Anti-lgr5 antibodies and uses thereof

Abstract

The application discloses an anti-LGR5 monoclonal antibody. Further, the application discloses an anti-LGR5 humanized antibody. The antibody of the application can be combined with LGR5 with high specificity and high affinity, and meanwhile, exhibits excellent endocytosis activity, so that the antibody can be applied to the treatment of LGR5 overexpression diseases.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and medical immunology. Specifically, it relates to antibodies that specifically bind to the LGR5 protein, their antigen-binding fragments, and immunoconjugates and pharmaceutical compositions containing such antibodies. This invention also relates to the use of said antibodies in the diagnosis, prevention, or treatment of cancers, particularly LGR5-positive solid tumors such as colorectal cancer, gastric cancer, and liver cancer. Background Technology

[0002] LGR5 (leucine-rich G protein-coupled receptor 5) is an important receptor in the Wnt signaling pathway and has been identified as a specific surface marker for stem cells in various solid tumors. Studies have shown that LGR5 is highly expressed in various malignant tumors such as colorectal cancer, gastric cancer, and ovarian cancer, and is closely related to tumor occurrence, development, drug resistance, recurrence, and metastasis. Therefore, antibody drugs targeting LGR5 have great potential for clinical application.

[0003] Although anti-LGR5 antibodies have been reported, for example (WO2013149159A9) discloses the use of antibody-drug conjugates to target LGR5-positive cells for the treatment of colorectal cancer, developing antibodies with higher affinity, stronger endocytosis capacity or better antibody-dependent cell-mediated cytotoxicity remains a technical problem that needs to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to provide an isolated monoclonal antibody or its antigen-binding fragment, wherein the antibody can specifically bind to human LGR5 protein with high affinity and has excellent internalization activity, thereby delivering cytotoxic drugs into tumor cells.

[0005] Another object of the present invention is to provide an immunoconjugate and a pharmaceutical composition comprising the antibody.

[0006] Another objective of this invention is to provide the use of the antibody in the preparation of medicaments for the diagnosis, prevention or treatment of cancer (especially LGR5-positive solid tumors such as colorectal cancer, gastric cancer, and liver cancer).

[0007] In a first aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof targeting LGR5, said monoclonal antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising a complementarity-determining region (CDR) as follows: CDR1 contains the amino acid sequence shown in SEQ ID NO: 33, 39, 47 or 51; CDR2 contains the amino acid sequence shown in SEQ ID NO: 34, 40, 45, 48 or 52; CDR3 contains the amino acid sequence shown in SEQ ID NO: 35, 41, 49 or 53.

[0008] In a preferred embodiment, the heavy chain variable region includes a complementary determination region (CDR) as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 33, CDR2 containing the amino acid sequence shown in SEQ ID NO: 34, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 35; CDR1 containing the amino acid sequence shown in SEQ ID NO: 39, CDR2 containing the amino acid sequence shown in SEQ ID NO: 40, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 41; CDR1 containing the amino acid sequence shown in SEQ ID NO: 39, CDR2 containing the amino acid sequence shown in SEQ ID NO: 45, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 41; CDR1 containing the amino acid sequence shown in SEQ ID NO: 47, CDR2 containing the amino acid sequence shown in SEQ ID NO: 48, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 49; or CDR1 containing the amino acid sequence shown in SEQ ID NO: 51, CDR2 containing the amino acid sequence shown in SEQ ID NO: 52, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 53.

[0009] In a specific implementation, the light chain variable region includes the following complementary determination region (CDR): CDR1 contains the amino acid sequence shown in SEQ ID NO: 36, 42, 46 or 54; CDR2 contains the amino acid sequence shown in SEQ ID NO: 37, 43, or 55; CDR3 contains the amino acid sequence shown in SEQ ID NO: 38, 44, 50 or 56.

[0010] In a preferred embodiment, the light chain variable region includes a complementary determinant region (CDR) as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 36, CDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 38; CDR1 containing the amino acid sequence shown in SEQ ID NO: 42, CDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 44; CDR1 containing the amino acid sequence shown in SEQ ID NO: 46, CDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 44; CDR1 containing the amino acid sequence shown in SEQ ID NO: 46, CDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 50; or CDR1 containing the amino acid sequence shown in SEQ ID NO: 54, CDR2 containing the amino acid sequence shown in SEQ ID NO: 55, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 56.

[0011] In a specific embodiment, the monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region complementarity-determining region (CDR) as shown below: (1) The complementary determination region (CDR) of the heavy chain variable region is as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 33, CDR2 containing the amino acid sequence shown in SEQ ID NO: 34, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 35; The complementary determinant regions (CDRs) of the light chain variable region are shown below: CDR1 containing the amino acid sequence shown in SEQ ID NO: 36, CDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 38. (2) The complementary determination region (CDR) of the heavy chain variable region is as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 39, CDR2 containing the amino acid sequence shown in SEQ ID NO: 45, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 41; The complementary determinant regions (CDRs) of the light chain variable region are shown below: CDR1 containing the amino acid sequence shown in SEQ ID NO: 42, CDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 44. (3) The complementary determination region (CDR) of the heavy chain variable region is as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 39, CDR2 containing the amino acid sequence shown in SEQ ID NO: 45, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 41; The complementary determinant regions (CDRs) of the light chain variable region are as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 46, CDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 44. (4) The complementary determination region (CDR) of the heavy chain variable region is as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 47, CDR2 containing the amino acid sequence shown in SEQ ID NO: 48, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 49; The complementary determinant regions (CDRs) of the light chain variable regions are as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 46, CDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 50; or (5) The heavy chain variable region complementarity-determining regions (CDRs) are as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 51, CDR2 containing the amino acid sequence shown in SEQ ID NO: 52, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 53. The complementary determinant regions (CDRs) of the light chain variable regions are as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 54, CDR2 containing the amino acid sequence shown in SEQ ID NO: 55, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 56.

[0012] In a specific embodiment, the monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and / or a light chain variable region as shown below: The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 7, 9, 11, 13, 15, 17 or 19; The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 8, 10, 12, 14, 16, 18 or 20.

[0013] In a preferred embodiment, the monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region as shown below: The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 8; The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 10; The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 11, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 12; The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 14; The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16; The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 18; The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 20.

[0014] In a preferred embodiment, the monoclonal antibody may be 13, 18, 20, or 22; antibody 13 is preferred.

[0015] In a second aspect, the present invention provides a humanized monoclonal antibody or antigen-binding fragment thereof targeting LGR5, wherein the humanized monoclonal antibody or antigen-binding fragment thereof is obtained by humanization of the monoclonal antibody or antigen-binding fragment thereof described in the first aspect.

[0016] In a preferred embodiment, the humanized monoclonal antibody or its antigen-binding fragment is obtained by humanization of a monoclonal antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region complementarity-determining region (CDR) as shown below: The complementary determinant regions (CDRs) of the heavy chain variable region are as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 39, CDR2 containing the amino acid sequence shown in SEQ ID NO: 45, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 41. The complementary determinant regions (CDRs) of the light chain variable regions are as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 42, CDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 44.

[0017] In a preferred embodiment, the heavy chain variable region complementarity-determining region CDR2 of the humanized monoclonal antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO: 57, 58, 59 or 60.

[0018] In a preferred embodiment, the monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and / or a light chain variable region as shown below: The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 21, 23, 25, 27, 29 or 31; The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 22 or 28.

[0019] In a preferred embodiment, the humanized monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region as shown below: The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 22; The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 22; The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 25, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 22; The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 28; The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 29, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 28; The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 31, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 28.

[0020] In a preferred embodiment, the humanized monoclonal antibody is B2, B2-NQ, or B2-SA; preferably B2-NQ or B2-SA.

[0021] In a third aspect, the present invention provides a recombinant protein having: (i) the monoclonal antibody or its antigen-binding fragment as described in the first aspect, or the humanized monoclonal antibody or its antigen-binding fragment as described in the second aspect; and (ii) Optional tag sequences to assist in expression and / or purification.

[0022] In a preferred embodiment, the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag.

[0023] In a preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0024] In a preferred embodiment, the recombinant protein is a fusion protein.

[0025] In a preferred embodiment, the fusion protein is a monospecific antibody (i.e., a monospecific antibody targeting LGR5), a bispecific antibody, or a multispecific antibody (such as a trispecific antibody).

[0026] In a preferred embodiment, the bispecific or multispecific antibody not only targets LGR5, but also specifically binds to additional target antigens (such as other tumor antigens).

[0027] In a preferred embodiment, the recombinant protein is a monomer, dimer, or polymer.

[0028] In a preferred embodiment, the recombinant protein further includes an additional fusion element (or fusion polypeptide fragment) fused together with the element (i).

[0029] In a third aspect, the present invention provides a chimeric antigen receptor (CAR) wherein the antigen-binding domain of the chimeric antigen receptor contains a single-chain variable region sequence scFv of an antibody targeting LGR5 prepared using the monoclonal antibody or its antigen-binding fragment described in the first aspect, or the humanized monoclonal antibody or its antigen-binding fragment described in the second aspect.

[0030] In a fourth aspect, the present invention provides an isolated polynucleotide molecule that encodes the monoclonal antibody or antigen-binding fragment thereof described in the first aspect, or the humanized monoclonal antibody or antigen-binding fragment thereof described in the second aspect, the recombinant protein described in the third aspect, or the chimeric antigen receptor CAR described in the fourth aspect.

[0031] In a fifth aspect, the present invention provides a carrier containing the polynucleotide molecule described in the fourth aspect.

[0032] In a preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof.

[0033] In a preferred embodiment, the vector is a retroviral vector.

[0034] In a sixth aspect, the present invention provides a host cell containing an exogenous polynucleotide molecule as described in the fourth aspect integrated into a vector or chromosome as described in the fifth aspect.

[0035] In a preferred embodiment, the cells are isolated cells, and / or the cells are genetically engineered cells.

[0036] In a preferred embodiment, the cell is a mammalian cell.

[0037] In a preferred embodiment, the cells are NK cells or T cells.

[0038] In a preferred embodiment, the host cell is an engineered immune cell.

[0039] In a seventh aspect, the present invention provides a method for preparing CAR-NK cells or CAR-T cells, wherein the CAR-NK cells or CAR-T cells express the chimeric antigen receptor of the present invention, comprising the following steps: The polynucleotides of the present invention or the vectors of the present invention are introduced into NK cells or T cells to obtain the CAR-NK cells or CAR-T cells.

[0040] In an eighth aspect, the present invention provides a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof of the present invention, a humanized monoclonal antibody or an antigen-binding fragment thereof, a recombinant protein, a chimeric antigen receptor, or a host cell, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0041] In a preferred embodiment, the pharmaceutical composition is a formulation, preferably a liquid formulation.

[0042] In a preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

[0043] In a preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the monoclonal antibody or antigen-binding fragment thereof of the present invention, or a humanized monoclonal antibody or antigen-binding fragment thereof, recombinant protein, chimeric antigen receptor, or host cell, or a combination thereof, and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentage is a percentage by mass of the pharmaceutical composition.

[0044] In a preferred embodiment, the pharmaceutical composition is used for the prevention and / or treatment of diseases with LGR5 overexpression.

[0045] In a preferred embodiment, the disease in which LGR5 is overexpressed is a tumor that overexpresses LGR5.

[0046] In a preferred embodiment, the tumors overexpressing LGR5 include, but are not limited to, colorectal cancer, gastric cancer, ovarian cancer, and liver cancer.

[0047] In a ninth aspect, the present invention provides an immunoconjugate comprising: (a) The antibody portion, wherein the antibody portion is selected from the group consisting of: the monoclonal antibody of the present invention or an antigen-binding fragment thereof, humanized monoclonal antibody or an antigen-binding fragment thereof, recombinant protein, or combinations thereof; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0048] In a preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0049] In a tenth aspect, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof, humanized monoclonal antibody or antigen-binding fragment thereof, recombinant protein, chimeric antigen receptor, host cell, pharmaceutical composition or immunoconjugate of the present invention. (a) Preparation of detection reagents or kits; and / or (b) To prepare drugs or formulations for the prevention and / or treatment of diseases associated with LGR5 overexpression.

[0050] In a preferred embodiment, the disease in which LGR5 is overexpressed is a tumor that overexpresses LGR5.

[0051] In a preferred embodiment, the tumors overexpressing LGR5 include, but are not limited to, colorectal cancer, gastric cancer, ovarian cancer, and liver cancer.

[0052] In an eleventh aspect, the present invention provides a method for in vitro detection of LGR5 protein in samples (including diagnostic or non-diagnostic samples), the method comprising the steps of: (1) Contact the sample with the monoclonal antibody or its antigen-binding fragment, humanized monoclonal antibody or its antigen-binding fragment, or recombinant protein of the present invention; (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of LGR5 protein in the sample.

[0053] In a preferred embodiment, the detection is for in vitro, non-therapeutic, and non-diagnostic purposes.

[0054] In a preferred embodiment, the method is a cell immunochemical detection method, or an immunohistochemical detection method, or a whole-cell ELISA detection method, or a cell lysate ELISA detection method.

[0055] In a twelfth aspect, the present invention provides a method for treating a disease associated with LGR5 overexpression, comprising: administering to a subject requiring treatment an effective amount of the monoclonal antibody or antigen-binding fragment thereof described in the present invention, a humanized monoclonal antibody or antigen-binding fragment thereof, a recombinant protein, or a pharmaceutical composition.

[0056] In a preferred embodiment, the disease in which LGR5 is overexpressed is a tumor that overexpresses LGR5.

[0057] In a preferred embodiment, the tumors overexpressing LGR5 include, but are not limited to, colorectal cancer, gastric cancer, ovarian cancer, and liver cancer.

[0058] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0059] Figure 1 The results of the four immune titers test in mice were displayed; Figure 2 The results of hybridoma antibody EC50 detection are shown; Figure 3 The results of ELISA species detection were displayed; Figure 4 The results of hybridoma antibody FACS detection are shown; Figure 5 The results of hybridoma antibody and overexpressing cell-specific detection are shown; Figure 6 The results of hybridoma antibody endocytosis detection were displayed; Figure 7The results of humanized antibody ELISA EC50 assay were displayed; Figure 8 The results of the detection of binding of humanized antibodies to KEK293-human LGR5 cells are shown; Figure 9 The SPR detection results are displayed; and Figure 10 The results of humanized antibody endocytosis detection are shown. Detailed Implementation

[0060] Through extensive and in-depth research and screening, the inventors obtained several murine antibodies with excellent properties, including high affinity for human LGR5. Based on these murine antibodies with high affinity and high antitumor activity, humanized antibodies were further prepared. The antibodies of this invention can effectively bind to LGR5 and exhibit excellent activity, making them suitable as monoclonal antibody drugs for targeted therapy. This invention was completed based on these findings.

[0061] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0062] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0063] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0064] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0065] As used in this article, the terms “light chain variable zone” and “VL” are used interchangeably.

[0066] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0067] In this invention, the terms "antibody of the present invention", "protein of the present invention" or "peptide of the present invention" are used interchangeably and all refer to antibodies that specifically bind to LGR5, such as proteins or peptides having heavy chain variable regions and / or light chain variable regions similar to those of the antibodies of the present invention.

[0068] Antibody As used in this article, the term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.

[0069] Existing antibody numbering schemes include: 1. The Kabat scheme (Kabat et al., 1991) is based on the location of highly variable regions between sequences of the same domain type, with different numbering for antibody heavy (VH) and light (Vλ and Vκ) variable domains.

[0070] 2. Chothia's scheme (Al-Lazikani, 1997) is similar to Kabat's scheme, but corrects the placement of the annotations around the first VH complementary determinant region (CDR) to correspond to the structural loop. Similarly, the enhanced Chothia scheme (Abhinandan and Martin, 2008) further structurally modifies the insertion position.

[0071] 3. In contrast to these Kabat-like schemes, IMGT (Lefranc, 2003) and AHo (Honegger and Plückthun, 2001) have defined unique schemes for variable domains of antibodies and T-cell receptors (TCRs) (Vα and Vβ). Therefore, equivalent residue positions can be readily compared between domain types. IMGT and AHo differ in the number of positions they annotated (128 and 149, respectively) and in the locations where they believe indels occur.

[0072] Immunoglobulins differ in the amino acid composition and sequence of their heavy chain constant regions, thus exhibiting different antigenicities. Based on this, immunoglobulins can be classified into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the heavy chain region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ or λ chains based on their constant regions. Each of the five classes of Ig can possess either a κ or λ chain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well-known to those skilled in the art.

[0073] The antibody light chain of the present invention may further include a light chain constant region, wherein the light chain constant region comprises a human or mouse κ, λ chain or a variant thereof.

[0074] In this invention, the antibody heavy chain may further include a heavy chain constant region, which contains human or mouse IgG1, IgG2, IgG3, IgG4, or variants thereof. The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains varies considerably and is called the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is called the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the terminal terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. In embodiments of the present invention, the Kabat numbering rule is adopted to divide the six CDRs of the LGR5 antibody.

[0075] In this invention, the term "mouse antibody" refers to a monoclonal antibody against LGR5 prepared according to the knowledge and skills in the art. Preparation involves injecting the test subject with the LGR5 antigen, followed by isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. In a preferred embodiment of the invention, the mouse LGR5 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 mouse IgG1, IgG2, IgG3, or a variant thereof.

[0076] 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, which can reduce the immune response induced by murine antibodies.

[0077] 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, i.e., a different type of human germline antibody framework sequence. Humanized antibodies can overcome the heterologous response induced by chimeric antibodies, which carry a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. To avoid a decrease in immunogenicity along with a decrease in activity, minimal reverse or reversion mutations can be performed on the human antibody variable region framework sequence to maintain activity.

[0078] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that maintain its ability to specifically bind to antigens. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include... (i)Fab fragment, a monovalent fragment composed of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment, which is a divalent fragment containing two Fab fragments connected by a disulfide bridge on the chain region; (iii) Fd fragments composed of VH and CH1 domains; (iv) The Fv fragment consisting of the VH and VL domains of the single arm of the antibody.

[0079] Fv antibodies contain variable regions on the heavy and light chains, but no constant regions, and are the smallest antibody fragments with all antigen-binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and can form the structure required for antigen binding.

[0080] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of these six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immune interest. NIH Publication 91-3242.

[0081] The term "epitope" or "antigenic determinant" refers to a site on an antigen where an immunoglobulin or antibody specifically binds (e.g., a specific site on the LGR5 molecule). Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation.

[0082] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example, approximately less than 10 -8 M, 10 -9 M or l0 -10 M or lower affinity (KD) binding.

[0083] The term "competitive binding" refers to an antibody that recognizes the same epitope (also called an antigenic determinant) or a portion of the same epitope on the extracellular region of LGR5 as the monoclonal antibody of the present invention and binds to said antigen. An antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of LGR5 recognized by the monoclonal antibody of the present invention.

[0084] The term "KD" or "Kd" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies of this invention have a dissociation equilibrium constant of less than approximately 10. -7 M, for example, less than approximately 10 -8 M, 10 -9 M or l0 -10 M or a smaller dissociation equilibrium constant (KD) is combined with LGR5.

[0085] As used herein, the term "antigen determinant" refers to a discontinuous three-dimensional spatial site on an antigen that is recognized by the antibody or antigen-binding fragment of the present invention.

[0086] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0087] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, can be prepared using DNA recombination techniques well known in the art.

[0088] As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone derived from a single cell. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. The cell may be a eukaryotic, prokaryotic, or phage clone.

[0089] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0090] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to the table below. Anti-LGR5 humanized antibody In another preferred embodiment, the heavy chain constant region and / or light chain constant region of the antibody of the present invention may be a humanized heavy chain constant region or a light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is the heavy chain constant region of human IgG1, IgG2, etc., or the light chain constant region of human kappa, Lambda.

[0091] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or substituting at least one amino acid sequence preferably has a homology of at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the amino acid sequence.

[0092] The antibodies of the present invention can be double-chain or single-chain antibodies, and are preferably fully humanized antibodies.

[0093] The antibody derivatives described in this invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2, or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes of antibodies.

[0094] The antibody of this invention may be a humanized antibody targeting LGR5, a CDR grafted and / or modified antibody.

[0095] In the above-described content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0096] Antibody preparation Any method suitable for producing monoclonal antibodies can be used to produce the LGR5 antibody of this invention. For example, animals can be immunized with the linked or naturally occurring LGR5 protein or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.

[0097] Any suitable form of LGR5 can serve as an immunogen (antigen) for generating non-human antibodies specific to LGR5 and screening for the biological activity of said antibodies. The immunogen can be used alone or in combination with one or more immunogenic enhancers known in the art. The immunogen can be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA). The DNA encoding the immunogen can be expressed using suitable genetic vectors, including but not limited to adenovirus vectors, baculovirus vectors, plasmids, and nonviral vectors.

[0098] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0099] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0100] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences are obtained by first synthesizing multiple small fragments and then ligating them. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0101] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0102] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one implementation, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked.

[0103] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0104] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant or animal cells (such as mammalian cells).

[0105] The steps of transforming host cells with recombinant DNA as described in this invention can be performed using techniques well known in the art. The obtained transformants can be cultured using conventional methods, and the transformants express the polypeptide encoded by the gene of this invention. Depending on the host cell used, the cells are cultured in a conventional culture medium under suitable conditions.

[0106] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well known to those skilled in the art.

[0107] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0108] antibody preparations Antibodies exhibit varying degrees of stability in different formulation buffers, manifesting as changes in charge heterogeneity, antibody molecule degradation, and polymerization. These changes in quality properties are related to the antibody's inherent physicochemical properties. Therefore, in antibody drug development, it is necessary to screen suitable formulation buffers based on the physicochemical properties of different antibodies. Currently, commonly used antibody formulation buffer systems include phosphate buffer, citrate buffer, and histidine buffer. Depending on the antibody's properties, different concentrations of salt ions or excipients such as sorbitol, trehalose, and sucrose, as well as appropriate amounts of surfactants such as Tween, are added to maintain antibody stability.

[0109] Pharmaceutical Composition The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the aforementioned antibody or its active fragment or fusion protein or its ADC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0110] The antibody described in this invention can also be expressed in cells by a nucleotide sequence for cell therapy, such as for chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0111] The pharmaceutical compositions of the present invention can be directly used to bind LGR5, and are therefore used for the prevention and treatment of LGR5-related diseases. Furthermore, other therapeutic agents can be used concurrently.

[0112] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described monoclonal antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0113] When using a pharmaceutical composition, a safe and effective amount of the composition is administered to a mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0114] Detection uses and kits The antibodies of this invention can be used in detection applications, such as for testing samples, to provide diagnostic information.

[0115] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of organs.

[0116] The samples used in this invention include fixed or preserved cell or tissue samples.

[0117] This invention also provides a kit containing the antibody (or a fragment thereof) of this invention. In a preferred embodiment of this invention, the kit further includes a container, instructions for use, a buffer, etc. The antibody of this invention can also be immobilized on a detection plate.

[0118] Advantages of this invention: (a) The inventors obtained highly specific and high-affinity multiclonal antibodies against LGR5 through hybridoma fusion technology; (b) Humanized antibodies were prepared based on the above-mentioned monoclonal antibodies; (c) The antibody of the present invention has high affinity and high specificity for LGR5; (d) The antibodies of the present invention exhibit superior endocytic activity compared to control antibodies, thereby enabling their application in the treatment of diseases overexpressing LGR5.

[0119] 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. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0120] Example 1. Synthesis and preparation of LGR5 protein and control antibody Based on the human LGR5 sequence information (uniport: O75473, SEQ ID NO: 1), three antigens—huLGR5-his (SEQ ID NO: 2), huLGR5-mFc (SEQ ID NO: 3), and huLGR5-huFc (SEQ ID NO: 4)—were synthesized and expressed in eukaryotes to obtain three purified proteins: LGR5-his, LGR5-mFc, and LGR5-huFc. The control antibody was named LGAB. The VH (SEQ ID NO: 5) and VL (SEQ ID NO: 6) antibody sequences were derived from Petosemtamab (CN104411721A). Gene synthesis was performed by Universal Biotechnology, and expression and purification were carried out by Xichang Biotechnology.

[0121] Example 2. Animal Immunization and Potency Detection Animal immunization was performed using synthetic LGR5-mFc as the immunogen. The specific procedure was as follows: 6-8 week old Balb / c mice (Vitalliwa) were selected. For the first immunization, 100 µg of LGR5-mFc antigen was emulsified with Freund's complete adjuvant (Sigma) and injected subcutaneously at multiple sites. Two weeks later, a second immunization was performed using 50 µg of LGR5-mFc emulsified with Freund's incomplete adjuvant (Sigma) and injected intraperitoneally. Two weeks later, a third immunization was performed using 50 µg of LGR5-mFc emulsified with Freund's incomplete adjuvant (Sigma) and injected intraperitoneally. Two weeks later, a third immunization was performed using 50 µg of LGR5-mFc emulsified with Freund's incomplete adjuvant (Sigma) and injected intraperitoneally. One week later, tail blood was collected from the mice for titer testing. Suitable mice were then selected for a booster immunization with 100 µg of LGR5-mFc intraperitoneally.

[0122] ELISA detection of mouse immunotiter: LGR5-huFc was diluted to 2 µg / mL with PBS, and 100 µL / well was added to the microplate and incubated overnight at 4°C. After washing three times with PBST, 200 µL / well of 3% BSA was added, and the plate was incubated at room temperature for 2 h. After washing three times with PBST, 100 µL / well of mouse immune serum (serialized 9 times at a dilution of 1:400) was added, and the plate was incubated at room temperature for 1 h. After washing three times with PBST, 100 µL / well of 1:5000 diluted goat anti-mouse secondary antibody (Sigma, A0168) was added, and the plate was incubated at room temperature for 1 h. After washing three times with PBST, 100 μL / well of TMB solution was added for color development. After 10 min of development, 50 μL / well of 1 M H2SO4 was added to terminate the reaction. The absorbance (OD450 nM) of each well was measured using a microplate reader.

[0123] The results are as follows Figure 1 As shown, all mice produced antibodies after immunization, with mouse #1 having a titer of 1:102400 (OD450 > 2NC), which can be used for fusion.

[0124] Example 3. Cell fusion and antibody screening After booster immunization, the spleen of mice was aseptically harvested, ground, and prepared into a spleen cell suspension. This suspension was then mixed with myeloma cells SP2 / 0 (ATCC, CRL1581) in a certain ratio and fused using an electrofusion instrument (BTX, ECM2001+). The cells were then cultured in HAT selective medium, and the medium was changed to HT medium after 3-5 days. After 7-10 days, the culture supernatant was collected for ELISA detection.

[0125] Screening of fusion cell supernatant: LGR5-huFc was diluted to 1 µg / mL with PBS, and 100 µL / well was added to the microplate and incubated overnight at 4°C; after washing 3 times with PBST, 200 µL / well of 3% BSA was added and incubated at room temperature for 2 h; after washing 3 times with PBST, 100 µL / well of cell fusion culture supernatant was added and incubated at room temperature for 1 h; after washing 3 times with PBST, 100 µL / well of 1:5000 diluted goat anti-mouse secondary antibody (Sigma, A0168) was added and incubated at room temperature for 1 h; after washing 3 times with PBST, 100 μl / well of TMB solution was added for color development, and after 10 min of color development, 50 μl / well of 1 M H2SO4 was added to terminate the reaction. The absorbance (OD450 nM) of each well was measured using a microplate reader.

[0126] Fusion screening yielded 31 positive clones. The top 10 clones (4-1H8, 10-6G5, 13-7C4, 18-8B10, 20-9A1, 22-9E8, 23-10E4, 25-10G2, 26-11C9, 31-15G2) were selected for subcloning and cryopreservation, ultimately obtaining 8 hybridoma cell lines (4-1H8, 13-7C4, 18-8B10, 20-9A1, 22-9E8, 25-10G2, 26-11C9, 31-15G2).

[0127] Example 4. Purification and detection of hybridoma antibodies Eight antibodies (4 / 13 / 18 / 20 / 22 / 25 / 26 / 31) were selected and used in serum-free medium for the production and purification of hybridoma cells. After purification of the cell culture supernatant with Protein G, hybridoma antibodies with a purity > 90% were obtained. Human LGR5-his (ACRO, LG5-H52H3) and cyno LGR5-his (ACRO, LG5-C52H7) were diluted to 2 µg / mL with PBS, and 100 µL / well was added to the microplate and incubated overnight at 4°C. After washing three times with PBST, 200 µL / well of 3% BSA was added, and the plate was incubated at room temperature for 2 h. After washing three times with PBST, 100 µL / well of hybridoma antibody (serialized to 30 µg / mL) was added, and the plate was incubated at room temperature for 1 h. After washing three times with PBST, 100 µL / well of 1:5000 diluted goat anti-mouse secondary antibody (Sigma, A0168) was added, and the plate was incubated at room temperature for 1 h. After washing three times with PBST, 100 μL / well of TMB solution was added for color development. After 10 min of development, 50 μL / well of 1 M H2SO4 was added to terminate the reaction. The absorbance (OD) of each well was measured using a microplate reader.

[0128] The results are as follows Figure 2As shown, all eight hybridoma antibodies bound the LGR5 antigen, with EC50 values ​​ranging from 0.02957 to 1.12 µg / mL. Species detection results are as follows... Figure 3 As shown, all eight hybridoma antibodies bound to human and monkey antigens.

[0129] Example 5. Detection of hybridoma antibody binding to cells Flow cytometry was performed using Lovo cells (Chinese Academy of Sciences Cell Bank, SCSP-514) expressing LGR5 and HEK293-LGR5 / HEK293-LGR6 / HEK293-LGR4 / HEK293-LGR4 / HEK293-LGR5 / HEK293-LGR5 / HEK293-LGR6 / HEK293-LGR4 / HEK293-LGR5 / well in U-shaped plates. Hybridoma antibody and control antibody LGAB were added, initially at 30 μg / mL, serially diluted 10 times in 3-fold increments, 100 μL / well, and incubated at 4°C for 45 min. Cells were washed twice with 1% FBS-PBS, and anti-mouse IgG (H+L)-FITC secondary antibody (Kangcheng Biotechnology, KC-MM-095) was added, diluted 1:200, 100 μL / well. Anti-human antibodies were added to the PC control wells. FC-FITC secondary antibody (Jackson, 109-095-098) was diluted 1:200 and incubated at 4°C for 45 min. Cells were washed twice with 1% FBS-PBS, resuspended in 150 uL of 1% FBS-PBS, and then analyzed by flow cytometry. Data were processed using GraphPad Prism 8.3.

[0130] The results are as follows Figure 4 As shown, the hybridoma antibodies have binding activity with LOVO cells, with 13 showing the strongest binding, while the binding abilities of 18 / 20 / 22 / 26 are similar to those of the control antibody LGAB.

[0131] Specific results such as Figure 5 As shown, except for antibody 18, all other antibodies specifically bind to HEK-human LGR5 cells.

[0132] Example 6. Hybridoma Antibody Internalization Detection Antibody endocytosis efficiency was detected using the CCK8 method: Day 1: Cells were plated; Day 2: The drug was diluted to 1.6 μg / mL with "Medium + 2% FBS" containing 1.6 μg / mL rat anti-mouse or mouse anti-human antibody-drug conjugate, and 50 μl / well was added; At the same time, a control group with only rat anti-mouse or mouse anti-human antibody-drug conjugate ("2ndAb-ADC") and a Blank group with only culture medium were set up; Day 5: 20 μL CCK8 chromogenic solution was added to each well for color development, and OD450 data were detected using an ELISA reader. Data were processed using GraphPad Prism 8.3.

[0133] The results are as follows Figure 6 As shown, five antibodies (13 / 18 / 20 / 22 / 26) exhibited endocytosis-guided indirect cell killing effects, while three antibodies (4 / 25 / 31) showed no endocytosis-guided indirect cell killing effects.

[0134] Example 7. Antibody humanization and activity detection The amino acid sequences of seven hybridoma antibodies were obtained by hybridoma sequencing (4-VH SEQ ID NO: 7, 4-VL SEQ ID NO: 8, 13-VH SEQ ID NO: 9, 13-VL SEQ ID NO: 10, 18-VH SEQ ID NO: 11, 18-VL SEQ ID NO: 12, 20-VH SEQ ID NO: 13, 20-VL SEQ ID NO: 14, 22-VH SEQ ID NO: 15, 22-VL SEQ ID NO: 16, 25-VH SEQ ID NO: 17, 25-VL SEQ ID NO: 18, 31-VH SEQ ID NO: 19, 31-VL SEQ ID NO: 20), where sequence number 26 is the same as sequence number 22. Antibody 13 was humanized using the CDR transplantation method. VH was aligned with the human IGHV germline sequence, and IGHV1-3*04 and IGHV1-2*02 were selected for IGHV and IGHJ1*01 was selected for IGHJ. VL was aligned with the human IGKV germline sequence, and FM164408_IGKV2-30*02_Homo and IGKV2-29*02 were selected for IGKV and IGKJ4*02 was selected for IGKJ. Because the 13 antibody VH CDR2 has a potential glycosylation motif, to avoid glycosylation affecting antibody binding activity, six antibodies were ultimately obtained through engineering modification (hu13-A1-H SEQ ID NO: 21, hu13-A1-L SEQ ID NO: 22), hu13-A1-NQ-H SEQ ID NO: 23, hu13-A1-NQ-L SEQ ID NO: 24), hu123-A1-SA-H (SEQ ID NO: 25, hu123-A1-SA-L SEQ ID NO: 26), hu13-B2-H (SEQ ID NO: 27, hu13-B2-L SEQ ID NO: 28), hu12-B2-NQ-H (SEQ ID NO: 29, hu12-B2-NQ-L SEQ ID NO: 30), and hu13-B2-SA-H (SEQ ID NO: 31, hu13-B2-SA-L SEQ ID NO: 28). NO: 32).

[0135] The binding activity of humanized antibodies to antigens was detected by ELISA. LGR5-his was diluted to 1 µg / mL with PBS, and 100 µL / well was added to the ELISA plate and incubated overnight at 4°C. After washing three times with PBST, 200 µL / well of 3% BSA was added, and the plate was incubated at room temperature for 2 h. After washing three times with PBST, 100 µL / well of humanized antibody was added, initially at 10 µg / mL, and serially diluted 3-fold, and incubated at room temperature for 1 h. After washing three times with PBST, 100 µL / well of 1:5000 diluted goat anti-human huFc secondary antibody (Jackson, 109-035-098) was added, and the plate was incubated at room temperature for 1 h. After washing three times with PBST, 100 μL / well of TMB solution was added for color development. After 10 min of development, 50 μL / well of 1 M H2SO4 was added to terminate the reaction. The absorbance (OD450 nM) of each well was measured using a microplate reader, and data were processed using GraphPad Prism 8.3.

[0136] The results are as follows Figure 7 As shown, all humanized antibodies bound to the antigen, among which three antibodies (hu13-B2, hu13-B2-NQ, and hu13-B2-SA) exhibited superior binding activity compared to LGAB.

[0137] Example 8. Detection of binding of humanized antibody to 293T-LGR5 cells Flow cytometry was performed using HEK293-LGR5 cells (Xichang Biotechnology) that expressed the antibody. 1E+05 cells / well were added to a U-shaped plate, along with humanized antibody and control antibody LGAB, initially at 30 μg / mL, serially diluted 10 times in 3-fold increments, 100 μL / well, and incubated at 4°C for 45 min. Cells were washed twice with 1% FBS-PBS, and anti-Human FC-FITC secondary antibody (Jackson, 109-095-098) was added, diluted 1:200, and incubated at 4°C for 45 min. Cells were washed twice with 1% FBS-PBS, resuspended in 150 μL of 1% FBS-PBS, and then analyzed by flow cytometry. Data were processed using GraphPad Prism 8.3.

[0138] The results are as follows Figure 8 As shown, the humanized LGR5 antibodies hu13-A1, hu13-A1-NQ, and hu13-A1-SA bound weakly to HEK293-humanLGR5 cells compared to LGAB, while hu13-B2, hu13-B2-NQ, and hu13-B2-SA bound better to HEK293-humanLGR5 cells than LGAB.

[0139] Example 9. Humanized Antibody Affinity Detection Humanized antibodies were diluted to 15 μg / mL using HBS-EP + Running buffer and captured using a protein A chip for 30 s. The antigen was serially diluted 2-fold using HBS-EP + Running buffer (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 0 nM). The sample injection sequence, binding time (180 s), dissociation time (180 s), and regeneration reagent (50 mM NaOH) were then configured. Data analysis was performed using Biacore T200 Evaluation Software (Version 3.2, GE Healthcare, USA) after sample injection.

[0140] The results are as follows Figure 9 As shown, the two antibodies (B2-NQ and B2-SA) showed an affinity of 280-826 pM for the antigen, which was superior to the control antibody LGAB (1.29 nM).

[0141] Table 1: SPR Detection Data Example 10. Detection of humanized antibody endocytosis efficiency The endocytosis efficiency of humanized antibodies was detected using a temperature difference method. HEK293-LGR5-1 cells (a LGR5 high-expression cell line, Xichang Biotechnology), at 1E+05 cells / well, were incubated with 10 μg / mL of humanized antibody at 4°C for 1 h. After washing twice with pre-chilled PBS, the cells were resuspended in pre-warmed PBS and incubated at 37°C. Cells were collected at 0 h, 1 h, and 4 h, respectively. Goat anti-human huFc FITC secondary antibody (Jackson, 109-095-098) was added, and the cells were incubated at 4°C for 30 min. After washing twice with pre-chilled PBS, the cell surface antibody signal was detected by flow cytometry. Data processing: Internalization% = 100 – (MFIt=x – MFIt=x background) / (MFIt=0 – MFIt=0 background) * 100 The results are as follows Figure 10 As shown, all antibodies exhibit endocytosis activity, with an endocytosis efficiency greater than 80% within 1 hour.

[0142] Table 2: Results of Humanized Antibody Endocytosis Detection The sequences used in this invention are summarized as follows: Note: The sequence of antibody 22 is the same as that of antibody 26; Antibodies 18, 20, and 22 have the same CDR region, but differ by one amino acid residue in their FR region.

[0143] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A monoclonal antibody targeting LGR5 or an antigen-binding fragment thereof, said monoclonal antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising a complementarity-determining region (CDR) as follows: CDR1 contains the amino acid sequence shown in SEQ ID NO: 33, 39, 47 or 51; CDR2 contains the amino acid sequence shown in SEQ ID NO: 34, 40, 45, 48 or 52; CDR3 contains the amino acid sequence shown in SEQ ID NO: 35, 41, 49 or 53.

2. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein, The light chain variable region includes the following complementary determinant region (CDR): CDR1 contains the amino acid sequence shown in SEQ ID NO: 36, 42, 46 or 54; CDR2 contains the amino acid sequence shown in SEQ ID NO: 37, 43, or 55; CDR3 contains the amino acid sequence shown in SEQ ID NO: 38, 44, 50 or 56.

3. The monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region complementarity-determining region (CDR) as shown below: (1) The complementary determination region (CDR) of the heavy chain variable region is as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 33, CDR2 containing the amino acid sequence shown in SEQ ID NO: 34, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 35; The complementary determinant regions (CDRs) of the light chain variable region are shown below: CDR1 containing the amino acid sequence shown in SEQ ID NO: 36, CDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and CDR3 containing the amino acid sequence shown in SEQ ID NO:

38. (2) The complementary determination region (CDR) of the heavy chain variable region is as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 39, CDR2 containing the amino acid sequence shown in SEQ ID NO: 45, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 41; The complementary determinant regions (CDRs) of the light chain variable region are shown below: CDR1 containing the amino acid sequence shown in SEQ ID NO: 42, CDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR3 containing the amino acid sequence shown in SEQ ID NO:

44. (3) The complementary determination region (CDR) of the heavy chain variable region is as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 39, CDR2 containing the amino acid sequence shown in SEQ ID NO: 45, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 41; The complementary determinant regions (CDRs) of the light chain variable region are as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 46, CDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR3 containing the amino acid sequence shown in SEQ ID NO:

44. (4) The complementary determination region (CDR) of the heavy chain variable region is as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 47, CDR2 containing the amino acid sequence shown in SEQ ID NO: 48, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 49; The complementary determinant regions (CDRs) of the light chain variable regions are as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 46, CDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 50; or (5) The heavy chain variable region complementarity-determining regions (CDRs) are as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 51, CDR2 containing the amino acid sequence shown in SEQ ID NO: 52, and CDR3 containing the amino acid sequence shown in SEQ ID NO:

53. The complementary determinant regions (CDRs) of the light chain variable regions are as follows: CDR1 containing the amino acid sequence shown in SEQ ID NO: 54, CDR2 containing the amino acid sequence shown in SEQ ID NO: 55, and CDR3 containing the amino acid sequence shown in SEQ ID NO:

56.

4. The monoclonal antibody or its antigen-binding fragment as described in claim 3, wherein, The monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and / or a light chain variable region as shown below: The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 7, 9, 11, 13, 15, 17 or 19; The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 8, 10, 12, 14, 16, 18 or 20.

5. A humanized monoclonal antibody or antigen-binding fragment thereof targeting LGR5, wherein the humanized monoclonal antibody or antigen-binding fragment thereof is obtained by humanization of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4.

6. A recombinant protein, said recombinant protein having: (i) the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-4, or the humanized monoclonal antibody or antigen-binding fragment thereof as described in claim 5; and (ii) Optional tag sequences to assist in expression and / or purification.

7. A chimeric antigen receptor (CAR) wherein the antigen-binding domain of the chimeric antigen receptor contains a single-chain variable region sequence scFv of an antibody targeting LGR5 prepared using the monoclonal antibody or its antigen-binding fragment according to any one of claims 1-4, or the humanized monoclonal antibody or its antigen-binding fragment according to claim 5.

8. An isolated polynucleotide molecule, said polynucleotide molecule encoding a monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-4, or a humanized monoclonal antibody or antigen-binding fragment thereof as described in claim 5, a recombinant protein as described in claim 6, or a chimeric antigen receptor CAR as described in claim 7.

9. A carrier comprising the polynucleotide molecule of claim 8.

10. A host cell containing, in which the vector of claim 9 or the chromosome has an integrated exogenous polynucleotide molecule of claim 8.

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