Anti-lrrc15 antibodies and uses thereof

Abstract

The present application provides anti-LRRC15 antibodies, nucleic acid molecules encoding the antibodies, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or vectors, and conjugates comprising the anti-LRRC15 antibodies and pharmaceutical compositions thereof. The present application also provides methods of making the anti-LRRC15 antibodies and uses thereof.

Description

[0001] Related Applications

[0002] This application claims priority to Chinese application CN202510542089.2, filed on April 27, 2025. The entire contents of the aforementioned application are expressly incorporated herein by reference. Technical Field

[0003] This application relates to the field of biomedicine; specifically, it relates to anti-LRRC15 antibodies and their uses. Background Technology

[0004] LRRC15 (Leucine-Rich Repeat-Containing 15) is a type I transmembrane protein belonging to the leucine-rich repeat (LRR) superfamily. It is highly expressed in the tumor microenvironment (TME) and mesenchymal-derived tumors, and has become an emerging target for cancer therapy. In recent years, some progress has been made in the development of antibody drugs targeting LRRC15, such as antibody-drug conjugates (ADCs) and bispecific antibody drugs (BsAbs).

[0005] Nanobodies, also known as VHH antibodies, offer numerous advantages over traditional antibodies due to their unique structure. These advantages include smaller molecular size and stronger tissue penetration, superior stability, multivalent / multifunctional design, ease of humanization, and wide applicability. Therefore, nanobodies hold significant value in disease treatment and diagnosis, and are gradually becoming an emerging force in next-generation therapeutic biopharmaceuticals and clinical diagnostic reagents.

[0006] There is a need in this field to develop nanobody drugs targeting the LRRC15 protein. Summary of the Invention

[0007] In view of this, in a first aspect, this application provides an anti-LRRC15 antibody or an antigen-binding fragment thereof.

[0008] In some embodiments, the anti-LRRC15 antibody or its antigen-binding fragment provided in this application comprises a VHH chain, wherein the VHH chain comprises CDR1, CDR2, and CDR3, wherein: The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 3; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 7; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 9; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 12; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 14, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 15; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 15; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 15; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 21, the amino acid sequence of CDR2 is shown in SEQ ID NO: 22, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 23; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 21, the amino acid sequence of CDR2 is shown in SEQ ID NO: 25, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 26; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 21, the amino acid sequence of CDR2 is shown in SEQ ID NO: 22, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 28; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 30, the amino acid sequence of CDR2 is shown in SEQ ID NO: 25, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 31. The amino acid sequence of CDR1 is shown in SEQ ID NO: 21, the amino acid sequence of CDR2 is shown in SEQ ID NO: 25, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 33; or The amino acid sequence of CDR1 is shown in SEQ ID NO:35, the amino acid sequence of CDR2 is shown in SEQ ID NO:36, and the amino acid sequence of CDR3 is shown in SEQ ID NO:37; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 30, the amino acid sequence of CDR2 is shown in SEQ ID NO: 25, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 39. The above CDR sequence is defined according to Kabat.

[0009] In some embodiments, the anti-LRRC15 antibody comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 4, 5, 8, 10, 13, 16, 17, 18, 19, 20, 24, 27, 29, 32, 34, 38, or 40.

[0010] In some embodiments, the anti-LRRC15 antibody consists of the amino acid sequence shown in SEQ ID NO: 4, 5, 8, 10, 13, 16, 17, 18, 19, 20, 24, 27, 29, 32, 34, 38 or 40.

[0011] In some embodiments, the anti-LRRC15 antibody comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 43.

[0012] In some embodiments, the anti-LRRC15 antibody consists of the amino acid sequence shown in SEQ ID NO: 43.

[0013] In some embodiments, the anti-LRRC15 antibody is a VHH antibody.

[0014] In other embodiments, the anti-LRRC15 antibody is in monovalent or multivalent (e.g., bivalent, trivalent, or quadrivalent).

[0015] In some embodiments, the anti-LRRC15 antibody is a VHH-Fc antibody.

[0016] In some specific implementations, the Fc fragment is derived from human IgG.

[0017] In some other specific embodiments, the Fc fragment is derived from human IgG1.

[0018] In some other specific embodiments, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 42.

[0019] In some implementations, the VHH-Fc antibody is in a multivalent (e.g., bivalent) form.

[0020] Secondly, this application provides isolated nucleic acid molecules that encode the anti-LRRC15 antibody or its antigen-binding fragment as described in the first aspect.

[0021] In some implementations, this application also provides a vector, such as an expression vector, which contains the aforementioned nucleic acid molecules.

[0022] In some embodiments, this application further provides a host cell containing the aforementioned nucleic acid molecules or vectors.

[0023] Thirdly, this application provides an antibody conjugate comprising the above-mentioned anti-LRRC15 antibody or its antigen-binding fragment, linker and effector.

[0024] In some implementation schemes, the effectors are selected from: radionuclides, antitumor agents, immunomodulators, bioreaction modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, imaging agents, chemiluminescent compounds, enzymes, and metal ions.

[0025] In some embodiments, the effector in the antibody conjugate provided in this application is a cytotoxic drug.

[0026] Fourthly, this application provides a radionuclide-labeled antibody conjugate comprising the above-mentioned anti-LRRC15 antibody or its antigen-binding fragment and a radionuclide-chelating agent.

[0027] In some embodiments, the aforementioned radionuclides include alpha-ray nuclides, gamma-ray nuclides, beta-ray nuclides, or positron-emitting nuclides.

[0028] Fifthly, this application provides a pharmaceutical composition comprising the above-mentioned anti-LRRC15 antibody or its antigen-binding portion, the above-mentioned nucleic acid molecule, vector or host cell, the above-mentioned antibody conjugate, or the above-mentioned radionuclide-labeled antibody conjugate.

[0029] Sixthly, this application provides the use of the above-mentioned anti-LRRC15 antibody or its antigen-binding portion, the above-mentioned nucleic acid molecule, the above-mentioned carrier or host cell, the above-mentioned antibody conjugate, the above-mentioned radionuclide-labeled antibody conjugate, or the above-mentioned pharmaceutical composition in the preparation of a medicament for the prevention, diagnosis and / or treatment of diseases characterized by LRRC15 expression (e.g., LRRC15-positive tumors).

[0030] In addition, this application also provides a method for preventing, diagnosing and / or treating diseases characterized by LRRC15 expression in an individual (e.g., LRRC15-positive tumors), comprising administering the above-mentioned antibody conjugate, the above-mentioned radionuclide-labeled antibody conjugate, or a pharmaceutical composition thereof to an individual in need.

[0031] In some implementations, the diseases characterized by LRRC15 expression involved in the above uses or methods are selected from: sarcoma, glioblastoma, melanoma, breast cancer (e.g., HER2-negative breast cancer, HER2-positive breast cancer, HR+ breast cancer, triple-negative breast cancer), head and neck cancer, head and neck squamous cell carcinoma, lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), testicular cancer, and squamous cell carcinoma, etc. Attached Figure Description

[0032] Figure 1 The results show the cell binding activity assay results for the candidate antibodies.

[0033] Figure 2 The results show the cell binding activity of the VHH antibody in this application.

[0034] Figure 3 This is the result of the endocytic activity test of the VHH antibody in this application.

[0035] Figure 4 The results show the cell-binding activity of the mature humanized VHH antibody for this application.

[0036] Figure 5 The results show the endocytic activity assay of the mature humanized VHH antibody with affinity as described in this application.

[0037] Figure 6 The results show the cell binding activity of the VHH antibody of this application conjugated with a chelating agent.

[0038] Figure 7 The results show the cell binding activity of the VHH antibody of this application conjugated with a chelating agent.

[0039] Figure 8 PET / CT images at different time points after administration of [68Ga]5A12-h2m9-06609-GSC-NOTA.

[0040] Figure 9 PET / CT images at different time points after administration of [68Ga]11E06-h8-107-GSC-NOTA.

[0041] Figure 10PET / CT images at different time points after administration of [68Ga]11E06-h8-m7-GSC-H3RESCA.

[0042] Figure 11 PET / CT images at different time points after administration of [68Ga]11E06-h8-107-H3RESCA.

[0043] Figure 12 PET / CT images at different time points after administration of [18F]11E06-m7-GSC-H3RESCA.

[0044] Figure 13 The results of tumor volume measurement in experimental animals after drug administration.

[0045] Figure 14 The results of weight measurement of experimental animals after administration of the drug.

[0046] Sequence Description

[0047] SEQ ID NO: 1-3 show the amino acid sequences of CDR1, CDR2 and CDR3 of the parental antibody clone 5A12 against LRRC15, respectively.

[0048] SEQ ID NO: 4 shows the amino acid sequence of the VHH chain of the maternal antibody clone 5A12 against LRRC15.

[0049] SEQ ID NO: 5, 8 and 10 show the amino acid sequences of the VHH chains of humanized antibody clones 5A12-h2m1, 5A12-h2m8 and 5A12-h2m9 based on the maternal antibody clone 5A12, respectively. SEQ ID NO: 1-3 show the amino acid sequences of CDR1, CDR2 and CDR3 of 5A12-h2m1, respectively; SEQ ID NO: 1, 6 and 7 show the amino acid sequences of CDR1, CDR2 and CDR3 of 5A12-h2m8, respectively; SEQ ID NO: 1, 6 and 9 show the amino acid sequences of CDR1, CDR2 and CDR3 of 5A12-h2m9, respectively.

[0050] SEQ ID NO: 13 and 16 show the amino acid sequences of the VHH chains of the affinity-matured molecules 5A12-h2m9-066 and 5A12-h2m9-088 based on the humanized antibody clone 5A12-h2m9, respectively. SEQ ID NO: 1, 11 and 12 show the amino acid sequences of CDR1, CDR2 and CDR3 of 5A12-h2m9-066, respectively. SEQ ID NO: 1, 14 and 15 show the amino acid sequences of CDR1, CDR2 and CDR3 of 5A12-h2m9-088, respectively.

[0051] SEQ ID NO: 17-20 show the amino acid sequences of the VHH chains of the variant molecules 5A12-h2m9-066A, 5A12-h2m9-06605, 5A12-h2m9-06607 and 5A12-h2m9-06609, respectively, based on the affinity-matured molecule 5A12-h2m9-066. SEQ ID NO: 1, 11 and 15 show the amino acid sequences of CDR1, CDR2 and CDR3 of 5A12-h2m9-066A, 5A12-h2m9-06605 and 5A12-h2m9-06609, respectively; SEQ ID NO: 1, 6 and 15 show the amino acid sequences of CDR1, CDR2 and CDR3 of 5A12-h2m9-06607, respectively.

[0052] SEQ ID NO: 21-23 show the amino acid sequences of CDR1, CDR2 and CDR3 of the parental antibody clone 11E06 against LRRC15, respectively.

[0053] SEQ ID NO: 24 shows the amino acid sequence of the VHH chain of the maternal antibody clone 11E06 against LRRC15.

[0054] SEQ ID NO: 27 and 29 show the amino acid sequences of the VHH chains of humanized antibody clones 11E06-h8 and 11E06-h6m1 based on the maternal antibody clone 11E06, respectively. SEQ ID NO: 21, 25 and 26 show the amino acid sequences of CDR1, CDR2 and CDR3 of 11E06-h8, respectively. SEQ ID NO: 21, 22 and 28 show the amino acid sequences of CDR1, CDR2 and CDR3 of 11E06-h6m1, respectively.

[0055] SEQ ID NO: 32, 34 and 43 show the amino acid sequences of the VHH chains of molecules 11E06-h8-102, 11E06-h8-107 and 11E06-h8-m7 after affinity maturation based on the humanized antibody clone 11E06-h8, respectively. SEQ ID NO: 30, 25 and 31 show the amino acid sequences of CDR1, CDR2 and CDR3 of 11E06-h8-102, respectively; SEQ ID NO: 21, 25 and 33 show the amino acid sequences of CDR1, CDR2 and CDR3 of 11E06-h8-107, respectively; SEQ ID NO: 30, 25 and 39 show the amino acid sequences of CDR1, CDR2 and CDR3 of 11E06-h8-m7, respectively.

[0056] SEQ ID NO: 38 and 40 show the amino acid sequences of the VHH chains of 11E06-h8-m3-2C and 11E06-h8-m7-2C, respectively, of the variant molecules of the humanized antibody clone 11E06-h8 (with the introduction of 2 cysteine ​​molecules), wherein SEQ ID NO: 35-37 show the amino acid sequences of CDR1, CDR2 and CDR3 of 11E06-h8-m3-2C, respectively; SEQ ID NO: 30, 25 and 39 show the amino acid sequences of CDR1, CDR2 and CDR3 of 11E06-h8-m7-2C, respectively.

[0057] SEQ ID NO: 41 shows the amino acid sequence of human LRRC15 protein, wherein amino acid sequence 22-538 is an extracellular epitope for VHH antibody binding.

[0058] SEQ ID NO: 42 shows the amino acid sequence of a human IgG1 Fc fragment linked to a VHH antibody against LRRC15.

[0059] SEQ ID NO: 44 shows the amino acid sequence of VHH in VHH-Fc-Isotype-DXd.

[0060] SEQ ID NO: 45 shows the amino acid sequence of the Fc fragment in 5A12-h2m9-088-Fc-DXd, 11E06-h8-m7-Fc-DXd, and 11E06-h8-107-Fc-DXd. Detailed Implementation

[0061] The inventors of this application have prepared novel antibodies against the LRRC15 protein using antibody engineering technology. In various aspects of this application, novel antibodies against the LRRC15 protein, nucleic acid molecules encoding said antibodies, vectors containing said nucleic acid molecules, cells containing said nucleic acid molecules or vectors, conjugates containing said antibodies, methods for preparing and purifying antibodies, and medical and biological uses of said antibodies are provided.

[0062] Unless otherwise specified, this application employs conventional molecular biology, microbiology, cell biology, biochemistry, and immunology techniques, and the terms used have the meanings commonly understood by those skilled in the art.

[0063] In this paper, the abbreviations of 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. The antibody variable region sequence adopts the Kabat coding scheme.

[0064] In this article, “anti-LRRC15 antibody” is an antibody against the LRRC15 protein (e.g., human LRRC15 protein), that is, an antibody that specifically binds to the LRRC15 protein (e.g., human LRRC15 protein), including antibodies containing the VHH chain, such as VHH antibody and VHH-Fc antibody.

[0065] In this article, “VHH antibody”, also known as “nanobody (Nb)” or “sdAb”, is a variable region (VHH) of a heavy chain antibody that is naturally missing a light chain. Its antigen binding site depends only on three complementarity-determining regions (CDR1, CDR2, and CDR3).

[0066] In this paper, “VHH-Fc antibody” refers to an engineered fusion protein of VHH and the Fc fragment of immunoglobulin G (IgG), which has both the targeting function of small molecule antibodies and the effector function mediated by Fc. The Fc fragment is derived from the constant region of IgG (e.g., human IgG1) and contains CH2 and CH3 domains.

[0067] In the first aspect, this application provides an anti-LRRC15 antibody or antigen-binding fragment.

[0068] In some embodiments, the anti-LRRC15 antibody provided in this application can be prepared by the following methods: immunizing alpacas with human LRRC15 protein to construct a phage display library; screening target phage clones by liquid-phase cell panning; identifying positive clones using FACS; and expressing and purifying the anti-LRRC15 antibody.

[0069] In some embodiments, the nano-anti-LRRC15 antibody provided in this application can be prepared by the following methods: immunizing alpacas with human LRRC15 protein to construct a yeast display library; screening target yeast clones using magnetic bead sorting technology; identifying positive clones using FACS; and expressing and purifying the anti-LRRC15 antibody.

[0070] In some embodiments, the screened positive clone antibody 5A12 (parental) contains a heavy chain variable region comprising complementarity-determining regions CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 is shown in SYTMG (SEQ ID NO: 1), the amino acid sequence of CDR2 is shown in GIYSDGSTYYADSVKG (SEQ ID NO: 2), and the amino acid sequence of CDR3 is shown in EYYSDPKLLTDFGS (SEQ ID NO: 3). In a specific embodiment, the parental antibody 5A12 has the amino acid sequence shown in SEQ ID NO: 4.

[0071] In a specific implementation scheme, the positive clone antibody 5A12 obtained through screening is humanized to obtain humanized antibody 5A12-h2m1. The amino acid sequence of its CDR1 is shown in SYTMG (SEQ ID NO: 1), the amino acid sequence of its CDR2 is shown in GIYSDGSTYYADSVKG (SEQ ID NO: 2), and the amino acid sequence of its CDR3 is shown in EYYSDPKLLTDFGS (SEQ ID NO: 3). In a specific implementation scheme, humanized antibody 5A12-h2m1 has the amino acid sequence shown in SEQ ID NO: 5.

[0072] In a specific implementation scheme, the positive clone antibody 5A12 obtained through screening is humanized to obtain humanized antibody 5A12-h2m8. The amino acid sequence of its CDR1 is shown in SYTMG (SEQ ID NO: 1), the amino acid sequence of its CDR2 is shown in GIYSDASTYYADSVKG (SEQ ID NO: 6), and the amino acid sequence of its CDR3 is shown in EYYSDAKLLTDFGS (SEQ ID NO: 7). In a specific implementation scheme, humanized antibody 5A12-h2m8 has the amino acid sequence shown in SEQ ID NO: 8.

[0073] In a specific implementation scheme, the positive clone antibody 5A12 obtained through screening is humanized to obtain humanized antibody 5A12-h2m9. The amino acid sequence of its CDR1 is shown in SYTMG (SEQ ID NO: 1), the amino acid sequence of its CDR2 is shown in GIYSDASTYYADSVKG (SEQ ID NO: 6), and the amino acid sequence of its CDR3 is shown in EYYSDARLLTDFGS (SEQ ID NO: 9). In a specific implementation scheme, humanized antibody 5A12-h2m9 has the amino acid sequence shown in SEQ ID NO: 10.

[0074] In a specific implementation scheme, the humanized antibody 5A12-h2m9 undergoes affinity maturation to obtain affinity-matured antibody 5A12-h2m9-066. The amino acid sequence of its CDR1 is shown in SYTMG (SEQ ID NO: 1), the amino acid sequence of its CDR2 is shown in GIFSDASTYYADSVKG (SEQ ID NO: 11), and the amino acid sequence of its CDR3 is shown in EYYLDARLLTDFGS (SEQ ID NO: 12). In a specific implementation scheme, affinity-matured antibody 5A12-h2m9-066 has the amino acid sequence shown in SEQ ID NO: 13.

[0075] In a specific implementation scheme, the humanized antibody 5A12-h2m9 undergoes affinity maturation to obtain affinity-matured antibody 5A12-h2m9-088. The amino acid sequence of its CDR1 is shown in SYTMG (SEQ ID NO: 1), the amino acid sequence of its CDR2 is shown in GIYSDARTYYADSVKG (SEQ ID NO: 14), and the amino acid sequence of its CDR3 is shown in EYYADARLLTDFGS (SEQ ID NO: 15). In a specific implementation scheme, affinity-matured antibody 5A12-h2m9-088 has the amino acid sequence shown in SEQ ID NO: 16.

[0076] In specific embodiments, variants 5A12-h2m9-066A, 5A12-h2m9-06605, and 5A12-h2m9-06609 of the affinity maturation antibody 5A12-h2m9-066 have the following amino acid sequences: CDR1 as shown in SYTMG (SEQ ID NO: 1), CDR2 as shown in GIFSDASTYYADSVKG (SEQ ID NO: 11), and CDR3 as shown in EYYADARLLTDFGS (SEQ ID NO: 15). In a specific embodiment, variant 5A12-h2m9-066A has the amino acid sequence shown in SEQ ID NO: 17. In a specific embodiment, variant 5A12-h2m9-06605 has the amino acid sequence shown in SEQ ID NO: 18. In a specific embodiment, variant 5A12-h2m9-06609 has the amino acid sequence shown in SEQ ID NO: 20.

[0077] In a specific embodiment, variant 5A12-h2m9-06607, based on the affinity maturation antibody 5A12-h2m9-066, has the amino acid sequence of CDR1 as shown in SYTMG (SEQ ID NO: 1), the amino acid sequence of CDR2 as shown in GIYSDASTYYADSVKG (SEQ ID NO: 6), and the amino acid sequence of CDR3 as shown in EYYADARLLTDFGS (SEQ ID NO: 15). In a specific embodiment, variant 5A12-h2m9-06607 has the amino acid sequence shown in SEQ ID NO: 19.

[0078] In some embodiments, the screened positive clone antibody 11E06 (parent) contains a heavy chain variable region comprising complementarity-determining regions CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 is shown as INTMG (SEQ ID NO: 21), the amino acid sequence of CDR2 is shown as EITTGGITNYADSVKG (SEQ ID NO: 22), and the amino acid sequence of CDR3 is shown as GDGMWYGRSEYDY (SEQ ID NO: 23). In a specific embodiment, parent antibody 11E06 has the amino acid sequence shown in SEQ ID NO: 24.

[0079] In a specific implementation scheme, the positive clone antibody 11E06 obtained through screening is humanized to obtain humanized antibody 11E06-h8. The amino acid sequence of its CDR1 is shown in INTMG (SEQ ID NO: 21), the amino acid sequence of its CDR2 is shown in EITTGGITNYADSVRG (SEQ ID NO: 25), and the amino acid sequence of its CDR3 is shown in GDTLWYGRSEYDY (SEQ ID NO: 26). In a specific implementation scheme, humanized antibody 11E06-h8 has the amino acid sequence shown in SEQ ID NO: 27.

[0080] In a specific implementation scheme, the positive clone antibody 11E06 obtained through screening is humanized to obtain humanized antibody 11E06-h6m1. The amino acid sequence of its CDR1 is shown in INTMG (SEQ ID NO: 21), the amino acid sequence of its CDR2 is shown in EITTGGITNYADSVKG (SEQ ID NO: 22), and the amino acid sequence of its CDR3 is shown in GDTMWYGRSEYDY (SEQ ID NO: 28). In a specific implementation scheme, humanized antibody 11E06-h6m1 has the amino acid sequence shown in SEQ ID NO: 29.

[0081] In a specific embodiment, the humanized antibody 11E06-h8 is subjected to affinity maturation to obtain affinity-matured antibody 11E06-h8-102. The amino acid sequence of its CDR1 is shown as YNTMG (SEQ ID NO: 30), the amino acid sequence of its CDR2 is shown as EITTGGITNYADSVRG (SEQ ID NO: 25), and the amino acid sequence of its CDR3 is shown as RDTLWYGRSGYDY (SEQ ID NO: 31). In a specific embodiment, affinity-matured antibody 11E06-h8-102 has the amino acid sequence shown in SEQ ID NO: 32.

[0082] In a specific embodiment, the humanized antibody 11E06-h8 is subjected to affinity maturation to obtain affinity-matured antibody 11E06-h8-107. The amino acid sequence of its CDR1 is shown in INTMG (SEQ ID NO: 21), the amino acid sequence of its CDR2 is shown in EITTGGITNYADSVRG (SEQ ID NO: 25), and the amino acid sequence of its CDR3 is shown in LDTDWYGRSEYDY (SEQ ID NO: 33). In a specific embodiment, affinity-matured antibody 11E06-h8-107 has the amino acid sequence shown in SEQ ID NO: 34.

[0083] In a specific embodiment, the humanized antibody 11E06-h8 is subjected to affinity maturation to obtain affinity-matured antibody 11E06-h8-m7, the amino acid sequence of which is shown as YNTMG (SEQ ID NO: 30), the amino acid sequence of which is shown as EITTGGITNYADSVRG (SEQ ID NO: 25), and the amino acid sequence of which is shown as RDTLWYGRPEYDY (SEQ ID NO: 39). In a specific embodiment, affinity-matured antibody 11E06-h8-m7 has the amino acid sequence shown in SEQ ID NO: 43.

[0084] In a specific embodiment, variant 11E06-h8-m3-2C, based on the humanized antibody 11E06-h8, has the following amino acid sequence: CDR1 as shown in IETMG (SEQ ID NO: 35), CDR2 as shown in EILTGGITNYADSVRG (SEQ ID NO: 36), and CDR3 as shown in LDTDWYGRPEYDY (SEQ ID NO: 37). In another specific embodiment, variant 11E06-h8-m3-2C has the amino acid sequence shown in SEQ ID NO: 38.

[0085] In a specific embodiment, variant 11E06-h8-m7-2C, based on the humanized antibody 11E06-h8, has the following amino acid sequence: CDR1 as shown in YNTMG (SEQ ID NO: 30), CDR2 as shown in EITTGGITNYADSVRG (SEQ ID NO: 25), and CDR3 as shown in RDTLWYGRPEYDY (SEQ ID NO: 39). In another specific embodiment, variant 11E06-h8-m7-2C has the amino acid sequence shown in SEQ ID NO: 40.

[0086] The term "identity" is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and vacancy introduction, reaching the maximum percentage of identity if desired. The methods and computer programs used for alignment are well known in the art.

[0087] In some embodiments, the amino acid sequence of the antibody provided in this application may be modified in various ways known in the art, such as introducing site-directed amino acid deletions, insertions or substitutions (1-25 residues) in the CDR region or frame region (FR) of VHH, or fusing the VHH antibody with functional elements, such as Fc tags, His tags or flexible linker peptides (e.g., G4SC, GSC).

[0088] In this paper, when measuring cell binding activity, the "half-maximum effective concentration (EC50)" was used. 50 "Value" refers to the antibody concentration at which 50% binding occurs to a cell surface target antigen (e.g., LRRC15 protein). EC 50 The lower the value, the stronger the antibody binding ability and the greater the affinity. "Bmax" represents the maximum capacity that the antibody can bind to the target antigen on the cell surface, reflecting the total receptor density or number of binding sites of the target antigen on the cell surface. The higher the Bmax, the higher the density of the target antigen on the cell surface. When measuring antibody affinity, "binding constant Ka" represents the rate at which the antibody binds to the antigen. The larger the Ka, the higher the affinity. "Dissociation constant Kd" represents the dissociation rate of the antibody-antigen complex into free antibody and antigen. The smaller the Kd, the higher the affinity. "Equilibrium dissociation constant KD" is the ratio of the binding rate constant (kon) to the dissociation rate constant (koff). It is a global indicator of the antibody-antigen binding strength in equilibrium. The smaller the KD, the higher the affinity.

[0089] In a second aspect, this application provides an isolated nucleic acid molecule encoding an anti-LRRC15 antibody or antigen-binding fragment, a vector containing the nucleic acid molecule, such as an expression vector, and a host cell containing the nucleic acid molecule or the vector.

[0090] In some embodiments, the nucleic acid molecule is operatively linked to a regulatory sequence that can be recognized by a host cell transformed with the vector. The choice of vector depends on the choice of host cell. In some embodiments, the host cell is a eukaryotic cell, such as yeast cells, mammalian cells, or other cells suitable for preparing antibodies or their antigen-binding fragments. In some embodiments, the host cell is a prokaryotic cell, such as *Escherichia coli*.

[0091] In a third aspect, this application provides antibody conjugates comprising anti-LRRC15 antibody or antigen-binding fragments.

[0092] In this paper, "antibody conjugates" are a class of composite molecules that conjugate a target carrier (e.g., an antibody) with an effector payload through a specific linking technology, thereby precisely delivering the effector payload through the targeted recognition of the antibody.

[0093] In some implementations, the anti-LRRC15 antibody or antigen-binding fragment provided in this application can be conjugated with a variety of different effectors to form antibody conjugates.

[0094] In some implementation schemes, effectors that can be conjugated with the antibodies provided in this application include, but are not limited to: radionuclides, antitumor agents, immunomodulators, bioreaction modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, imaging agents, chemiluminescent compounds, enzymes, metal ions, etc.

[0095] In some embodiments, the anti-LRRC15 antibody or antigen-binding fragment provided in this application is linked to the effector via a linker. In some embodiments, the linker includes cleavable linkers and non-cleavable linkers. In specific embodiments, those skilled in the art can select a suitable linker based on the type of effector.

[0096] In some embodiments, the antibody conjugate provided in this application is: VHH-Fc-Mal-GGFG-DXd. Wherein, Mal is maleimide, GGFG is a tetrapeptide short chain, and DXd is an eczemacon derivative used in antibody-drug conjugates, with the following structural formula: .

[0097] In some implementations, the antibody conjugate provided in this application is: 11E06-h8-m7-Fc-Mal-GGFG-DXd.

[0098] In a fourth aspect, this application provides an antibody conjugate comprising the anti-LRRC15 antibody or antigen-binding fragment of this application and a radionuclide, also known as a radionuclide-labeled antibody conjugate or radionuclide conjugate (RDC), which obtains diagnostic and / or therapeutic functions by linking different types of radionuclides to the anti-LRRC15 antibody or antigen-binding fragment.

[0099] In some embodiments, the radionuclide-labeled antibody conjugate provided in this application comprises the anti-LRRC15 antibody or antigen-binding fragment of this application and a radionuclide-chelating agent.

[0100] In some embodiments, the radionuclide-labeled antibody conjugates provided in this application further include a linker.

[0101] Any radionuclide known in the art that can be used for treatment or diagnosis can be conjugated to the antibody of this application. For example, diagnostic radionuclides that are mainly gamma-ray or positron-emitting radionuclides, and therapeutic radionuclides that are mainly beta-ray or alpha-ray radionuclides.

[0102] In some implementations, the applicable radionuclides include, but are not limited to: 11 C 13 N、 15 O、¹ 8 F, 32 P, 44 Sc、 47 Sc、 51 Mn, 52m Mn, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 68 Ge 72 As、 75 Br、 76 Br、 82m Rb、 83 Sr、 86 Y、 89 Sr、 89 Zr、 90 Y、 99m Tc, 105 Rh、 110 In、 111 In、 117 mSn, 120 I, 123 I, 124 I, 125 I、¹³¹I、 134 Ce、 148 Gd, 149 Tb, 149 Pm, 152 Tb, 153 Sm、 154-158 Gd, 160 Gd, 161 Tb, 165 Ho、 166 Ho、 167 Tm、 175 Yb、¹ 77 Lu、 186 Re、 188 Re、 197 Au、 198 Au、 199 Au、 201 Ti、 209Bi、 211 At、 212 Pb, 212 Bi、 213 Bi、 213 Po、²²³Ra、 223 Fr、²² 5 Ac、 227 Th and 229 Th.

[0103] Any chelating agent known in the art that can be used to coordinate with a radionuclide can be used in this application. The chelating agent stably binds the radionuclide to the antibody, preventing premature dissociation of the radionuclide during in vivo circulation and avoiding damage to non-target tissues.

[0104] In some embodiments, suitable chelating agents include, but are not limited to: 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), N,N'-bis(2-hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED-CC), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) or triazacyclononane phosphate (TRAP), EDTA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, AAZTA, BAT, BAT-TM, Crown, Cyclen, DO2A, CB-DO2A, DO3A, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, MACROPA, HEHA, DOTA-GA, p-BnDOTA, p-Bn-SCN-DOTA, NH2-DOTA, NH2-DOTA-GA, p-NC S-Bn-DOTA-GA, p-NH2-Bn-OxODO3A, p-SCN-Bn-oxo-DO3A, NODA-GA, NH2-NODA-GA, p-NCS-Bn-NODA-GA, p-NH2Bn-NOTA, p-SCN-Bn-NOTA, NCS-MP-NOD A, NH2-MPAA-NODA, PCTA, p-NH2-Bn-PCTA, pSCN-Bn-PCTA, p-SCN-Bn-HEHA, H2-MACROPA-NCS, H1-MACROPA, H2-MACROPA-NH2, H4OCTAPA, tetra-(S,S,S,S)-Me-DOTA, SHBED, (HBED-CC)TFP, DMSA, DMPS, DHLA, tetra-(S,S,S,S)-Et-DOTA, tetra-(S,S,S,S)-iBu-DOTA, and maleimide-nBu-DOTA and their derivatives.

[0105] In some implementations, suitable chelating agents include, but are not limited to: H3RESCA, (+-) H3RESCA-TFP, and (+-) H3RESCA-Mal.

[0106] In some embodiments, the radionuclide-labeled antibody conjugates provided in this application are: radionuclide-VHH-GSC-NOTA, radionuclide-VHH-NOTA, radionuclide-VHH-GSC-H3RESCA, or radionuclide-VHH-H3RESCA. In this document, "radionuclide" refers to a radionuclide; VHH-GSC-NOTA, VHH-GSC-H3RESCA, VHH-NOTA, and VHH-H3RESCA are radionuclide-labeled precursors, with the radionuclide labeled onto the chelating agents NOA and H3RESCA, chelating with the multidentate chelating agents.

[0107] In this document, "VHH-chelating agent" is an abbreviation for "VHH-Mal-chelating agent," "VHH-GSC-chelating agent," or "VHH-GSC-Mal-chelating agent," and these terms can be used interchangeably. For example, "VHH-NOTA" is an abbreviation for "VHH-Mal-NOTA," "VHH-GSC-NOTA," or "VHH-GSC-Mal-NOTA," and "VHH-H3RESCA" is an abbreviation for "VHH-Mal-H3RESCA," "VHH-GSC-H3RESCA," or "VHH-GSC-Mal-H3RESCA." For the abbreviations "VHH-NOTA" and "VHH-Mal-NOTA," the specific linker used can be determined according to the specific embodiment. Variant sequences obtained by introducing one or more cysteine ​​pairs into a VHH sequence can be directly coupled to a Mal-chelating agent, such as 11E06-h8-m3-2C-NOTA (an abbreviation of 11E06-h8-m3-2C-Mal-NOTA). VHH sequences without two cysteine ​​pairs can generally be coupled to a Mal-chelating agent via a linker (e.g., G4SC, GSC), such as 11E06-h8-m7-GSC-H3RESCA (an abbreviation of 11E06-h8-m7-GSC-Mal-NOTA).

[0108] In the specific implementation scheme, the radionuclide-labeled antibody conjugates provided in this application are selected from: 68 Ga-anti-LRRC15 antibody-GSC-Mal-NOTA 68 Ga-anti-LRRC15 antibody-GSC-Mal-H3RESCA, 18 F-anti-LRRC15 antibody-GSC-Mal-NOTA 18 F-anti-LRRC15 antibody-GSC-Mal-H3RESCA.

[0109] In the specific implementation scheme, the radionuclide-labeled antibody conjugates provided in this application are selected from: 68Ga-5A12-h2m9-06609-GSC-NOTA, 68 Ga-11E06-h8-107-GSC-NOTA 68 Ga-11E06-h8-m7-GSC-NOTA 68 Ga-11E06-h8-107-GSC-H3RESCA, 68 Ga-11E06-h8-m7-GSC-H3RESCA 18 F-11E06-h8-m7-GSC-H3RESCA 18 F-11E06-h8-107-GSC-H3RESCA.

[0110] In a fifth aspect, this application provides pharmaceutical compositions comprising the different forms of antibodies described above. For example, pharmaceutical compositions comprising any one of the antibodies described above, pharmaceutical compositions comprising nucleic acid molecules encoding any one of the antibodies described above, pharmaceutical compositions comprising a carrier or host cell comprising the nucleic acid molecule described above, and pharmaceutical compositions comprising antibody conjugates described above (such as radionuclide-labeled antibody conjugates).

[0111] In some embodiments, the pharmaceutical composition further comprises pharmaceutical excipients.

[0112] In a sixth aspect, this application provides the use of the aforementioned different forms of antibodies, for example, for the prevention, diagnosis and / or treatment of diseases characterized by LRRC15 expression, or for use in the preparation of medicaments for the prevention, diagnosis and / or treatment of diseases characterized by LRRC15 expression, such as LRRC15-positive cancers / tumors.

[0113] Accordingly, this application provides a method for the prevention, diagnosis and / or treatment of diseases characterized by LRRC15 expression in an individual, comprising administering the above-described different forms of antibody conjugates or pharmaceutical compositions thereof to an individual in need.

[0114] In this article, "LRRC15-positive cancer / tumor" is understood in the conventional sense in the art, generally referring to cancer / tumor cells expressing LRRC15. The degree of LRRC15 positivity in cancer / tumor can be qualitatively, quantitatively, or semi-quantitatively analyzed using various methods in the art. An exemplary method is to perform immunohistochemical analysis on solid tumor samples (e.g., biopsy or postoperative sections), for example, semi-quantitatively expressed using H-scores, where cancer / tumor with an H-score > 100 can generally be defined as "LRRC15-positive cancer / tumor".

[0115] According to the TCGA dataset, LRRC15 is overexpressed in patients with glioma, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, urothelial carcinoma, prostate cancer, testicular cancer, breast cancer, cervical cancer, endometrial cancer, and melanoma.

[0116] In principle, the antibody conjugates, the radionuclide-labeled antibody conjugates, or the pharmaceutical compositions thereof provided in this application can be used to treat any cancer involving LRRC15-expressing cells.

[0117] In some implementations, diseases characterized by LRRC15 expression are cancers, including but not limited to: sarcoma, glioblastoma, melanoma, breast cancer, HER2-negative breast cancer, HER2-positive breast cancer, HR+ breast cancer, triple-negative breast cancer, head and neck cancer, head and neck squamous cell carcinoma, lung cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, pancreatic ductal adenocarcinoma, ovarian cancer, colorectal cancer, testicular cancer, and squamous cell carcinoma, etc.

[0118] The anti-LRRC15 antibody provided in this application has a stable structure, high affinity, strong specificity, strong tissue penetration, and good tolerability, making it widely applicable in scientific research, diagnosis, and treatment. For example, radionuclide conjugates containing the anti-LRRC15 antibody of this application combine the advantages of nanobodies and radionuclides, exhibiting high uptake at tumor sites and showing broad application prospects in tumor imaging and the diagnosis and / or treatment of related diseases (e.g., therapeutic integration).

[0119] It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments.

[0120] Example

[0121] The following embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application shall fall within the scope of this application.

[0122] Unless otherwise specified, all reagents used in the examples are commercially available and all technical means used in the examples are conventional means well known to those skilled in the art.

[0123] Example 1: Generation and screening of LRRC15 antibodies

[0124] 1. Preparation of LRRC15 antigen

[0125] The His-tagged human LRRC15 protein was used as an immunogenic antigen, the Biotin-tagged human LRRC15 was used as a panning antigen, and the His-tagged human LRRC15 was used as a screening antigen. All of these were prepared by Aikon Biotechnology Co., Ltd.

[0126] The sequence of human LRRC15 protein (Accession # Q8TF66-1) is as follows, where the underlined part is the extracellular region sequence, and the amino acid sequence is Tyr 22 - Gly 538.

[0127] Human LRRC15 protein (SEQ ID NO: 41): MPLKHYLLLLVGCQAWGAGLA YHGCPSECTCSRASQVECTGARIVAVPTPLPWNAMSLQILNTHITELN ESPFLNISALIALRIEKNELSRITPGAFRNLGSLRYLSLANNKLQVLPIGLFQGLDSLESLLLSSNQLLQIQPAHFS QCSNLKELQLHGNHLEYIPDGAFDHLVGLTKLNLGKNSLTHISPRVFQHLGNLQVLRLYENRLTDIPMGTFDGLVNL QELALQQNQIGLLSPGLFHNNHNLQRLYLSNNHISQLPPSVFMQLPQLNRLTLFGNSLKELSPGIFGPMPNLRELWL YDNHISSLPDNVFSNLRQLQVLILSRNQISFISPGAFNGLTELRELSLHTNALQDLDGNVFRMLANLQNISLQNNRL RQLPGNIFANVNGLMAIQLQNNQLENLPLGIFDHLGKLCELRLYDNPWRCDSDILPLRNWLLLNQPRLGTDTVPVCF SPANVRGQSLIIINVNVAVPSVHVPEVPSYPETPWYPDTPSYPDTTSVSSTTELTSPVEDYTDLTTIQVTDDRSVWG MTQAQSG LAIAAIVIGIVALACSLAACVGCCCCKKRSQAVLMQMKAPNEC

[0128] 2. Alpaca Immunization

[0129] Two healthy alpacas were immunized. On day 0, the initial immunization was performed by injecting 0.5 mg of human LRRC15 antigen mixed with Gerbu adjuvant. On day 14, 0.25 mg of human LRRC15 antigen mixed with Gerbu adjuvant was injected. On days 28 and 42, 0.125 mg of human LRRC15 antigen and mouse LRRC15 antigen mixed with Gerbu adjuvant were injected. Blood samples (5 ml, 50 ml, and 50 ml) were collected on days 21, 35, and 49, respectively. Serum titers were measured, and alpaca peripheral lymphocytes meeting the library construction criteria were used to construct the display library.

[0130] 3. Construction and screening of yeast display libraries

[0131] 3.1 Construction of the yeast display library

[0132] A large-capacity yeast display library was constructed using multi-round immunization and molecular cloning techniques. The specific implementation steps are as follows: 3.1.1 Preparation of Immunosamples (1) 50 mL of peripheral blood samples were collected after the third and fourth immunizations, respectively; (2) Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation. (3) Extract total RNA using TRIzol® reagent according to standard procedures.

[0133] 3.1.2 cDNA Synthesis and VHH Amplification

[0134] (1) Double-stranded cDNA was synthesized using the PrimeScript™ II First-Strand cDNA Synthesis Kit (TaKaRa, CAT# 6210B); (2) Design specific primers for triple PCR amplification: 3.1.3 Document Construction and Transformation (1) The purified PCR product was mixed with the linearized pYDisplay vector (iCareab) at a certain molar ratio; (2) Electroconvert the ligation product into EYB100 competent yeast cells; (3) Spread the transformed bacterial solution evenly on a solid culture medium and incubate at 30°C for 48-72 hours until single colonies are formed.

[0135] 3.1.4 Library Quality Assessment

[0136] (1) Library capacity determination: Five culture plates were randomly selected to count the clonal density, and the total library capacity was calculated to be 2.22 × 10⁻⁶. 9 CFU (Alpaca A) and 2.41×10 9 CFU (Alpaca B); (2) Insertion efficiency detection: Fifty random clones were validated by Colony PCR, and the positive rate of inserted fragments was analyzed; (3) Diversity analysis: Sanger sequencing was performed on positive clones to analyze the CDR region sequence.

[0137] 3.2 Magnetic bead sorting of yeast display library

[0138] The specific steps for screening target antibodies to display yeast clones using magnetic bead sorting technology are as follows: 3.2.1 Negative Filtering Preprocessing (1) Take a suspension of yeast cells (2×10^8 cells) cultured in SG-CAA medium to the logarithmic phase, add pre-blocked Dynabeads M-270 magnetic beads, and incubate at 4℃ using a rotary mixer (20 rpm) for 30 minutes; (2) Place the mixture on a magnetic rack and let it stand at room temperature for 10 minutes, then discard the supernatant containing non-specifically bound cells. (3) Repeat the negative screening operation: Transfer the retained cell suspension to a fresh closed magnetic bead system, incubate twice under the same conditions, and then magnetically separate to obtain pre-screened cells free of non-specific binding.

[0139] 3.2.2 Preparation of Antigen Magnetic Beads

[0140] (1) Dilute Biotin-LRRC15 antigen with 0.5% PBSA to a working concentration of 50 μg / mL, and mix it with streptavidin magnetic beads (NEB, CAT# S1420S) at a volume ratio of 1:3. (2) The antigen-magnetic bead complex was continuously incubated at low speed in a rotary mixer at 4℃ for 60 minutes to complete the directional coupling of the antigen-magnetic bead complex; (3) Remove free antigens using magnetic separation technology: Add 1 mL of pre-cooled 0.5% PBSA buffer, let stand for 5 minutes, discard the supernatant, and repeat the washing twice.

[0141] 3.2.3 Positive screening and isolation

[0142] (1) Mix the antigen magnetic bead complex with the pre-screened bacterial cells and incubate at 4°C by rotating the mixture (15 rpm) for 60 minutes; (2) After separation by magnetic beads, discard the supernatant of unbound cells, add 1 mL of 0.5% PBSA buffer and gently resuspend, let stand at room temperature for 5 minutes, and repeat the washing three times; (3) Collect the magnetic bead-bound cells, resuspend them in 1 mL of SD-CAA medium, and then aliquot them for further processing: ① Sample preservation: Mix with an equal volume of 50% sterile glycerol and freeze at -80℃ for long-term storage; ② Amplify the sample: Add 2 mL of SD-CAA medium and incubate at 30℃ on a shaker (220 rpm) for 16-32 hours.

[0143] 3.2.4 Secondary treatment and amplification

[0144] (1) Magnetic separation to remove free magnetic beads from the culture medium: After standing at room temperature for 15 minutes, transfer the supernatant to a sterile centrifuge tube; (2) Collect the bacterial cells by centrifugation at 3000×g for 3 minutes, and resuspend them twice in 2 mL SG-CAA medium (1 mL each time); (3) Transfer to a shake tube and culture at 30°C and 220 rpm for 12 hours to complete the recovery and amplification.

[0145] 3.3 Flow Cytometry Screening of Yeast Monoclonal Antibodies

[0146] The specific steps for screening positive yeast clones and obtaining antibody genes by flow cytometry are as follows: 3.3.1 Monoclonal Culture and Induction The sorted yeast culture was evenly spread on the surface of SD-CAA solid medium and incubated at 30℃ for 48 hours. Discrete single colonies were picked and inoculated into 5 mL of SG-CAA liquid medium and cultured at 30℃ with shaking for 24 hours until the logarithmic growth phase. The culture was then transferred to an induction medium containing 2% galactose and induced to express for 48 hours at 20℃.

[0147] 3.3.2 Antigen binding detection

[0148] After induction, the bacterial cells were mixed with 100 nM Biotin-LRRC15 antigen solution and incubated at 25°C in the dark for 60 minutes. Then, APC-labeled streptavidin (eBioscience, CAT# 17-4317-82) secondary antibody (1:200 dilution) was added and incubated again at 25°C in the dark for 30 minutes. Flow cytometry (Thermo, Attune Nxt flow cytometer) was used for detection, and the APC channel detection threshold was set to screen clones with high fluorescence intensity.

[0149] 3.3.3 Positive clone processing and gene acquisition

[0150] Collect target antigen-binding positive bacteria, add 0.2% SDS lysis buffer, and treat at 95℃ for 10 minutes; centrifuge at 12000×g for 5 minutes at 4℃, and take 0.5 μL of supernatant as template; amplify the VH / VL gene using high-fidelity DNA polymerase, and send the amplified product for sequencing analysis after purification; retain 1 mL of bacterial culture and mix with 20% glycerol, and store at -20℃ for long-term use.

[0151] 4. Construction and selection of phage display libraries

[0152] 4.1 Construction of Phage Display Library

[0153] 100 mL of peripheral blood was collected, and PBMCs were isolated using lymphocyte separation medium. RNA was extracted and processed using PrimeScript. TM II. Reverse transcription was performed using the 1st Strand cDNA synthesis kit to prepare cDNA. The cDNA was used as the template for the first round of PCR. After two rounds of PCR amplification, the second-round PCR product fragment was ligated into the phage library vector pDisplay. The ligation product was then electroporated into *E. coli* competent cells SS320. 100 μl of transformation buffer was titrated, and the library size was calculated based on the number of clones. Twenty clones were randomly selected for sequencing to assess library quality. Sequencing results, based on library insertion rate and library diversity analysis, indicated that the library met library quality standards, with a library volume of 1.13 × 10⁻⁶. 9 .

[0154] 4.2 Liquid-phase panning of phage display libraries

[0155] The target phage clones were screened using a three-round liquid-phase cell panning method, as detailed below: 4.2.1 Pre-experimental treatment (1) Block the EP tubes with 3% PBSA solution and leave them at 4°C overnight; (2) Prepare 1×10 7 CHO-S cells (negative control) and CHO-S-LRRC15 Cells (antigen expression lines) were each added with 3% PBSA blocking solution and blocked at 37°C. Processing time: 60 minutes; (3) Phage library pretreatment: Mix 150 μL of the original library with 850 μL of 3% PBSA and block at 4°C for 60 minutes.

[0156] 4.2.2 First Round of Selection

[0157] (1) Negative screening treatment: The pretreated phage library was mixed with CHO-S cell suspension and adsorbed at low speed for 30 minutes at 4°C. This negative screening process was repeated twice. (2) Positive screening: The phage mixture treated by negative screening was transferred to CHO-S-LRRC15 cell suspension and adsorbed at low speed at 4°C for 60 minutes. (3) Separation and washing: Collect cell complexes by centrifugation at 600×g for 5 minutes at 4℃, and wash 6 times with pre-cooled PBS buffer; (4) Phage elution: After resuspending the cell pellet in 200 μL of pre-cooled PBS, add 200 μL of 2×TEA elution buffer and react for 10 minutes. Immediately neutralize the reaction with 400 μL of 1M Tris-HCl buffer (pH 7.6).

[0158] 4.2.3 Amplification and Repeated Screening

[0159] After phage amplification culture of the elution buffer obtained in the first round, the second and third rounds of screening were carried out under the same experimental conditions, and the screening conditions and parameters were kept consistent between each round.

[0160] 5. Eukaryotic expression of antibodies

[0161] Antibody sequences were extracted from positive clones selected from yeast and bacteriophage screening, digested with enzymes, ligated into the pcDNA3.4 vector, and transiently transfected into HEK 293F cells. The VHH-Fc antibody expression supernatant was collected, and the binding of candidate antibodies to cell surface antigen proteins was detected by FACS. The binding of purified antibodies to the LRRC15 protein displayed on the cell surface was verified, and clones were further screened. Fourteen clones were selected as candidate antibodies and named 12A05, 11E06, 15C02, 1G02, 2D04, 2B02, 2G05, 3G05, 4G11, 5A12, 5B10, 5C03, 5D06, and 5D12.

[0162] 6. Detection of cell-binding activity of candidate antibodies

[0163] Flow cytometry was used to analyze the binding characteristics of candidate antibodies to target cells. The specific steps are as follows: Frozen human osteosarcoma cells Saos-2 (Pronosai) were revived and cultured in medium containing 10% FBS until the logarithmic growth phase. After digestion, the cells were counted and the cell density was adjusted to 5 × 10⁶ cells / year. 5 Cells / mL, 100 μL of cell suspension per well. Candidate antibodies were diluted to 10 μg / mL, 2 μg / mL, and 1 μg / mL with PBS buffer (pH 7.4) containing 1% BSA, respectively. 100 μL of each solution was added to the cell suspension to ensure a final antibody concentration of 5 μg / mL, 1 μg / mL, and 0.5 μg / mL in the wells. The plates were incubated at 4°C in the dark for 60 minutes. After centrifugation at 800×g for 3 minutes (4°C), the supernatant was discarded, and the plates were washed three times with pre-chilled PBS buffer (pH 7.4). 100 μL of PE-labeled goat anti-human IgG Fc secondary antibody was added and diluted 1:1000 with blocking buffer. The plates were incubated at 4°C in the dark for 30 minutes. After centrifugation at 800×g for 3 minutes (4°C), the plates were washed three times with pre-chilled PBS buffer (pH 7.4). Cells were gently resuspended in 200 μL PBS buffer (containing 0.5% BSA); analysis was performed using a BD FACSCanto II flow cytometer. Cell data were collected, and the proportion of PE-positive cells was analyzed using FlowJo software. The cell binding activity assay results of the 14 candidate antibodies screened are shown in the figure. Figure 1 .

[0164] Based on the test results, two clones with the best cell binding activity were selected as anti-LRRC15 VHH antibodies. The variable region of the antibody sequence was encoded using the Kabat encoding scheme, as shown below (where CDRs are shown in bold and underlined, and the same applies below): 5A12 (SEQ ID NO: 4): AVQLVDSGGGLVQPGGSLRLSCAASGFTFS SYTMG WARQVPGKGLEWVS GIYSDGSTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAMYYCAT EYYSDPKLLTDFGS WGQGTQVTVSS 11E06 (SEQ ID NO: 24): QVQLVESGGGLVQPGGSMRLSCTASGSIFS INTMG WYRQTPGKQRELVA EITTGGITNYADSVKG RFAISRDDAQNTLYLQMASLKPEDTGVYYCAA GDGMWYGRSEYDY WGQGTQVTISS The VHH-Fc antibody sequence was further constructed, wherein the Fc sequence adopted was the human IgG1 Fc sequence (SEQ ID NO: 42), as follows: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0165] Example 2: Activity detection of anti-LRRC15 antibody

[0166] 1. Cell-binding activity of anti-LRRC15 antibody EC 50 Measurement

[0167] Resuscitate cryopreserved human osteosarcoma cells Saos-2 (Pronosai), and adjust the cell count density to 4 × 10⁻⁶ cells in the logarithmic growth phase. 5Cells / mL, 100 μL of cell suspension was taken from each well. Candidate antibodies were diluted to 200 nM with PBS buffer (pH 7.4) containing 1% BSA, then diluted 10-fold, and 100 μL was added to each well of the cell suspension; the antibody concentration in the starting well was 100 nM. Incubate at 4°C in the dark for 60 minutes; centrifuge at 800×g for 3 minutes (4°C), discard the supernatant, and wash three times with pre-chilled PBS buffer (pH 7.4). Add 100 μL of PE-labeled goat anti-human IgG Fc secondary antibody, diluted 1:1000 with blocking buffer; incubate at 4°C in the dark for 30 minutes; centrifuge at 800×g for 3 minutes (4°C), and wash three times with pre-chilled PBS buffer (pH 7.4). Gently resuspend the cells in 200 μL of PBS buffer (containing 0.5% BSA); analyze using a BD flow cytometer, collect cell data, and analyze using FlowJo software.

[0168] See results Figure 2 And Table 1. This shows that both selected antibodies bind to LRRC15 expressed in saos-2 cells.

[0169] Table 1. Alpaca antibody cell-binding activity EC 50 value

[0170] 2. Intracellular endocytic activity of anti-LRRC15 antibody

[0171] CHO-S overexpressing cells were seeded into 8-well plates until the cells reached 70%-80% confluence. Some wells required pre-cooling at 4°C for 30 minutes (to inhibit endocytosis). Zenon was used. TM pHrodo TM Incubate the labeled antibody with iFL IgG labeling reagent (catalog number Z25611). Experimental group: Add pHrodo labeled antibody (final concentration 1-10 μg / mL) to pre-cooled culture medium. Negative control is isotype control antibody.

[0172] Endocytosis induction: Incubate at 37℃ in a 5% CO2 incubator for 0.5-6 hours. Termination and washing: Remove the culture medium and wash three times with pre-cooled DPBS (to terminate endocytosis). Flow cytometry quantification: After collecting cells, resuspend them in DPBS and detect the fluorescence intensity of the FL2 or PE channel. Calculate the MFI (mean fluorescence intensity) from the flow cytometry data and compare the experimental and control groups. Calculate endocytosis efficiency: Endocytosis efficiency (%) = (Experimental group MFI - 4℃ control group MFI) / (Maximum MFI at 37℃ - 4℃ control group MFI) × 100. Results are shown in [link to results]. Figure 3 .

[0173] The results showed that both antibodies had endocytic activity, with the 5A12 antibody exhibiting the best endocytic activity.

[0174] 3. Affinity assay of anti-LRRC15 antibody

[0175] Molecular affinity was determined using ForteBio Octet's Biolayer Interference (BLI) technique to measure intermolecular affinity (antibody-antigen binding). Humanized antibodies were immobilized using an AHC2 sensor at a concentration of 5 μg / ml for 200 s. The buffer solution was PBST (PBS + 0.02% Tween 20 + 0.1% BSA), diluted with Human-LRRC15-His (Acro, LR5-H52H3) to 25, 12.5, 6.25, 3.13, 1.56, and 0 nM. Affinity testing was performed at equilibration for 120 s, binding for 180 s, and dissociation for 180 s, at a detection temperature of 30 °C. Octet data analysis software was used to select a fitting model (commonly the 1:1 Langmuir binding model) and calculate the binding rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD = koff / kon). The affinity determination results are shown in Table 2.

[0176] Table 2. Affinity assay results of anti-LRRC15 antibody

[0177] Example 3: Humanization of anti-LRRC15 antibody

[0178] 1. Humanization of anti-LRRC15 antibody

[0179] Humanization of the anti-LRRC15 antibody was performed using the CDR-grafting method. The parental anti-LRRC15 antibody was compared with fully human germline genes in the IMGT or NCBI / Igblast databases using the IMGT or NCBI website. The human germline gene IGHV3-23 (IGHV3-23), which is highly homologous to the anti-LRRC15 antibody, was selected. 01; IGHV3-23 04) As a humanization template, CDR was transplanted, and four key residues (37, 44, 45, 47) in the FR2 of the nanobody were retained. The key core residues near the CDR region and the Vernier position residues that interact with the CDR were reversed to obtain the humanized antibody.

[0180] The variable region of the antibody sequence uses the Kabat coding scheme. The following is an example of the humanized antibody molecule design sequence for the 5A12 molecule: 5A12-h2m1 (SEQ ID NO: 5): EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYTMG WARQAPGKGLEWVS GIYSDGSTYYADSVKG RFTISRDNAKNTVYLQMNSLRAEDTAVYYCAT EYYSDPKLLTDFGS WGQGTQVTVSS 5A12-h2m8 (SEQ ID NO: 8): EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYTMG WARQAPGKGLEWVS GIYSDASTYYADSVKG RFTISRDNAKNTVYLQMNSLRAEDTAVYYCAT EYYSDAKLLTDFGS WGQGTQVTVSS 5A12-h2m9 (SEQ ID NO: 10): EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYTMG WARQAPGKGLEWVS GIYSDASTYYADSVKG RFTISRDNAKNTVYLQMNSLRAEDTAVYYCAT EYYSDARLLTDFGS WGQGTQVTVSS The assay protocol for detecting cell binding activity using flow cytometry can be found in Example 2. Exemplary detection results are shown in Table 3.

[0181] Table 3. Results of cell-binding activity assays for the first round of humanized 5A12 antibody molecules.

[0182] The second round of humanized antibody sequences was designed using 5A12-h2m1 as a template. FACS cell binding activity assays were performed, and exemplary results are shown in Table 4, demonstrating that all humanized antibodies retained cell binding activity.

[0183] Table 4. Results of cell-binding activity assays for the second round of humanized 5A12 antibody molecules.

[0184] The affinity of the antibody with the best cell binding activity was simultaneously determined, and the affinity determination method was performed according to Example 2. The affinity data of 5A12-h2m1-Fc and 5A12-h2m8-Fc are shown in Table 5; the affinity data of 5A12-h2m9-Fc are shown in Table 6 for 5A12-h2m9-Fc (parent parent).

[0185] Table 5. Affinity assay results of humanized 5A12 antibody molecules

[0186] Based on cell activity, expression levels, and affinity data, 5A12-h2m8 and 5A12-h2m9 were selected as humanized lead molecules.

[0187] The following is an example of a humanized antibody molecule design sequence for the 11E06 molecule: 11E06-h6m1 (SEQ ID NO: 29): EVQLVESGGGLVQPGGSLRLSCAASGSIFS INTMG WYRQAPGKQRELVA EITTGGITNYADSVKG RFTISRDDAQNTLYLQMNSLRAEDTAVYYCAA GDTMWYGRSEYDY WGQGTTVTVSS 11E06-h8 (SEQ ID NO: 27): EVQLVESGGGLVQPGGSLRLSCAASGSIFS INTMG WYRQAPGKGRELVS EITTGGITNYADSVRG RFTISRDDAQNTLYLQMNSLRAEDTAVYYCAA GDTLWYGRSEYDY WGQGTQVTVSS Test results showed that all designed humanized antibodies retained cell-binding activity. Based on cell activity, expression levels, and affinity data, 11E06-h6m1 and 11E06-h8 were selected as humanized lead molecules.

[0188] 2. Affinity maturation of humanized anti-LRRC15 antibody

[0189] Humanized lead antibody molecules were used to design a mutant library based on a humanized lead antibody requiring affinity maturation. A combination of single-point and combined saturation mutations was employed to target the complementarity-determining region (CDR) and construct a phage mutant library. Following phage library panning procedures, solid-phase and liquid-phase methods using human LRRC15-His Tag and human LRRC15-His Tag-Biotin were employed. After three rounds of panning and rigorous elution, clones with high OD450 were initially screened using phage ELISA, and secondary screening was performed using biomembrane interference (BLI, such as Octet) to determine the dissociation constant (KD). The effective library capacity of the 11E06 antibody is approximately 1 × 10⁻⁶. 8 The effective library capacity of the 5A12 antibody is approximately 1×10⁻⁶. 8 .

[0190] The sequence of the molecule after affinity maturation of the 5A12-h2m9 antibody is as follows: 5A12-h2m9-066 (SEQ ID NO: 13): EVQLVESGGGLVQPGGSLRLSCAASGFRFS SYTMG WARQAPGKGLEWVS GIFSDASTYYADSVKG RFTISRDNAKNTVYLQMNSLRAEDTAVYYCAT EYYLDARLLTDFGS WGQGTQVTVSS 5A12-h2m9-088 (SEQ ID NO: 16): EVQLVESGGGLVQPGGSLRLSCAASGFRFS SYTMG WARQAPGKGLEWVS GIYSDARTYYADSVKG RFTISRDNAKNTVYLQMNSLRAEDTAVYYCAT EYYADARLLTDFGS WGQGTQVTVSS The sequences of the exemplary 11E06-h8 antibody affinity-matured molecules are as follows: 11E06-h8-102 (SEQ ID NO: 32): EVQLVESGGGLVQPGGSLRLSCAASGSIFS YNTMG WYRQAPGKGRELVS EITTGGITNYADSVRG RFTISRDDAQNTLYLQMNSLRAEDTAVYYCAA RDTLWYGRSGYDY WGQGTQVTVSS 11E06-h8-107 (SEQ ID NO: 34): EVQLVESGGGLVQPGGSLRLSCAASGSIFS INTMG WYRQAPGKGRELVS EITTGGITNYADSVRG RFTISRDDAQNTLYLQMNSLRAEDTAVYYCAA LDTDWYGRSEYDY WGQGTQVTVSS 11E06-h8-m7 (SEQ ID NO: 43): EVQLVESGGGLVQPGGSLRLSCAASGSIFS YNTMG WYRQAPGKGRELVS EITTGGITNYADSVRG RFTISRDDAQNTLYLQMNSLRAEDTAVYYCAA RDTLWYGRPEYDY WGQGTQVTVSS After designing and constructing a mutant library, screening and selection, and affinity testing (using the BLI method), the results showed that after affinity maturation, the affinity of the 5A12 candidate molecule was approximately 10-fold higher than that of the parent antibody 5A12-h2m9. The affinity of the 11E06 candidate molecule was approximately 10-fold higher than that of the parent antibody 11E06-h8. Exemplary affinity test results are shown in Tables 6 and 7.

[0191] Table 6. Affinity test results of 5A12 antibody molecules with mature affinity.

[0192] Table 7. Affinity test results of 11E06 antibody molecules with maturation affinity.

[0193] The humanized or affinity-matured VHH antibody or its variants were further constructed into an Fc-containing molecular form, wherein the Fc sequence is shown in SEQ ID NO: 42. Cell binding activity was assessed using the experimental protocol of Example 2; exemplary cell binding activity detection results are shown below. Figure 4 See Table 8.

[0194] Table 8 Cell-binding activity of EC 50 value

[0195] The results showed that the molecules 5A12-h2m9-066-Fc, 5A12-h2m9-088-Fc, 11E06-h8-102-Fc, and 11E06-h8-107-Fc all bound to U118MG cells expressing LRRC15.

[0196] The endocytosis activity assay followed the protocol described in Example 2. The endocytosis activity detection results are shown in [Figure 2]. Figure 5 This indicates that the molecules 5A12-h2m9-066-Fc, 5A12-h2m9-088-Fc, 11E06-h8-102-Fc, and 11E06-h8-107-Fc all have endocytic activity.

[0197] The exemplary variable region sequence of the 5A12-h2m9-066 variant after affinity maturation is as follows: 5A12-h2m9-066A(SEQ ID NO: 17): EVQLVESGGGLVQPGGSLRLSCAASGFRFS SYTMG WARQAPGKGLEWVS GIFSDASTYYADSVKG RFTISRDNAKNTVYLQMNSLRAEDTAVYYCATEYYADARLLTDFGS WGQGTQVTVSS 5A12-h2m9-06605(SEQ ID NO: 18): EVQLLESGGGLVQPGGSLRLSCAASGFRFS SYTMG WARQAPGKGLEWVS GIFSDASTYYADSVKG RFTISRDNAKNTVYLQMNSLRAEDTAVYYCAT EYYADARLLTDFGS WGQGTQVTVSS 5A12-h2m9-06607(SEQ ID NO: 19): EVQLLESGGGLVKPGGSLRLSCAASGFRFS SYTMG WARQAPGKGLEWVS CLOTHING RFTISRDNAKNTVYLQMNSLRAEDTAVYYCAT EYYADARLLTDFGS WGQGTQVTVSS 5A12-h2m9-06609(SEQ ID NO: 20): EVQLLESGGGLVKPGGSLRLSCAASGFRFS SYTMG WARQAPGKGLEWVS GIFSDASTYYADSVKG RFTISRDNAKNTVYLQMNSLRAEDTAVYYCAT EYYADARLLTDFGS WGQGTQVTVSS The variable region sequence of the variant obtained after introducing two cysteine ​​residues into the exemplary variant sequence based on 11E06-h8 after affinity maturation is as follows: 11E06-h8-m3-2C(SEQ ID NO: 38): EVQLVESGGGLVQPGGSLRLSCAASGSIFS IETMG WYRQAPGKGRELVS EILTGGITNYADSVRG RFTISRDDAQNTLYLQMNSLRCEDTAVYYCAA LDTDWYGRPEYDY WGQGTQVTVSC 11E06-h8-m7-2C(SEQ ID NO: 40): EVQLVESGGGLVQPGGSLRLSCAASGSIFS YNTMG WYRQAPGKGRELVS EITTGGITNYADSVRG RFTISRDDAQNTLYLQMNSLRCEDTAVYYCAA RDTLWYGRPEYDY WGQGTQVTVSC

[0198] Example 4: Application of anti-LRRC15 antibody

[0199] 1. Expression and purification of VHH antibody

[0200] Selected antibody sequences were inserted into the eukaryotic expression vector pcDNA3.4 and transfected into CHO cells for antibody expression. The supernatant was added to Amsphere A3 Protein A purification medium and incubated at room temperature for 2 hours on a rotary incubator. The Amsphere A3 Protein A affinity column was equilibrated with 3-5 times the column volume using equilibration buffer. The sample supernatant was loaded, binding the antibody to Protein A medium. The column was washed with 5-10 CV of equilibration buffer until the UV signal dropped to baseline. The Amsphere A3 Protein A affinity column was eluted with elution buffer, and the elution was collected based on the A280 value of the monitored UV absorbance. The collected elution was neutralized with neutralization buffer to adjust the sample pH to approximately 5.0. After ultrafiltration to replace the buffer, the eluted and neutralized antibody was filtered through a 0.22 μm filter in a biosafety cabinet, and the sample volume was recorded. The concentration was measured and recorded using Nanodrop One.

[0201] 2. VHH antibody conjugated with maleimide-NOTA / H3RESCA

[0202] VHH antibody was added to TCEP at a molar ratio of VHH:TCEP = 1:10, and incubated at 37°C for 60 minutes to release free cysteine. PBS was used as the buffer in the experiment. After the reaction was complete, Mal-NOTA (or MAL-H3RESCA) was added at a molar ratio of VHH:Mal-NOTA (or MAL-H3RESCA) = 1:10, and incubated at room temperature for 2 hours. The experiment was performed in PBS buffer. The reacted samples were purified using a Superdex 75pg molecular sieve, with PBS as the mobile phase at a flow rate of 1 ml / min. The average conjugation ratio (CAR) of the chelating agent to the antibody was calculated using mass spectrometry. The results showed that the average CAR values ​​of the conjugated VHH-Mal-NOTA and VHH-Mal-H3RESCA (abbreviated as VHH-NOTA and VHH-H3RESCA) were close to 1.

[0203] The cell binding activity assay followed the experimental protocol of Example 2. The cell binding activities of 11E06-h8-m3-2C-NOTA, 11E06-h8-m7-2C-NOTA, 11E06-h8-107-GSC-NOTA, 5A12-h2m9-06609-GSC-NOTA, and 11E06-h8-m7-GSC-H3RESCA are shown in Table 9. Figure 6 and Figure 7 .

[0204] Table 9 Cell-binding activity of EC 50 value

[0205] The results showed that 11E06-h8-m3-2C-NOTA, 11E06-h8-m7-2C-NOTA, 11E06-h8-107-GSC-NOTA, 5A12-h2m9-06609-GSC-NOTA, and 11E06-h8-m7-GSC-H3RESCA all bound to U118MG cells expressing LRRC15 after being conjugated with either Nota or H3RESCA.

[0206] 3. Stability testing of VHH-NOTA and VHH-H3RESCA

[0207] Stability testing of the RDC precursors of VHH-Fc and VHH-NOTA / H3RESCA was conducted under three pH conditions and with varying incubation temperatures and times. Incubation was performed at 37°C for 30 minutes at three pH conditions (pH 3.5; 4.5; 5.5). For pH 3.5, 4.5, and 5.5, an acetate-sodium acetate system was used, with acetate-sodium acetate concentrations ranging from 0.5 to 0.7 M. After incubation, the antibody peaks were determined by SEC-HPLC to assess antibody aggregation and other factors. The turbidity of the antibody solutions under different pH conditions was also observed.

[0208] The results showed that the 11E06-h8-107-GSC-NOTA and 11E06-h8-m7-GSC-H3RESCA molecules did not exhibit significant aggregation or fragmentation at pH 4.5; the 5-A12-h2m9-066-Fc, 5-A12-h2m9-088-Fc, 11E06-h8-102-Fc, and 11E06-h8-107-Fc molecules remained stable at pH 3.5. Exemplary results are shown in Tables 10 and 11.

[0209] Table 10. Detection results of VHH-NOTA antibody at low pH

[0210] Table 11. SEC-HPLC analysis of molecular low pH stability after affinity maturation.

[0211] 4. Accelerated stability testing of VHH-NOTA and VHH-H3RESCA

[0212] Preliminary accelerated stability analysis of the purified RDC precursors VHH-NOTA and VHH-H3RESCA was conducted using a high-temperature accelerated method to evaluate whether VHH-NOTA and VHH-H3RESCA would precipitate, aggregate, or degrade.

[0213] The samples were placed at 40°C, and two samples were taken for analysis of supernatant content and SEC-HPLC purity. The data were then compared with those of samples not placed at 40°C.

[0214] SEC-HPLC analysis showed that at 2 weeks, both 11E06-h8-107-GSC-NOTA and 11E06-h8-m7-GSC-H3RESCA exhibited weak aggregation and fragmentation. Exemplary results are shown in Tables 12 and 13.

[0215] Table 12. Accelerated stability of VHH-NOTA determined by SEC-HPLC.

[0216] Table 13 Accelerated stability of VHH-H3RESCA by SEC-HPLC

[0217] The same method was used to perform accelerated stability experiments on affinity maturation candidate antibodies (with Fc tags), and the results are shown in Table 14.

[0218] Table 14 Results of accelerated stability experiments for affinity maturation candidate antibodies

[0219] The results showed that the content of the supernatant of samples 5-A12-h2m9-088-Fc, 11E06-h8-102-Fc, and 11E06-h8-107-Fc did not change significantly after being placed at 40℃ for 2 weeks. SEC-HPLC analysis showed no significant change in the purity of the SEC main peak.

[0220] 5. VHH-NOTA / VHH-H3RESCA precursor 68 Ga / 18 F marking and quality control

[0221] 5.1 VHH-NOTA precursor 68 Ga labeling and quality control

[0222] Achieving this through chelation coupling technology 68 The specific steps for Ga radioactive labeling are as follows: (1) Preparation of NOTA-VHH coupling solution: Take VHH-NOTA precursor (5 nmol), add 0.5 mL of 0.35 mol / L sodium acetate buffer (containing 1 mg / mL gentian acid) to redissolve, mix well and transfer all to reaction flask.

[0223] (2) Preparation of radionuclides: Rinse with 0.1 mol / L sterile hydrochloric acid solution 68 Ge / 68 Ga generator, collection 68 Pour the GaCl3 eluent into a vial.

[0224] (3) Construction of chelation reaction system (labeling reaction): Take 1 mL 68 Add GaCl3 eluent to the reaction flask; place the reaction flask in a constant temperature metal bath and react at 37°C for 15-30 minutes.

[0225] (4) Purification and displacement: The reaction solution was purified by a PD-10 desalting column. Column equilibration: 15 mL sodium chloride injection containing 15 mg / mL sodium ascorbate and 1 mg / mL gentic acid; loading volume: 1.5 mL; elution buffer: sodium chloride injection containing 15 mg / mL sodium ascorbate and 1 mg / mL gentic acid, and 1.5-3.0 mL of elution peak were collected; the solution was filtered using a 0.22 μm PVDF sterile filter.

[0226] (5) Quality control analysis: Radioactivity purity was detected using a high performance liquid chromatograph with a radioactivity detector and an SEC column. The mobile phase was adjusted to pH 6.8 with 10 mmol / L phosphate buffer (containing 150 mmol / L sodium chloride) using phosphoric acid or 1 mol / L sodium hydroxide. The radiochemical purity was ≥90%.

[0227] free 68 Ga ions and 68 Ga colloids were detected using a radiometric scanner. iTLC-SG was used as the thin-layer plate, with 0.1 mol / L sodium citrate, 1 mol / L citric acid (adjusted to pH 5.0), and 1 mol / L ammonium acetate solution-DMSO = 90:10 as the developing solvents for detection. 68 Ga ions and 68 Ga colloid content ≤ 5%.

[0228] Table 15 provides examples of VHH-NOTA precursors. 68 Ga labeling test results. The results show that all percentage contents meet the requirements.

[0229] Table 15 VHH-NOTA precursors 68 Ga marker detection results

[0230] 5.2 VHH-H3RESCA precursor 68 Ga labeling and quality control

[0231] Achieving this through chelation coupling technology 68 The specific steps for Ga radioactive labeling are as follows: (1) Preparation of H3RESCA-VHH coupling solution: Take VHH-H3RESCA precursor (5 nmol), add 1.5 mL of 0.35mol / L sodium acetate buffer (containing 1 mg / mL gentian acid and appropriate amount of Tween 80) to reconstitute, mix well and transfer all to reaction flask.

[0232] (2) Preparation of radionuclides: Rinse with 0.1 mol / L sterile hydrochloric acid solution 68 Ge / 68 Ga generator, collection 68 Pour the GaCl3 eluent into a vial.

[0233] (3) Construction of chelation reaction system (labeling reaction): Take 3 mL 68 Add GaCl3 eluent to the reaction flask; place the reaction flask in a constant temperature metal bath and react at 37°C for 20 minutes.

[0234] (4) Sterilization filtration: Filtration is carried out using a 0.22 μm PVDF sterile filter.

[0235] (5) Quality control analysis: Table 16 shows the precursors of VHH-H3RESCA. 68 Ga labeling test results. The results show that all percentage contents meet the requirements.

[0236] Table 16 VHH-H3RESCA precursors 68 Ga marker detection results

[0237] 5.3 VHH-H3RESCA precursor 18 F marking and quality control

[0238] Achieving this through chelation coupling technology 18 The specific steps for F radioactive labeling are as follows: (1) Preparation of aluminum chloride solution: Take 10 mg of aluminum chloride or aluminum chloride hydrate and add it to 10-50 mL of 0.1 mol / L sodium acetate buffer. After complete dissolution, take 0.2 mL of the solution and add it to the reaction flask.

[0239] (2) Preparation of radioactive nuclides: fluorine-containing [ 18 F] ions H2 18 After O was enriched by QMA column, fluoride was removed using sodium chloride injection. 18 F] ions were eluted into the reaction flask and reacted at 37°C for 1-5 minutes.

[0240] (3) Preparation of H3RESCA-VHH coupling solution: Take VHH-H3RESCA precursor (10-500 nmol), add 0.5 mL of 0.1 mol / L sodium acetate buffer (containing 1-5 mg / mL gentian acid and appropriate amount of Tween 80) to redissolve, mix well and transfer all to reaction flask.

[0241] (4) Construction of chelation reaction system (labeling reaction): Place the reaction flask in a constant temperature metal bath or synthesizer and react at 37°C for 15-30 minutes.

[0242] (5) Purification and replacement: The reaction solution was purified by an antibody purification column. Column equilibration: 15 mL sodium chloride injection containing 15 mg / mL sodium ascorbate and 1 mg / mL gentianic acid; loading volume: 1.5 mL; elution buffer: sodium chloride injection containing 15 mg / mL sodium ascorbate and 1 mg / mL gentianic acid, and 1.5-3.0 mL of elution peak were collected; the solution was filtered using a 0.22 μm PVDF sterile filter.

[0243] (6) Quality control analysis: Table 17 shows the precursors of VHH-H3RESCA. 18 F-labeling test results. The results show that all percentage contents meet the requirements.

[0244] Table 17 VHH-H3RESCA precursors 18 F-mark detection results

[0245] 6. 68 Ga-NOTA-VHH / 68 In vivo distribution of Ga-H3RESCA-VHH

[0246] 6.1 68 In vivo imaging of Ga-NOTA-VHH

[0247] A U118MG cell line overexpressing the LRRC15 antigen was constructed using the original U118MG cell line, yielding a clone, U118MG_clone13, with high expression levels, which was then used to establish a mouse model. In vivo studies were conducted using 6-8 week old female NCG mice (18-20 g). Xenografting was performed, with U118MG_clone13 cells subcutaneously implanted into the upper right back of the mice. The cell seeding density was approximately 8 × 10⁶ cells / mL. 6 Cells / mouse. Usable tumors (150-200 mm) were obtained after 3 weeks. 3 ).

[0248] Radiolabeled VHH antibody was administered to mice via tail vein injection at approximately 100 μCi. Imaging studies were conducted at 30 min, 1 h, 2 h, and 4 h post-administration to investigate drug targeting and analyze radioactive uptake in tumors, heart, muscle, and kidneys at multiple time points. Simultaneously, corresponding regions of the kidneys, tumors, muscles, and heart were delineated on PET / CT fusion images to obtain the %ID / g of the respective organs. In the blocking experiment, mice were pre-administered VHH-NOTA antibody, and after drug distribution was complete, they were injected via tail vein. Imaging studies were performed at 30 min, 1 h, 2 h, and 4 h post-administration.

[0249] The PET-CT imaging results at different time points after drug administration in the 5A12-h2m9-06609-GSC-NOTA group and the 11E06-h8-107-GSC-NOTA group are as follows: Figure 8 and Figure 9 As shown, animal imaging data are presented in Tables 18 and 19. PET-CT images of U118MG_clone13 tumor-bearing mice from the 5A12-h2m9-06609-GSC-NOTA and 11E06-h8-107-GSC-NOTA groups showed that both molecules performed well on LRRC15-positive tumors. The 11E06-h8-107-GSC-NOTA group showed significant uptake at the tumor site, with lower uptake in the kidneys, resulting in an excellent tumor / kidney ratio. Uptake in non-target organs was rapidly cleared over time.

[0250] Table 18 68 Ga-5A12-h2m9-06609-GSC-NOTA Animal Imaging Data

[0251] (%ID / g and its ratios, average ± SD, n = 3)

[0252] Table 19 68 Ga-11E06-h8-107-GSC-NOTA Animal Imaging Data

[0253] (%ID / g and its ratios, average ± SD, n = 3)

[0254] 6.2 68 In vivo imaging of Ga-H3RESCA-VHH

[0255] Using the U118MG mouse model 68 In vivo imaging studies of Ga-H3RESCA-VHH were conducted using 6-8 week old female NCG mice (18-20 g). Xenografting was employed, with U118MG cells subcutaneously implanted into the upper right back of the mice. The cell seeding density was approximately 5 × 10⁻⁶ cells / mL. 6 Cells / mouse. Usable tumors (150-200 mm) were obtained after 3 weeks. 3 ).

[0256] Radiolabeled VHH antibody was administered to mice via tail vein injection at approximately 100 μCi. Imaging studies were conducted at 30 min, 1 h, 2 h, and 4 h post-administration to investigate drug targeting and analyze radioactive uptake in tumors, heart, muscle, and kidneys at multiple time points. Simultaneously, corresponding regions of the kidneys, tumors, muscles, and heart were delineated on PET / CT fusion images to obtain the %ID / g of the respective organs. In the blocking experiment, mice were pre-administered VHH antibody, and after drug distribution was complete, they were injected via tail vein. Imaging studies were performed at 30 min, 1 h, 2 h, and 4 h post-administration.

[0257] [ 68 Ga]11E06-h8-m7-GSC-H3RESCA and [ 68 PET-CT imaging results at different time points after administration of Ga]11E06-h8-107-H3RESCA are as follows: Figure 10 and Figure 11 As shown, the animal imaging data are shown in Tables 20 and 21.

[0258] pass[ 68 Ga]11E06-h8-m7-GSC-H3RESCA and [ 68 In PET-CT images of U118MG tumor-bearing mice, Ga]11E06-h8-107-H3RESCA showed good imaging effects on LRRC15-positive tumors, with significant uptake in tumor tissues and low uptake in the kidneys, resulting in excellent tumor / kidney ratio. Uptake in non-target organs was rapidly cleared over time.

[0259] Table 20 [ 68 Ga]11E06-h8-m7-GSC-H3RESCA Animal Imaging Data

[0260] (%ID / g and its ratios, average ± SD, n = 3)

[0261] Table 21 [ 68 Ga]11E06-h8-107-H3RESCA Animal Imaging Data

[0262] (%ID / g and its ratios, average ± SD, n = 3)

[0263] 6.3 18 In vivo imaging of F-H3RESCA-VHH

[0264] Using the U118MG mouse model 18 In vivo imaging studies of F-H3RESCA-VHH were conducted using 6-8 week old female NCG mice (18-20 g). Xenografting was employed, with U118MG cells subcutaneously implanted into the upper right back of the mice. The cell seeding density was approximately 5 × 10⁻⁶ cells / mL. 6 Cells / mouse. Usable tumors (150-200 mm) were obtained after 3 weeks. 3 ).

[0265] Radiolabeled VHH antibody was administered to mice via tail vein injection at approximately 100 μCi. Imaging studies were conducted at 30 min, 1 h, 2 h, and 4 h post-administration to investigate drug targeting and analyze radioactive uptake in tumors, heart, muscle, and kidneys at multiple time points. Simultaneously, corresponding regions of the kidneys, tumors, muscles, and heart were delineated on PET / CT fusion images to obtain the %ID / g of the respective organs. In the blocking experiment, mice were pre-administered VHH antibody, and after drug distribution was complete, they were injected via tail vein. Imaging studies were performed at 30 min, 1 h, 2 h, and 4 h post-administration.

[0266] [ 18 PET-CT imaging results at different time points after administration of F]11E06-m7-GSC-H3RESCA are as follows Figure 12 And as shown in Table 22. By [ 18 In PET-CT images of U118MG tumor-bearing mice, F]11E06-m7-GSC-H3RESCA showed good imaging effect on LRRC15-positive tumors, with significant uptake in all tumor tissue sites and low uptake in the kidneys, resulting in excellent tumor / kidney ratio. Uptake in non-target organs was rapidly cleared over time.

[0267] Table 22 [ 18F]11E06-m7-GSC-H3RESCA Animal Imaging Data

[0268] (%ID / g and its ratios, average ± SD, n = 3)

[0269] 7. Preparation of VHH-Fc antibody conjugated with maleimide-GGFG-DXd

[0270] VHH-Fc antibody was mixed with TCEP (tris(2-carboxyethyl)phosphine) at a molar ratio of 1:10 and incubated at 37°C for 40 minutes for reduction. PBS was used as the buffer in the experiment. After the reaction, Mal-DXd (abbreviation for Mal-GGFG-DXd) was added at a molar ratio of VHH-Fc:linker-payload = 1:10, and the mixture was incubated at room temperature for 1 hour. The reacted sample was purified using a Superdex 200pg molecular sieve to remove unreacted linker-payload, with PBS as the mobile phase at a flow rate of 1 ml / min. The average coupling ratio (DAR) of the payload to the antibody was calculated using mass spectrometry. The results showed that the average DAR values ​​of the conjugated VHH-Fc-Isotype-DXd, 5A12-h2m9-088-Fc-DXd, 11E06-h8-m7-Fc-DXd, and 11E06-h8-107-Fc-DXd were all approximately 4. Purity analysis was performed using SEC-HPLC, and the purity was greater than 95%.

[0271] The amino acid sequence of VHH in VHH-Fc-Isotype-DXd is shown in SEQ ID NO: 44, and the amino acid sequence of its Fc fragment is shown in SEQ ID NO: 42. VHH-Fc was prepared according to the method described in this application. Furthermore, the amino acid sequences of the Fc fragments of 5A12-h2m9-088-Fc-DXd, 11E06-h8-m7-Fc-DXd, and 11E06-h8-107-Fc-DXd are shown in SEQ ID NO: 45.

[0272] 8. In vivo efficacy of VHH-Fc antibody conjugated with maleimide-GGFG-DXd

[0273] B-hLRRC15 MC38 cells (Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd., catalog number 321867) were cultured at a rate of 5 × 10⁻⁶. 60.1 mL / mouse was injected into the right axilla of 60 C57BL / 6J mice (Peking University School of Medicine, strain C57BL / 6J, female, 6-7 weeks old, weighing 16-18 g, housed in an SPF-grade barrier facility) until the average tumor volume reached 160 mm. 3 Around 21 days after administration, 45 mice were randomly selected based on tumor volume and body weight, with 9 mice per group, for a total of 5 groups: G1: PBS, G2: VHH-Fc Isotype-Dxd (7 mg / kg), G3: 5A12-h2m9-088-Fc-Dxd (7 mg / kg), G4: 11E06-h8-m7-Fc-Dxd (7 mg / kg), and G5: 11E06-h8-107-Fc-Dxd (7 mg / kg). All groups received intraperitoneal (ip) injection twice weekly for 5 consecutive weeks. The experiment ended on day 21 after administration. Body weight and tumor volume were measured and recorded twice weekly during the administration and observation period.

[0274] The results of tumor volume measurement in experimental animals after drug administration are as follows: Figure 13 As shown in Table 23, on day 21 after drug administration, the mean tumor volume in the G1 PBS group was 1731.9 ± 126.2 mm. 3 The average tumor volume in groups G2 through G5 was 1701.8 ± 180.5 mm, respectively. 3 659.7±275.3 mm 3 0±0 mm 3 and 1.9±1.9 mm 3 Tumor growth inhibition rate (TGI) TV The rates were 1.92%, 68.30%, 110.50%, and 110.43%, respectively. The complete tumor regression rates in each group were 0% (0 / 9), 0% (0 / 9), 44.4% (4 / 9), 100% (9 / 9), and 88.9% (8 / 9), respectively. Significant antitumor therapeutic effects were observed in the 11E06-h8-m7-Fc-Dxd and 11E06-h8-107-Fc-Dxd groups, with complete tumor regression rates of 100% and 88.9%, respectively. TV The tumor regression rates were 110.5% and 110.4%, respectively, showing highly statistically significant differences compared to the PBS and Isotype-DXd groups (P < 0.01). The 5A12-h2m9-088-Fc-DXd group exhibited some inhibitory effect on tumor growth, with a complete tumor regression rate of 44.4% (4 / 9). TGI... TV The tumor volume was 68.3%, with significant individual variation, but not statistically significant compared to the PBS group (P>0.05); post-drug body weight measurements of the experimental animals are as follows: Figure 14As shown in Table 24, mice did not experience weight loss during the administration period, maintained good activity and appetite, and tolerated the test products well in each group.

[0275] Table 23 Effects of the test substance on tumor volume of B-hLRRC15 MC38 cells transplanted into C57BL / 6J mice

[0276] a: Mean ± standard error; b: Statistical comparison of tumor volume between the drug-treated group and the PBS control group on day 21 after drug administration, using one-way ANOVA analysis and Dunnett's test. p < 0.05, p < 0.01.

[0277] Table 24 Effects of the test product on the body weight of C57BL / 6J mice inoculated with B-hLRRC15 MC38 cells

[0278] a: Mean ± standard error; b: Statistical comparison of body weight between the treatment group and the G1 control group on day 21 of drug administration, using one-way ANOVA analysis and Dunnett's test.

[0279] All patents, patent application publications, and non-patent documents mentioned and / or listed in this application are incorporated herein by reference in their entirety. Exemplary embodiments of the inventions described above have been described; however, those skilled in the art can modify or improve the exemplary embodiments described herein without departing from the spirit and scope of this application, and such variations or equivalents also fall within the scope of this application.

Claims

1. An anti-LRRC15 antibody or its antigen-binding fragment, comprising a VHH chain, wherein the VHH chain comprises CDR1, CDR2, and CDR3, wherein: The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 3; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 7; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 9; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 12; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 14, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 15; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 15; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 15; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 21, the amino acid sequence of CDR2 is shown in SEQ ID NO: 22, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 23; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 21, the amino acid sequence of CDR2 is shown in SEQ ID NO: 25, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 26; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 21, the amino acid sequence of CDR2 is shown in SEQ ID NO: 22, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 28; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 30, the amino acid sequence of CDR2 is shown in SEQ ID NO: 25, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

31. The amino acid sequence of CDR1 is shown in SEQ ID NO: 21, the amino acid sequence of CDR2 is shown in SEQ ID NO: 25, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 33; or The amino acid sequence of CDR1 is shown in SEQ ID NO:35, the amino acid sequence of CDR2 is shown in SEQ ID NO:36, and the amino acid sequence of CDR3 is shown in SEQ ID NO:37; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 30, the amino acid sequence of CDR2 is shown in SEQ ID NO: 25, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

39. The above CDR sequence is defined according to Kabat.

2. The anti-LRRC15 antibody or its antigen-binding fragment as described in claim 1, wherein: The VHH chain comprises an amino acid sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NO: 4, 5, 8, 10, 13, 16, 17, 18, 19, 20, 24, 27, 29, 32, 34, 38, and 40, or consists of any of the amino acid sequences shown in SEQ ID NO: 4, 5, 8, 10, 13, 16, 17, 18, 19, 20, 24, 27, 29, 32, 34, 38, and 40; or The VHH chain contains an amino acid sequence that has at least 85% identity with the amino acid sequence shown in SEQ ID NO: 43, or is composed of the amino acid sequence shown in SEQ ID NO:

43.

3. The anti-LRRC15 antibody or its antigen-binding fragment as described in claim 1, wherein the anti-LRRC15 antibody is a VHH antibody or a VHH-Fc antibody; Optionally, the Fc fragment is derived from IgG; Optionally, the Fc fragment is derived from human IgG1; and / or Optionally, the anti-LRRC15 antibody may be in monovalent or multivalent form.

4. An isolated nucleic acid molecule encoding the anti-LRRC15 antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the nucleic acid molecule of claim 4 or the vector of claim 5.

7. An antibody conjugate comprising the anti-LRRC15 antibody as described in any one of claims 1 to 3, or its antigen-binding fragment, linker, and effector; Optionally, the effector is selected from: radionuclides, antitumor agents, immunomodulators, bioresponse modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, imaging agents, chemiluminescent compounds, enzymes, and metal ions; Optionally, the effector is a cytotoxic drug; Optionally, the antibody conjugate is VHH-Fc-Mal-GGFG-DXd; Optionally, the antibody conjugate is 11E06-h8-m7-Fc-Mal-GGFG-DXd.

8. A radionuclide-labeled antibody conjugate comprising the anti-LRRC15 antibody or its antigen-binding fragment as described in any one of claims 1 to 3 and a radionuclide-chelating agent; Optionally, the radionuclide-labeled antibody conjugate further comprises a linker; Optionally, the radioactive nuclides include alpha-ray nuclides, gamma-ray nuclides, beta-ray nuclides, and positron-emitting nuclides; Optionally, the radionuclide is selected from: 11 C 13 N、 15 O、¹ 8 F, 32 P, 44 Sc、 47 Sc、 51 Mn, 52m Mn, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 68 Ge 72 As、 75 Br、 76 Br、 82m Rb、 83 Sr、 86 Y、 89 Sr、 89 Zr、 90 Y、 99m Tc, 105 Rh、 110 In、 111 In、 117 mSn, 120 I, 123 I, 124 I, 125 I、¹³¹I、 134 Ce、 148 Gd, 149 Tb, 149 Pm, 152 Tb, 153 Sm、 154-158 Gd, 160 Gd, 161 Tb, 165 Ho、 166 Ho、 167 Tm、 175 Yb、¹ 77 Lu、 186 Re、 188 Re、 197 Au、 198 Au、 199 Au、 201 Ti、 209 Bi、 211 At、 212 Pb, 212 Bi、 213 Bi、 213 Po、²²³Ra、 223 Fr、²² 5 Ac, 227 Th and 229 Th; Optionally, the chelating agent is selected from: 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), N,N'-bis(2-hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED-CC), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), or triazacyclononane phosphate (TRAP), EDTA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, AAZTA, BAT, BAT-TM, Crown, Cyclen, DO2A, CB-DO2A, DO3A, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, MACROPA, HEHA, DOTA-GA, p-BnDOTA, p-Bn-SCN-DOTA, NH2-DOTA, NH2-DOTA-GA, p-N CS-Bn-DOTA-GA, p-NH2-Bn-OxODO3A, p-SCN-Bn-oxo-DO3A, NODA-GA, NH2-NODA-GA, p-NCS-Bn-NODA-GA, p-NH2Bn-NOTA, p-SCN-Bn-NOTA, NCS-MP-N ODA, NH2-MPAA-NODA, PCTA, p-NH2-Bn-PCTA, pSCN-Bn-PCTA, p-SCN-Bn-HEHA, H2-MACROPA-NCS, H1-MACROPA, H2-MACROPA-NH2, H4OCTAPA, tetra-(S,S,S,S)-Me-DOTA, SHBED, (HBED-CC)TFP, DMSA, DMPS, DHLA, tetra-(S,S,S,S)-Et-DOTA, tetra-(S,S,S,S)-iBu-DOTA and maleimide-nBu-DOTA and their derivatives; Optionally, the chelating agent is selected from H3RESCA, (+-)H3RESCA-TFP, and (+-)H3RESCA-Mal; Optionally, the radionuclide-labeled antibody conjugate is: radionuclide-VHH-GSC-Mal-NOTA, radionuclide-VHH-Mal-NOTA, radionuclide-VHH-GSC-Mal-H3RESCA, or radionuclide-VHH-Mal-H3RESCA. Optionally, the radionuclide-labeled antibody conjugate is selected from 68Ga-5A12-h2m9-06609-GSC-NOTA, 68Ga-11E06-h8-107-GSC-NOTA, 68Ga-11E06-h8-m7-GSC-NOTA, 68Ga-11E06-h8-107-GSC-H3RESCA, 68Ga-11E06-h8-m7-GSC-H3RESCA, 18F-11E06-h8-m7-GSC-H3RESCA, and 18F-11E06-h8-107-GSC-H3RESCA.

9. A pharmaceutical composition comprising the anti-LRRC15 antibody or its antigen-binding fragment as described in any one of claims 1 to 3, the nucleic acid molecule as described in claim 4, the vector as described in claim 5, the host cell as described in claim 6, the antibody conjugate as described in claim 7, or the radionuclide-labeled antibody conjugate as described in claim 8.

10. Use of the anti-LRRC15 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, the nucleic acid molecule as described in claim 4, the vector as described in claim 5, the host cell as described in claim 6, the antibody conjugate as described in claim 7, the radionuclide-labeled antibody conjugate as described in claim 8, or the pharmaceutical composition as described in claim 9 in the preparation of a medicament for the prevention, diagnosis, and / or treatment of diseases characterized by LRRC15 expression; Optionally, the diseases characterized by LRRC15 expression are selected from: sarcoma, glioblastoma, melanoma, breast cancer, HER2-negative breast cancer, HER2-positive breast cancer, HR+ breast cancer, triple-negative breast cancer, head and neck cancer, head and neck squamous cell carcinoma, lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, ovarian cancer, colorectal cancer, non-small cell lung cancer, testicular cancer, and squamous cell carcinoma.

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