Anti-human ROR1 antibodies, genes, and uses thereof

Abstract

The application discloses anti-human ROR1 antibodies, genes and application thereof, and a heavy chain variable region and a light chain variable region of the anti-human ROR1 antibodies, which are shown as SEQ ID NO. 1-17. Anti-human ROR1 antibodies in some examples of the application can be combined with the extracellular region of ROR1 with high efficiency and specificity, and the affinity is as high as 1.329 nM, and the anti-human ROR1 antibodies can be used for preparing detection reagents and targeted drugs.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to an anti-human ROR1 antibody, gene, and its applications. Background Technology

[0002] The type I receptor-tyrosine-kinase-like orphan receptor ROR1 is a transmembrane protein in the tyrosine kinase receptor family, which includes two closely related type I transmembrane proteins, ROR1 and ROR2 (Casaletto JB, et al. Nat. Rev. Cancer (2012) 12:387–400). The ROR family belongs to the Wnt signaling pathway and is closely associated with MuSK (muscle-specific kinase) and Trk (tropomyosin) family receptors. The extracellular domain of ROR1 contains an immunoglobulin-like domain (IgG-like domain), a cysteine-rich domain (Fz), and a Kringle domain. A single transmembrane helix connects the extracellular domain to a cytoplasmic region containing a tyrosine kinase-like or pseudokinase domain, two serine / threonine (Ser / Thr)-rich domains, and a proline-rich (Pro) domain inserted between them. ROR1 plays a crucial role in various physiological processes by mediating signal transduction through non-canonical Wnt pathways (Karvonen H, et al. Oncogene (2019) 38:3288–300.), including regulating cell division, proliferation, migration, and chemotaxis, especially Wnt5a. Wnt5a is a typical activator of non-canonical Wnt signaling pathways, participating in the phosphorylation of the NF-κB subunit p65 (Chen Y, et al. Blood (2019) 134:1084–94), activating the NF-κB pathway in tumor cells, and promoting cell migration and invasion. As a receptor for Wnt5a, ROR1 is involved in activating the NF-κB pathway in tumor cells (Nusse R, et al. Cell (2017) 169:985–99). ROR1 is primarily present during embryonic development and is not expressed in most normal tissues after birth. However, studies have shown that ROR1 is overexpressed in many hematologic and solid malignancies, such as chronic lymphocytic leukemia (CLL), breast cancer, ovarian cancer, melanoma, and lung adenocarcinoma, and tends to be expressed in poorly differentiated tumors (Borcherding N, et al. Protein Cell (2014) 5:496–502.). Cancer cells expressing ROR1 exhibit stronger invasive, metastatic, and recurrent abilities, and the level of expression is associated with poor prognosis. Using siRNA to interfere with ROR1 expression in metastatic breast cancer and melanoma cell lines, or applying anti-ROR1 monoclonal antibodies, can inhibit tumor cell migration and invasion and induce tumor cell apoptosis.Therefore, ROR1 can serve as a potential target for the treatment of hematologic malignancies and solid tumors. ROR1 is a highly conserved molecular target, highly homologous in animals such as humans and primates, and exhibits similar tissue expression distribution.

[0003] To date, numerous therapeutic strategies targeting ROR1 have been developed and evaluated in clinical trials and preclinical studies. Most targeted therapies utilize small molecule drugs or mAb-based strategies (Ferguson FM, et al. Nat. Rev. Drug Discovery (2018) 17:353–77; Carter PJ, et al. Nat. Rev. Drug Discov. (2018) 17:197–223). Small molecule tyrosine kinase inhibitors (TKIs) are ATP-competitive inhibitors that target the catalytic domain of tyrosine kinases (Imai K, et al. Nat. Rev. Cancer (2006) 6:714–27). Monoclonal antibodies, on the other hand, directly block ligand binding, activating the immune system to eliminate tumor cells. Currently, various monoclonal antibodies targeting ROR1 have been developed. In malignant cells expressing ROR1, these monoclonal antibodies mediate antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), ROR1 internalization, and apoptosis. Cirmtuzumab is the only ROR1-targeting monoclonal antibody (mAb) evaluated in clinical trials. Cirmtuzumab showed promising efficacy in a Phase I clinical trial in CLL patients (Choi MY, et al. Cell Stem Cell (2018) 22:951–59.e953). Phase Ib and I / II trials of Cirmtuzumab in combination with paclitaxel or ibrutinib for breast cancer or CLL / MCL patients are also underway. Based on the promising efficacy of ROR1-targeting monoclonal antibody therapy, antibody-drug conjugates (ADCs) and bispecific T-cell engagers (BiTEs) have been developed, some of which are currently being evaluated in clinical trials. Antibody-drug conjugates (ADCs) chemically link biologically active small molecule drugs to mAbs or scFvs, with the antibody molecule acting as the primary carrier to deliver the small molecule drug to target cells or molecules, allowing the small molecule drug to selectively exert its antitumor toxicity. In a model of antiROR1 scFv-endog protein, when a therapeutic immunotoxin targeting the extracellular region of ROR1—a non-immunogenic human endonuclease G—is selectively delivered to ROR1+ tumor cells, this immunotoxin, which belongs to the terminal apoptosis molecule, can rapidly induce tumor cell apoptosis (Peng H, et al. J Mol Biol. 2017 Sep 15;429(19):2954-2973).Antibody-drug conjugates (ADCs) may have toxic side effects on the heart (such as anthracyclines) and liver. In addition, they can induce tumor resistance in some treatment regimens. For example, when antibody-drug conjugate T-DM1 is used to treat human epidermal growth factor receptor 2 (Her2) positive breast cancer, it activates the Hippo transcriptional coactivator YAP3 through ROR1, thus inducing tumor resistance.

[0004] In ROR1-targeted therapy, bispecific antibodies have gradually become a research hotspot. Bispecific antibodies are composed of two different heavy chains (VH) and light chains (VL), which can bind to two different specific antigenic epitopes, selectively activating the immune response and mediating apoptosis. Studies have shown that R11BiAb binding to the proximal membrane epitope of ROR1 in blood and solid malignant tumor cells has greater selectivity and anti-tumor effect on T cells than R12 BiAb binding to the distal membrane epitope. This is mainly due to the distance between the ROR1 and CD3 binding arms of the bispecific antibody and the position of its epitope on ROR1 and CD3 (Qi J, et al.Proc. Natl. Acad. Sci. USA. 2018 Jun 12;115(24):E5467-E5476). Based on the bispecific antibody BiAb, the bispecific T-cell engager (BiTE) expands the scope of its application in clinical treatment. BiTE has no Fc domain, targets tumor antigens at one end and activates the CD3 subunit of T cells at the other end. Its small molecular weight makes it easy to infiltrate into the tumor microenvironment to exert its anti-tumor function. Researchers have successfully constructed ROR1 BiTE by preparing specific scFvs and mouse CD3 scFvs using anti-ROR1 antibodies that bind to the distal immunoglobulin-like (Ig-like) or proximal frizzled (Fr) domain (FzD) of ROR1 membrane. These CD3 scFvs can specifically bind to human T cells. Moreover, BiTE targeting ROR1 FzD produces superior and sustained cytotoxicity in ROR1+ cancer cell lines compared to BiTE targeting Ig-like. Furthermore, this BiTE targeting ROR1-FzD can activate T cell-mediated cytotoxic effects in ROR1+ pancreatic cancer cells, ovarian cancer cells, and a range of solid tumor cells with different histological morphologies. (Gohil SH, et al. Oncoimmunology. 2017May 17;6(7):e1326437). In summary, the ROR1-BiTE regimen is showing great promise for development and treatment in ROR1-targeted cancer therapy. It has the functions of promoting T cell infiltration, enhancing their effector function, and reversing the immunosuppressive microenvironment. It has shown good therapeutic effects in a range of malignant tumors, such as chronic lymphocytic leukemia (CLL), breast cancer, ovarian cancer, melanoma, and lung adenocarcinoma. Summary of the Invention

[0005] In tumor cells, the expression level of ROR1 antigen is low, limiting its potential for development as a monoclonal and bispecific antibody. Therefore, antibodies with high affinity for the ROR1 tumor antigen are needed to prevent tumor escape due to low binding ability. In addition, ROR1 bispecific antibodies based on bispecific T-cell engagers (BiTEs) still have potential for further optimization, such as optimizing the relative structural positions of ROR1 and CD3.

[0006] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide an anti-human ROR1 antibody, gene and its application.

[0007] The technical solution adopted in this invention is:

[0008] A first aspect of the present invention provides: an anti-human ROR1 antibody, comprising a heavy chain variable region and a light chain variable region.

[0009] The amino acid sequence of the heavy chain CDR1 in the heavy chain variable region is selected from one of the sequences shown in SEQ ID NO. 1 to 2;

[0010] The amino acid sequence of the heavy chain CDR2 in the heavy chain variable region is selected from one of the sequences shown in SEQ ID NO.3 to 5;

[0011] The amino acid sequence of the heavy chain CDR3 in the heavy chain variable region is selected from one of the sequences shown in SEQ ID NO. 6 to 9;

[0012] The amino acid sequence of the light chain CDR1 in the light chain variable region is selected from one of the sequences shown in SEQ ID NO. 10 to 12;

[0013] The amino acid sequence of the light chain CDR2 in the light chain variable region is selected from one of the sequences shown in SEQ ID NO. 13-14;

[0014] The amino acid sequence of the light chain CDR3 in the light chain variable region is selected from one of the sequences shown in SEQ ID NO.15-17.

[0015] In some examples of anti-human ROR1 antibodies, the heavy chain variable region and light chain variable region are as follows:

[0016]

[0017] Among them, CDR-H1, CDR-H2, and CDR-H3 refer to heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, respectively; CDR-L1, CDR-L2, and CDR-L3 refer to light chain CDR1, light chain CDR2, and light chain CDR3, respectively.

[0018] In some examples of anti-human ROR1 antibodies, a heavy chain framework region and a light chain framework region are also included, wherein the amino acid sequence of the heavy chain FR1 in the heavy chain framework region is selected from one of the sequences shown in SEQ ID NO. 18 to 20;

[0019] The amino acid sequence of the heavy chain FR2 in the heavy chain framework region is selected from one of the sequences shown in SEQ ID NO. 21 to 23;

[0020] The amino acid sequence of the heavy chain FR3 in the heavy chain framework region is selected from one of the sequences shown in SEQ ID NO. 24 to 27;

[0021] The amino acid sequence of the heavy chain FR4 in the heavy chain framework region is either empty or selected from one of the sequences shown in SEQ ID NO.28-31;

[0022] The amino acid sequence of the light chain FR1 in the light chain framework region is selected from one of the sequences shown in SEQ ID NO.32-35;

[0023] The amino acid sequence of the light chain FR2 in the light chain framework region is selected from one of the sequences shown in SEQ ID NO. 36-37;

[0024] The amino acid sequence of the light chain FR3 in the light chain framework region is selected from one of the sequences shown in SEQ ID NO.38-40;

[0025] The amino acid sequence of the light chain FR4 in the light chain framework region is selected from one of the sequences shown in SEQ ID NO.41-43.

[0026] In some examples of anti-human ROR1 antibodies, the heavy chain framework region and light chain framework region are as follows:

[0027]

[0028] Among them, FR-H1, FR-H2, FR-H3, and FR-H4 refer to heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4, respectively; FR-L1, FR-L2, FR-L3, and FR-L4 refer to light chain FR1, light chain FR2, light chain FR3, and light chain FR4, respectively.

[0029] In some examples of anti-human ROR1 antibodies, the amino acid sequence is selected from one of the amino acid sequences described in SEQ ID NO.44-53.

[0030] In some instances of anti-human ROR1 antibodies, the antibody is a single-chain antibody.

[0031] A second aspect of the invention provides: a gene encoding the anti-human ROR1 antibody described in the first aspect of the invention.

[0032] The specific coding genes can be optimized with corresponding codons depending on the expression system.

[0033] A third aspect of the invention provides: a recombinant expression vector or recombinant bacterial strain capable of expressing the anti-human ROR1 antibody described in the first aspect of the invention.

[0034] A fourth aspect of the present invention provides: a bispecific or polyclonal antibody, characterized in that it comprises a heavy chain variable region and a light chain variable region of the antibody described in the first aspect of the present invention.

[0035] A fifth aspect of the present invention provides: a reagent formed by directly or indirectly conjugating a tracer molecule to an anti-human ROR1 antibody as described in the first aspect of the present invention; or obtained by directly or indirectly conjugating a drug molecule to an anti-human ROR1 antibody as described in the first aspect of the present invention.

[0036] The beneficial effects of the present invention are: the anti-human ROR1 antibody of some examples of the present invention can bind to the extracellular region of ROR1 efficiently and specifically, with an affinity as high as 1.329 nM, and can be used to prepare detection reagents and targeted drugs.

[0037] The anti-human ROR1 antibodies in some examples of this invention, when constructed into bispecific antibodies, can still bind efficiently and specifically to the extracellular region of ROR1. Attached Figure Description

[0038] Figure 1 These are ELISA results for different monoclonal bacteriophages.

[0039] Figure 2 These are ELISA test results for different clones of CD3 / ROR1.

[0040] Figure 3 The results show the killing effect of different CD3 / ROR1 clones on MDA MB 468 tumor cells. Detailed Implementation

[0041] The technical solution of the present invention will be further explained below with reference to experiments.

[0042] Example 1: Eukaryotic expression and purification of ROR1 ECD (Extra-Cellular Domain) protein

[0043] Plasmid preparation: The DH5α strain transformed with pFUSE-Human ROR1 ECD-IgG Fc Fusion was cultured in 100 mL of Low salt LB containing 0.1% Zeocin and then the plasmid was extracted using the GenStar StarPure Endo-free PlasmidMaxiprep Kit and the concentration was determined.

[0044] Inoculation 1.5×10 6 293 suspension cells were placed in 200 mL of culture medium in a 500 mL shake flask and cultured at 37 °C, 165 rpm, and 5% CO2 concentration in a shaker incubator for 24 h. After 24 h, cell counting was performed, and the cell density was adjusted to 3 × 10⁹ / mL. 6 For each step, mix the light and heavy chain plasmids constructed in the above steps in 10 mL of Opti-MEM and let stand at room temperature for 5 min. Add 500 µL (1:2.5) PEI 40000 to 10 mL of Opti-MEM, mix gently, and let stand at room temperature for 5 min. Mix the plasmid and PEI mixture, mix gently, and let stand at room temperature for 20 min. Add the above mixture dropwise to 200 mL of 293 cell suspension culture medium, gently shaking the culture flask while adding. After mixing well, place on a shaker and transfect for 72 h. After transfection, centrifuge at 100 g for 5 min, collect the supernatant, add 100 mL of culture medium to the culture flask to resuspend the cells, and culture for 48 h. Centrifuge at 3000 rpm for 5 min, collect the supernatant, and mix the two collections of 293 cell suspension supernatant. Store at -20℃.

[0045] Example 2: Immunization of mice using ROR1 ECD antigen

[0046] Mouse immunization: The initial immunization was performed by subcutaneous injection of ROR1 antigen protein mixed with Freund's complete adjuvant at a 1:1 ratio, at multiple sites, at a dose of 100 μg. A second immunization was performed two weeks later using the same method. Two weeks after that, immunization was performed again using ROR1 antigen protein mixed with Freund's incomplete adjuvant at a 1:1 ratio, at a dose of 50 μg. One week later, blood was collected from the orbital venous plexus to measure serum antibody titers. Spleens were harvested if the titer was higher than 1:10000.

[0047] Example 3: Construction of scFv library from spleen of immunized mice

[0048] Total RNA extraction and reverse transcription from mouse spleen: Total RNA was extracted and cDNA was synthesized using the Tiangen RNA prep Pure RNA extraction and cDNA first-strand synthesis kit.

[0049] Takara primer MAX DNA amplification system

[0050]

[0051] (The overlap PCR system is the same as above)

[0052] SfiI enzyme digestion reaction

[0053]

[0054] Enzyme digestion reaction conditions: incubation at 50℃ for 4 hours. 1.2% agarose gel electrophoresis: samples were mixed with 6× Loading Buffer and loaded onto the gel. After electrophoresis, the gel was cut and the gel was recovered for quantification.

[0055] DNA Ligation Kit Ligation Reaction

[0056]

[0057] Incubate the ligation product sample and electroporation cuvette on ice for 10 min. Simultaneously, thaw 300 µL of electrotransformed competent *E. coli* on ice (do not allow the thawed *E. coli* to remain on ice for more than 20 min). Using a 1 mL pipette tip, transfer the 300 µL of electrotransformed competent *E. coli* to the sample, mix thoroughly by pipetting once, then transfer to the electroporation cuvette and incubate on ice for 1 min. Electroporate at 2.5 kV, 25 µF, and 200 Ω. Immediately rinse the electroporation cuvette at room temperature with 1 mL of SOC medium, followed by two more rinses with 2 mL of SOC medium. Add this 5 mL mixture to a 50 mL centrifuge tube and incubate at 37°C and 250 rpm for 1 h with shaking. Add 10 mL of preheated SB medium, 3 µL of 100 mg / mL Ampicillin, and 30 µL of 5 mg / mL Tetracycline. (If titration of transformed bacteria is required, pipette 2 µL of culture into 200 µL of SB medium (1:100), and spread 10 µL and 100 µL onto Ampicillin+LB plates, respectively. Incubate overnight at 37°C and count the total number of transformed positive clones.) Incubate 15 mL of culture at 37°C with shaking at 250 rpm for 1 h, then add 4.5 µL of 100 mg / mL Ampicillin and shake for another 1 h. Add 2 mL of VCSM13 helper phage (10... 12 -10 13The culture (pfu / mL) was transferred to a 500mL centrifuge bottle. 183mL of preheated SB medium (37℃) and 92.5µL of 100mg / mL Ampicillin were added, followed by 370µL of 5mg / mL Tetracycline. This 200mL culture was incubated at 37℃ with shaking at 300rpm for 1.5-2 hours. 280µL of 50mg / mL Kanamycin was added, and the culture was incubated overnight at 37℃ with shaking at 300rpm. The culture was centrifuged at 3000g for 15 minutes at 4℃, and the bacterial pellet was retained for phage DNA extraction. The supernatant was transferred to a new 500mL centrifuge bottle, and 8g of PEG-8000 (4% w / v) and 6g of NaCl (3% w / v) were added. The pellet was dissolved by shaking at 37℃ and 300rpm for 5 minutes, then incubated on ice for 30 minutes. Centrifuge at 15000g for 15 min at 4℃, discard the supernatant, and thoroughly drain the water for 10 min. Resuspend the phage particles in 2 mL of TBS containing 1% (w / v) BSA and transfer to a 2 mL centrifuge tube. Mix well by pipetting and aspiration, then centrifuge at full speed at 4℃ for 5 min. Filter the supernatant through a 0.2 µm filter membrane.

[0058] Example 4: Construction and selection of phage antibody display library

[0059] Phage library amplification and concentration: The method is the same as that for phage amplification and concentration in Example 3.

[0060] Remove the screening antigen from the -80℃ freezer and place it on ice to thaw. 25.5 µL (1.96 mg / mL) of ROR1 antigen was coated onto immunotubes (50 µg / tube, coating buffer: CBS, pH 9.6, 2 mL / tube), and incubated overnight at 4°C with slow rotation. Simultaneously, 1 µL (50 µg of 5% non-fat milk powder, diluted to 100 mL with coating buffer, 50 mg / mL) was coated as a control. The liquid in the overnight coated immunotubes was discarded, and 2 mL of PBS buffer was added to wash the immunotubes three times at room temperature, rotating for 5 min each time. 2 mL of blocking buffer (3% BSA-PBST) was added, and the tubes were blocked by rotation at room temperature for 2 h. The liquid in the blocked immunotubes was discarded, and 1 mL of PBS buffer was added to wash the immunotubes three times at room temperature, rotating for 5 min each time. The washing buffer was discarded, and 2 mL of PBS buffer was added. The prepared phage library was added as the first-round screening input phage library, and the tubes were incubated by rotation at room temperature for 1 h. The liquid in the immunotubes was discarded, and 2 mL of PBS buffer was added to wash the tubes. Wash the immunotubes 20 times with PBST (1×PBS plus 0.1% Tween 20, the same below) buffer at room temperature, rotating for 5 min each time; discard the liquid in the immunotubes, removing as much residual liquid as possible, add 1 mL of 0.25 mg / mL LTrypsin solution, and elute by rotating at room temperature for 30 min; add 10 µL of 10% AEBSF to stop elution, and transfer the solution in the immunotubes to a new 1.5 mL centrifuge tube, which is the elution buffer for the first round of phage selection.

[0061] First-round amplification of phage eluent: The XL1-Bule strain, stored at -80℃, was streaked onto 2×YT solid medium (Tet resistant). A single colony was picked from the streak plate and transferred to 5 mL of 2×YT medium containing 10 µg / mL Tet, and incubated overnight at 37℃. 250 µL of the overnight culture was transferred to 5 mL of 2×YT liquid medium containing 10 µg / mL Tet, and incubated at 37℃ and 250 rpm for approximately 45-60 minutes until the OD600 value reached 0.5-0.55. 500 µL of the phage eluent obtained from the first round of selection was added to the culture with an OD600 of 0.5-0.55 (the remaining eluent was stored at 4℃). The culture was further incubated at 37℃ and 220 rpm for 30 minutes. The entire culture was then evenly spread onto a plate containing 100 µg / mL Amp... Incubate overnight at 37°C on 2% glucose and 2% agarose medium plates. Take the overnight plates and add 6 mL of 2×YT liquid medium (containing 10 µg / mL Tet) to the surface. Gently scrape the colonies off the plate with a spreader and collect the bacterial solution into a 15 mL centrifuge tube; this is the amplified bacterial library. Simultaneously, measure the OD600 value of the bacterial solution using a spectrophotometer; this is the OD600 value of the eluted bacterial library. Add 20% glycerol to a final concentration to obtain the first-round bacterial library. Transfer the eluted bacterial library to 100 mL of 2×YT liquid medium (containing 10 µg / mL Tet and 100 µg / mL Amp) to achieve an initial OD600 of 0.1; incubate at 37°C and 250 rpm until the OD600 reaches 0.5-0.55.

[0062] Add helper phages to make the bacterial-to-phage ratio 1:20. Continue culturing at 37℃ and 220rpm for 30 min. Add Kanamycin to a final concentration of 50µg / mL and IPTG to a final concentration of 0.2μM, respectively, and incubate overnight at 30℃ and 250rpm.

[0063] Example 5: Selection and Affinity Identification of Monoclonal Bacteriophages

[0064] Take 10 µL of phage elution buffer from the second round of screening and serially dilute it 10-fold in 1.5 mL centrifuge tubes, for a total of 12 dilutions, and mix thoroughly by vortexing. Add 90 µL of bacterial suspension with an OD600 value of 0.5-0.55 to each dilution centrifuge tube and mix well. Incubate at 37°C and 220 rpm for 30 min. Spread the bacterial suspension evenly onto solid medium plates containing 100 µg / mL Amp and incubate overnight at 37°C. Randomly pick single colonies from the overnight culture plates and place them into sterile 96-well cell culture plates. Add 200 µL of 2×YT medium (containing 100 µg / mL Amp and 10 µg / mL Tet) to each well and incubate statically at 37°C overnight. Transfer 2 µL of the overnight culture to 200 µL of 2×YT liquid medium (containing 100 µg / mL Amp and 10 µg / mL Tet) to each well. In a new 96-well cell culture plate, incubate at 37°C for 3-5 hours. Store the overnight cultured bacterial solution at 4°C before transfer. Add helper phage M13K07 to each well to make the bacterial count: phage count = 1:20.Incubate at 37℃ for 30 min, add Kanamycin to a final concentration of 50 µg / mL and 0.2 µM IPTG, and incubate overnight at 30℃. Centrifuge the 96-well plate at 4℃, 4000 rpm for 10 min, and store at 4℃ for later use. Coat the microplate with ROR1 antigen (1 ng / µL, coating buffer: CBS, pH 9.6, 100 µL / well), and simultaneously coat with BSA as a control, and incubate overnight at 4℃. Discard the liquid in the overnight coated microplate, add 200 µL PBS buffer to each well, and wash the microplate 3 times at room temperature for 10 min each time. Add 200 µL blocking buffer (3% BSA) to each well to block the microplate, and block at room temperature for 1 h. Discard the blocking buffer, add 200 µL PBST buffer (1×PBS plus 0.1% Tween 20, the same below) to each well, and wash the microplate 3 times at room temperature. Each step involves three incubation cycles, 10 min each. Add 120 µL of 3% BSA to each well, followed by 80 µL of the supernatant from step 6.6.11. Incubate at room temperature for 2 h. Discard the liquid in the ELISA plate. Wash each well three times with 200 µL of PBST buffer, 10 min each time. Add 100 µL of M13 Bacteriophage Antibody (HRP), diluted 1:8000 in blocking buffer, to each well. Incubate at room temperature for 1 h. Discard the liquid in the ELISA plate. Wash each well six times with 200 µL of PBST buffer, 5 min each time. Add 100 µL of TMB single-component chromogenic solution to each well. Incubate in the dark for 2-3 min. Stop incubation with 100 µL of 2M H2SO4. Read the OD450 value using a microplate reader, record and save the results. Figure 1 As shown, positive ELISA reactions were observed in the positive antigen (ROR1) group, meaning the OD450 value of the positive antigen well was greater than 5 compared to the OD450 value of the negative antigens (BSA, Her2). These positive clones were selected for the next sequencing step.

[0065] Example 6: Sequencing of positive clones

[0066] Positive monoclonal antibodies were selected based on ELISA test data and secondary validation data.

[0067] Take 5 µL of positive clone bacterial culture from the monoclonal ELISA detection plate and inoculate it into 2 mL of 2×YT medium (containing 100 µg / mL Amp and 10 µg / mL Tet). Incubate at 37°C and 250 rpm until OD600 reaches 0.8-1.0 (approximately 6-8 h). Take 1 mL of the bacterial culture for sequencing, and store the remaining bacterial culture at 4°C.

[0068] Example 7: Sequence Analysis

[0069] The sequenced sequences were analyzed using GENtle software for sequence alignment, and the antibody sequences were translated into amino acids using GENtle software.

[0070] Antibodies P3-A7, P3-B11, P3-C7, P3-D4, P3-D5, P3-G6, P3-G10, P3-G11, P3-H6, and P3-H9 (SEQ ID NOs 44–53) with good affinity were obtained through screening. Analysis revealed:

[0071] The amino acid sequence of the heavy chain CDR1 in the heavy chain variable region is one of the sequences shown in SEQ ID NO. 1-2;

[0072] The amino acid sequence of the heavy chain CDR2 in the heavy chain variable region is one of the sequences shown in SEQ ID NO. 3 to 5;

[0073] The amino acid sequence of the heavy chain CDR3 in the heavy chain variable region is one of the sequences shown in SEQ ID NO. 6 to 9;

[0074] The amino acid sequence of the light chain CDR1 in the light chain variable region is one of the sequences shown in SEQ ID NO. 10-12;

[0075] The amino acid sequence of the light chain CDR2 in the light chain variable region is one of the sequences shown in SEQ ID NO. 13-14;

[0076] The amino acid sequence of the light chain CDR3 in the light chain variable region is one of the sequences shown in SEQ ID NO. 15-17;

[0077] The amino acid sequence of the heavy chain FR1 in the heavy chain framework region is one of the sequences shown in SEQ ID NO. 18-20;

[0078] The amino acid sequence of the heavy chain FR2 in the heavy chain framework region is one of the sequences shown in SEQ ID NO. 21-23;

[0079] The amino acid sequence of the heavy chain FR3 in the heavy chain framework region is one of the sequences shown in SEQ ID NO. 24-27;

[0080] The amino acid sequence of the heavy chain FR4 in the heavy chain framework region is either empty or one of the sequences shown in SEQ ID NO. 28–31;

[0081] The amino acid sequence of the light chain FR1 in the light chain framework region is one of the sequences shown in SEQ ID NO.32-35;

[0082] The amino acid sequence of the light chain FR2 in the light chain framework region is one of the sequences shown in SEQ ID NO. 36-37;

[0083] The amino acid sequence of the light chain FR3 in the light chain framework region is one of the sequences shown in SEQ ID NO.38-40;

[0084] The amino acid sequence of the light chain FR4 in the light chain framework region is one of the sequences shown in SEQ ID NO.41-43.

[0085] The heavy chain variable regions and light chain variable regions of different antibodies are as follows:

[0086]

[0087] Among them, CDR-H1, CDR-H2, and CDR-H3 refer to heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, respectively; CDR-L1, CDR-L2, and CDR-L3 refer to light chain CDR1, light chain CDR2, and light chain CDR3, respectively.

[0088] The heavy chain and light chain framework regions of different antibodies are as follows:

[0089]

[0090] Among them, FR-H1, FR-H2, FR-H3, and FR-H4 refer to heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4, respectively; FR-L1, FR-L2, FR-L3, and FR-L4 refer to light chain FR1, light chain FR2, light chain FR3, and light chain FR4, respectively.

[0091] Example 8: Construction of CD3 / ROR1 bispecific antibody

[0092] Construction of CD3-HC-ROR1 and CD3-LC expression vectors:

[0093] The heavy chain of the ROR1 / CD3 bispecific antibody is composed of CD3-HC (SP34) and ROR1-scFv linked as follows: ROR1-scFv is linked to the C-terminus 228C of the constant region (CH1) domain of CD3-HC via a flexible linker (G4S)3 Linker; the light chain CD3-LC is left unmodified. The encoding genes for CD3-HC-ROR1-scFv and CD3-LC were synthesized using conventional molecular biology methods, and the synthesized genes were inserted into the Amp-resistant pcAGGS eukaryotic expression vector via homologous recombination. Depending on the linking method, the bispecific antibody expressed later is labeled CD3 / ROR1. The anti-ROR1-scFv can be linked to CD3-HC via the (G4S)3 Linker selected in this experiment, or alternative linkers known to those skilled in the art can be used.

[0094] Example 9: Eukaryotic expression of CD3 / ROR1 protein

[0095] Inoculation 1.5×10 6 293 suspension cells were placed in 200 mL of culture medium in a 500 mL shake flask and cultured at 37 °C, 165 rpm, and 5% CO2 concentration in a shaker incubator for 24 h. After 24 h, cell counting was performed, and the cell density was adjusted to 3 × 10⁹ / mL. 6 For each step, mix the light and heavy chain plasmids constructed in the above steps in 10 mL of Opti-MEM and let stand at room temperature for 5 min. Add 500 µL (1:2.5) PEI 40000 to 10 mL of Opti-MEM, mix gently, and let stand at room temperature for 5 min. Mix the plasmid and PEI mixture, mix gently, and let stand at room temperature for 20 min. Add the above mixture dropwise to 200 mL of 293 cell suspension culture medium, gently shaking the culture flask while adding. After mixing well, place on a shaker and transfect for 72 h. After transfection, centrifuge at 100 g for 5 min, collect the supernatant, add 100 mL of culture medium to the culture flask to resuspend the cells, and culture for 48 h. Centrifuge at 3000 rpm for 5 min, collect the supernatant, and mix the two collections of 293 cell suspension supernatant. Store at -20℃.

[0096] Example 10: Purification of CD3 / ROR1 antibody

[0097] Take 400 mL of cell supernatant from Example 10 above, centrifuge at 15000 rpm and 4°C for 30 min, collect the supernatant, filter through a 0.45 μm filter membrane, and store on ice for later use. Add 4 mL of Protein G (20% ethanol / Protein G 1:1) to the chromatography column, wash three times with binding buffer, and then press the resin surface with a pad. Equilibrate the Protein G column with 20 mL of binding buffer. Load 10 mL of sample at a constant rate (approximately 0.5 mL / min) through the Protein G column. Wash the Protein G column with 40 mL of binding buffer at a constant rate (approximately 1 mL / min). First, add 10% of the elution buffer volume of neutralization buffer to the elution collection tube, then add Elution buffer to the column, elute 5 mL at a time until the protein concentration cannot be quantified. Concentrate the collected protein sample using an Amicon Ultra-15 centrifuge filter, centrifuge at 3000 rpm for 20 min at 4°C, and quantify the protein concentration.

[0098] Example 11: Validation of antigen binding of CD3 / ROR1 bispecific antibody

[0099] ROR1 ECD-Fc antigen protein was diluted to 10 µg / mL with PBS and coated onto 96-well ELISA plates at 100 µL / well, incubated overnight at 4°C. The liquid in the plate was discarded, and the plate was washed twice with PBST. Then, 200 µL / well of 5% skim milk PBST solution was added, and the plates were blocked at room temperature for 2 h. The blocking buffer was then drained from the wells. Eight gradients of CD3 / ROR1 bispecific antibodies from different clones were prepared using blocking buffer, with an initial antibody concentration of 100 nM. Each concentration gradient was applied in triplicate. 100 µL / well of the standard was added to each well, and the plates were incubated at room temperature for 2 h. After washing the plates three times with PBST and draining the buffer, HRP-labeled anti-kappa chain antibody was diluted 1:5000 with 5% skim milk PBST and added to each well at 100 µL / well, and incubated at room temperature for 1 h. After washing the plate three times with PBST and draining it, add 100 µL of TMB chromogenic solution to each well and incubate at room temperature in the dark for 10 min. Then, add 50 µL of 2M H2SO4 to each well to stop the color development reaction. Measure the absorbance at OD450 nm using a microplate reader. Plot a four-parameter nonlinear regression graph with sample concentration on the x-axis and absorbance on the y-axis, and calculate EC. 50 Values. The results are shown in Table 1 and Figure 2 As shown, the binding ability of different CD3 / ROR1 clones to immobilized ROR1 antigen was detected by ELISA. Among them, the clones with the best binding ability (P3-A7, P3-C7, P3-D4, P3-D5, P3-G6, P3-G10, P3-H9, P3-H6) could reach 1-3 nM EC50.50 The value can be used as a further preferred antibody clone.

[0100] Table 1 Comparison of the binding ability of CD3 / ROR1 bispecific antibodies to ROR1 antigen mediated by T cells

[0101]

[0102] Example 12: In vitro tumor-killing activity of different ROR1 / CD3 bispecific antibodies

[0103] Activated human T cells (PBMCs from healthy volunteers) were cultured in RPMI-1640 complete medium containing 300 IU / mL IL-2, and MDA-MB-468 breast cancer cells were cultured in DMEM medium containing 10% fetal bovine serum. T cells were cultured in RPMI-1640 medium (with IL-2 removed) for 24 hours prior to the experiment. The T cell density was adjusted to 2 × 10⁶ cells / mL. 6 The tumor cell density is 2×10⁻⁶. 5 After mixing the two cell types in equal volumes at an effector-to-target ratio of 10:1, 100 µL / well was added to each well of a 96-well cell plate. The bispecific antibody to be tested was diluted 10-fold with culture medium in six gradients, starting at a concentration of 100 nM, with two replicates for each concentration gradient, and 10 µL / well was added to each well. Positive controls (cell lysis buffer added directly during detection) and negative controls (tumor cells and T cells) were also included. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h. The degree of target cell lysis in the 96-well plate was detected using a lactate dehydrogenase assay kit, and the OD490 readings in each well were measured using a multi-mode microplate reader. Data analysis was performed using Graphpad Prism 6 software, with antibody concentration on the x-axis and absorbance on the y-axis, and IC50 was calculated. 50 Values. The results are shown in Table 2 and Figure 3 As shown, the killing ability of different CD3 / ROR1 clones was tested by their ability to kill MDA MB468 positive cells. The clones with the best killing ability (P3-C7, P3-D4, P3-D5, P3-G10, P3-H9, P3-H6) achieved IC50 values ​​of 1-4 pM. 50 The value can be used as a preferred antibody clone.

[0104] Table 2 Comparison of the killing ability of CD3 / ROR1 bispecific antibody-mediated T cells against MDA MB 468 tumor cells

[0105]

[0106] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. An anti-human ROR1 antibody, comprising a heavy chain variable region and a light chain variable region, characterized in that: The amino acid sequence of the heavy chain CDR1 in the heavy chain variable region is shown in SEQ ID NO.2; The amino acid sequence of the heavy chain CDR2 in the heavy chain variable region is shown in SEQ ID NO.4; The amino acid sequence of the heavy chain CDR3 in the heavy chain variable region is shown in SEQ ID NO.7; The amino acid sequence of the light chain CDR1 in the light chain variable region is shown in SEQ ID NO.11; The amino acid sequence of the light chain CDR2 in the light chain variable region is shown in SEQ ID NO.14; The amino acid sequence of the light chain CDR3 in the light chain variable region is shown in SEQ ID NO.

16.

2. The anti-human ROR1 antibody according to claim 1, characterized in that, It also includes heavy chain framework regions and light chain framework regions. The amino acid sequence of the heavy chain FR1 in the heavy chain framework region is selected from one of the sequences shown in SEQ ID NO. 18 to 20; The amino acid sequence of the heavy chain FR2 in the heavy chain framework region is selected from one of the sequences shown in SEQ ID NO. 21 to 23; The amino acid sequence of the heavy chain FR3 in the heavy chain framework region is selected from one of the sequences shown in SEQ ID NO. 24 to 27; The amino acid sequence of the heavy chain FR4 in the heavy chain framework region is selected from one of the sequences shown in SEQ ID NO. 28 to 31; The amino acid sequence of the light chain FR1 in the light chain framework region is selected from one of the sequences shown in SEQ ID NO.32-35; The amino acid sequence of the light chain FR2 in the light chain framework region is selected from one of the sequences shown in SEQ ID NO. 36-37; The amino acid sequence of the light chain FR3 in the light chain framework region is selected from one of the sequences shown in SEQ ID NO.38-40; The amino acid sequence of the light chain FR4 in the light chain framework region is selected from one of the sequences shown in SEQ ID NO.41-43.

3. The anti-human ROR1 antibody according to claim 2, characterized in that, Its heavy chain framework region and light chain framework region are as follows: the amino acid sequences of its heavy chain framework region FR1, FR2, FR3, and FR4 are shown in SEQ ID NO.19, SEQ ID NO.22, SEQ ID NO.25, and SEQ ID NO.30, respectively, and the amino acid sequences of its light chain framework region FR1, FR2, FR3, and FR4 are shown in SEQ ID NO.33, SEQ ID NO.37, SEQ ID NO.39, and SEQ ID NO.42, respectively.

4. The anti-human ROR1 antibody according to claim 1, characterized in that, Its amino acid sequence is selected from one of the amino acid sequences described in SEQ ID NO.45 or SEQ ID NO.

52.

5. The gene encoding the anti-human ROR1 antibody as described in any one of claims 1 to 4.

6. A recombinant expression vector or recombinant bacterial strain, characterized in that, It expresses the anti-human ROR1 antibody as described in any one of claims 1 to 4.

7. The use of the anti-human ROR1 antibody according to any one of claims 1 to 4 in the preparation of ROR1-targeting detection reagents.

8. A ROR1 / CD3 bispecific antibody, characterized in that, The heavy chain CD3-HC and ROR1-scFv of the CD3 antibody are linked to the C-terminus 228C of the constant region (CH1) domain of CD3-HC via a flexible linker peptide. The ROR1-scFv is constructed from the antibody according to any one of claims 1 to 4. The light chain CD3-LC of the CD3 antibody is not modified in any way. The clone number of the CD3 antibody is SP34.

9. A reagent, characterized in that, It is formed by directly or indirectly conjugating a tracer molecule with the anti-human ROR1 antibody as described in any one of claims 1 to 4.

Images