Anti-CD30 rabbit Fab antibody as well as preparation method and application thereof

Abstract

The invention provides an anti-CD30 rabbit Fab antibody as well as a preparation method and application thereof. Human CD30 protein is expressed through in-vitro recombination, a rabbit is immunized for three times by using the CD30 protein, peripheral blood lymphocytes are separated out, total RNA of the peripheral blood lymphocytes is extracted, the peripheral blood lymphocytes are reversely transcribed into cDNA, the cDNA is taken as a template, and a Fab sequence is amplified by using a specific primer; according to the invention, a rabbit Fc structural domain is used as an antigen, a Fab antibody capable of being combined with CD30 protein is obtained by screening through a yeast display technology, a Fab sequence is combined with the rabbit Fc structural domain to obtain high-purity fusion protein, and the fact that the Fab antibody has relatively strong activity with CD30 and has specific binding capacity with tumor cells is verified at the cellular level.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and specifically relates to an anti-CD30 rabbit-derived Fab antibody and a preparation method and application thereof. Background Art

[0002] Lymphoma is a malignant tumor originating from lymphatic tissue, including two major types: Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). The number of new cases of lymphoma is increasing worldwide. Data show that there were 589,600 new cases of lymphoma worldwide in 2018. Although many patients have the possibility of being cured, about 30% of patients will eventually face the challenge of disease recurrence or no response to existing treatment. 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] 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 use in adult patients, especially CD30-positive lymphoma patients. BV is suitable for a variety of lymphoma types, including but not limited to relapsed or refractory classical Hodgkin's 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 a new treatment option for lymphoma patients, which is expected to improve their treatment outcomes 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 proposes an anti-CD30 rabbit Fab antibody and its preparation method and application. The present invention screens and obtains a Fab antibody that can specifically bind to CD30 protein through yeast display technology, and verifies the activity of the Fab antibody binding to CD30 at the cellular level.

[0005] The technical solution of the present invention is achieved in this way:

[0006] The present invention provides an anti-CD30 rabbit Fab antibody, comprising a heavy chain and a light chain; the heavy chain comprises an antigen complementary determining region as shown in at least one of the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; the light chain comprises an antigen complementary determining region as shown in at least one of the amino acid sequences of 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; and the amino acid sequence of the light chain is as shown in SEQ ID NO:2.

[0008] On the basis of the above technical solution, 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, which encodes the rabbit-derived Fab antibody.

[0010] The present invention also provides an engineered strain comprising the encoding gene.

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

[0012] (1) Express CD30 protein to immunize rabbits;

[0013] (2) extracting blood from the rabbit ear vein and isolating lymphocytes, extracting total RNA from the lymphocytes, using the cDNA obtained by reverse transcription of the RNA as a template, amplifying the Fab antibody sequence, and then cloning the Fab antibody sequence into a yeast vector to obtain a recombinant product, which is the initial Fab antibody yeast library;

[0014] (3) transforming the recombinant product into a Saccharomyces cerevisiae competent cell to obtain an electroporation product, and then culturing the electroporation product to screen out yeast cells that can bind to the CD30 protein;

[0015] (4) Single-cell cloning of the screened yeast cells is performed, and clones with strong positive results are further screened to obtain Fab recombinant protein, which is sequenced to obtain the coding sequence of the Fab antibody;

[0016] (5) The coding sequence of the Fab antibody is connected to the coding sequence of the rabbit Fc domain, cloned into a mammalian expression vector, transfected into HEK293F cells, and purified after culture to obtain a Fab-Fc fusion protein.

[0017] Based on the above technical solution, preferably, in step (4), the positive clones are screened by flow cytometry, which is to add tumor lymphocytes to yeast cells labeled with fluorescently labeled antibodies and incubate them for detection, and sequence the positive clones with double positive signals of yeast and tumor cells specifically binding greater than 30%.

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

[0019] On the basis of the above technical solution, 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 encoding gene, 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 solution, preferably, the lymphoma is non-Hodgkin's 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, and thus has the advantages of high expression level, high affinity and high stability.

[0024] (2) In the present invention, the human CD30 antibody is expressed and secreted by mammalian cells (293F), Fab is fused with rabbit Fc, and 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 high purity.

[0025] (3) ELISA binding experiments show that the antibodies 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 antibodies of the present invention can specifically bind to tumor cell lines expressing human CD30, have specific binding ability, and have stronger binding force than known antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0029] Figure 2 The results of antibody purification are shown in Figure 1; Lane 1 is the Marker, and Lane 2 is the fusion protein of the Fab antibody 0115-1-Fc.

[0030] Figure 3 This is the result of ELISA to identify the binding ability of 0115-1-Fc antibody to human CD30 protein.

[0031] Figure 4 The results are as follows: specific binding of 0115-1-Fc antibody to lymphoma cell lines; Figure A is the result of Hut78 cells, Figure B is the result of L540 cells, and Figure C is the result of CHO cells.

[0032] Figure 5 This is the specific IP identification of 0115-1-Fc antibody and lymphoma cell line; Lane M: Marker, Lane 1: Hut78 lysate, Lane 2: Negative control 0115-1, Lane 3: Experimental group.

[0033] Figure 6 The results of flow cytometry detection of the binding activity of 0115-1-FC and known antibodies were compared. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0036] EC50: The full name is halfmaximal effective concentration, which refers to the concentration of drugs, antibodies or toxins that can achieve 50% of the maximum biological effect after a specific exposure time. It is usually used to measure the efficacy of drugs. The lower the EC50 value, the stronger the efficacy of the drug, that is, it can produce the same biological effect at a lower concentration. In quantitative reactions, EC50 refers to the drug dose that can cause 50% of the maximum reaction intensity; in qualitative reactions, it refers to the drug dose that only causes 50% of the experimental animals to have a positive reaction.

[0037] Example 1 Fab antibody screening

[0038] 1) Animal immunization

[0039] Rabbits were immunized with recombinant CD30 protein (Company: Aimi Technology, Catalog No.: PRO-1369). Freund's complete adjuvant (Company: Sigma, Catalog No.: F5881) was used for the first immunization, and Freund's incomplete adjuvant (Company: Sigma, Catalog No.: F5506) was used for the next two immunizations. The antigen and adjuvant were mixed in a volume ratio of 1:1 and injected subcutaneously for 3 times, with an interval of 2 weeks between each immunization.

[0040] 2) Construction of Fab antibody yeast display library

[0041] After immunization three times, blood was drawn from the rabbit ear vein and lymphocytes were isolated, and total RNA was extracted using the RNA extraction kit of Novazon. The RNA was then reverse transcribed into cDNA using the Novazon RNA reverse transcription kit (HiScript IIQ Select RT SuperMix forqPCR R232). Using cDNA as a template, rabbit-specific primers (synthesized by Qingke Biotech) were used to amplify the Fab light and heavy chains. The amplified Fab sequence was 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 idno:11);

[0044] rb-fabvh2:ccgccggaattccaggagcagctgrtggag(seq id no:12);

[0045] rb-fabvh3:ccgccggaattccaggagcagctggaggagtcc(seq idno:13);

[0046] rb-ch1-r:cgccgcggatcccgtgggcttgctgcatgtcg(seq id no:14);

[0047] Rabbit-specific primers for amplification of 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] Saccharomyces cerevisiae EBY100 was streaked on YPD solid medium and cultured at 30°C for about 48 h. A single clone was picked and transferred to 10 mL YPD medium (formula: 20 g glucose, 20 g peptone, 10 g yeast extract, dissolved in 1000 mL ddHO). 2 O, mixed and sterilized by high pressure), shaken and cultured overnight, all inoculated into 200 mL fresh YPD medium, and cultured overnight until OD 600 The cells were centrifuged at 4000 g for 3 min at about 1.6 and the supernatant was discarded. The cells were washed twice with 50 mL of ice water and then electroporated with 50 mL of ice-cold electroporation buffer (1 M sorbitol, 1 mM CaCl 2 ) Wash once. Resuspend the cells in 20mL LiAc buffer (0.1M LiAc, 10mM DTT) and incubate at 30℃, 180rpm for 30min. Centrifuge the cells at 4000g, 3min, and wash once with 50mL pre-cooled electroporation buffer. Resuspend the cells in 200μL of ice-cold electroporation buffer to a final volume of about 1mL, which can be used for two electroporation reactions.

[0053] Transform Saccharomyces cerevisiae EBY100 competent cells (prepared by yourself) using Gene PulserXcell total electroporator (BioRad): The yeast plasmid Fab antibody gene (the above homologous recombination product) was transformed into Saccharomyces cerevisiae EBY100 competent cells in a 0.1 cm electroporation cup. The electroporation product (500 μL) was resuspended in 20 mL SD-Trp medium and incubated at 30°C, 220 rpm for 1 h. Saccharomyces cerevisiae EBY100 was spread on 4 SD-Trp (formula: 10 g agar powder, 26.7 g mini SD-Base, 8.56 g NaH 2 PO 4 , 5.4 g Na 2 HPO 4 and 0.64 g of dropoutmix Trp dissolved in 1000 mL of ddHO 2O, autoclave after mixing) plates to amplify the yeast library and culture at 30°C for 48h. Scrape the yeast from the plate, vortex and thoroughly mix it with 25% glycerol at a final concentration, freeze it in liquid nitrogen in 1mL aliquots and store it at -80°C. At the same time, take 0.2μL and 0.02μL of yeast solution to coat 150mm SD-Trp plates, count the number of clones grown on the 0.2μL plate, and then multiply the number of clones by 10 5 , 0.02 μL plate colony number multiplied by 10 6 The two calculation results are averaged to get the library size. The results show that the library capacity is 2.3×10 8 .

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

[0055] Thaw the library at room temperature, take cells from the frozen yeast library and transfer them to SD-Trp medium to make the initial OD of the culture 600 The value was about 0.5. The culture was incubated at 30°C and 200 rpm overnight. The culture broth was centrifuged at 3000g for 5 min to obtain the amplified yeast culture. The SG-Trp (formula: 20 g galactose, 6.7 g YNB, 8.56 g NaH 2 PO 4 5.4gNa 2 HPO 4 , 0.64 g dropoutmix Trp, 100 g PEG8000 dissolved in 1000 mL ddH 2 O, mixed and autoclaved) medium to resuspend the cells to induce yeast to express Fab, and the initial induction OD 600 value is 1. Incubate at 20℃, 200rpm for 48h.

[0056] 4) Antibody screening

[0057] Take 50μL magnetic beads in a 1.5mL sterile EP tube, wash once with 1mL sterile 1×PBS (PBS is used later), add 5-10μg target antigen (human CD30), and rotate and incubate overnight at 4℃. Pour the induced yeast into a 50mL sterile centrifuge tube, collect the yeast by centrifugation at 3000g for 3min, and remove the supernatant. Wash 3 times with 50mL PBS, resuspend the yeast with 50mL 0.1% BSA-PBS, rotate at room temperature, and block for 1h. Wash the blocked yeast with 50mL PBS twice, resuspend the yeast with 50mL 0.1% BSA-PBS. At the same time, place the magnetic beads incubated overnight on a magnetic stand, wash twice with 1mL PBS, resuspend the magnetic beads with 1mL sterile PBS and add them to the yeast, rotate and incubate at room temperature for 1-2h. After incubation, place on a magnetic rack and adsorb for 30 minutes, discard the supernatant, wash five times with 10 mL PBS, and finally resuspend the antigen-bound yeast with 1 mL PBS. Dilute the selected yeast to 10 2 , 10 3 , 10 4 , coated on SD-Trp solid plates, and the reservoir capacity was determined. The remaining yeast was coated on 15 cm SD-Trp solid plates, and then collected for the next round of panning. The results showed that the first round of screening reservoir capacity was 4×10 5 , the second round of screening library capacity: 7.4×10 6 , the third storage capacity: 8.2×10 6 .

[0058] 5) Monoclonal yeast flow cytometry to identify positive clones

[0059] After three rounds of continuous panning, 96 individual yeast colonies were picked into a 96-well round-bottom culture dish containing 100 μL of SD-Trp medium. Incubate for 12 hours at 30°C and 200 rpm, centrifuge the dish at 2000 g for 3 min, discard the supernatant, and add fresh induction medium SG-Trp to the dish. Incubate for 36 hours at 20°C and 200 rpm. Take 25 μL of culture to a new 96-well plate (U-bottom), centrifuge at 2000 g for 3 min, discard the supernatant, block the yeast precipitate with 200 μL BSA for 30 min, wash twice with PBSA, add the prepared goat anti-rabbit Fab fluorescently labeled secondary antibody (diluted with BSA), and incubate at 4°C with shaking for 30 min. Wash twice with PBSA. At the same time, prepare the required tumor lymphocytes (L540 and Hut78), wash the cells twice with PBSA, add BSA diluted fluorescent dye (1:8000 dilution) to resuspend the cells, incubate at 4°C for 30min, wash twice with PBS, resuspend the cells with PBSA, add 100μL / well to the yeast culture dish labeled with fluorescent secondary antibody and mix well, incubate at 4°C for 30min. Then perform flow cytometry detection (the method for obtaining candidate antibody sequences is referred 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 monoclonal yeast are as follows Figure 1 As shown, the positive clones with double positive signals greater than 30% for yeast and tumor cells were sequenced, the sequence results were analyzed and verified by flow cytometry, and finally a clone with a strong positive signal was obtained, named 0115-1, and the light and heavy chain sequence information was obtained by sequencing. It can be seen that when 0115-1 was verified by flow cytometry, yeast and tumor cells ( Figure 1 The double positive signal of specific binding is relatively strong.

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

[0062] QSVKESGGRLVTPGGTLTLTCAASEFSFSGYAVNWVRQAPGKGLEWIGFIGINGDTYYASWAKGRFTISKTSSTTVELKMTSLAVADTATYFCSRGDLWGRGTLVTVS SGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPT(SEQ IDNO:1);

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

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

[0065] The three antigen complementary determining regions (CDR1, CDR2 and CDR3) of the heavy chain of the 0115-1 antibody 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 light chain of the 0115-1 antibody 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] In order 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 the Fab was connected 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), and the transfected mammalian cells were cultured in Freestyle TM293 expression medium under CO 2 The mammalian cells were cultured for 5 days in a shaker at 37°C and 150 rpm. The supernatant of the mammalian cell culture was collected by centrifugation at 2000 rpm for 10 min. The Fab antibody-FC fusion protein was purified using a protein A column and analyzed by SDS-PAGE electrophoresis. The results are shown in Figure 2 The results showed that the heavy chain size was about 50KD, the light chain size was about 28KD, and the sizes of the light and heavy chains were correct, indicating that a high-purity Fab-Fc fusion protein 0115-1-FC was obtained from the supernatant.

[0075] Among them, the rabbit Fc domain fragment is directly connected to the back of the Fab antibody heavy chain, and the amino acid sequence of the rabbit Fc domain fragment is:

[0076] PPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYIN NEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:9);

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

[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] The ELISA plate was coated with 2 μg / mL CD30 protein overnight, unbound sites were blocked with MPBS at room temperature for 2 h, and then washed 3 times with PBS-0.1% Tween 20 (PBST). A PBS solution with a concentration gradient of CD30 protein from 0.1 nM to 1 μM was established, and 0.5 μM of the 0115-1-FC fusion protein prepared in Example 2 was added to each gradient to make a total volume of 100 μL. After incubation at room temperature for 30 min, it was added to the ELISA plate coated with the antigen described above and incubated at 37°C for 30 min. After washing 4 times with PBST, 100 μL / well goat anti-rabbit Fab-HRP secondary antibody (Cat. No.: E-AB-1102) was added and incubated at 37°C for 1 h, 100 μL / well TMB was added for color development for 15 min, and then 50 μL 1M H 2 SO 4 The color development was stopped, and the absorbance at 450 nM was detected. The obtained data were used for nonlinear curve fitting using the graphics software GraphPad.

[0081] The results of the above ELISA for identifying the binding ability of 0115-1-FC antibody to human CD30 protein are as follows Figure 3 The results showed that 0115-1-FC antibody has a strong binding ability with human CD30 protein. 50 is 70nM.

[0082] Example 40115-1-FC binding activity with different lymphoma cell lines

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

[0084] Prepare the required tumor lymphocytes (L540 and Hut78) and negative cells CHO, wash twice with PBSA, and then add 5×10 5 / well was added to a U-bottom 96-well culture dish, followed by the addition of 0.2 μg of 0115-1-FC prepared in Example 2, incubated at 4°C for 30 min, washed twice with PBS, and then added with the prepared goat anti-rabbit Fab fluorescently labeled secondary antibody (diluted with BSA), incubated at 4°C for 30 min. Then, flow cytometry (manufacturer: Shenzhen Beida Technology, model: BeamCyte-1026M) was performed for detection. The detection results are shown in Figure 2. Figure 4 As shown, 0115-1-FC has strong binding to tumor cells L540 and Hut78, but has no binding to negative cells CHO.

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

[0086] Take 50μL ProteinG magnetic beads into a 1.5mL centrifuge tube, wash twice with PBS, add 5μg antibody 0115-1-FC, and rotate and incubate at 4℃ overnight. Place the centrifuge tube on a magnetic rack for 2 minutes, discard the supernatant, and wash twice with PBS. Add 1mL lymphoma cell Hut78 lysate and rotate and incubate at 4℃ for 1h. Place the centrifuge tube on a magnetic rack for 2 minutes, discard the supernatant, and wash twice with PBS. Resuspend the magnetic beads with 20μL PBS and add it to 2× reducing buffer, and separate it with SDS-PAGE (8% separation gel). The results are as follows. Figure 5 As shown, it was shown that 0115-1-Fc could specifically bind to human CD30 protein.

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

[0088] Prepare the required tumor lymphocyte Hut78 cells, wash them twice with PBSA, and then culture them at a rate of 5×10 5 / well was added to a U-bottom 96-well culture dish, followed by the addition of 0.2μg of 0115-1-FC and positive control TNFRSF8 CD30 (Ki-4), incubated at 4℃ for 30min, washed twice with PBS, and the prepared goat anti-rabbit Fab fluorescently labeled secondary antibody (diluted with BSA) was added, and incubated at 4℃ for 30min. Flow cytometry was then performed, and the test results were as follows Figure 6 As shown, 0115-1-FC has stronger binding activity to the positive control TNFRSF8 CD30 (Ki-4) than other antibodies.

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

Claims

1. An anti-CD30 rabbit Fab antibody, characterized in that: The rabbit-derived Fab antibody includes a heavy chain and a light chain; the heavy chain contains an antigen complementary determining region shown in at least one of the amino acid sequences such as SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; the light chain contains an antigen complementary determining region shown in at least one of the amino acid sequences such as SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:

8.

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

2.

3. The rabbit Fab antibody according to claim 1, characterized in that: The rabbit Fab antibody also includes a rabbit Fc domain having an amino acid sequence as shown in SEQ ID NO:

9.

4. A coding gene, characterized in that The coding 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 comprises the coding gene according to claim 4.

6. The method for preparing a rabbit-derived Fab antibody according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Express CD30 protein to immunize rabbits; (2) extracting blood from the rabbit ear vein and isolating lymphocytes, extracting total RNA from the lymphocytes, using the cDNA obtained by reverse transcription of the RNA as a template, amplifying the Fab antibody sequence, and then cloning the Fab antibody sequence into a yeast vector to obtain a recombinant product, which is the initial Fab antibody yeast library; (3) transforming the recombinant product into a Saccharomyces cerevisiae competent cell to obtain an electroporation product, and then culturing the electroporation product to screen out yeast cells that can bind to the CD30 protein; (4) Single-cell cloning of the screened yeast cells is performed, and clones with strong positive results are further screened to obtain Fab recombinant protein, which is sequenced to obtain the coding sequence of the Fab antibody; (5) The coding sequence of the Fab antibody is connected to the coding sequence of the rabbit Fc domain, cloned into a mammalian expression vector, transfected into HEK293F cells, and purified after culture to obtain a Fab-Fc fusion protein.

7. The preparation method according to claim 6, characterized in that: In step (4), positive clones are screened using flow cytometry, which is performed by adding tumor lymphocytes to yeast cells labeled with fluorescently labeled antibodies and incubating them for detection, and sequencing positive clones with double positive signals of yeast and tumor cells specifically binding greater than 30%.

8. The preparation method according to claim 6, characterized in that: In the step (5), the coding sequence of the Fab antibody is connected to the coding sequence of the rabbit Fc domain via a linker having an amino acid sequence as shown in SEQ ID NO:

10.

9. Use of the rabbit Fab antibody according to any one of claims 1 to 3, or the encoding gene according to claim 4, or the engineered strain according to claim 5 in the preparation of a drug, preparation or kit for preventing, treating and / or diagnosing lymphoma.

10. The use according to claim 9, characterized in that The lymphoma is non-Hodgkin's lymphoma.

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