quickly freeze it in liquid nitrogen in 1 mL aliquots, and store it at -80 °C. At the same time, take 0.2 μL and 0.02 μL of the yeast cell suspension and spread them on 150 mm SD-Trp plates, count the number of colonies grown on the 0.2 μL plate, and then multiply the number of colonies by 10 5 , multiply the number of colonies on the 0.02 μL plate by 10 6 , and take the average of the two calculation results to obtain the library size. The results show that the library capacity is 2.3×10 8 .

[0054] 3) Amplification of the Fab antibody yeast display library

[0055] Thaw the library at room temperature, take the cells from the cryopreserved yeast library into SD-Trp medium to make the initial OD 600 value of the culture approximately 0.5. Incubate overnight at 30 °C and 200 rpm. Centrifuge the culture solution at 3000 g for 5 min to obtain the amplified cultured yeast, and resuspend the cells in SG-Trp (formula: 20 g galactose, 6.7 g YNB, 8.56 g NaH2PO4, 5.4 g Na2HPO4, 0.64 g dropout mix Trp, 100 g PEG8000 dissolved in 1000 mL of ddH2O, autoclave after mixing) medium to induce yeast to express Fab, making the initial induction OD 600 value 1. Incubate at 20 °C and 200 rpm for 48 h.

[0056] 4) Antibody screening

[0057] Take 50 μL of magnetic beads into a 1.5 mL sterile EP tube, wash once with 1 mL of sterile 1×PBS (PBS will be used hereinafter), add 5 - 10 μg of the target antigen (human CD30), and incubate with rotation at 4°C overnight. Pour the induced yeast into a 50 mL sterile centrifuge tube, centrifuge at 3000 g for 3 min to collect the yeast, and discard the supernatant. Wash 3 times with 50 mL of PBS, resuspend the yeast with 50 mL of 0.1% BSA-PBS, rotate at room temperature, and block for 1 h. Wash the blocked yeast 2 times with 50 mL of PBS, and resuspend the yeast with 50 mL of 0.1% BSA-PBS. At the same time, place the magnetic beads incubated overnight on a magnetic stand, wash 2 times with 1 mL of PBS, resuspend the magnetic beads with 1 mL of sterile PBS and add them to the yeast, and incubate with rotation at room temperature for 1 - 2 h. After incubation, place on the magnetic stand, adsorb for 30 min, discard the supernatant, wash 5 times with 10 mL of PBS, and finally resuspend the yeast bound with the antigen with 1 mL of PBS. Dilute the panned yeast to 10 2 、10 3 、10 4 ,coat on SD-Trp solid plates, and determine the library size. Coat the remaining yeast on 15 cm SD-Trp solid plates, collect them, and perform the next round of panning. The results show that the library size in the first round of screening: 4×10 5 ,the library size in the second round of screening: 7.4×10 6 ,the library size in the third round: 8.2×10 6 .

[0058] 5) Flow cytometry identification of positive clones of monoclonal yeast

[0059] After three consecutive rounds of panning, 96 individual yeast colonies were picked into a 96-well round-bottom culture dish containing 100 μL of SD-Trp medium. Incubate continuously at 30 °C and 200 rpm for 12 hours, then centrifuge the culture dish at 2000 g for 3 min, discard the supernatant, and supplement the culture dish with fresh induction medium SG-Trp. Incubate continuously at 20 °C and 200 rpm for 36 h. Take 25 μL of the culture to a new 96-well plate (U-bottom), centrifuge at 2000 g for 3 min, discard the supernatant, and incubate the yeast pellet with 200 μL of BSA for 30 min, wash twice with PBSA, add the prepared fluorescein-labeled goat anti-rabbit Fab secondary antibody (diluted with BSA), and incubate with shaking at 4 °C for 30 min. Wash twice with PBSA. Meanwhile, prepare the required tumor lymphocytes (L540 and Hut78), wash the cells twice with PBSA, resuspend the cells with the fluorescein dye diluted with BSA (diluted 1:8000), incubate at 4 °C for 30 min, wash twice with PBS, resuspend the cells with PBSA, add 100 μL / well to the yeast culture dish labeled with the fluorescein secondary antibody and mix well, and incubate with shaking at 4 °C for 30 min. Subsequently, perform flow cytometry analysis (For the method of obtaining the candidate antibody sequence, refer to Sádio F, Stadlmayr G, Stadlbauer K, et al. Yeast Surface Display and Cell Sorting of Antigen-Binding Fc Fragments. Methods Mol Biol. 2019;1923:287-308. doi:10.1007 / 978-1-4939-9024-5-13).

[0060] The flow cytometry results of the monoclonal yeast are as shown in Figure 1 Figure [Figure number not provided in the original, please check]. The positive clones with a double-positive signal of more than 30% for the specific binding of yeast and tumor cells were sequenced, the sequence results were analyzed, and flow cytometry was repeated for verification. Finally, a clone with a strong positive signal was obtained, named 0115-1, and the heavy and light chain sequence information was obtained by sequencing. It can be seen that when 0115-1 was verified by flow cytometry, the double-positive signal for the specific binding of yeast and tumor cells ( Figure 1 Figure [Figure number not provided in the original, please check]) was relatively strong.

[0061] The amino acid sequence of the heavy chain of 0115-1 is:

[0062] QSVKESGGRLVTPGGTLTLTCAASEFSFSGYAVNWVRQAPGKGLEWIGFIGINGDTYYASWAKGRFTISKTSSTTVELKMTSLAVADTATYFCSRGDLWGRGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPT(SEQ IDNO:1);

[0063] The light chain amino acid sequence of 0115-1 is as follows:

[0064] AIDMTQTPPSVSAAVGGTVTINCQASQSVYNNNNLAWFQQKPGQPPKLLIYAASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCAGYKGSGIDVLA FGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQID NO:2);

[0065] The three antigen - complementary determining regions (CDR1, CDR2, and CDR3) of the 0115-1 antibody heavy chain are as follows:

[0066] CDR1: EFSFSGYA(SEQ ID NO:3);

[0067] CDR2: FIGINGDT(SEQ ID NO:4);

[0068] CDR3: SRGDL(SEQ ID NO:5);

[0069] The three antigen - complementary determining regions (CDR1, CDR2, and CDR3) of the 0115-1 antibody light chain are as follows:

[0070] CDR1: QSVYNNNN(SEQ ID NO:6);

[0071] CDR2: AAS(SEQ ID NO:7);

[0072] CDR3: CAGYKGSGIDVL(SEQ ID NO:8).

[0073] Example 2 Expression and Purification of Nanobody 0115-1-Fc Fusion Protein

[0074] To improve the in vivo stability of the antibody and enable the antibody to effectively exert antibody-dependent cell-mediated cytotoxicity (ADCC) to enhance the protective effect of the antibody, the coding sequence of Fab was designed to be linked to the coding sequence of the rabbit Fc domain fragment with a linker, and then cloned into the mammalian expression vector pTT5. The constructed vector was transfected into HEK293F (ATCC, CBP60437, density about 5.0×10 6 cells / mL) using polyethyleneimine (PEI). The transfected mammalian cells were cultured in Freestyle TM293 expression medium in a CO2 incubator shaker at 37 °C and 150 rpm for 5 days. The cells were centrifuged at 2000 rpm for 10 min, and the supernatant of the mammalian cell culture was collected. The Fab antibody-Fc fusion protein was purified using a protein A column and analyzed by SDS-PAGE electrophoresis. The results are as Figure 2 shown. The results showed that the heavy chain was about 50 KD in size and the light chain was about 28 KD in size, and the sizes of the heavy and light chains were correct, indicating that high-purity Fab-Fc fusion protein 0115-1-FC was obtained from the supernatant.

[0075] Among them, the rabbit Fc domain fragment was directly linked behind the Fab heavy chain, and the amino acid sequence of the rabbit Fc domain fragment was:

[0076] PPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO:9);

[0077] The amino acid sequence of the linker was:

[0078] PSTCSKPTCPP (SEQ ID NO:10).

[0079] Example 3 Analysis of the Binding Activity of Fab-Fc Fusion Protein 0115-1-FC to CD30 Protein

[0080] Coat an ELISA plate overnight with 2 μg / mL of CD30 protein. Block unbound sites with MPBS for 2 h at room temperature, then wash 3 times with PBS-0.1% Tween 20 (PBST). Prepare PBS solutions of CD30 protein with a concentration gradient from 0.1 nM to 1 μM. Add 0.5 μM of the 0115-1-FC fusion protein prepared in Example 2 to each gradient, making the total volume 100 μL. After incubating at room temperature for 30 min, add it to the aforementioned ELISA plate coated with antigen and incubate at 37 °C for 30 min. After washing 4 times with PBST, add 100 μL / well of goat anti-rabbit Fab-HRP secondary antibody (Catalog No.: E-AB-1102) and incubate at 37 °C for 1 h. Add 100 μL / well of TMB for color development for 15 min, then add 50 μL of 1 M H2SO4 to stop color development, and measure the absorbance at 450 nM. The obtained data is used for non-linear curve fitting with the graphing software GraphPad.

[0081] The results of the above ELISA for identifying the binding affinity of the 0115-1-FC antibody to human CD30 protein are as Figure 3 shown. The results indicate that the 0115-1-FC antibody has a strong binding ability to human CD30 protein, and the EC 50 for binding to human CD30 is 70 nM.

[0082] Example 4 Binding activity of 0115-1-FC to different lymphoma cell lines

[0083] 1) Flow cytometry detection of the binding activity of 0115-1-FC to different lymphoma cell lines

[0084] Prepare the required tumor lymphocytes (L540 and Hut78) and negative cells CHO, wash 2 times with PBSA, add them to a U-bottom 96-well culture dish at 5×10 5 / well, then add 0.2 μg of the 0115-1-FC prepared in Example 2, incubate at 4 °C for 30 min, wash 2 times with PBS, add the prepared goat anti-rabbit Fab fluorescently labeled secondary antibody (diluted with BSA), and incubate at 4 °C for 30 min. Subsequently, perform flow cytometry analysis (manufacturer: Shenzhen Bidak, model: BeamCyte-1026M). The detection results are as Figure 4 shown. 0115-1-FC has strong binding to both tumor cells L540 and Hut78, but no binding to negative cells CHO.

[0085] 2) IP detection of the binding activity of 0115-1-FC to lymphoma cell lines

[0086] Take 50 μL of ProteinG magnetic beads into a 1.5 mL centrifuge tube, wash twice with PBS, add 5 μg of antibody 0115-1-FC, and incubate with rotation at 4 °C overnight. Place the centrifuge tube on a magnetic stand for adsorption for 2 min, discard the supernatant, and wash twice with PBS. Add 1 mL of lymphoma cell Hut78 lysate and incubate with rotation at 4 °C for 1 h. Place the centrifuge tube on a magnetic stand for adsorption for 2 min, discard the supernatant, and wash twice with PBS. Resuspend the magnetic beads with 20 μL of PBS and add to 2× reducing buffer, and separate by SDS-PAGE (8% separating gel). The results are as Figure 5 shown, indicating that 0115-1-Fc can specifically bind to human CD30 protein.

[0087] 3) Flow cytometry detection of 0115-1-FC compared with known antibodies

[0088] Prepare the required tumor lymphocytes Hut78, wash twice with PBSA, add to a U-bottom 96-well culture dish at 5×10 5 / well, then add 0.2 μg of 0115-1-FC and the positive control TNFRSF8 CD30 (Ki-4), incubate at 4 °C for 30 min, wash twice with PBS, add the prepared goat anti-rabbit Fab fluorescently labeled secondary antibody (diluted with BSA), and incubate at 4 °C for 30 min. Subsequently, perform flow cytometry analysis. The detection results are as Figure 6 shown, 0115-1-FC has stronger binding activity than other antibodies compared with the positive control TNFRSF8 CD30 (Ki-4).

[0089] The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A rabbit-derived Fab antibody against CD30, characterized in that: The rabbit-derived Fab antibody includes a heavy chain and a light chain; the heavy chain contains heavy chain complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences are EFSFSGYA, FIGINGDT, and SRGDL, respectively; the light chain contains light chain complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences are QSVYNNNN, AAS, and CAGYKGSGIDVL, respectively.

2. The rabbit-derived Fab antibody according to claim 1, wherein: The amino acid sequence of the heavy chain is as shown in SEQ ID NO:1; the amino acid sequence of the light chain is as shown in SEQ ID NO:

2.

3. The rabbit-derived Fab antibody according to claim 1, characterized in that: The rabbit-derived Fab antibody further includes a rabbit Fc domain with the amino acid sequence as shown in SEQ ID NO:

9.

4. A coding gene, characterized in that, The encoding gene encodes the rabbit-derived Fab antibody according to any one of claims 1-3.

5. An engineered strain, characterized in that, The engineered strain contains the encoding gene according to claim 4.

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