Anti-ror1 antibody c1 and uses thereof

Abstract

The application provides an anti-ROR1 antibody or antigen binding fragment thereof and application thereof. The anti-ROR1 antibody or antigen binding fragment thereof comprises heavy chain variable region CDRs and light chain variable region CDRs; wherein the heavy chain variable region CDRs have an amino acid sequence as shown in SEQ ID NO: 2, 4 and 6 or a conservatively modified form of the amino acid sequence; and / or the light chain variable region CDRs have an amino acid sequence as shown in SEQ ID NO: 9, 11 and 13 or a conservatively modified form of the amino acid sequence. The anti-ROR1 antibody or antigen binding fragment thereof can be combined with human ROR1-ECD, has high affinity, can be combined with ROR1 positive tumor cells and has concentration-dependent ADCC activity, has high tumor cell killing activity, can be used for developing a drug for treating ROR1 positive cancer or tumor and a ROR1 detection product, meets clinical requirements of ROR1 positive cancer or tumor diagnosis, treatment response prediction, treatment, prognosis evaluation, curative effect evaluation, recurrence monitoring and the like, has high clinical value and wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically, this invention relates to anti-ROR1 antibodies or their antigen-binding fragments and their applications. Background Technology

[0002] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a type I transmembrane receptor tyrosine kinase with a molecular weight of approximately 104 kDa. ROR1 is highly expressed in various cancers and is associated with tumor malignancy and poor prognosis, while it is expressed at low levels or not at all in normal adult tissues, making it a potential target for oncology drug development.

[0003] There are some new research findings on anti-ROR1 antibodies in existing technologies. For example, Chinese patent application CN110590951A discloses a ROR1 monoclonal antibody. This monoclonal antibody G3 specifically binds to the frizzled domain of human ROR1. Compared with anti-ROR1 antibodies that bind to other domains of human ROR1, it has better therapeutic effects and outstanding therapeutic effects on non-small cell lung adenocarcinoma.

[0004] Therefore, there is an urgent need to develop anti-ROR1 antibodies or their antigen-binding fragments that have high affinity for ROR1 and can efficiently kill tumor cells, in order to develop more drugs for treating ROR1-positive cancers or tumors, as well as products for tumor diagnosis, prognostic assessment, efficacy monitoring, or recurrence monitoring. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides an anti-ROR1 antibody or its antigen-binding fragment and its applications. The anti-ROR1 antibody or its antigen-binding fragment of this invention can bind to human ROR1-ECD with high affinity, can bind to ROR1-positive tumor cells and exhibits concentration-dependent ADCC activity, demonstrating high tumor cell killing activity. It can be used to develop drugs for treating ROR1-positive cancers or tumors and ROR1 detection products, meeting clinical needs such as diagnosis, treatment response prediction, treatment, prognostic assessment, efficacy evaluation, and recurrence monitoring of ROR1-positive cancers or tumors. It has high clinical value and broad application prospects.

[0006] In a first aspect, the present invention provides an anti-ROR1 antibody or an antigen-binding fragment thereof. According to embodiments of the present invention, it comprises: heavy chain variable region CDRs and light chain variable region CDRs; wherein the heavy chain variable region CDRs have amino acid sequences as shown in SEQ ID NO: 2, 4, and 6 or amino acid sequences of conserved modified forms thereof; and / or the light chain variable region CDRs have amino acid sequences as shown in SEQ ID NO: 9, 11, and 13 or amino acid sequences of conserved modified forms thereof. The anti-ROR1 antibody or antigen-binding fragment thereof according to embodiments of the present invention can bind to human ROR1-ECD with high affinity, can bind to ROR1-positive tumor cells, exhibits high tumor cell killing activity, and can be used to develop drugs for treating ROR1-positive cancers or tumors and ROR1 detection products, meeting clinical needs such as diagnosis, treatment response prediction, treatment, prognostic assessment, efficacy evaluation, and recurrence monitoring of ROR1-positive cancers or tumors. It has high clinical value and broad application prospects.

[0007] In a second aspect, the present invention provides a multispecific antibody. According to embodiments of the present invention, it comprises: the aforementioned anti-ROR1 antibody or its antigen-binding fragment. According to embodiments of the present invention, the multispecific antibody based on the aforementioned anti-ROR1 antibody or its antigen-binding fragment possesses ROR1-positive tumor cell binding activity and high tumor cell killing activity, exhibiting high clinical application value and high drug development value.

[0008] In a third aspect, the present invention provides a conjugate. According to embodiments of the present invention, it comprises: the aforementioned antibody or its antigen-binding fragment or the aforementioned multispecific antibody; and a conjugate portion linked to the antibody or its antigen-binding fragment, or the multispecific antibody. According to embodiments of the present invention, the conjugate based on the aforementioned anti-ROR1 antibody or its antigen-binding fragment and the multispecific antibody possesses ROR1-positive tumor cell binding activity and enhanced tumor cell killing activity, thus possessing clinical application value and drug development value.

[0009] In a fourth aspect, the present invention provides a chimeric antigen receptor. According to embodiments of the invention, it comprises the aforementioned antibody or its antigen-binding fragment. According to embodiments of the invention, a chimeric antigen receptor capable of binding to human ROR1-ECD can be developed based on the aforementioned anti-ROR1 antibody or its antigen-binding fragment for the treatment of diseases such as ROR1-positive cancers or tumors.

[0010] In a fifth aspect, the present invention provides a genetically modified cell. According to an embodiment of the invention, the cell expresses the aforementioned chimeric antigen receptor. Thus, a transgenic modified cell with tumor cell killing activity is obtained for the treatment of ROR1-positive cancers or tumors.

[0011] In a sixth aspect, the present invention provides a nucleic acid. According to embodiments of the invention, the nucleic acid encodes the aforementioned antibody or its antigen-binding fragment, the aforementioned multispecific antibody, or the aforementioned chimeric antigen receptor. The nucleic acid encoding the aforementioned anti-ROR1 antibody or its antigen-binding fragment, or the aforementioned multispecific antibody, according to embodiments of the invention, is suitable for developing novel monoclonal antibodies, multispecific antibody drugs, conjugates, or chimeric antigen receptors with high ROR1 affinity and high tumor cell killing activity for treating ROR1-positive cancers or tumors. Further, the protein encoded by the nucleic acid can be used to treat ROR1-positive cancers or tumors.

[0012] In a seventh aspect, the present invention provides a vector or transformant. According to embodiments of the invention, it contains the aforementioned nucleic acid. Thus, using the constructed vector or transformant, the aforementioned antibody or its antigen-binding fragment, or the aforementioned multispecific antibody, can be effectively expressed, and the aforementioned conjugate can be obtained.

[0013] In an eighth aspect, the present invention provides a recombinant host cell. According to embodiments of the invention, it contains the aforementioned nucleic acid or the aforementioned vector; or expresses the aforementioned antibody or its antigen-binding fragment, the aforementioned multispecific antibody, or the aforementioned chimeric antigen receptor. According to embodiments of the invention, the cell can efficiently express the aforementioned antibody or its antigen-binding fragment or the aforementioned multispecific antibody under suitable conditions, further obtaining an anti-ROR1 antibody or its antigen-binding fragment or multispecific antibody that can bind to human ROR1-ECD protein, has high affinity, and exhibits high tumor cell killing activity.

[0014] In a ninth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, it comprises: the aforementioned antibody or its antigen-binding fragment, the aforementioned multispecific antibody, the aforementioned conjugate, the aforementioned chimeric antigen receptor, or the aforementioned genetically modified cells. The pharmaceutical composition according to embodiments of the invention can be used to treat ROR1-positive cancers or tumors.

[0015] In a tenth aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises: the aforementioned antibody or its antigen-binding fragment, and the aforementioned multispecific antibody. The kit according to embodiments of the present invention can be used for ROR1 detection, achieving purposes such as tumor diagnosis, treatment response prediction, prognostic assessment, efficacy evaluation, and recurrence monitoring, and can be widely used in ROR1-related research and disease diagnosis and treatment.

[0016] In an eleventh aspect of the invention, the present invention proposes the use of the aforementioned antibody or its antigen-binding fragment, or the aforementioned multispecific antibody, in the preparation of a product. According to an embodiment of the invention, the product is used for ROR1 detection.

[0017] Those skilled in the art will understand that the features and advantages described above for antibodies or their antigen-binding fragments, multispecific antibodies, and kits also apply to this application and will not be repeated here.

[0018] In a twelfth aspect of the invention, the invention proposes the use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antibody, the aforementioned conjugate, the aforementioned chimeric antigen receptor, or the aforementioned genetically modified cell in the preparation of a medicament. According to embodiments of the invention, the medicament is used to treat ROR1-positive cancers or tumors.

[0019] Those skilled in the art will understand that the features and advantages described above for antibodies or their antigen-binding fragments, multispecific antibodies, conjugates, chimeric antigen receptors, and genetically modified cells also apply to this application, and will not be repeated here.

[0020] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0021] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0022] Figure 1 This is a graph showing the electrophoresis results of antigen protein expression and purification in Example 1 of the present invention;

[0023] Figure 2 This is the result of mouse immune titer detection in Example 1 of the present invention; where "W" represents 10 4 ;

[0024] Figure 3 The results of flow cytometry detection of mouse immune serum and H1299-ROR1 in Example 1 of this invention are shown; where red represents the blank serum group and blue represents the immune serum group; the vertical axis represents fluorescence intensity and the horizontal axis represents cell number.

[0025] Figure 4 This is a graph showing the screening results of ROR1-ECD-His-Assay-beads for Beacon single B cell antibodies in Example 1 of this invention.

[0026] Figure 5 This is a graph showing the screening results of H1299-ROR1 Beacon single B cell antibody in Example 1 of the present invention;

[0027] Figure 6 The diagram shows the amplification results of heavy and light chains in positive B cells of Example 1 of the present invention; where (A) is the heavy chain amplification result; and (B) is the light chain amplification result.

[0028] Figure 7 This is an electrophoresis result of the C1 antibody expression and purification in Example 1 of the present invention, where NR represents non-reduction, R represents reduction, L represents loading, FT represents flow-through, and M represents marker.

[0029] Figure 8 This is a graph showing the C1 antibody ELISA detection results of Example 1 of the present invention; where the horizontal axis represents antibody concentration (ng / mL) and the vertical axis represents OD. 450 Absorbance value;

[0030] Figure 9 This is a graph showing the detection results of C1 antibody BLI in Example 2 of the present invention;

[0031] Figure 10 The image shows the flow cytometry results of C1 antibody detection in Example 3 of this invention; where (A) represents H1299-ROR1 cells (ROR1 overexpressing cell line); (B) represents 231 cells (human breast cancer cells); and (C) represents LM3 cells (human liver cancer cells); red represents the blank control group, and blue represents the C1 antibody group.

[0032] Figure 11 The graph shows the detection results of the killing activity of C1 antibody against A549 tumor cells (human non-small cell lung cancer cells) in Example 4 of the present invention; where the horizontal axis is the antibody concentration Log (ng / mL) and the vertical axis is the killing efficiency (%).

[0033] Figure 12 The graph shows the detection results of the killing activity of C1 antibody against 231 tumor cells (human breast cancer cells) in Example 4 of the present invention; where the horizontal axis is the antibody concentration Log (ng / mL) and the vertical axis is the killing efficiency (%).

[0034] Figure 13 The graph shows the detection results of the killing activity of C1 antibody against LM3 tumor cells (human liver cancer cells) in Example 4 of the present invention; where the horizontal axis is the antibody concentration Log (ng / mL) and the vertical axis is the killing efficiency (%).

[0035] Figure 14 The results of the C1 antibody binding epitope analysis in Example 5 of this invention are shown; where NTC is the antibody-free negative control. Detailed Implementation

[0036] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0037] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0038] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0039] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0040] Terms and Definitions

[0041] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0042] In this article, the term "ROR1" refers to receptor tyrosine kinase-like orphan receptor 1.

[0043] In this article, the term "ROR1-ECD" refers to the ROR1 extracellular domain (ECD), which is located on the outer side of the cell membrane and is responsible for recognizing and binding specific signaling molecules (ligands, such as Wnt5a).

[0044] In this article, the term "antibody" generally refers to an antibody that recognizes one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, triple-chain antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 4854-4861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy-chain, full-length light-chain, or intact immunoglobulin molecules; or the immunologically active portion of any of these polypeptides, i.e., molecules or portions thereof containing an antigen-binding site that specifically binds to a target antigen of interest, such targets including but not limited to cancer cells.

[0045] In this paper, certain regions within the variable region exhibit a higher degree of variation in amino acid composition and sequence, termed "hypervariable region (HVR)." The hypervariable region is the location where antigens and antibodies bind, and is therefore also called the complementarity-determining region (CDR). Both the heavy chain and light chain variable regions contain three CDR regions. For example, these typically include amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)); and / or amino acid residues from “high-variable rings” (e.g., amino acid residues near 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and near 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)).

[0046] In this document, the term "antigen-binding fragment" is equivalent to "antibody fragment" or "antigen-binding antibody fragment," and can include a portion of a complete antibody, generally a binding region or variable region. This includes, but is not limited to: Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, scFv-Fc fragments, or bispecific antibodies (BsAbs), linear antibodies, or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol ("PEGylated," "PEGylated") (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" stands for polyethylene glycol).

[0047] In this article, the term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the constant region amino acid sequence of a monoclonal antibody from one species (such as a mouse) with the constant region of an antibody from another species (such as a human).

[0048] In this article, the term "humanized antibody" refers to a recombinant antibody obtained by replacing all non-CDR (Fv backbone region (FR)) amino acid sequences in the constant and variable regions of a monoclonal antibody from one species (e.g., mouse) with non-CDR amino acid sequences in the constant and variable regions of an antibody from another species (e.g., human). That is, when the constant region of an antibody is humanized, it is called a chimeric antibody, while when all non-CDR amino acid sequences in both the constant and variable regions are humanized, it is called a humanized antibody. The humanization methods can be performed using conventional antibody engineering techniques and will not be elaborated upon here.

[0049] For nucleotides, the terms "homology," "identity," or "similarity" are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of "sequence homology" between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. This is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first sequence—is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings and known computer algorithms for sequence alignment, such as NCBI Blast v2.0. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0 967 284, EP 1 085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0050] For peptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more peptides or their designated sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The percentage of homology between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = number of identical positions / total number of positions × 100), where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and identity percentage determination between two sequences can be performed using mathematical algorithms, as described in the non-limiting examples below.

[0051] This invention proposes an anti-ROR1 antibody or its antigen-binding fragment, a multispecific antibody, a conjugate, a nucleic acid, a vector or transformant, a cell, a pharmaceutical composition, a kit, and their applications, which will be described in detail below.

[0052] Antibody or its antigen-binding fragment

[0053] This invention provides an anti-ROR1 antibody or its antigen-binding fragment. According to embodiments of the invention, it comprises: heavy chain variable region CDRs and light chain variable region CDRs; wherein the heavy chain variable region CDRs have amino acid sequences as shown in SEQ ID NO: 2, 4, and 6 or their conserved modified amino acid sequences; and / or the light chain variable region CDRs have amino acid sequences as shown in SEQ ID NO: 9, 11, and 13 or their conserved modified amino acid sequences. The anti-ROR1 antibody or its antigen-binding fragment according to embodiments of the invention can bind to human ROR1-ECD with high affinity, can bind to ROR1-positive tumor cells and has concentration-dependent ADCC activity, exhibiting high tumor cell killing activity. It can be used to develop drugs for treating ROR1-positive cancers or tumors, as well as for developing ROR1 detection products and ROR1 detection retrospective analysis, meeting the needs of ROR1-positive cancer or tumor diagnosis, treatment response prediction, treatment, prognostic assessment, efficacy evaluation, recurrence monitoring, etc., with high clinical value and broad application prospects.

[0054] In this document, the term "conservatively modified amino acid sequence" refers to an amino acid modification that does not significantly affect or alter the binding properties of an antibody containing that amino acid sequence. Such modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this invention using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine).

[0055] It should be noted that one or more amino acid residues in the CDR region of the anti-ROR1 antibody or its antigen-binding fragment of the present invention may be replaced by other amino acid residues from the same side chain family, and the modified antibody retention function can be tested using the functional assay methods described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0056] According to embodiments of the present invention, the above-mentioned anti-ROR1 antibody or its antigen-binding fragment may further include at least one of the following additional technical features:

[0057] According to an embodiment of the present invention, the heavy chain variable regions (CDRs) include: heavy chain variable regions CDR1, CDR2, and CDR3; the light chain variable regions (CDRs) include: light chain variable regions CDR1, CDR2, and CDR3; wherein, the heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 2, the heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 4, and the heavy chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 6; the light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 9, the light chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 11, and the light chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 13.

[0058] It should be noted that the antigen-binding fragment will consist of a partial sequence of the heavy chain variable region or the light chain variable region of its source antibody, or contain partial sequences of the light and heavy chain variable regions, wherein the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as its source antibody.

[0059] According to an embodiment of the present invention, the dissociation constant KD of the antibody or its antigen-binding fragment with ROR1-ECD is ≤10×10⁻⁶. -9 M, the KD value is determined by biomembrane interferometry (BLI).

[0060] According to an embodiment of the present invention, the antibody or its antigen-binding fragment comprises a frame region; wherein the frame region comprises a heavy chain frame region and / or a light chain frame region.

[0061] In some embodiments, at least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies, human antibodies, and primate antibodies.

[0062] According to embodiments of the present invention, the heavy chain framework region has an amino acid sequence as shown in SEQ ID NO: 1, 3, 5 and 7, or an amino acid sequence of a conserved modified form thereof.

[0063] According to embodiments of the present invention, the light chain framework region has an amino acid sequence as shown in SEQ ID NO: 8, 10, 12 and 14, or an amino acid sequence of a conserved modified form thereof.

[0064] According to an embodiment of the present invention, the heavy chain framework region includes heavy chain framework regions FR1, FR2, FR3, and FR4, and the light chain framework region includes light chain framework regions FR1, FR2, FR3, and FR4; wherein, the heavy chain framework region FR1 has the amino acid sequence shown in SEQ ID NO: 1, the heavy chain framework region FR2 has the amino acid sequence shown in SEQ ID NO: 3, the heavy chain framework region FR3 has the amino acid sequence shown in SEQ ID NO: 5, and the heavy chain framework region FR4 has the amino acid sequence shown in SEQ ID NO: 7; the light chain framework region FR1 has the amino acid sequence shown in SEQ ID NO: 8, the light chain framework region FR2 has the amino acid sequence shown in SEQ ID NO: 10, the light chain framework region FR3 has the amino acid sequence shown in SEQ ID NO: 12, and the light chain framework region FR4 has the amino acid sequence shown in SEQ ID NO: 14.

[0065] According to embodiments of the present invention, the antibody or its antigen-binding fragment comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence in a conserved modified form thereof; and / or, the antibody or its antigen-binding fragment comprises: a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence in a conserved modified form thereof.

[0066] According to an embodiment of the present invention, the antibody or its antigen-binding fragment further comprises a constant region; wherein the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0067] In some embodiments, at least a portion of the heavy chain constant region and / or light chain constant region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies, human antibodies, and primate antibodies.

[0068] It should be noted that the antibodies described herein can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules, including engineered subclasses with altered Fc moieties that provide reduced or enhanced effector cell activity. Immunoglobulins can be derived from any species.

[0069] In some embodiments, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0070] In some embodiments, the light chain constant region includes a light chain constant region selected from κ-type or λ-type.

[0071] It should be noted that the amino acid sequences mentioned in this invention are shown from the N-terminus to the C-terminus.

[0072] According to an embodiment of the present invention, the N end of the heavy chain constant region is connected to the C end of the heavy chain variable region; and / or, the N end of the light chain constant region is connected to the C end of the light chain variable region.

[0073] According to embodiments of the present invention, the heavy chain constant region has an amino acid sequence as shown in SEQ ID NO: 17 or an amino acid sequence having at least 80% identity with it; and / or, the light chain constant region has an amino acid sequence as shown in SEQ ID NO: 18 or an amino acid sequence having at least 80% identity with it.

[0074] It should be noted that, without substantially affecting the human ROR1-ECD binding activity of the antibody or its antigen-binding fragment (retaining at least 95% activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the heavy or light chain variable region of the antibody or its antigen-binding fragment of the present invention to obtain sequences of the antibody or its antigen-binding fragment. These variations are all considered to be included within the scope of protection of the present invention. For example, amino acids with similar properties can be substituted in the heavy or light chain variable region. The sequences of the above-mentioned variations of the present invention can have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence.

[0075] It should be noted that the sequence identity described in this invention can be measured using sequence analysis software. For example, the computer program BLAST with default parameters, especially BLASTP or TBLASTN.

[0076] According to embodiments of the present invention, the antibody comprises at least one of a full-length monoclonal antibody, a chimeric antibody, a humanized antibody, Fv, scFv, Fab, Fab', Fab'-SH, and F(ab')2; the antigen-binding fragment of the antibody comprises at least one of an F(ab')2 fragment, a Fab' fragment, a Fab fragment, an F(ab)2 fragment, an Fv fragment, a scFv fragment, a scFv-Fc fusion protein, and a scFv-Fv fusion protein.

[0077] Multispecific antibodies

[0078] This invention provides a multispecific antibody. According to embodiments of the invention, it comprises: the aforementioned anti-ROR1 antibody or its antigen-binding fragment. According to embodiments of the invention, the multispecific antibody based on the aforementioned anti-ROR1 antibody or its antigen-binding fragment possesses ROR1-positive tumor cell binding activity and high tumor cell killing activity, exhibiting high clinical application value and high drug development value.

[0079] Conjugate

[0080] This invention provides a conjugate. According to embodiments of the invention, it comprises: the aforementioned antibody or its antigen-binding fragment or the aforementioned multispecific antibody; and a conjugate portion linked to the antibody or its antigen-binding fragment, or the multispecific antibody. According to embodiments of the invention, the conjugate based on the aforementioned anti-ROR1 antibody or its antigen-binding fragment and multispecific antibody exhibits ROR1-positive tumor cell binding activity and enhanced tumor cell killing activity, possessing clinical application value and drug development value.

[0081] In this article, the term "conjugate" is understood in context to refer to an antibody or its antigen-binding fragment that is conjugated to a coupling part such as a carrier substance, drug, toxin, cytokine, protein tag, modifier, therapeutic agent, or chemotherapeutic agent using any covalent or non-covalent biological conjugation strategy.

[0082] In some embodiments, the conjugation portion includes at least one selected from carriers, drugs, toxins, cytokines, protein tags, modifiers, therapeutic agents, and chemotherapeutic agents. Thus, by utilizing the targeting properties of the aforementioned antibodies, effector molecules (drugs, toxins, cytokines, etc.) are precisely delivered to ROR1-positive tumor cells at the target site, thereby improving therapeutic efficacy and reducing systemic toxicity.

[0083] Chimeric antigen receptor

[0084] This invention provides a chimeric antigen receptor. According to embodiments of the invention, it includes the aforementioned antibody or its antigen-binding fragment. According to embodiments of the invention, based on the aforementioned anti-ROR1 antibody or its antigen-binding fragment, a chimeric antigen receptor capable of binding to human ROR1-ECD can be developed for the treatment of ROR1-positive cancers or tumors.

[0085] Gene-modified cells

[0086] This invention provides a genetically modified cell. According to an embodiment of the invention, the cell expresses the aforementioned chimeric antigen receptor. Thus, a transgenic modified cell with tumor cell killing activity is obtained for the treatment of ROR1-positive cancers or tumors.

[0087] In some implementations, the genetically modified cells are transgenic immune cells.

[0088] For example, the transgenic immune cells may be selected from: T cells, NK cells, and macrophages. These transgenic immune cells may be derived from peripheral blood, umbilical cord blood, or iPSCs.

[0089] In some implementations, the aforementioned chimeric antigen receptor can be transduced onto immune cells via an expression vector (lentiviral vector) to express it on the surface of these immune cells.

[0090] Nucleic acid

[0091] This invention provides a nucleic acid. According to embodiments of the invention, the nucleic acid encodes the aforementioned antibody or its antigen-binding fragment, the aforementioned multispecific antibody, or the aforementioned chimeric antigen receptor. The nucleic acid encoding the aforementioned anti-ROR1 antibody or its antigen-binding fragment, or the aforementioned multispecific antibody, according to embodiments of the invention, is suitable for developing novel monoclonal antibodies, multispecific antibody drugs, conjugates, or chimeric antigen receptors with high ROR1 affinity and high tumor cell killing activity for treating ROR1-positive cancers or tumors. Further, the protein encoded by the nucleic acid can be used to treat ROR1-positive cancers or tumors.

[0092] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases, the complementary strand is also disclosed. Furthermore, the molecular sequences in this invention include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0093] Vector or transformant

[0094] This invention provides a vector or transformant. According to embodiments of the invention, it contains the aforementioned nucleic acid. Thus, using the constructed vector or transformant, the aforementioned antibody or its antigen-binding fragment, or the aforementioned multispecific antibody, can be effectively expressed, and the aforementioned conjugate can be obtained.

[0095] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0096] When linking the aforementioned nucleic acid molecules to the vector or transformant, such as an expression vector, the nucleic acid molecules can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. Of course, these control elements can be directly derived from the vector itself or be exogenous, i.e., not derived from the vector itself. The nucleic acid molecules and control elements simply need to be operatively linked.

[0097] According to embodiments of the present invention, the vector may refer to a cloning vector or an expression vector, which can be obtained by operatively linking the nucleic acid to a commercially available vector (such as a plasmid or viral vector).

[0098] In this paper, the term "transformant" refers to a host cell that has received exogenous nucleic acid. A transformant is a host cell that contains non-hereditary genetic material by introducing exogenous nucleic acid into the host cell through any means such as transformation, transfection, transduction, electroporation, or microinjection.

[0099] In this paper, the term "operably ligated" refers to ligating a foreign gene to a vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. The vectors used in this invention are not particularly limited; commonly used plasmids (such as pSeTag2, PEE14, pMH3, etc.), viral vectors (such as adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus), bacteriophages, etc., can all be used. After these vectors enter the host cell, they can express the target gene.

[0100] In some implementation schemes, after the expression vector is introduced into suitable recipient cells, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of a regulatory system, thereby enabling the in vitro large-scale production of the proteins encoded by the nucleic acid molecules.

[0101] According to an embodiment of the present invention, the carrier is a eukaryotic carrier or a prokaryotic carrier.

[0102] Recombinant host cells

[0103] This invention provides a recombinant host cell. According to embodiments of the invention, it contains the aforementioned nucleic acid or the aforementioned vector; or expresses the aforementioned antibody or its antigen-binding fragment, the aforementioned multispecific antibody, or the aforementioned chimeric antigen receptor. According to embodiments of the invention, the cell can efficiently express the aforementioned antibody or its antigen-binding fragment or the aforementioned multispecific antibody under suitable conditions, further obtaining an anti-ROR1 antibody or its antigen-binding fragment or multispecific antibody that can bind to human ROR1-ECD protein, has high affinity, and exhibits high tumor cell killing activity.

[0104] In this paper, the term "recombinant host cell" refers to a host cell modified by recombinant DNA technology, whose genome or episomes contain foreign nucleic acid sequences and can replicate or express foreign genes.

[0105] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the antibody or its antigen-binding fragment or multispecific antibody described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the antibody or its antigen-binding fragment or multispecific antibody include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the recombinant antibody based on the specific environment of their laboratory.

[0106] In some embodiments, the cells are selected from at least one of prokaryotic host cells, eukaryotic host cells, and bacteriophages.

[0107] For example, the prokaryotic host cells include, but are not limited to: Escherichia coli, Streptomyces, Bacillus subtilis, or Mycobacterium; the eukaryotic host cells include, but are not limited to: animal cells, plant cells, or fungi.

[0108] In some embodiments, the animal cells include mammalian cells, insect cells, or Caenorhabditis elegans.

[0109] For example, the mammalian cells include, but are not limited to: 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, Vero, BHK cells, CV1 cells, HeLa cells, MDCK cells, Hep-2 cells, and Per6 cells.

[0110] In some embodiments, the mammalian cells are preferably 293 series cells, Per6 cells, or CHO cells.

[0111] Pharmaceutical Composition

[0112] This invention provides a pharmaceutical composition. According to embodiments of the invention, it comprises: the aforementioned antibody or its antigen-binding fragment, the aforementioned multispecific antibody, the aforementioned conjugate, the aforementioned chimeric antigen receptor, or the aforementioned genetically modified cells. The pharmaceutical composition according to embodiments of the invention can be used to treat ROR1-positive cancers or tumors.

[0113] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0114] In the specific implementation plan, pharmaceutically acceptable excipients are further included.

[0115] In this article, the term "pharmaceuticalally acceptable" refers to an ingredient that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0116] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0117] In some embodiments, the excipients include one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers.

[0118] Exemplary examples include, but are not limited to, water, saline, buffer solutions, glucose, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration. These are well known to those skilled in the art.

[0119] It should be noted that the pharmaceutical composition includes combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the present invention. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0120] The medicament of the present invention contains a safe and effective amount of the active ingredient of the present invention and pharmaceutically acceptable excipients. The active ingredient includes: an antibody or an antigen-binding fragment thereof, the aforementioned multispecific antibodies, and / or the aforementioned conjugates.

[0121] The effective amount of the active ingredient of this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0122] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, conjugates, or pharmaceutical compositions of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are anticipated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Generally, the pharmaceutical formulation should be matched with the route of administration.

[0123] In some embodiments, the pharmaceutical compositions of the present invention are administered by intravenous or subcutaneous injection.

[0124] According to an embodiment of the present invention, the pharmaceutical composition is an injection.

[0125] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0126] In this document, the term "treatment" means the use of a drug or compound to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to disease but not yet diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0127] Reagent test kit

[0128] This invention proposes a kit. According to embodiments of the invention, it comprises: the aforementioned antibody or its antigen-binding fragment, and the aforementioned multispecific antibody. The kit according to embodiments of the invention can be used for ROR1 detection, achieving purposes such as tumor diagnosis, treatment response prediction, prognostic assessment, efficacy evaluation, and recurrence monitoring, and can be widely used in ROR1-related research and disease diagnosis and treatment.

[0129] Exemplarily, the kit can be used for: tumor diagnosis, treatment response prediction, prognostic assessment, efficacy evaluation, or recurrence monitoring; according to an embodiment of the present invention, the tumor expresses ROR1, and ROR1 is a biomarker for tumor diagnosis, tumor prognostic assessment, tumor efficacy evaluation, and / or tumor recurrence monitoring.

[0130] In some implementations, the antibody or its antigen-binding fragment is labeled with a detectable marker.

[0131] In some embodiments, the kit includes a carrier comprising the aforementioned antibody or its antigen-binding fragment; preferably, the carrier is nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, or porous materials.

[0132] In some embodiments, the detectable marker is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and colloids.

[0133] application

[0134] This invention proposes the application of the aforementioned antibody or its antigen-binding fragment or the aforementioned multispecific antibody in the preparation of a product for ROR1 detection.

[0135] Those skilled in the art will understand that the features and advantages described above for antibodies or their antigen-binding fragments, and for multispecific antibodies, also apply to this application, and will not be repeated here.

[0136] In some implementations, the product is used for tumor diagnosis, treatment response prediction, prognostic assessment, efficacy evaluation, or recurrence monitoring, wherein the tumor expresses ROR1.

[0137] In some implementations, ROR1 serves as a biomarker for tumor diagnosis, prognostic assessment, efficacy evaluation, or recurrence monitoring. ROR1 is highly expressed during embryonic development, expresses very low levels in normal adult tissues (present only in small amounts in adipocytes, immature B cells, etc.), but exhibits abnormally high expression in various malignant tumors (such as CLL, TNBC, lung adenocarcinoma, and ovarian cancer), making it a potential tumor-specific biomarker. Studies have shown that ROR1 promotes tumor proliferation, migration, and drug resistance by activating the Wnt5a / NF-κB pathway, and its expression level is positively correlated with tumor progression. Therefore, detecting ROR1 expression levels in clinical samples (such as peripheral blood) can be used for tumor screening and diagnosis.

[0138] In CAR-T or ADC therapy clinical trials, a decrease in sROR1 levels after ROR1-targeted therapy is significantly associated with tumor shrinkage (e.g., after Zilovertamab Vedotin treatment for DLBCL, sROR1 decreased, resulting in a 100% CR rate). Studies show that sROR1 significantly increases before recurrence (earlier than radiographic recurrence), especially in CLL and TNBC. That is, a sustained decrease in sROR1 concentration in the patient's blood after treatment is significantly associated with treatment response, while a rebound or persistently high concentration may indicate tumor residue or early recurrence. Therefore, detecting ROR1 expression levels in clinical samples (such as peripheral blood) can be used for tumor diagnosis, treatment response prediction, prognostic assessment, and recurrence monitoring.

[0139] In some implementations, the product includes: reagents, reagent kits, or chips.

[0140] The present invention also proposes the use of the aforementioned antibodies or antigen-binding fragments thereof, the aforementioned multispecific antibodies, the aforementioned conjugates, the aforementioned chimeric antigen receptors, or the aforementioned genetically modified cells in the preparation of a drug for the treatment of ROR1-positive cancers or tumors.

[0141] Those skilled in the art will understand that the features and advantages described above for antibodies or their antigen-binding fragments, multispecific antibodies, conjugates, chimeric antigen receptors, and genetically modified cells also apply to this application, and will not be repeated here.

[0142] According to embodiments of the present invention, the cancer or tumor is selected from solid tumors and / or hematologic malignancies.

[0143] According to embodiments of the present invention, the solid tumor includes at least one of breast cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, and melanoma.

[0144] According to embodiments of the present invention, the hematologic malignancy includes at least one of chronic lymphocytic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, Hodgkin's lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, multiple myeloma, and diffuse large B-cell lymphoma.

[0145] Methods of treating diseases

[0146] This invention provides a method for treating ROR1-positive cancers or tumors.

[0147] According to an embodiment of the present invention, the method includes administering to a subject a pharmaceutically acceptable amount of the antibody or its antigen-binding fragment described above, the multispecific antibody described above, the conjugate described above, the pharmaceutical composition described above, the chimeric antigen receptor described above, or the genetically modified cell described above.

[0148] It should be noted that the terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one implementation, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. Subjects can be humans, but also include other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0149] The effective amount of the antibody or its antigen-binding fragment, multispecific antibody conjugate, or pharmaceutical composition described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0150] According to embodiments of the present invention, the cancer or tumor is selected from solid tumors and / or hematologic malignancies.

[0151] According to embodiments of the present invention, the solid tumor includes at least one of breast cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, and melanoma.

[0152] According to embodiments of the present invention, the hematologic malignancy includes at least one of the following: chronic lymphocytic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, Hodgkin's lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, multiple myeloma, and diffuse large B-cell lymphoma.

[0153] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0154] Example 1: Antibody Acquisition, Expression, and Purification

[0155] This embodiment provides a method for obtaining the antibody of the present invention. The amino acid sequence of the antibody is sequenced to determine the antibody sequence, and the antibody is expressed and purified to obtain a pure antibody protein, which provides a basis for subsequent functional verification and performance evaluation.

[0156] 1. Antigen expression and immunity

[0157] Antigen expression: ROR1-ECD-hFc and ROR1-ECD-His proteins were expressed using a eukaryotic expression system. ROR1-ECD-hFc and ROR1-ECD-His proteins were then separated and purified by Protein A affinity chromatography. Figure 1 (For later use.) ROR1-ECD-hFc is the extracellular domain of human ROR1 (UniProt ID: Q01973, amino acids 24-403) fused with a human Fc fragment, and ROR1-ECD-His is the extracellular domain of human ROR1 (UniProt ID: Q01973, amino acids 24-403) linked with 6 histidine tags.

[0158] Immunization method: 11 immunizations on the foot pads and 2 subcutaneous immunizations. The specific immunization schedule is as follows:

[0159] Footpad immunization: 10 μg ROR1-ECD-hFc (1 mg / mL, 10 μL) + 10 μg CpG (CpG oligonucleotide (CpGODN) is a special type of DNA oligonucleotide, usually ranging from 10 to 30 bases in length, where "CpG" represents a cytosine and a guanine linked together. These molecules play an important role in the immune system because they can mimic the DNA sequences of bacteria and viruses, thereby triggering the body's immune response) (1 mg / mL, 10 μL) + 10 μL aluminum adjuvant + 20 μL PBS. 25 μL was administered to each of the two hind footpads of each mouse twice a week.

[0160] Subcutaneous immunization: 40 μg ROR1-ECD-hFc (1 mg / mL, 40 μL) + 35 μL physiological saline + 75 μL Freund's complete / incomplete adjuvant, emulsified, and injected subcutaneously at multiple points on the back of mice (4-6 week old female Balb / c mice) once every 2 weeks.

[0161] The immune titer of mice was detected by ELISA, and the binding performance of mouse immune serum to H1299-ROR1 (ROR1 overexpressing cell line) was evaluated by flow cytometry.

[0162] Mouse immune titer test results as follows Figure 2 As shown, the flow cytometry results of mouse immune serum and H1299-ROR1 are as follows: Figure 3 As shown.

[0163] The results showed that, compared with the blank control group, the serum titer of immunized mice reached 20 w (in this article, "w" or "W" represents 10). 4 Compared with the blank serum group, the fluorescence intensity of the mouse immune serum group was significantly increased, indicating that the mouse immune serum can specifically bind to H1299-ROR1 cells that highly express ROR1.

[0164] The above results indicate that the mouse immune response was successful and Beacon single B cell antibody screening can be performed.

[0165] 2. Beacon single B cell antibody screening

[0166] Mouse spleen cells and bone marrow cells were collected, and highly purified mouse CD138+ cells were enriched and isolated using the EasySep™ Mouse CD138 Positive Selection Kit. Beacon single B cell antibody screening was performed, and positive single B cells that bound both ROR1-ECD-His and H1299-ROR1 were exported and subsequently amplified for antibody light and heavy chains.

[0167] The ROR1-ECD-His-Assay-beads screening results are as follows: Figure 4 As shown, the H1299-ROR1 screening results are as follows: Figure 5 As shown.

[0168] The results show: Figure 4 , Figure 5 The signal intensity of both ROR1-ECD-His and H1299-ROR1 double-positive cell populations exceeded the threshold required for single B cell light and heavy chain amplification, indicating a high proportion of double-positive cells.

[0169] The above results indicate that further amplification of the light and heavy chains of single B cells can be performed to screen for anti-ROR1 antibodies.

[0170] 3. Single B-cell light and heavy chain amplification and sequencing

[0171] The exported positive B cells were amplified using the Opto Plasma B Discovery Sanger Prep Kit, Mouse, and Opto Plasma BDiscovery cDNA Synthesis Kit, and then sent for sequencing.

[0172] Results of heavy and light chain amplification in positive B cells as follows Figure 6 As shown.

[0173] Sequencing and screening yielded an anti-ROR1 antibody with excellent ROR1-ECD affinity, designated as antibody C1.

[0174] The heavy and light chain sequencing results of anti-ROR1 antibody C1 are as follows:

[0175] 1 DVQFQESGPGLVKPSQSLSLTCTVTGYSIT HFR1 2 SDYAWN CDR-H1 3 WIRQFPGKKLEWMG HFR2 4 FITFRGFISYNPSLKS CDR-H2 5 RISITRDTSKNQFFLYLNSMTAGDTATYYCAR HFR3 6 GSGTYYFDV CDR-H3 7 WGAGTSVTVSS HFR4 8 ETTVTQSPASLSVSIGEKVTIRC LFR1 9 ITSTDIDDDLN CDR-L1 10 WYQQKPGEPPKLLIS LFR2 11 EDNILRP CDR-L2 12 GVPSRFSSGYGTDFLFSIENVLSEDVADYYC LFR3 13 LQSNDLPLT CDR-L3 14 FGAGTKLELN LFR4

[0176] 3. Expression, purification, and ELISA detection of anti-ROR1 antibody C1

[0177] The anti-ROR1 antibody protein, designated C1, from Example 1 was expressed using a eukaryotic expression system. The C1 antibody protein was purified by Protein A affinity chromatography, and the ROR1-ECD-His binding activity of the purified C1 antibody was detected by ELISA.

[0178] In this embodiment of the invention, the light and heavy chain constant regions of the C1 antibody are selected from the human IgG1 subtype.

[0179] The amino acid sequence of the heavy chain variable region of the C1 antibody is as follows:

[0180] DVQFQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGKKLEWMGFITFRGFFISYNPSLKSRISITRDTSKNQFFLYLNSMTAGDTATYYCARGSGTYYFDVWGAGTSVTVSS (SEQ ID NO: 15).

[0181] The amino acid sequence of the light chain variable region of the C1 antibody is as follows:

[0182] ETTVTQSPASLSSVSIGEKVTIRCITSTDIDDDLNWYQQKPGEPPKLLISEDNILRPGVPSRFSSSGYGTDFLFSIENVLSEDVADYYCLQSNDLPLTFGAGTKLELN (SEQ ID NO: 16).

[0183] In this embodiment of the invention, the amino acid sequence of the heavy chain constant region of the C1 antibody is as follows:

[0184] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:17).

[0185] In this embodiment of the invention, the amino acid sequence of the light chain constant region of the C1 antibody is as follows:

[0186] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18).

[0187] After purification of C1 antibody expression, the electrophoresis results of non-reducing and reducing gels are as follows: Figure 7 As shown, the ROR1-ECD binding activity ELISA detection results are as follows: Figure 8 As shown.

[0188] The results showed that: (1) Non-reducing gel results showed that the purified C1 antibody band was single and accounted for ≥90%, while reducing gel results showed that the heavy chain and light chain bands were clear and the heavy chain / light chain density ratio was moderate. (2) ELISA results showed that the purified C1 antibody could generate a strong binding reaction with ROR1-ECD-His.

[0189] The above results indicate that the C1 antibody, which was expressed in eukaryotes and purified by Protein A affinity chromatography, can be further used to evaluate the affinity and other properties of anti-ROR1 antibodies.

[0190] Example 2: Investigation of C1 affinity of anti-ROR1 antibody

[0191] The binding of the purified C1 antibody expressed in Example 1 to ROR1-ECD-His was detected using bio-layer interferometry (BLI). The specific detection method is as follows:

[0192] Protein A sensor was used to immobilize C1 antibody (concentration: 5 µg / mL), achieving an immobilization signal of 0.7–0.9 nm. ROR1-ECD-His was diluted to 4.2 µg / mL, 2.1 µg / mL, 1.05 µg / mL, 0.53 µg / mL, 0.26 µg / mL, 0.13 µg / mL, 0.07 µg / mL, and 0 µg / mL, respectively. The sensors containing different concentrations of ROR1-ECD-His were then reacted with the immobilized C1 antibody. Kinetic curves were fitted using Octet Analysis Studio 13 software, and the dissociation constant KD was calculated.

[0193] BLI test results are as follows Figure 9 As shown.

[0194] BLI assay results showed that the C1 antibody and ROR1-ECD-His had a dissociation constant KD≤10×10⁻⁶. -9 M (KD=6.35×10 -9 It binds to M with an affinity and has a good affinity for ROR1-ECD.

[0195] Example 3: Investigation of the binding performance of anti-ROR1 antibody to C1 solid tumor cells

[0196] The binding of the purified C1 antibody from Example 1 to H1299-ROR1 (ROR1 overexpressing cell line), 231 (human breast cancer cells), and LM3 (human liver cancer cells) cells was detected by flow cytometry. The specific detection method is as follows:

[0197] Cells were incubated with 5 µg / mL C1 antibody at 37°C for 30 min, washed three times with PBS, and then incubated with Alexa Fluor 647-conjugated goat anti-human IgG secondary antibody (1:200 dilution) at 37°C for 30 min, followed by three washes with PBS. Flow cytometry was used to analyze the binding of C1 antibody to each cell.

[0198] Flow cytometry results as follows Figure 10 As shown.

[0199] Flow cytometry results showed that the C1 antibody could bind to a variety of solid tumor cells, demonstrating excellent potential against solid tumor cells.

[0200] Example 4: Detection of anti-ROR1 antibody C1 activity against solid tumors

[0201] Healthy human PBMCs were mixed with ROR1-highly expressed A549 (human non-small cell lung cancer cells), 231 (human breast cancer cells), and LM3 (human liver cancer cells) cells (effect cell:target cell ratio = 5:1), and then incubated with different concentrations of C1 antibody. The lactate dehydrogenase (LDH) released after tumor cell death was detected by CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (romega), and the killing rate of the antibody against tumor cells (100%) was calculated.

[0202] Tumor cell killing activity test results as follows Figures 11-13 As shown.

[0203] The results showed that the C1 antibody of this invention could produce an antibody concentration-dependent ADCC effect on various solid tumor cells, including human non-small cell lung cancer cells, human breast cancer cells, and human liver cancer cells.

[0204] The above results indicate that the C1 antibody of the present invention has good killing activity against solid tumors and can be further used to develop drugs for the treatment of ROR1-positive tumors, products for detecting ROR1, diagnosing tumors, assessing tumor prognosis, evaluating efficacy, or monitoring recurrence.

[0205] Example 5: Analysis of C1 binding epitopes of anti-ROR1 antibody

[0206] To clarify the epitope differences between the C1 antibody of this invention and Zilovertamab (formerly known as Cirmtuzumab, a positive control antibody targeting ROR1), this embodiment used ELISA to detect the binding activity of the two antibodies to different extracellular domains of ROR1. The specific scheme is as follows:

[0207] The ROR1 extracellular domain was segmented into IG domain, FZ domain, KR domain, IGFZ tandem domain, FZKR tandem domain and full-length extracellular domain (ECD) and coated onto an ELISA plate. The results were then analyzed.

[0208] The results are as follows Figure 14 As shown.

[0209] The results showed that: (1) C1 antibody could specifically bind to all proteins containing the IG domain (IG domain alone, IGFZ tandem domain, and full-length ECD), but had no significant binding to the FZ, KR domains alone or the FZKR tandem domain alone, confirming that its binding epitope is located in the IG domain of ROR1. (2) Zilovertamab only produced significant binding signals to the IGFZ tandem domain and the full-length ECD, and could not bind to the IG or FZ domains alone, suggesting that its binding epitope is located in the IGFZ tandem region, which is a complex epitope dependent on the co-conformation of the IG and FZ domains.

[0210] The above results indicate that the binding epitope of the C1 antibody of the present invention is in the ROR1-IG domain, and the binding epitope of Zilovertamab is in the ROR1-IGFZ domain. The binding epitopes of the C1 antibody and Zilovertamab do not overlap.

[0211] The above results suggest that the C1 antibody of this invention can exert its anti-tumor effect through a dual mechanism: on the one hand, it inhibits tumor cell proliferation by blocking the ROR1-mediated pro-survival signaling pathway; on the other hand, it recruits immune effector cells to mediate specific killing of tumor cells. This dual-functionality, combining signal blocking and immune killing, provides a more effective candidate drug for the immunotherapy of ROR1-positive tumors.

[0212] 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. An anti-ROR1 antibody or its antigen-binding fragment, characterized in that, It comprises: heavy chain variable regions (CDRs) and light chain variable regions (CDRs); wherein the heavy chain variable regions (CDRs) have amino acid sequences as shown in SEQ ID NO: 2, 4 and 6 or amino acid sequences of their conserved modified forms; and / or the light chain variable regions (CDRs) have amino acid sequences as shown in SEQ ID NO: 9, 11 and 13 or amino acid sequences of their conserved modified forms.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable regions (CDRs) comprise: heavy chain variable regions CDR1, CDR2, and CDR3; the light chain variable regions (CDRs) comprise: light chain variable regions CDR1, CDR2, and CDR3; wherein, the heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 2, the heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 4, and the heavy chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 6; the light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 9, the light chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 11, and the light chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 13; optionally, the dissociation constant KD of the antibody or its antigen-binding fragment with ROR1-ECD is ≤10 × 10⁻⁶. -9 M, the KD value is determined by biomembrane interferometry (BLI).

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, characterized in that, The antibody or its antigen-binding fragment comprises a frame region; wherein the frame region includes a heavy chain frame region and / or a light chain frame region; optionally, at least a portion of the heavy chain frame region and / or the light chain frame region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies, human antibodies, and primate antibodies; optionally, the heavy chain frame region has an amino acid sequence as shown in SEQ ID NO: 1, 3, 5, and 7, or an amino acid sequence of a conserved modified form thereof; optionally, the light chain frame region has an amino acid sequence as shown in SEQ ID NO: The amino acid sequences shown in SEQ ID NO: 1, 10, 12, and 14, or their conserved modified forms, are further wherein the heavy chain framework region comprises heavy chain framework regions FR1, FR2, FR3, and FR4, and the light chain framework region comprises light chain framework regions FR1, FR2, FR3, and FR4; wherein the heavy chain framework region FR1 has the amino acid sequence shown in SEQ ID NO: 1, the heavy chain framework region FR2 has the amino acid sequence shown in SEQ ID NO: 3, the heavy chain framework region FR3 has the amino acid sequence shown in SEQ ID NO: 5, and the heavy chain framework region FR4 has the amino acid sequence shown in SEQ ID NO: 7; the light chain framework region FR1 has the amino acid sequence shown in SEQ ID NO: 8, the light chain framework region FR2 has the amino acid sequence shown in SEQ ID NO: 10, the light chain framework region FR3 has the amino acid sequence shown in SEQ ID NO: 12, and the light chain framework region FR4 has the amino acid sequence shown in SEQ ID NO:

8. The amino acid sequence shown in SEQ ID NO: 14; optionally, the antibody or its antigen-binding fragment comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence in a conserved modified form thereof; and / or, the antibody or its antigen-binding fragment comprises: a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence in a conserved modified form thereof; optionally, the antibody or its antigen-binding fragment further comprises a constant region; wherein the constant region comprises: a heavy chain constant region and / or a light chain constant region;Optionally, at least a portion of the heavy chain constant region and / or light chain constant region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably mouse antibodies, human antibodies, and primate antibodies. At least one of the target antibodies; optionally, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; optionally, the light chain constant region includes a light chain constant region selected from κ-type or λ-type; optionally, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or, the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region; optionally, the heavy chain constant region has as shown in SEQ The amino acid sequence shown in SEQ ID NO: 17 or having at least 80% identity with it; and / or, the light chain constant region has an amino acid sequence shown in SEQ ID NO: 18 or having at least 80% identity with it; optionally, the antibody comprises at least one of a full-length monoclonal antibody, a chimeric antibody, a humanized antibody, Fv, scFv, Fab, Fab', Fab'-SH, and F(ab')2; the antigen-binding fragment of the antibody comprises at least one of the following: F(ab')2 fragment, Fab' fragment, Fab fragment, F(ab)2 fragment, Fv fragment, scFv fragment, scFv-Fc fusion protein, and scFv-Fv fusion protein.

4. A multispecific antibody, characterized in that, It includes: the anti-ROR1 antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

5. A conjugate, characterized in that, include: The antibody or its antigen-binding fragment according to any one of claims 1 to 3 or the multispecific antibody according to claim 4; a conjugated portion, said conjugated portion being linked to the antibody or its antigen-binding fragment, or the multispecific antibody; optionally, said conjugated portion comprising at least one selected from carriers, drugs, toxins, cytokines, protein tags, modifiers, therapeutic agents and chemotherapeutic agents.

6. A chimeric antigen receptor, characterized in that, Includes the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3.

7. A gene-modified cell, characterized in that, The cells express the chimeric antigen receptor as described in claim 6.

8. A nucleic acid, characterized in that, The nucleic acid encodes the antibody or its antigen-binding fragment as described in any one of claims 1 to 3, the multispecific antibody as described in claim 4, or the chimeric antigen receptor as described in claim 6.

9. A vector or transformant, characterized in that, It contains the nucleic acid as described in claim 8.

10. A recombinant host cell, characterized in that, Contains the nucleic acid of claim 8 or the vector of claim 9; or expresses the antibody or its antigen-binding fragment of any one of claims 1 to 3, the multispecific antibody of claim 4, or the chimeric antigen receptor of claim 6.

11. A pharmaceutical composition, characterized in that, include: The pharmaceutical composition comprises, optionally, an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, a multispecific antibody as described in claim 4, a conjugate as described in claim 5, a chimeric antigen receptor as described in claim 6, or a gene-modified cell as described in claim 7; optionally, further comprising pharmaceutically acceptable excipients; optionally, the excipients comprise: one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers; optionally, the pharmaceutical composition is an injection.

12. A reagent kit, characterized in that, It comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, or the multispecific antibody as described in claim 4.

13. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the multispecific antibody according to claim 4, in the preparation of a product, characterized in that, The product is used for ROR1 detection; optionally, the product is used for tumor diagnosis, prognostic assessment, efficacy evaluation, or recurrence monitoring, wherein the tumor expresses ROR1; optionally, the product includes: reagents, kits, or chips.

14. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the multispecific antibody according to claim 4, the conjugate according to claim 5, the chimeric antigen receptor according to claim 6, or the gene-modified cell according to claim 7 in the preparation of a drug, characterized in that, The drug is used to treat ROR1-positive cancers or tumors; optionally, the cancers or tumors include: solid tumors and / or hematologic malignancies; further, the solid tumors include at least one of: breast cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, and melanoma; further, the hematologic malignancies include at least one of: chronic lymphocytic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, Hodgkin's lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, multiple myeloma, and diffuse large B-cell lymphoma.

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