Anti-ROR1 nano antibody

Abstract

The invention provides an anti-ROR1 nano antibody and an application thereof. Through alpaca immunization and a phage library, a high-affinity nano antibody HCAbs1 is screened out, and through flow cytometry and a pull-down experiment, it is determined that the HCAbs1 can be effectively combined with ROR1. The HBABs1 can reduce the ERK phosphorylation level stimulated by Wnt5a in ROR1 stably transfected cells, and has huge potential in the aspects of cancer diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to the field of antibody engineering pharmaceuticals, specifically to the anti-ROR1 nanobody HCAbs1, its preparation method, and its applications. Background Technology

[0002] According to global statistics in 2020, an estimated 19.3 million new cancer cases and nearly 10 million cancer deaths were reported worldwide, with breast cancer, lung cancer, and colorectal cancer ranking as the top three [1]. Among them, breast cancer has surpassed lung cancer to become the most common cancer; triple-negative breast cancer accounts for 10%-20%, and it has special biological behavior and clinicopathological characteristics, with a poorer prognosis than other cancer types. Lung cancer is also one of the main causes of cancer death. In the past few decades, traditional cancer treatments have included surgery, chemotherapy, and radiotherapy. Traditional surgical treatments may lead to incomplete tumor resection, metastasis, recurrence, etc., while chemotherapy and radiotherapy also have significant limitations and toxic side effects. With the gradual advancement of tumor research, new treatment methods are rapidly developing. Immunotherapy achieves specific killing effects by targeting tumor cells, and has the advantages of high specificity and low side effects. Currently, there are five main types of immunotherapy: immune checkpoint inhibitors (PD-1 / L1 and CTLA-4 inhibitors), adoptive immunotherapy (CAR-T, TCR-T, TIL, CAR-NK, CAR-M, etc.), immune cell conjugates (TCE, NKCE, etc.), cytokine therapy, and cancer vaccines. The continuous development of immunotherapy has also led to the discovery of an increasing number of new targets.

[0003] Receptor tyrosine kinases (RTKs) are a large family of proteins that play crucial roles in cell migration, survival, proliferation, and differentiation, and are also involved in the development and progression of various cancer types. To date, the RTK superfamily comprises 58 members, divided into 20 subfamilies. Among them, the receptor tyrosine kinase-like orphan receptor (ROR) family is a type I transmembrane receptor family, including ROR1 and ROR2. Because the ligands were unknown when the ROR receptor family was first discovered, it was initially defined as an orphan receptor. Many studies have shown that ROR1 plays a vital role in various physiological processes by recognizing Wnt proteins, particularly the non-canonical Wnt signaling pathway mediated by Wnt5a, including regulating cell division, proliferation, migration, and chemotaxis. Wnt5a is a typical activator of the non-canonical Wnt signaling pathway. Wnt5a participates in the phosphorylation of the NF-κB subunit p65, activating the NF-κB pathway in tumor cells and promoting cell migration and invasion, epithelial-mesenchymal transition (EMT), and cancer metastasis. In the presence of Wnt5a, ROR1 participates in the activation of the NF-κB pathway in tumor cells. ROR1 is expressed at low levels or not at all in normal human tissues, but is highly expressed in a variety of malignant tumors or tissues, such as chronic lymphocytic leukemia (CLL), breast cancer, ovarian cancer, melanoma, and lung adenocarcinoma [1]. Tumor-selective expression makes ROR1 a potential therapeutic target.

[0004] The discovery of camel nanobodies ushered in a new era in antibody research. Caplacizumab was the first nanobody drug approved for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) in adults. Nanobodies consist only of the heavy chain, composed of CH2, CH3, and VHH, lacking the CH1 of traditional antibodies. Nanobodies have a diameter of 2.5 nm, a height of 4 nm, and a molecular weight of only 15 kDa. Nanobodies can penetrate dense tissues, such as the blood-brain barrier. Although VHH contains only three CDRs of the heavy chain, it has similar affinity to mAbs. Its CDR3 region has approximately 18 amino acids, 4-6 more than the CDR3 region of mAbs, allowing nanobodies to penetrate deeply into antigens and bind to epitopes that are difficult for traditional mAbs to bind, such as G protein-coupled receptors (GPCRs). Therefore, nanobodies have advantages such as small molecular weight, high specificity, high stability, and good solubility, and are widely used in the medical field, including cancer treatment, inflammation treatment, and immunotherapy. In addition, nanobodies play an important role in the development of biotechnology, imaging technology, and diagnostic tools.

[0005] Currently, no anti-ROR1 nanobodies have been approved for the treatment of herpes simplex virus (HSV) infection. Herpes simplex virus infection is widespread and prolonged, and can easily lead to fatal infections in certain populations. This severe disease burden underscores the urgent need for enhanced clinical intervention for HSV infection. Therefore, developing a nanobody targeting HSV and screening for highly druggable therapeutic HSV nanobodies could contribute to the treatment of herpes simplex infection, reduce the socioeconomic burden, and has broad market application prospects. Summary of the Invention

[0006] To address the problems in the prior art, one of the objectives of this invention is to prepare anti-ROR1 nanobodies.

[0007] This invention involves immunizing alpacas with the prepared ROR1 antigen to obtain alpaca antibodies, which are then used to construct a phage library. After phage library enrichment and screening, six VHH sequences were obtained. ROR1-stable cells were then constructed, and the expression and identification of anti-ROR1 antibodies were performed. HCAbs1 effectively binds to the surface of ROR1-stable cells, and HCAbs1 exhibits a high binding affinity for ROR1.

[0008] The present invention provides a nanobody HCAbs1, characterized in that the sequences of the antigen complementarity-determining regions CDR1, CDR2 and CDR3 of the nanobody HCAbs1 are shown as SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0009] The present invention provides a nanobody HCAbs1, characterized in that the amino acid sequence of the nanobody HCAbs1 is shown in SEQ ID NO:3.

[0010] The present invention also provides a polynucleotide encoding the aforementioned nanobody HCAbs1.

[0011] The polynucleotide has the nucleotide sequence shown in SEQ ID NO:4.

[0012] The present invention provides an expression vector comprising the aforementioned polynucleotides.

[0013] The present invention provides a host cell comprising the expression vector described above.

[0014] The present invention also provides a conjugate comprising the aforementioned antibody HCAbs1; preferably, the conjugate further comprises a marker, a drug, a cytokine, an enzyme, gold nanoparticles / nanorobars, magnetic nanoparticles, liposomes, or a viral capsid protein.

[0015] Another object of the present invention is to provide the application of the anti-ROR1 nanobody HCAbs1.

[0016] The present invention also provides the use of the nanobody HCAbs1 or conjugates containing the above-described nanobody HCAbs1 in the preparation of vaccines or drugs for treating and / or preventing ROR1-positive diseases, wherein the ROR1-positive diseases are cancer and diseases caused by herpesviruses; preferably, the cancer is chronic lymphocytic leukemia (CLL), breast cancer, ovarian cancer, melanoma, or lung adenocarcinoma.

[0017] The present invention provides the use of the above-described nanobody HCAbs1 or a conjugate containing the above-described nanobody HCAbs1 in the preparation of compositions for detecting ROR1 in biological samples.

[0018] The present invention provides the application of the above-mentioned nanobody HCAbs1 or conjugates containing the above-mentioned nanobody HCAbs1 in the preparation of vaccines or drugs for diagnosing ROR1-positive diseases, wherein the ROR1-positive diseases are cancer and diseases caused by herpesviruses; preferably, the cancer is chronic lymphocytic leukemia (CLL), breast cancer, ovarian cancer, melanoma, or lung adenocarcinoma.

[0019] The beneficial effects of this invention are as follows:

[0020] The anti-ROR1 nanobody lacks the CH1 constant region in its VHH region, resulting in a simple structure and small molecular weight, which is beneficial for expression and use. The highly variable region CDR1 increases the extension of CDR3, enabling the nanobody to penetrate deeply into the antigen and bind to the latent epitope, thus exhibiting stronger specificity. The nanobody provided by this invention can be used to prepare vaccines or drugs for the detection, diagnosis, and treatment of diseases related to ROR1 overexpression, and has high application value. Attached Figure Description

[0021] Figure 1 A shows the peak pattern of ROR1 purified by SEC after nickel column purification; B shows the SDS-PAGE electrophoresis image of purified ROR1; C shows the schematic diagram of alpaca immunization and sampling protocol; D shows the workflow for screening ROR1 nanobody phage display library.

[0022] Figure 2 A shows the sequencing results of positive monoclonal antibodies selected from the phage library; B shows protein band imaging using enhanced chemiluminescence (ECL); C shows the flow cytometry verification of antibodies HCAbs1 and HCAbs4 with ROR1-transfected cells; D shows the SPR affinity assay of HCAbs1 and ROR1.

[0023] Figure 3After adding HCAbs1 to ROR1 stable cell lines, the cells were treated with Wnt5a stimulation or no stimulation, and the intracellular ERK phosphorylation level was detected by Western Blot.

[0024] Figure 4 A shows the nickel column purification of the extracellular region of ROR1, where band M is the molecular weight marker, band 1 is the buffer elution, band 2 is the elution with 30 mM imidazole, band 3 is the elution with 300 mM imidazole, and band 4 is the elution with 500 mM imidazole; B shows the alpaca immunotiter analysis; C shows the pull-down binding verification of HCAbs1 and ROR1, where M is the molecular weight marker, band 1 is ROR1, band 2 is HCAbs1, and band 3 is the eluted sample after ROR1 and HCAbs1 binding; D shows the ELISA affinity determination of HCAbs1 and ROR1. Detailed Implementation

[0025] Example 1: Preparation of anti-ROR1 antibody

[0026] (1) Antigen preparation

[0027] The full-length human ROR1 contains 937 amino acids, with an extracellular region of 1-403 amino acids, as shown in SEQ ID NO: 1. The extracellular ROR1(ECD) region was constructed into the PTT5 vector (purchased from Beijing Solarbio Science & Technology Co., Ltd.) to obtain the pTT5-hROR1(ECD)-HRV3C-His plasmid. The C-terminus of the ROR1(ECD) contains an 8-His tag for affinity purification, and an HRV 3C cleavage site for tag removal is located between the ROR1(ECD) and the 8-His tag. Expi-293F cells (purchased from Beijing Solarbio Science & Technology Co., Ltd.) were transfected with the pTT5-hROR1(ECD)-HRV3C-His plasmid. After culturing at 5% CO2, 150 rpm, and 37°C for 5 days, the supernatant was collected by centrifugation at 5000 rpm for 10 min, purified using a nickel column, and then purified by size exclusion chromatography (SEC) using a Superdex 200 column. Figure 1 A) The purified ROR1 was identified by SDS-PAGE to obtain high-purity antigen ROR1 protein. Figure 1 B), hereinafter referred to as ROR1.

[0028] (2) Alpaca Immunization

[0029] Purified ROR1 was used to immunize alpacas. For the first immunization, 300 μg of ROR1 was thoroughly mixed with an equal volume of Freund's complete adjuvant BioFroxx 2203ML010, emulsified, and injected into the alpaca's cervical lymph nodes. Immunizations were repeated every 3 weeks for a total of 6 times. For the 2nd to 6th immunizations, 300 μg of ROR1 was thoroughly mixed with an equal volume of Freund's incomplete adjuvant BioFroxx 1643ML010, emulsified, and injected. Figure 1 C). After six immunizations, blood was drawn from the jugular vein of the alpaca, and peripheral blood lymphocytes were separated using Ficoll cell separation medium (Solepro). The alpaca serum was then subjected to ELISA to determine its titer. The serum was first diluted 25-fold as the initial concentration, and then tripled 11 times, resulting in a titer of 164025 (…). Figure 4 B).

[0030] (3) Construction of phage VHH display library

[0031] RNA was extracted from alpaca blood PBMCs after immunization. Using mRNA as a template, cDNA was reverse transcribed from mRNA using CALL001 and CALL002 primers, respectively. The CALL001 sequence is shown in SEQ ID NO:5, and the CALL002 sequence is shown in SEQ ID NO:6. The cDNA was used as a template for PCR amplification to amplify various alpaca antibody sequences, and the corresponding bands were recovered by gel electrophoresis. Using the product recovered from the first round of PCR as a template, a second round of PCR was performed using VHH1-F and VHH1-R primers to amplify the VHH variable region of the alpaca antibody heavy chain, and the corresponding fragments were recovered by gel electrophoresis. The VHH1-F sequence is shown in SEQ ID NO:10, and the VHH1-R sequence is shown in SEQ ID NO:11. The recovered VHH sequences and Pcomb3XSS plasmid (purchased from Beijing Solarbio Science & Technology Co., Ltd.) were digested with SfiI restriction enzyme (purchased from New England Biotech, Inc., USA) and ligated with T4 ligase (purchased from New England Biotech, Inc., USA). The concentration of the ligation product was 40 ng / μl.

[0032] Add 2 μL of ligation product (40 ng / μL) to 100 μL of competent cells, gently stir several times, and then transfer to an electroporation cuvette (Bio-Rad, wipe off the outside with paper). The electroporation program is Ec2 (2.5 kV, 5 ms). Immediately after completion, remove the cells, add 900 μL of SB medium (32 g tryptone, 20 g yeast extract, 5 g sodium chloride dissolved in 1000 mL distilled water for sterilization), gently pipet twice, aspirate dry, and transfer to EP tubes. Dilute the samples 10⁻⁶ times. -4 10 -5 and 10 -6A 100 μL sample (containing tetracycline resistance and ampicillin resistance) was plated to calculate the library size. After a total of 3 electroporations, the final library size was 8 × 10⁻⁶. 8 .

[0033] (4) Phage library enrichment and screening

[0034] The process and diagram for phage library construction, enrichment, and screening are shown below. Figure 1 As shown in D. After library construction, phage libraries were enriched using ROR1-coated microplates (the plates were purchased from Thermo Fisher Scientific). Antigen was added at a concentration of 40 μg / ml (100 μl / well) and incubated overnight at 4°C. 300 μl / well of 3% skim milk powder + 0.5% BSA was added to each well of a 96-well plate and blocked at 37°C for 1 hour. The amplified phage library resuspended in PBS was added at 100 μl / well of the 96-well plate and incubated at 37°C for 1 hour. 100 μL of Glycine-HCl was added, and the mixture was mixed by pipetting or allowed to stand for at least 10 minutes to elute the antigen-bound phages. The phages were neutralized to pH 7 by adding 1M Tris-HCl (pH = 8.8) to the glycine-HCl wash buffer, completing one round of enrichment. The antigen concentration was gradually decreased, and screening was performed after four rounds of enrichment. The enrichment concentrations for each round were 40 μg / ml, 20 μg / ml, 2 μg / ml and 0.5 μg / ml, for a total of 4 rounds.

[0035] After enrichment, single clones were picked from the plates and screened by phage ELISA. The fourth round of enriched phages were used to infect logarithmically growing ER2738 competent cells (purchased from CellRaytech (Beijing) Life Science Technology Co., Ltd., K-12 genotype), diluted 10⁻⁶ times. 7 The infected ER2738 cells were spread twice on a plate, and single colonies were picked and transferred to 96-well plates. The plates were incubated at 37°C and 250 rpm for 4-5 hours with shaking. Diluted helper phages were added, and the plates were incubated at room temperature for 20 minutes. Then, 100 μL of 2YT medium (containing 16 g tryptone, 10 g yeast extract, and 5 g sodium chloride dissolved in 1000 mL distilled water, sterilized) containing 100 mg / mL ampicillin and 50 mg / mL kanamycin was added, and the plates were incubated overnight. After 16 hours, the culture was centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant was collected. 100 μL of the supernatant was added to a 96-well microplate and incubated at 37°C for 1 hour. The plates were washed three times with PBST. Add 100 μL (0.1 μg / mL) of HRP-labeled anti-M13 phage secondary antibody (Thermo Fisher Scientific), incubate at 37°C for 40 min, and wash three times with PBST. Develop color with 100 μL / well TMB chromogenic buffer for 15 min. Stop the reaction with 60 μL of dilute sulfuric acid, and read the OD values ​​using a microplate reader. 450After screening, clones with an OD value greater than 3 were considered positive. Eight clones (A4, D7, E5, F10, F11, G2, H1, and H7) were selected as positive clones. These positive wells were then further cultured and sequenced, yielding six VHH sequences. Figure 2 A).

[0036] (5) Construction of ROR1 stable cells

[0037] Human embryonic kidney 293T cells (purchased from Beijing Solarbio Science & Technology Co., Ltd.) were seeded at 60% density into six-well plates and transfected the next day in DMEM complete medium (containing 10% FBS and 1% PS). Two sterile 1.5 mL EP tubes were prepared. In one tube, 6 μg of LVX-Kozak-hROR1(1-937) plasmid was added to 250 μl of Opti-MEM (Gibco), and the mixture was thoroughly mixed with a pipette and incubated at room temperature for 10 min. In the other tube, 18 μg of PEI was added to 250 μl of Opti-MEM, and the mixture was thoroughly mixed with a pipette and incubated at room temperature for 10 min. The solutions from both EP tubes were combined and thoroughly mixed, and incubated at room temperature for 10 min. The solution was then added dropwise to the cells in the six-well plate, avoiding adding it to the same spot to prevent uneven titer distribution. The medium (DMEM complete medium) was changed every 4-6 hours. 24 hours later, cells were transferred to 10cm culture dishes. The old culture medium was discarded, and 300μl of trypsin was added for 20s digestion. The trypsin was discarded, and the cells were quickly resuspended in DMEM complete medium containing 2μg / mL puromycin (AB11L241). The cells were pipetted 4-5 times to achieve a single, free state, and then transferred to 10cm dishes for further culture. Untransfected cells died within 1-2 days, while successfully transfected cells grew normally in puromycin at 2μg / mL.

[0038] (6) Preparation of ROR1 stable cell monoclonal cells

[0039] Discard the culture medium from the stable cells constructed in the 6-well plate, add 300 μl of trypsin for 20 seconds, pipette off the trypsin, and immediately resuspend the cells in 500 μl of DMEM complete medium containing 2 μg / mL puromycin. Swish the cells 4-5 times to achieve a single, free state. Mix 10 μl of cells with 10 μl of trypan blue staining solution, and add 10 μl to a cell counting plate for counting. Take 100 cells at the desired density, dilute with 20 mL of DMEM complete medium containing 2 μg / mL puromycin, and seed 200 μl into each well of a 96-well plate. One week later, observe the number of single colonies in each well under a microscope, and label wells with a single colony count of 1. Two weeks later, transfer the cells from wells with a single colony count of 1 to separate them for further culture to obtain stable monoclonal cells.

[0040] Example 2: Expression and Identification of Anti-ROR1 Antibody

[0041] (1) Expression of anti-ROR1 antibody

[0042] The six unique VHH sequences obtained in Example 1 were constructed into the PTT5 vector for expression and named HCAbs1, HCAbs2, HCAbs3, HCAbs4, HCAbs5, and HCAbs6, respectively. Specifically, a human Fc sequence was added to the C-terminus of the VHH sequence for subsequent purification and identification, constructing the plasmid PTT5-VHH-HRV3C-Fc, which was transformed into TOP10 competent cells (Beijing Solarbio Science & Technology Co., Ltd.). The plasmid was extracted through mass culture at a concentration of 3 × 10⁻⁶ cells. 6 HEK293F cells (Wuhan Pronosei Life Sciences Co., Ltd.) were transfected at a density of / ml, with DNA:PEI = 1:1. Expression was performed at 37°C for 4 days in 5% CO2 at 150 rpm. The supernatant was collected, filtered through a 0.22 μm filter membrane, passed through a Protein A column, and eluted with pH 4 citrate buffer. The purification results showed that HCAbs1, HCAbs3, HCAbs4, and HCAbs6 were successfully expressed. Figure 2 B), while the expression levels of HCAbs2 and HCAbs5 were low.

[0043] (2) FACS validation of expression

[0044] The full-length ROR1 stably transfected cell line constructed in Example 1(5) was used. Flow cytometry was used to verify that HCAbs1 could effectively bind to the surface of ROR1-stably transfected cells. Specifically, an appropriate amount of the constructed stably transfected ROR1-overexpressing cells was taken and washed three times with PBS. 100 nM HCAbs1 / HCAbs3 / HCAbs4 / HCAbs6 (primary antibody) was added, and the cells were incubated on ice for 1 hour, followed by washing three times with PBS. Anti-human IgG FC-PE secondary antibody (purchased from Thermo Fisher Scientific) was added, and the cells were incubated on ice for 1 hour. Stably transfected cells that had not been incubated with primary and secondary antibodies were used as a control group, and the binding of the antibody to ROR1 on the cell surface was detected by flow cytometry. The results showed that HCAbs4 exhibited low binding ( Figure 2 C) In FACS, neither HCAbs3 nor HCAbs6 was detected to be bound to cells.

[0045] Example 3: Binding affinity analysis of HCAbs1

[0046] (1) ELISA analysis

[0047] The antigen obtained in Example 1(1) was coated onto an ELISA plate at a concentration of 1 μg / ml, 100 μl per well, and incubated overnight at 4°C. After washing the plate with PBST buffer, 300 μl of 4% BSA was added to each well, and the plate was incubated at 37°C for 1 hour. 100 μg / ml of HCAbs1 / HCAbs3 / HCAbs4 / HCAbs6 (primary antibody) was added to each well, starting with a 4-fold to 11-fold gradient, and incubated at 37°C for 2 hours. The plate was washed 4 times with PBST. HRP-labeled anti-human Fc secondary antibody (purchased from Thermo Fisher Scientific) was diluted 1:5000 with PBST, 100 μl per well, and incubated at 37°C for 1 hour. After washing the plate 4 times with PBST, 100 μl of TMB chromogenic solution was added to each well, and the plate was incubated at room temperature in the dark for 10 minutes. The reaction was terminated with 60 μl of dilute sulfuric acid, and the OD was read using an ELISA reader. 450 Value. When OD 450 When the value is half of the maximum value, the corresponding concentration represents the binding affinity between the antigen and the antibody. The binding affinity of ROR1 and HCAbs1 measured by ELISA was 4.42 nM. Figure 4 D).

[0048] (2) Pull-down analysis

[0049] Add 1 ml of PBS to 20 μl of nickel column resin, mix well, centrifuge, and discard the supernatant. Repeat the washing three times. Add 20 μg of ROR1, incubate for 30 min, wash three times with 1 ml of PBS, centrifuge, and discard the supernatant. Add an equimolar amount of antibody HCAbs1, incubate for 1 h, wash three times with 1 ml of PBS, and centrifuge. Discard the supernatant, add reducing 5× loading buffer, boil for 10 minutes, and run the gel. The pull-down experiment also qualitatively confirmed that HCAbs1 can bind to the extracellular region of ROR1 and be pulled down (…). Figure 4 C).

[0050] (3) SPR analysis

[0051] Surface plasmon resonance (SPR) was also used to analyze the binding affinity of HCAbs1 to ROR1. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were mixed in a 1:1 ratio to activate the CM5 chip. The ROR1 antigen protein was diluted to 10 μg / ml with sodium acetate solution at pH 4.5 and immobilized on the chip. HCAbs1 antibody was injected at five concentrations ranging from 50 nM to 400 nM. Binding isotherms were fitted based on instrumental analysis data, and SPR determined the binding affinity of ROR1 to HCAbs1 to be 12.9 nM. Figure 2 D).

[0052] In summary, both ELISA and pull-down analyses indicate that HCAbs1 has a high binding affinity for ROR1.

[0053] Example 4: HCAbs1 inhibits Wnt5a-stimulated ERK phosphorylation levels

[0054] The full-length ROR1 stably transfected cell line constructed in Example 1(5) was used. The full-length ROR1 is highly expressed on the cell membrane and has a molecular weight of 105 kDa. To verify whether HCAbs1 plays an important role in intracellular signal transduction, functional assessment was performed using this stably transfected cell line.

[0055] ROR1 stable cell lines were incubated with HCAbs1 for 2 hours, followed by stimulation with 200 ng / μl Wnt5a or no stimulation. The treated cells were centrifuged at 2000 rpm for 5 minutes at 4°C and washed twice with PBS. The supernatant was discarded. Cell lysis buffer containing phosphatase / protease inhibitors was added, and cells were lysed on ice for 30 minutes. The lysates were centrifuged at 12500 rpm for 15 minutes at 4°C. 5× reduction loading buffer was added to the supernatant sample, and the mixture was boiled at 95°C for 5 minutes for SDS-PAGE, followed by transfer to a PVDF membrane. The membrane was blocked with TBST containing 3% skim milk powder for 1 hour. Anti-ERK phosphorylation primary antibody (1:2000 dilution, Thermo Fisher Scientific) was added, and the membrane was incubated on a shaker at room temperature for 2 hours. Anti-mouse IgG FC secondary antibody (1:5000 dilution, Thermo Fisher Scientific) was added, and the membrane was incubated on a shaker at room temperature for 1 hour. Protein band imaging using enhanced chemiluminescence (ECL).

[0056] Adding ROR1 ligand Wnt5a to stimulate cells increases ERK phosphorylation levels, while HCAbs1 decreases ERK phosphorylation levels. Figure 3 This indicates that HCAbs1 can inhibit Wnt5a-stimulated ERK phosphorylation. Many processes involved in ERK pathway activation are also related to ROR1 function; ERK overactivation is present in many human diseases, especially cancer and herpesvirus-related diseases. Since the nanobody HCAbs1 can inhibit Wnt5a-stimulated ERK phosphorylation, it shows promising potential for the detection, diagnosis, and treatment of diseases associated with high ROR1 expression.

[0057] References

[0058] 1. Lee, B.K.; Wan, Y.; Chin, Z.L.; Deng, L.; Deng, M.; Leung, T.M.; Hua, J.; Zhang, H., Developing ROR1 Targeting CAR-T Cells against Solid Tumors in Preclinical Studies. Cancers (Basel) 2022, 14(15).

[0059] Sequence Listing

[0060] SEQ ID No.1 Amino acid sequence of ROR1

[0061] MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNIS

[0062] SELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPV

[0063] VQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLF

[0064] VKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQ

[0065] GEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRD

[0066] LCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANC

[0067] IRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHT

[0068] HTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDS

[0069] KDSKEKNKMESEQ ID No.2 Nucleotide sequence of ROR1

[0070] ATGCACAGACCCAGAAGAAGAGGCACAAGACCCCCTCTGCTGGCCC

[0071] TGCTGGCCGCTCTGCTGCTGGCTGCTAGAGGCGCCGCCGCTCAGGA

[0072] GACAGAGCTGTCCGTGTCCGCCGAGCTGGTGCCCACCAGCTCCTGG

[0073] AATATCAGCTCCGAGCTGAACAAGGATTCCTACCTGACCCTGGACGA

[0074] GCCCATGAACAACATCACAACATCCCTGGGCCAGACAGCCGAGCTG

[0075] CACTGTAAGGTGTCCGGCAATCCCCCTCCTACCATCAGATGGTTTAA

[0076] GAATGACGCCCCTGTGGTGCAGGAGCCTAGGAGACTGAGCTTTAGG

[0077] AGCACAATCTACGGCTCCAGGCTGAGAATCAGAAATCTGGACACAA

[0078] CCGACACCGGCTACTTCCAGTGTGTGGCCACAAATGGCAAGGAGGT

[0079] GGTGAGCAGCACCGGCGTGCTGTTCGTGAAGTTCGGCCCTCCCCCC

[0080] ACCGCCAGCCCTGGATATAGCGATGAGTACGAGGAGGATGGCTTCTG

[0081] TCAGCCCTACAGAGGCATCGCCTGCGCCAGGTTTATCGGCAACAGAA

[0082] CAGTGTACATGGAGTCCCTGCACATGCAGGGCGAGATCGAGAACCA

[0083] GATCACCGCCGCCTTCACAATGATCGGCACAAGCTCCCACCTGAGCG

[0084] ACAAGTGCAGCCAGTTTGCCATCCCCAGCCTGTGCCACTACGCCTTT

[0085] CCTTACTGTGACGAGACAAGCTCCGTGCCTAAGCCCAGAGATCTGTG

[0086] TAGAGACGAGTGCGAGATCCTGGAGAACGTGCTGTGTCAGACCGAG

[0087] TACATCTTCGCCAGAAGCAACCCCATGATCCTGATGAGGCTGAAGCT

[0088] GCCCAATTGTGAGGATCTGCCTCAGCCTGAGAGCCCCGAGGCCGCT

[0089] AACTGCATCAGAATCGGCATCCCCATGGCCGACCCCATCAATAAGAA

[0090] CCACAAGTGCTACAACTCCACCGGCGTGGACTACAGAGGCACCGTG

[0091] AGCGTGACCAAGTCCGGCAGACAGTGTCAGCCCTGGAATAGCCAGT

[0092] ACCCTCACACCCACACATTCACAGCCCTGAGGTTTCCTGAGCTGAAT

[0093] GGCGGCCACAGCTACTGTAGGAATCCTGGCAATCAGAAGGAGGCCC

[0094] CCTGGTGTTTTACCCTGGATGAGAACTTTAAGAGCGACCTGTGTGAC

[0095] ATCCCCGCCTGTGATAGCAAGGACTCCAAGGAGAAGAACAAGATGG

[0096] AG

[0097] Amino acid sequence of nanobody HCAbs1, SEQ ID No.3 (注:原文中“纳米抗体HCAbs1氨基酸序列”直接翻译为“Amino acid sequence of nanobody HCAbs1”,这里添加了“SEQ ID No.3”使句子更完整符合语境)

[0098] DVQLQESGGGLVQPGGSLRLDCAASASTVNITIMGWFRQAPGNEREFL

[0099] AAVRMMGGATWYADSVRGRFTISRDSATNMVSLQMNSLTPEDTANYY

[0100] CAAIRGKVSFSKSPRHWNTWGQGTQVTVSS

[0101] SEQ ID No.4 Nanobody HCAbs1 Nucleotide Sequence

[0102] GACGTGCAGCTGCAGGAGAGCGGAGGTGGGCTGGTGCAGCCAGGG

[0103] GGCTCCCTTCGATTGGATTGTGCTGCTTCCGCTTCTACAGTCAACATC

[0104] ACTATCATGGGCTGGTTCCGCCAGGCCCCCGGCAACGAGAGGGAGT

[0105] TCCTGGCGGCGGTACGCATGATGGGAGGCGCCACCTGGTACGCGGA

[0106] CTCCGTGCGCGGTCGCTTCACCATCTCTCGGGACAGTGCAACCAACA

[0107] TGGTGAGCTTGCAGATGAATTCCCTCACTCCTGAAGACACCGCCAAC

[0108] TATTACTGCGCCGCCATCCGTGGCAAGGTGTCCTTTTCAAAGTCCCC

[0109] GCGCCACTGGAACACGTGGGGCCAGGGCACCCAGGTCACCGTGTCG

[0110] TCG

[0111] SEQ ID NO:5 Primer CALL001 Nucleotide Sequence

[0112] GTCCTGGCTGCTCTTCTACAAGG

[0113] SEQ ID NO:6 Primer CALL002 Nucleotide Sequence

[0114] GGTACGTGCTGTTGAACTGTTCC

[0115] The antigen complementarity-determining region (CDR1) amino acid sequence of SEQ ID No. 7 nanobody HCAbs1 ASTVNITI

[0116] The antigen complementarity-determining region (CDR2) amino acid sequence of SEQ ID No. 8 nanobody HCAbs1 is VRMMGGAT.

[0117] The antigen complementarity-determining region (CDR3) amino acid sequence of SEQ ID No. 9 nanobody HCAbs1: AAIRGKVSFSKSPRHWNT

[0118] SEQ ID NO:10VHH1-F nucleotide sequence

[0119] ATGGCTSAKGTGCAGCTGGTGGAGTCTGG

[0120] SEQ ID NO:11VHH1-R nucleotide sequence

[0121] GGAGACGGTGACCTGGGT

[0122] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Nanobody HCAbs1, characterized in that, The sequences of the antigen complementarity-determining regions CDR1, CDR2, and CDR3 of the nanobody HCAbs1 are shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.

2. Nanobody HCAbs1, characterized in that, The amino acid sequence of the nanobody HCAbs1 is shown in SEQ ID NO:

3.

3. A polynucleotide encoding the nanobody HCAbs1 as described in any one of claims 1 or 2.

4. The polynucleotide of claim 3, wherein the nucleotide sequence is shown in SEQ ID NO:

4.

5. An expression vector comprising the polynucleotide of claim 3 or 4.

6. A host cell comprising the expression vector of claim 5.

7. A conjugate comprising the antibody HCAbs1 as described in claim 1 or 2; preferably, the conjugate further comprises a marker, a drug, a cytokine, an enzyme, gold nanoparticles / nanorobars, magnetic nanoparticles, liposomes, or a viral capsid protein.

8. The use of the nanobody HCAbs1 of claim 1 or 2 or the conjugate of claim 7 in the preparation of vaccines or pharmaceuticals for the treatment and / or prevention of ROR1-positive diseases, wherein the ROR1-positive diseases are cancer and diseases caused by herpesviruses. Preferably, the cancer is chronic lymphocytic leukemia (CLL), breast cancer, ovarian cancer, melanoma, or lung adenocarcinoma.

9. The use of the nanobody HCAbs1 of claim 1 or 2 or the conjugate of claim 7 in the preparation of compositions for the detection of ROR1 in biological samples.

10. The use of the nanobody HCAbs1 of claim 1 or 2 or the conjugate of claim 7 in the preparation of a vaccine or medicament for diagnosing ROR1-positive diseases, wherein the ROR1-positive diseases are cancer and diseases caused by herpesviruses. Preferably, the cancer is chronic lymphocytic leukemia (CLL), breast cancer, ovarian cancer, melanoma, or lung adenocarcinoma.

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