Anti-ror1 antibodies and uses thereof

Abstract

The present application relates to an anti-ROR1 antibody and its application, and belongs to the field of biological medicine. Specifically, the application discloses an antibody or an antigen-binding fragment thereof specifically binding to ROR1, a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof, a vector expressing the nucleic acid molecule, a method for preparing and purifying the antibody, a recombinant cell, a composition, and a use thereof, in particular, in the preparation of a drug for treating and / or preventing a tumor. Meanwhile, the provided antibody has high specificity, high affinity, high binding capacity, and can mediate intracellular endocytosis.

Description

Technical Field

[0001] This disclosure pertains to the field of biomedicine. Specifically, it relates to anti-ROR1 antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding said antibodies or their antigen-binding portions, vectors expressing nucleic acid molecules, methods for preparing and purifying antibodies or their antigen-binding fragments, recombinant cells, compositions, and their use in the preparation of medicaments for the treatment and / or prevention of tumors. Background Technology

[0002] ROR1 belongs to the receptor tyrosine kinase-like orphan receptor (ROR) family, which also includes ROR2. ROR1 is a type I transmembrane glycoprotein composed of 937 amino acids. Its extracellular region includes an immunoglobulin-like domain (Ig) at the distal end, a Kringle domain (KRD) at the proximal end, and a frizzled domain (FZD) located in the middle of the extracellular region. Human and mouse ROR1 share 96.8% homology, and the extracellular region sequence of human and monkey ROR1 is completely identical. ROR1 is highly expressed in early embryonic development but almost not expressed in mature tissues. Conversely, ROR1 is abnormally highly expressed in various solid tumors and hematologic malignancies and is negatively correlated with prognosis. Mechanistically, Wnt5a can bind to the FZD of ROR1, transmitting extracellular signals into the cell and activating the Wnt-NF-κB pathway, thereby regulating cell polarization, proliferation, motility, and migration. Furthermore, ROR1 crosses with signaling pathways such as EGFR and Met and jointly participates in the occurrence and development of tumors, therefore, targeting ROR1 to treat tumors has great potential.

[0003] Given the important role of ROR1 antibodies in the disease, more therapeutic agents targeting ROR1 need to be developed to further clarify the therapeutic potential of this target. Summary of the Invention

[0004] This invention relates to anti-ROR1 antibodies, their antigen-binding fragments, and their applications.

[0005] The present invention provides an anti-ROR1 antibody or its antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0006] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7.

[0007] In some embodiments, the heavy chain variable region comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:7.

[0008] In some embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8.

[0009] In some embodiments, the light chain variable region comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:8.

[0010] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0011] In some embodiments, the antibody or its antigen-binding fragment is a whole antibody, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a single-chain Fv fragment (scFv).

[0012] In some embodiments, the antibody or its antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a single-chain Fv fragment (scFv).

[0013] In some implementations, the antibody is a murine antibody, a chimeric antibody, a human antibody, or a humanized antibody.

[0014] In some implementations, the antibody is a murine antibody.

[0015] In some implementations, the antibody is a chimeric antibody.

[0016] In some implementations, the antibody is a human antibody.

[0017] In some implementations, the antibody is a humanized antibody.

[0018] In some embodiments, the antibody or its antigen-binding fragment contains a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4.

[0019] In some embodiments, the antibody or its antigen-binding fragment comprises human IgG1.

[0020] In some embodiments, the antibody or its antigen-binding fragment comprises the IgG1 heavy chain constant region shown in SEQ ID NO:9.

[0021] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region derived from the human κ chain and λ chain.

[0022] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region derived from the human λ chain.

[0023] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region derived from the human κ chain.

[0024] In some embodiments, the antibody or its antigen-binding fragment comprises the light chain constant region of the human κ chain shown in SEQ ID NO:10.

[0025] In some embodiments, the antibody or its antigen-binding fragment has a heavy chain as shown in SEQ ID NO:11 and a light chain as shown in SEQ ID NO:12.

[0026] In some embodiments, the antibody or its antigen-binding fragment has high affinity and binding capacity.

[0027] In some embodiments, the antibody or its antigen-binding fragment mediates endocytosis.

[0028] In another aspect, the present invention provides biological materials related to the aforementioned anti-ROR1 antibody or its antigen-binding fragment, said biological materials being 1), 2), or 3) as follows.

[0029] 1) A nucleic acid molecule encoding the antibody or its antigen-binding fragment as described in this invention;

[0030] 2) A carrier containing the nucleic acid molecule described in 1);

[0031] 3) A host cell containing the nucleic acid molecule described in 1) or the vector described in 2).

[0032] In another aspect, the present invention provides a nucleic acid molecule encoding an anti-ROR1 antibody or an antigen-binding fragment thereof as described in any one of the present invention.

[0033] Another aspect of the present invention provides a carrier comprising the nucleic acid molecule described herein.

[0034] In some embodiments, the vector includes bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0035] Another aspect of the present invention provides a host cell comprising the nucleic acid molecules described in the present invention, or the vector described in the present invention.

[0036] Another aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof described in the present invention, the method comprising culturing the cells described in the present invention under conditions that cause the expression of the antibody or antigen-binding fragment thereof that specifically binds to ROR1 as described in the present invention.

[0037] Another aspect of the present invention provides a pharmaceutical composition comprising: the antibody or antigen-binding fragment thereof described in the present invention, the nucleic acid molecule described therein, the carrier described therein and / or the cell described therein, and optionally a pharmaceutically acceptable carrier.

[0038] Another aspect of the present invention provides a pharmaceutical composition comprising: the antibody or antigen-binding fragment thereof described herein, and / or the biological material described herein, and optionally a pharmaceutically acceptable carrier.

[0039] Another aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in any one of the present invention, the nucleic acid molecule, the carrier, the cell, and / or the composition described herein in the preparation of a medicament for the prevention and / or treatment of diseases.

[0040] Another aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in any one of the present invention, the biological material described therein, and / or the composition described therein in the preparation of a medicament for the prevention and / or treatment of diseases.

[0041] In some implementations, the disease is a ROR1-mediated disease.

[0042] In some implementations, the disease is cancer, transplant rejection, autoimmune disease, or infectious disease.

[0043] In some implementations, the disease is cancer.

[0044] In some implementations, the disease is breast cancer, colon cancer, or B-cell lymphoma.

[0045] Another aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in any one of the present invention, the nucleic acid molecule, the vector, the cell, and / or the composition described herein in the preparation of a diagnostic kit for detecting anti-ROR1 antibodies in biological samples.

[0046] Another aspect of the present invention provides a kit comprising the antibody or antigen-binding fragment thereof as described in any one of the present invention, the nucleic acid molecule, the vector, the cell, and / or the composition described herein.

[0047] In some embodiments, the kit is a diagnostic kit for detecting anti-ROR1 antibodies in biological samples.

[0048] The present invention provides an antibody that specifically binds to ROR1 and a method for preparing the same. The antibody provided has high specificity, high affinity and binding ability; at the same time, the antibody provided has the ability to mediate endocytosis. Attached Figure Description

[0049] Figure 1 This is a diagram showing the endocytic activity of the antibody on CHO-K1-hROR1 cells. Detailed Implementation

[0050] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0051] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. When a trademark name is used herein, unless the context otherwise indicates, the trademark name includes the product formulation, generic medicine, and active ingredient of the product for which the trademark name is used.

[0052] Certain terms in this invention

[0053] The term "CDR" refers to the complementarity-determining region within the variable sequence of an antibody. For each variable region, there are three CDRs in each variable region of the heavy and light chains, which are called CDR1, CDR2, and CDR3. The exact boundaries of these CDRs are defined differently depending on the system. The system described by Kabat et al. (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a definitive residue numbering system applicable to antibody variable regions but also provides residue boundaries that define the three CDRs. These CDRs can be called Kabat CDRs. Each complementarity-determining region can contain amino acid residues from what is defined as a "complementarity-determining region" as by Kabat. Chothia et al. (Chothia & Lesk, J. Mol. Biol, 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (-1989)) found that Kabat Certain sub-regions within a CDR adopt nearly identical peptide skeletons, despite significant diversity at the amino acid sequence level. These sub-regions are referred to as L1, L2, and L3, or H1, H2, and H3, where "L" and "H" denote the light and heavy chain regions, respectively. These regions may be termed Chothia CDRs, with boundaries overlapping with the Kabat CDR. Other CDR boundary definitions may not strictly adhere to one of the aforementioned systems but will still overlap with the Kabat CDR. The methods used herein can utilize CDRs defined according to any of these systems, although the preferred embodiment uses CDRs defined by Kabat or Chothia.

[0054] The term "antibody" refers to immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs. Based on this, immunoglobulins can be classified into five classes, or different types of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding constant regions of the heavy chain designated α, δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins, and due to differences in chemical structure and biological function, it can be further divided into four subclasses: IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in their constant regions. The subunit structure and three-dimensional conformation of different immunoglobulin classes are well-known to those skilled in the art. The term "chimeric antibody" refers to an antibody containing sequences derived from two different antibodies, typically from different species. For example, a chimeric antibody contains fragments of human and rodent antibodies, typically the human constant region and the mouse variable region. Methods for generating chimeric antibodies include conventional recombinant DNA and gene transfection techniques known to those skilled in the art.

[0055] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antibody fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parent antibody's binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments (scFv), linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies.

[0056] In this invention, the term "mouse antibody" refers to a monoclonal antibody against human ROR1 prepared according to the knowledge and skills in the art. Preparation involves injecting the test subject with the ROR1 antigen, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. In a preferred embodiment of the invention, the mouse ROR1 antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a mouse IgG1, IgG2, IgG3, or IgG4 or a variant thereof.

[0057] The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" does not include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human backbone sequence (i.e., "humanized antibody").

[0058] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into the variable region framework of a human antibody. Humanized antibodies overcome the drawback of chimeric antibodies, which, due to carrying a large amount of mouse protein components, induce a strong immune response. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the variable region of the human antibody to maintain its activity.

[0059] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can mitigate the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies is selected. The variable region gene is then cloned from mouse hybridoma cells, followed by the desired human antibody constant region gene. The mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or their variants, preferably containing the heavy chain constant region of human IgG1, IgG2, or IgG4. Alternatively, an IgG1 heavy chain constant region that has been mutated to enhance ADCC (antibody-dependent cell-mediated cytotoxicity) can be used.

[0060] The term "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

[0061] The term "expression vector" or "vector" refers to a delivery system that allows for the operative insertion of polynucleotides or nucleic acids encoding a protein, thereby enabling the expression of that protein. Vectors can be used to transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within the host cells.

[0062] The term "host cell" refers to a cell into which a foreign polynucleotide or nucleic acid and / or vector has been introduced. Host cells include "transformers" and "transformed cells," which include the initially transformed cell and its progeny, regardless of the number of passages. Progeny cells may not be identical to parental cells in their nucleic acid content and may contain mutations. This application includes screening or selecting mutant progeny with the same function or biological activity from the initially transformed cells.

[0063] The term “treatment” refers to alleviating a disease or symptom, slowing the onset or development of a disease or symptom, reducing the risk of developing a disease or symptom, or delaying the development of symptoms associated with a disease or symptom, reducing or terminating symptoms associated with a disease or symptom, producing a complete or partial reversal of a disease or symptom, curing a disease or symptom, or a combination of the above.

[0064] The term "prevention" includes the suppression of the occurrence or development of a disease or condition, or symptoms of a particular disease or condition. In some implementations, subjects with a family history of the disease are candidates for preventative programs. Generally, the term "prevention" refers to the administration of a drug before the onset of symptoms or signs, particularly in subjects at risk.

[0065] The term "pharmaceutical composition" herein refers to a composition in which an active substance is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to human or animal subjects. In some embodiments, the active substance is present in a unit dose that is suitable for administration in a treatment regimen and for demonstrating a statistically significant probability of achieving the intended therapeutic effect when used in the relevant population.

[0066] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0067] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages, proportions, ratios, or parts are by weight.

[0068] Example 1: Preparation and purification of anti-ROR1 antibody

[0069] 1.1 Preparation and purification of antigens

[0070] The nucleotide sequence (SEQ ID NO: 15) of the his-tagged fusion protein encoding the extracellular region AA30-403 (uniprotID: Q01973) of human ROR1 protein was inserted into the pcDNA3.4 expression vector with appropriate restriction sites. HEK293 cells were transfected with PEI, and the cell culture supernatant was harvested after expression. The his-tagged protein was purified by affinity purification using a Ni column to obtain the hROR1-his protein.

[0071] 1.2 Construction of overexpression cell lines

[0072] The nucleotide sequence encoding the full-length human ROR1 (uniprot ID: Q01973, SEQ ID NO: 16) was constructed into the pLVX-IRES-puro vector and co-transfected with the backbone vectors pMD2G and psPAX2 into 293T cells. After culture, the culture medium was discarded, and fresh complete F12K medium was added for further culture. 48 h after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter to obtain the viral supernatant.

[0073] Add all viral supernatant to a solution containing 3×10 5 CHO-K1 cells were cultured in 6-well plates with polybrene added for 12 hours. The supernatant was then discarded, and fresh puromycin-containing complete F12K medium was added, followed by 48 hours of further culture. Subsequently, cells were seeded at 0.5 cells / well in two 96-well plates and cultured for 2 weeks in puromycin-containing F12K complete medium. Single clones were selected for scale-up, resulting in the CHOK1-hROR1 cell line.

[0074] 1.3 Obtaining monoclonal antibodies

[0075] Several 6-8 week old Balb / c, ICR, and KM mice were used. Recombinant human ROR1 extracellular domain hROR1-his protein was emulsified with either complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and administered subcutaneously or intraperitoneally, once every two weeks for a total of four immunizations. Tail blood was collected for ELISA and flow cytometry (FACS) detection. Mice with the best ELISA titer and positive FACS were selected for sprint immunization. Mouse spleen cells were obtained aseptically, fused with SP2 / 0 myeloma cells, and plated for culture. Monoclonal hybridoma-positive clones were obtained through limiting dilution, ELISA, and FACS screening. Sequencing yielded the heavy chain variable region nucleotide sequences and light chain variable region nucleotide sequences of the candidate molecule (SEQ ID NO: 17 and SEQ ID NO: 18). After expression and purification, a monoclonal antibody named G2 was obtained. The amino acid sequences of the heavy chain variable region of the G2 monoclonal antibody are shown in SEQ ID NO:7, the amino acid sequences of the light chain variable region are shown in SEQ ID NO:8, the amino acid sequences of the heavy chain constant region are shown in SEQ ID NO:9, and the amino acid sequences of the light chain constant region are shown in SEQ ID NO:10; the CDR region sequences are shown in SEQ ID NO:1-6 (as defined by the Kabat numbering system). See Table 1.

[0076] Table 1. Heavy chain variable region, light chain variable region, and CDR sequence of G2 monoclonal antibody

[0077]

[0078] 1.4 Preparation of control antibodies

[0079] The control antibody was prepared according to the contents described in patent document CN112262157A, and its heavy chain amino acid sequence is shown in SEQ ID NO:13 and its light chain amino acid sequence is shown in SEQ ID NO:14.

[0080] Example 2: Detection of antibody binding ability to antigen protein

[0081] The binding ability of antibodies to antigen proteins was detected by ELISA. 96-well ELISA plates were coated with 1 μg / mL hROR1-His (100 μl / well) overnight. The next day, the plates were washed with 0.05% PBST, blocked with 2% BSA at 37 °C, washed with 0.05% PBST, and then serially diluted (120, 30, 7.5, 1.875, 0.469, 0.117, 0.029, 0.007, 0.002, 0.0005, 0.0001 μg / mL) of each antibody and incubated at 37 °C. After incubation, the plates were washed with 0.05% PBST, and then incubated with HRP-labeled goat anti-human IgG secondary antibody. After incubation, the plates were washed with 0.05% PBST, and the incubation was terminated with stop solution. Finally, the OD value was measured at 450 nm using a microplate reader (TECAN, Spark). The results are shown in Table 2.

[0082] Table 2. ECs bound to ROR1-his protein by antibodies 50 value

[0083]

[0084] The results showed that antibody G2 could bind to the antigen protein hROR1-His, and the binding ability was comparable to that of the control antibody.

[0085] Example 3: Detection of antibody binding ability to overexpressing cells

[0086] Human ROR1-overexpressing CHO-K1-hROR1 cells in logarithmic growth phase were collected, washed, and then used to prepare a single-cell suspension with 1xPBS. The cell density was adjusted to 5 × 10⁶ cells / year. 6 Cells / mL were added at a rate of 20 μL per well to 96-well round-bottom plates. Serially diluted antibodies were added to the corresponding wells, mixed, and incubated at 4 °C for 30 min. After incubation, the cells were washed three times with 1×PBS, and then 20 μL of 1:50 diluted PE anti-human IgG Fc Recombinant Antibody (Biolegend, 366904) was added. The cells were incubated at 4 °C for 30 min in the dark. After washing three times with 1×PBS, the cells were analyzed by flow cytometry (Agilent, Advanteon). The results are shown in Table 3.

[0087] Table 3. Antibody binding to ECs overexpressing cells 50 value

[0088]

[0089] The results show that antibody G2 can bind to CHO-K1-hROR1 cells, and the binding ability is comparable to that of the control antibody.

[0090] Example 4: Antibody endocytosis efficiency detection

[0091] CHO-K1-hROR1 cells were treated and resuspended in RPMI-1640 complete medium. 20 μL of the cell suspension was collected for cell counting and viability determination. The cell density was adjusted to 4E6 cells / mL, and 70 μL / well was added to 96-well U-plates. The antibody was diluted to 40 nM with RPMI-1640 complete medium, and pHrodo™ iFL Green Human IgG Labeling Reagent (Thermo, Z25611) was diluted to 120 nM with RPMI-1640 complete medium. An equal volume of antibody (35 μL) and an equal volume of pHrodo (35 μL) were mixed and incubated at room temperature in the dark for 30 min. Then, an equal volume (70 μL) was added to a 96-well U-plate containing CHO-K1-hROR1 cells and cultured in CO2 for 2 h. 40 μL of the cell suspension was then used for flow cytometry to detect the green fluorescence signal; the signal intensity represents the strength of endocytosis.

[0092] The results are as follows Figure 1 As shown, antibody G2 can be endocytosed on CHO-K1-hROR1 cells, and its endocytosis capacity is comparable to that of the control antibody.

[0093] Example 5 Antibody affinity kinetics detection

[0094] The affinity between the antibody and the antigen hROR1-His was detected using a Biacore T200 instrument. An S-series Protein A sensor chip was used. 2 μg / mL of candidate antibody and different concentration gradients (100-0.390625 nM, 9 concentration points with 2-fold dilution) of human ROR1 antigen were prepared using running buffer. Samples were placed according to the set parameters and the sample order automatically set by the instrument, and the method was run. Specific parameter settings were as follows: 2 μg / mL antibody was captured at 10 μL / min for 30 s (control antibody for 12 s), and stabilized for 100 s; ROR1 antigen was used as the analyte at a flow rate of 30 μL / min, with a binding time of 180 s and a dissociation time of 300 s, followed by regeneration with Glycine 1.5 (Cytiva# BR100354) at 30 μL / min for 30 s. After the operation was completed, data analysis was performed using Biacore Evaluation Software. A 1:1 binding method was used to fit the complete curve using a model to obtain the kinetic parameters ka and kd, as well as the affinity data KD. The results are shown in Table 4.

[0095] Table 4. Results of antibody affinity kinetics assay

[0096]

[0097] The results show that the affinity of antibody G2 for hROR1-his is 1.38E-09 M, which is better than that of the control antibody.

[0098] This invention has been illustrated through various specific embodiments. However, those skilled in the art will understand that this invention is not limited to the specific embodiments, and various modifications or variations can be made within the scope of this invention. Furthermore, the various technical features mentioned throughout this specification can be combined with each other without departing from the spirit and scope of this invention. Such modifications and variations are all within the scope of this invention.

Claims

1. An anti-RORl antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 in sequence, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 in sequence.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 7, and the light chain variable region amino acid sequence is shown in SEQ ID NO:

8.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein, the antibody or antigen-binding fragment thereof is a Fab fragment, a Fab' fragment, a F(ab')2 fragment or a single-chain Fv fragment.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein, the antibody is a murine antibody, a chimeric antibody, a human antibody or a humanized antibody.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein, the antibody or antigen-binding fragment thereof further comprises a light chain constant region selected from kappa subtype or lambda subtype.

7. The antibody or antigen-binding fragment thereof of claim 1, wherein, the antibody or antigen-binding fragment thereof has a heavy chain shown in SEQ ID NO: 11, and a light chain shown in SEQ ID NO:

12.

8. A biological material related to the anti-ROR1 antibody or antigen-binding fragment thereof of any one of claims 1-7, which is 1), 2) or 3) as follows: 1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-7; 2) a vector containing the nucleic acid molecule of 1); 3) a host cell containing the nucleic acid molecule of 1) or containing the vector of 2).

9. A pharmaceutical composition comprising: the antibody or antigen-binding fragment thereof of any one of claims 1-7, and / or the biological material of claim 8, and optionally a pharmaceutically acceptable carrier.

10. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-7, the biological material of claim 8 or the composition of claim 9 in the preparation of a diagnostic kit for detecting ROR1 protein in a biological sample.

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