Anti-GPC4 antibodies

Abstract

Provided herein are antibodies, including antigen-binding fragments thereof, that bind all or part of GPC4; compositions containing the antibodies or antigen-binding fragments thereof; combinations of the antibodies or antigen-binding fragments thereof and methods of use. In particular embodiments, antibodies or antigen-binding fragments thereof are used in methods for detecting the presence of GPC4 by imaging, including molecular, medical, and diagnostic imaging.

Description

[0001] Related patent applications This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 540,896, filed September 27, 2023, entitled “ANTI-GPC4 ANTIBODIES,” inventored by Susannah Kassmer et al., with agent number 102738-1379830-003000US. The entire contents of the aforementioned patent application (including all text, tables, and figures) are incorporated herein by reference for all purposes.

[0002] field In some aspects, this disclosure relates to antibodies or antigen-binding fragments thereof that bind to phosphatidylinositol proteoglycan 4 (GPC4), and methods, systems, and kits for detecting GPC4. In some aspects, this disclosure relates to antibodies or antigen-binding fragments thereof for determining the level of GPC4 in samples containing or suspected of containing GPC4. In some aspects, this disclosure relates to antibodies or antigen-binding fragments thereof for diagnosing or treating individuals who have or are suspected of having a disease or condition related to GPC4.

[0003] background Phosphatidylinositol proteoglycan 4 (GPC4) is a phosphatidylinositol proteoglycan belonging to the heparan sulfate proteoglycan (HSPG) family of cell surface proteoglycans that may be involved in cell division and growth regulation. HSPG family members can act as reservoirs or regulators of several growth factors and signaling molecules. HSPG molecules contain core proteins anchored to the cell membrane via GPI anchors. HSPGs function as co-receptors or regulators of Wnt activation. GPCs are R-spondin co-receptors that regulate Wnt signaling. GPC3 enhances Wnt signaling, while GPC6 inhibits it. The binding of Wnt3a to frizzled protein and LRP6 stabilizes cytoplasmic β-catenin, which translocates to the nucleus to activate the TCF / LEF transcription factor. Cells expressing GPC4 show a higher degree of activation of the β-catenin pathway in response to Wnt3a. Knockdown of GPC4 inhibits activation of the β-catenin pathway. GPC4 is upregulated in pancreatic and colorectal cancers and is associated with resistance to fluorouracil (5FU) chemotherapy.

[0004] Overview This document provides antibodies that bind all or part of GPC4, including their antigen-binding fragments; compositions containing said antibodies or their antigen-binding fragments; combinations of said antibodies or their antigen-binding fragments; and methods of use. In a particular embodiment, the antibody or its antigen-binding fragment is used in a method for detecting the presence of GPC4 by imaging (including molecular, medical, and diagnostic imaging).

[0005] This document provides antibodies or antigen-binding fragments thereof, including antibodies or antigen-binding fragments thereof that specifically bind to GPC4 (e.g., human GPC4), wherein the antibody or antigen-binding fragment contains specific complementarity-determining regions (CDRs), including heavy chain CDRs (i.e., CDRH1, CDRH2, and / or CDRH3) and light chain CDRs (i.e., CDRL1, CDRL2, and / or CDRL3), such as any CDRs described herein. In some embodiments, the antibody or antigen-binding fragment thereof includes a heavy chain variable domain and a light chain variable domain, such as any variable domain described herein.

[0006] This document provides antibodies or antigen-binding fragments thereof that bind to GPC4 or a portion thereof, the antibodies or antigen-binding fragments thereof comprising a) an immunoglobulin heavy chain variable domain comprising: (i) a heavy chain complementarity-determining region 1 (CDRH1) comprising SEQ ID NO:2; (ii) a heavy chain complementarity-determining region 2 (CDRH2) comprising SEQ ID NO:3; (iii) a heavy chain complementarity-determining region 3 (CDRH3) comprising SEQ ID NO:4; and b) an immunoglobulin light chain variable domain comprising: (i) a light chain complementarity-determining region 1 (CDRL1) comprising SEQ ID NO:5; (ii) a light chain complementarity-determining region 2 (CDRL2) comprising SEQ ID NO:6; and (iii) a light chain complementarity-determining region 3 (CDRL3) comprising SEQ ID NO:7.

[0007] In some embodiments, the immunoglobulin heavy chain variable domain comprises: CDRH1, wherein CDRH1 comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:2; CDRH2, wherein CDRH2 comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:3; and CDRH3, wherein CDRH3 comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:2; and CDRH3, wherein CDRH3 comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or more sequence identity with SEQ ID NO:2; and CDRH3, wherein CDRH3 comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, NO:4 shows an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0008] In some embodiments, the immunoglobulin heavy chain variable domain comprises: CDRH1, which contains the amino acid sequence shown in SEQ ID NO:2; CDRH2, which contains the amino acid sequence shown in SEQ ID NO:3; and CDRH3, which contains the amino acid sequence shown in SEQ ID NO:4.

[0009] In some embodiments, the immunoglobulin light chain variable domain comprises: CDRL1, wherein CDRL1 comprises the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:5; CDRL2, wherein CDRL2 comprises the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:6; and CDRL3, wherein CDRL3 comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:5; and CDRL3, wherein CDRL3 comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or more sequence identity with SEQ ID NO:5; and CDRL3, wherein CDRL3 comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, NO:7 shows an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0010] In some embodiments, the immunoglobulin light chain variable domain comprises: CDRL1, which contains the amino acid sequence shown in SEQ ID NO:5; CDRL2, which contains the amino acid sequence shown in SEQ ID NO:6; and CDRL3, which contains the amino acid sequence shown in SEQ ID NO:7.

[0011] In some embodiments, CDRH1 contains the amino acid sequence shown in SEQ ID NO:2; CDRH2 contains the amino acid sequence shown in SEQ ID NO:3; CDRH3 contains the amino acid sequence shown in SEQ ID NO:4; CDRL1 contains the amino acid sequence shown in SEQ ID NO:5; CDRL2 contains the amino acid sequence shown in SEQ ID NO:6; and CDRL3 contains the amino acid sequence shown in SEQ ID NO:7.

[0012] In some embodiments, CDRH1 comprises the sequence shown in SEQ ID NO:2 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:2; CDRH2 comprises the sequence shown in SEQ ID NO:3 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:2; CDRH3 comprises the sequence shown in SEQ ID NO:4 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:2; NO:4 shows an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity; CDRL1 contains the sequence shown in SEQ ID NO:5 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:5; CDRL2 contains the sequence shown in SEQ ID NO:6 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:5; NO:6 shows an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identical; and CDRL3 contains the sequence shown in SEQ ID NO:7 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identical to SEQ ID NO:7.

[0013] In some embodiments, the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO:15 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:15.

[0014] In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence shown in any of SEQ ID NO:15.

[0015] In some embodiments, the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:17 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:17.

[0016] In some embodiments, the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:17.

[0017] In some embodiments, the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO:15 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:15; and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:17 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:17.

[0018] In some embodiments, the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO:15; and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:17.

[0019] In some implementations, the antibody or antigen-binding fragment includes an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain.

[0020] In some implementations, the antibody or antigen-binding fragment comprises two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.

[0021] In some implementations, the antibody or its antigen-binding fragment is isolated.

[0022] In some implementations, the antibody or its antigen-binding fragment is humanized.

[0023] In some implementations, the antibody or its antigen-binding fragment is conjugated.

[0024] In some embodiments, the antibody or antigen-binding fragment further comprises an oligonucleotide. In some embodiments, the oligonucleotide comprises a sample barcode sequence. In some embodiments, the oligonucleotide comprises a binding site for primers and anchors.

[0025] In some embodiments, the antibody or its antigen-binding fragment is conjugated to a detectable biomarker or label. In some embodiments, the detectable biomarker or label is directly conjugated to an antigen or its antigen-binding fragment. In some embodiments, the detectable biomarker or label is conjugated to an oligonucleotide. In some embodiments, the detectable biomarker or label comprises a detectable portion. In some embodiments, the detectable portion is a radioisotope, a fluorescent label, or an enzyme substrate label.

[0026] In some implementations, the antibody or its antigen-binding fragment is non-diffusely immobilized on a solid support.

[0027] In some embodiments, this disclosure provides isolated antibodies that specifically bind to GPC4, wherein the isolated antibodies compete with the antibodies described herein for binding to GPC4.

[0028] In some embodiments of the antibody described herein, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody comprises one or more personal framework regions. In some embodiments, the antibody or antigen-binding fragment is a single-chain fragment. In some embodiments, the single-chain fragment is a single-chain variable fragment (scFv).

[0029] In some aspects, this document provides combinations of antibodies or antigen-binding fragments thereof, wherein the combination comprises two or more anti-GPC4 antibodies or antigen-binding fragments described herein. In some embodiments, the two or more antibodies or antigen-binding fragments comprise one or more first antibodies or antigen-binding fragments thereof that bind to a first epitope or region within GPC4; and one or more second antibodies or antigen-binding fragments thereof that bind to a second epitope or region within GPC4. In some further embodiments, one or more first antibodies or antigen-binding fragments thereof and one or more second antibodies or antigen-binding fragments thereof bind to and / or do not compete for binding to GPC4 with non-overlapping epitopes or regions of GPC4 (e.g., human GPC4). Furthermore, in some embodiments, the antibody is conjugated to a detectable biomarker or label. In some embodiments, at least one antibody or antigen-binding fragment in the combination of two or more anti-GPC4 antibodies or antigen-binding fragments described herein, optionally one or more first antibodies or antigen-binding fragments thereof or one or more second antibodies or antigen-binding fragments thereof, is conjugated to a label. In some embodiments, at least one antibody or antigen-binding fragment, optionally one or more first antibodies or their antigen-binding fragments, or one or more second antibodies or their antigen-binding fragments, is attached or immobilized to a solid support. In some embodiments, one or more first antibodies or second antibodies or antigen-binding fragments are attached or immobilized to a solid support, and another of said one or more first antibodies or second antibodies or antigen-binding fragments is conjugated to a label. In some embodiments, the label is a fluorescent dye, fluorescent protein, radioisotope, chromophore, metal ion, gold particle, silver particle, magnetic particle, polypeptide, enzyme, streptavidin, biotin, luminescent compound, or oligonucleotide. In some embodiments, the solid support is a bead, column, array, assay plate, microwell, rod, filter, or strip. In some embodiments, the antibody is non-diffusely immobilized on the solid support. In other embodiments, the device is a rapid detection device or a rapid diagnostic device.

[0030] On the other hand, this disclosure features isolated nucleic acids encoding isolated antibodies described herein. This disclosure also provides expression vectors containing the nucleic acids described herein. Furthermore, this disclosure provides isolated host cells containing the expression vectors described herein.

[0031] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can be used to detect GPC4 in a sample. In some embodiments, the antibodies or antigen-binding fragments thereof bind to cells expressing GPC4 in the sample. In some embodiments, the sample includes immune cells. In some embodiments, the sample includes a heterogeneous population of immune cells. In some embodiments, the immune cells are selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and peripheral blood mononuclear cells (PBMCs). In some embodiments, the sample includes cells with a disease or condition. In some embodiments, the disease or condition is cancer, an autoimmune disease, an inflammatory disease, a neurological disease, or an infection. In some embodiments, the cancer is acute myeloid leukemia, acute lymphoblastic leukemia, colorectal cancer, ovarian cancer, breast cancer, gynecological cancer, liver cancer, glioblastoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, esophageal cancer, gastric cancer, pancreatic cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, and melanoma. In some embodiments, the detection includes using a single antibody or an antigen-binding fragment thereof to bind a portion of GPC4. In some embodiments, the detection includes using two antibodies or antigen-binding fragments thereof, each capable of binding a different portion of GPC4. In some embodiments, the detection of GPC4 is performed on the cell surface. In some embodiments, the detection of GPC4 is performed intracellularly. In some embodiments, the detection of GPC4 indicates the presence or absence of a disease or condition. In some embodiments, the detection is performed in vitro. In some embodiments, the detection is performed in vivo.

[0032] In some implementations, the antibody or its antigen-binding fragment binds to cells expressing GPC4.

[0033] This document provides diagnostic antibodies or antigen-binding fragments thereof, including any antibodies or antigen-binding fragments thereof described herein. This document also provides kits containing antibodies or antigen-binding fragments thereof from any of the embodiments described herein. In some embodiments, the kit is a diagnostic kit configured to detect GPC4 in biological samples.

[0034] This document provides compositions comprising an antibody or an antigen-binding fragment thereof of any of the embodiments described herein and a pharmaceutically acceptable excipient. In some embodiments, the antibody or antigen-binding fragment thereof is used as an adjuvant or in combination with an adjuvant.

[0035] This document provides isolated nucleic acids containing nucleotide sequences of immunoglobulin heavy chain variable domains encoding an agent of any embodiment described herein. This document also provides isolated nucleic acids containing nucleotide sequences of immunoglobulin light chain variable domains encoding an agent of any embodiment described herein. The sequences encoding the immunoglobulin heavy chain variable domains of an agent of any embodiment described herein and the sequences encoding the immunoglobulin light chain variable domains of an agent of any embodiment described herein may be on the same isolated nucleic acid or on different isolated nucleic acids. Therefore, this document also provides isolated nucleic acids containing nucleotide sequences of immunoglobulin heavy chain variable domains and immunoglobulin light chain variable domains encoding an antibody or antigen-binding fragment thereof of any embodiment described herein.

[0036] This document provides a recombinant expression vector comprising isolated nucleic acids of any embodiment described herein. The document also provides a recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette contains a nucleic acid molecule having a nucleotide sequence encoding a variable domain of an immunoglobulin heavy chain of any embodiment described herein, and the second expression cassette contains a nucleic acid molecule having a nucleotide sequence encoding a variable domain of an immunoglobulin light chain of an antibody or antigen-binding fragment thereof of any embodiment described herein.

[0037] This document provides a recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette contains a nucleic acid molecule having a nucleotide sequence containing any embodiment described herein, and the second expression cassette contains a nucleic acid molecule having a nucleotide sequence containing any embodiment described herein. In some embodiments, the first and second expression cassettes contain a promoter.

[0038] This article provides host cells transfected with recombinant expression vectors using any of the embodiments described herein.

[0039] This document provides antibody-drug conjugates comprising an antibody or an antigen-binding fragment thereof of any embodiment described herein. This document also provides compositions comprising an antibody-drug conjugate and a pharmaceutically acceptable carrier.

[0040] This document provides a method for detecting GPC4, comprising: a) contacting the sample with the antibody or antigen-binding fragment thereof under conditions that bind the antibody or antigen-binding fragment thereof of any embodiment described herein to a GPC4 receptor on the sample, wherein the binding results in the formation of a receptor / antibody or antigen-binding fragment thereof complex; b) detecting the presence of the receptor / antibody or antigen-binding fragment thereof complex; and c) wherein the detection includes the presence or absence of the GPC4 receptor on the sample.

[0041] This article provides methods for treating or preventing GPC4-related diseases or conditions in subjects, comprising: a) contacting a sample known to contain or suspected to contain GPC4 with an antibody or antigen-binding fragment thereof of any embodiment described herein; b) detecting the presence of a complex comprising GPC4 and an antibody or antigen-binding fragment thereof; wherein the presence of the complex indicates the presence of a disease or condition; and c) administering to a subject an antibody or antigen-binding fragment thereof of any embodiment described herein.

[0042] This document provides methods for diagnosing diseases or conditions, comprising: a) isolating a sample from a subject; b) incubating the sample with an antibody or an antigen-binding fragment thereof of any embodiment described herein for a period of time sufficient to generate a GPC4:anti-GPC4 complex; c) detecting the presence or absence of the GPC4:anti-GPC4 complex from the isolated tissue; and d) correlating the presence or abundance of GPC4 with the site of interest in the tissue sample. In some embodiments, an increase in GPC4 at the site of interest in the tissue sample exceeding control levels indicates a disease or condition in the subject.

[0043] In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo. In some embodiments, the assay includes intracellular assay. In some embodiments, the assay includes cell surface assay. In some embodiments, the assay includes hybridization of the detectable portion with an antibody or an antigen-binding fragment thereof. In some embodiments, the sample is contacted with a second antibody. In some embodiments, the second antibody is an antibody containing the detectable portion. In some embodiments, the detectable portion contains an oligonucleotide. In some embodiments, the detectable portion contains a fluorescent label. In some embodiments, the assay includes sequencing. In some embodiments, the detectable portion includes immunofluorescence. In some embodiments, the sample is a formalin-fixed paraffin-embedded sample. In some embodiments, the sample includes cells. In some embodiments, the sample includes a tissue sample.

[0044] In some embodiments, the sample includes immune cells. In some embodiments, the immune cells are selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and peripheral blood mononuclear cells (PBMCs). In some embodiments, the sample includes tissues or cells associated with a disease or condition. In some embodiments, the disease or condition is cancer, an autoimmune disease, an inflammatory disease, or an infection. In some implementation schemes, the disease or condition is selected from non-viral cancer, virus-associated cancer, cancer associated with HBV infection, cancer associated with Epstein-Barr virus (EBV) infection, cancer associated with polyomavirus infection, erythema nodosum leprosy (ENL), autoimmune diseases, autoimmune inflammation, autoimmune thyroid diseases, B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, B-cell leukemia, myelodysplastic syndrome, solid phase cancer, herpes virus infection, and / or rejection of transplanted tissues or organs.

[0045] In some embodiments, antibodies or antigen-binding fragments thereof may be used in methods for associating the presence or abundance of GPC4 with a location of interest in a tissue sample.

[0046] In some embodiments, antibodies or antigen-binding fragments thereof may be used in methods for detecting GPC4 in tissue samples. In some embodiments, the method includes generating nucleic acid molecules comprising all or part of the sequence of an oligonucleotide or its complement.

[0047] In some embodiments, antibodies or antigen-binding fragments thereof may be used for the construction of protein libraries. In some embodiments, the construction of protein libraries includes sequencing. In some embodiments, the construction of protein libraries includes the use of flow cytometry. Brief description of the attached diagram The accompanying drawings illustrate some embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not drawn to scale, and in some cases, aspects may be exaggerated or enlarged to facilitate understanding of a particular embodiment.

[0049] Figure 1This is an image showing NTERA-2 cells stained with GPC4 antibody AB2.

[0050] Figure 2 This is a diagram showing that GPC4 antibody AB2 shows negative staining on U-937 cells.

[0051] Figures 3A-3C This indicates that GPC4 antibody AB2 does not interact with GPC1 ( Figure 3A ), GPC2 ( Figure 3B ) or GPC6 ( Figure 3C (Diagram of cross-reaction)

[0052] Figure 4 This is a bar graph showing that the GPC4 antibody AB2 can reduce Wnt3a-induced translocation of β-catenin to the cell nucleus.

[0053] Figure 5 The image shows that the GPC4 antibody AB2 can reduce Wnt3a-induced translocation of β-catenin to the cell nucleus, while commercially available GPC4 antibodies do not show this effect.

[0054] Figure 6 The bar graph shows that the GPC4 antibody AB2 reduced Wnt3a-induced translocation of β-catenin to the cell nucleus at all tested concentrations, while commercially available antibodies did not show this effect.

[0055] Detailed Explanation This document provides antibodies that bind to GPC4, including their antigen-binding fragments; nucleic acids encoding said antibodies and antigen-binding fragments; and cells, such as recombinant cells for expressing and producing these antibodies and antigen-binding fragments that can bind GPC4 under physiological and / or in vitro conditions. Methods for producing and using the antibodies and antigen-binding fragments are also provided, for example, in methods for detecting GPC4 in samples from individuals, including methods for laboratory / research purposes (e.g., flow cytometry, ELISA, and / or Western blotting), and / or for using and treating and / or preventing various diseases or conditions by delivering a drug or other composition containing said antibody or its antigen-binding fragment.

[0056] All references cited in this article, including patent applications, patent publications, and scientific literature and databases, are incorporated herein by reference in their entirety for all purposes, to the extent that each individual reference is specifically and individually indicated by inclusion.

[0057] For clarity of the content and without limitation, the details are divided into the following subsections. The section headings used in this document are for organizational purposes only and should not be construed as limiting the topics described.

[0058] I. Definition Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this subject belongs. In some cases, terms with their commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein is not necessarily construed as representing a material difference from the commonly understood meaning in the art. It should be understood that the disclosure provided herein is not limited to specific compositions or biological systems. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and should not be construed as limiting.

[0059] As used herein, the term "antibody" includes antigen-binding fragments that retain their binding specificity. For example, many well-characterized antigen-binding fragments exist. Thus, for instance, pepsin digests an antibody at the C-terminus of a disulfide bond in its hinge region to produce F(ab)'2, a dimer of Fab, which itself is a light chain linked to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions to break the disulfide bond in the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a partially hinged region (for a more detailed description of other antigen-binding fragments, see Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993)). Although the various antigen-binding fragments are defined based on the digestion of intact antibodies, those skilled in the art will understand that fragments can be synthesized de novo by chemical methods or using recombinant DNA methods. Therefore, the term antibody, as used herein, also includes antigen-binding fragments generated by modifying intact antibodies or synthesized using recombinant DNA methods.

[0060] Antibodies as described herein may consist of one or more polypeptides encoded essentially by immunoglobulin genes or segments of immunoglobulin genes. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as a large number of immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which, in turn, define the types of immunoglobulins: IgG, IgM, IgA, IgD, and IgE. In some embodiments, the antibody is IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, or IgE.

[0061] Typical immunoglobulin (antibody) structural units are known to consist of tetramers. Each tetramer contains two identical pairs of polypeptide chains, each pair having a "light" chain (approximately 25 kD) and a "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

[0062] In antibodies, substitution variants remove at least one amino acid residue and insert a different residue at its position. The most interesting sites for substitution mutagenesis include hypervariable regions, but framework alterations are also considered. Examples of conserved substitutions are described in this paper.

[0063] Substantial modifications to antibody biological properties are achieved by selecting substitutions that significantly differ in maintaining the following: (a) the polypeptide backbone structure of the substituted region (e.g., β-sheet or helical conformation), (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Naturally occurring residues are grouped according to their common side chain properties as follows: (1) Nonpolar: Leucine, Met, Ala, Val, Leu, Ile; (2) Polar and uncharged: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged): Asp, Glu; (4) Alkaline (positively charged): Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatics: Trp, Tyr, Phe, His.

[0064] Non-conservative substitution is performed by replacing members of one of these categories with members of another category.

[0065] One type of substitution that can be performed is changing one or more cysteine ​​residues in the antibody (which can be chemically reactive) to another residue, such as, but not limited to, alanine or serine. For example, non-canonical cysteine ​​substitutions can be present. Substitutions can be made in the CDR or frame regions of the variable domains of the antibody or in constant regions. In some embodiments, the cysteine ​​is canonical (e.g., involved in disulfide bond formation). Any cysteine ​​residue that does not participate in maintaining the correct conformation of the antibody can also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bonds can be added to the antibody to improve its stability, particularly when the antibody is an antigen-binding fragment such as an Fv fragment.

[0066] Antibodies contain VH -V L Dimers, including single-chain antibodies (antibodies that exist as a single polypeptide chain), such as single-chain Fv antibodies (sFv or scFv), in which variable heavy chain and variable light chain domains (directly or via peptide linkers) are linked together to form a continuous polypeptide. Single-chain Fv antibodies are covalently linked V... H -V L It can be expressed by nucleic acids, which contain V proteins directly linked or linked via peptide-coding linkers. H -and V L - Encoded sequence (e.g.) ， Huston, et al.Proc.Nat. Acad.Sci.USA ,85:5879-5883, 1988). Although V H and V L Interconnected as a single polypeptide chain, but V H and V L The domains are non-covalently associated. Optionally, the antibody can be another fragment. Other fragments can also be generated (e.g., using recombinant techniques) as soluble proteins or as fragments obtained from the display method. Antibodies can also include diantibodies and miniantibodies. The antibodies disclosed herein also include heavy chain dimers, such as antibodies derived from camels. In some embodiments, the antibody is dimer. In other embodiments, the antibody can be in monomeric form having an active isotype. In some embodiments, the antibody is in a multivalent form, such as a trivalent or tetravalent form.

[0067] "Antibody fragment" or "its antigen-binding fragment" includes a portion of a complete antibody, the antigen-binding region of a complete antibody, and / or the variable region. Antibody fragments or their antigen-binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv(dsFv), Fd fragments, Fd' fragments; diabody; linear antibody (see U.S. Patent No. 5,641,870, Example 2; Zapata et al, Protein Eng. 8(10): 1057-1062

[1995] ); single-chain antibody molecules, including single-chain Fv(scFv) or single-chain Fab(scFab); antigen-binding fragments of any of the above and multispecific antibodies derived from antibody fragments.

[0068] The “Fv” contains a heavy chain variable region domain and a light chain variable region domain linked by non-covalent association. Folding of these two domains produces six complementarity-determining regions (CDRs) (three from the heavy chain and three from the light chain), which facilitate the binding of amino acid residues to the antigen and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind antigens, although in some cases, the affinity is lower than that of the intact binding site.

[0069] “dsFv” refers to an engineered intermolecular disulfide bond with a stable VH-VL pair.

[0070] The “Fd fragment” is an antibody fragment containing a variable domain (VH) and a constant region domain (CHI) of the antibody heavy chain.

[0071] A “Fab fragment” is an antibody fragment produced by digesting a full-length immunoglobulin with papain, or a fragment with the same structure produced synthetically (e.g., through recombinant methods). A Fab fragment consists of a light chain (containing VL and CL) and another chain, which contains a variable domain (VH) of the heavy chain and a constant region domain (CHI) of the heavy chain.

[0072] The “F(ab')2 fragment” is an antibody fragment produced by digesting immunoglobulins with pepsin at pH 4.0–4.5, or a fragment with the same structure produced synthetically (e.g., by recombination methods). The F(ab')2 fragment essentially contains two Fab fragments, each of which contains several additional amino acids, including cysteine ​​residues that form the disulfide bond connecting the two fragments.

[0073] A “Fab’ fragment” is a fragment containing half of an F(ab’)2 fragment (one heavy chain and one light chain).

[0074] The “Fd’ fragment” is an antibody fragment containing a heavy chain portion of the F(ab’)2 fragment.

[0075] The “Fv” fragment is a fragment that contains only the VH and VL domains of the antibody molecule.

[0076] "scFv fragment" refers to an antibody fragment containing a variable light chain (VL) and a variable heavy chain (VH) covalently linked in any order via a peptide linker. The linker length allows the two variable domains to be bridged without significant interference. An exemplary linker is (Gly-Ser)n residues, with some Glu or Lys residues scattered throughout to improve solubility.

[0077] "Die" is a dimerized scFv; dienes typically have shorter peptide linkers than scFv and preferentially dimerize.

[0078] As used herein, the terms "variable region" and "variable domain" refer to the amino acid sequence portions of the antibody's light and heavy chains that contain complementarity-determining regions (CDRs, such as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) and framework regions (FRs). The variable domains of the heavy and light chains are typically named V1 and V2, respectively. H and V L Variable domains are contained in the Fab, F(ab')2, Fv, and scFv antigen-binding fragments described herein and participate in specific antigen recognition.

[0079] As used in this article, "complementarity-determining regions (CDRs)" refer to the three hypervariable regions in each chain that disrupt the four framework regions established by the variable regions of the light and heavy chains. CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (numbered sequentially from the N-terminus) and are usually identified by the chain in which the specific CDR resides. Therefore, VH CDR3 is located in the variable domain of its host antibody heavy chain, while VL CDR1 is the CDR1 of its host antibody light chain variable domain.

[0080] The sequences of different light chain or heavy chain framework regions are relatively conserved within a species. The framework region of an antibody, which is the combined framework region of the light and heavy chains that form its components, is used to locate and arrange CDRs in three-dimensional space.

[0081] The amino acid sequences of the CDR and framework regions can be determined using various well-known definitions in the art, such as the Kabat, North method (see, e.g., North et al., J Mol Biol. 406(2):228-256, 2011), the Chothia, the international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et al., ibid.; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VHsegments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)).The definition of antigen binding sites is also described in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219–221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268–9272 (1989); Martin, et al, Methods Enzymol., 203, 121–153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141–172 1996).

[0082] As used herein, a “chimeric antibody” refers to an immunoglobulin molecule in which (a) a constant region or a portion thereof is altered, substituted, or exchanged such that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, effector function, and / or species, or to a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that imparts novel properties to the chimeric antibody; or (b) a variable region or a portion thereof is altered, substituted, or exchanged with a variable region or a portion thereof that has a different or altered antigen specificity, or with a corresponding sequence from another species or from another antibody class or subclass.

[0083] As used herein, a "humanized antibody" refers to an immunoglobulin molecule in which a CDR derived from a donor antibody has been grafted onto a human frame sequence. Humanized antibodies may also contain donor-derived residues within the frame sequence. Humanized antibodies may also contain at least a portion of the human immunoglobulin constant region. Humanized antibodies may also contain residues that are not present in the recipient antibody or in the input CDR or frame sequence. Humanization can be achieved using methods known in the art (e.g., Jones et al., Nature 321:522-525; 1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988); Presta, Curr. Op. Struct. Biol. 2:593-596, 1992; U.S. Patent No. 4,816,567), including "hyperhumanized" antibodies (Tan et al., J. Immunol. 169: 1119, 2002) and "surface remodeling" (e.g., Staelens et al., Mol. Immunol. 43:1243, 2006; and Roguska et al., Proc. Natl. Acad. Sci. USA 91: 969, ). The technology was implemented in 1994.

[0084] The term "recombinant," when used to refer to, for example, cells or nucleic acids, proteins, or vectors, indicates that the cell, nucleic acid, protein, or vector has been modified by introducing a heterologous nucleic acid or protein or by altering the native nucleic acid or protein, or that the cell is derived from a cell that has been so modified. Thus, for example, recombinant cells express genes that are not present in the natural (non-recombinant) form of the cell, or express native genes that are abnormally expressed, underexpressed, or not expressed at all.

[0085] The terms “antigen,” “immunogen,” “antibody target,” and “target analyte,” etc., are used herein to refer to molecules, compounds, or complexes that are recognized by antibodies (i.e., can be specifically bound by antibodies). This term can refer to any molecule that can be specifically recognized by antibodies, such as peptides, polynucleotides, carbohydrates, lipids, chemical moieties, or combinations thereof (e.g., phosphorylated or glycosylated peptides). Those skilled in the art will understand that this terminology does not imply that the molecule is immunogenic in every case, but only that it can be targeted by antibodies.

[0086] Antibodies bind to “epitopes” on antigens. An epitope is a localization site on an antigen that is recognized and bound by an antibody. An epitope may comprise several amino acids or a portion of several amino acids, such as five or six or more (e.g., 20 or more) amino acids, or a portion of those amino acids. In some cases, epitopes include non-protein components, such as components from carbohydrates, nucleic acids, or lipids. In some cases, an epitope is a three-dimensional part. Thus, for example, when the target is a protein, an epitope may contain consecutive amino acids or amino acids from different parts of a protein that are close together by protein folding (e.g., discontinuous epitopes). The same is true for other types of target molecules that form three-dimensional structures. Epitopes typically contain at least three, and more commonly at least five or eight to ten, amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

[0087] A “label” or “detectable component” is a diagnostic agent or component that can be detected by spectroscopic, radiological, photochemical, biochemical, immunochemical, chemical, or other physical means. Exemplary labels include radiolabels (e.g., 111 In、 99m Tc, 131 I, 67 Ga) and other FDA-approved imaging agents. Additional markers include 32 P, fluorescent dyes, electron-dense reagents, enzymes, biotin, digoxigenin or haptens and proteins or other detectable entities, such as by incorporating radiolabels into the targeting agent. Any method known in the art for conjugating nucleic acids or nanocarriers with labels can be used, such as the method described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0088] "Labeled" or "tagged" antibodies or agents are antibodies or agents that are covalently bound to a label by a linker or chemical bond, or non-covalently bound to a label by an ionic bond, van der Waals bond, electrostatic bond, or hydrogen bond, so that the presence of the antibody or agent can be detected by detecting the presence of the label bound to the antibody or agent.

[0089] The technique of conjugating detectable and therapeutic agents with antibodies is well known (see, for example...). ，Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", inMonoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery" in Controlled Drug Delivery (2 nd Ed.), Robinson et al.(eds.), pp. 623-53 (MarcelDekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In CancerTherapy: A Review" in Monoclonal Antibodies '84: Biological And ClinicalApplications, Pinchera et al.(eds.), pp. 475-506 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., 62:119-58 (1982)).

[0090] The terms “specific to,” “specific binding,” and similar terms refer to a molecule (e.g., an antibody or antigen-binding fragment) that binds to a target molecule (e.g., an antibody or antigen-binding fragment) with at least twice the affinity of the non-target compound (e.g., at least 4, 5, 6, 7, 8, 9, 10, 20, 25, 50, or 100 times the affinity of the non-target compound). For example, an antibody that specifically binds to a target (e.g., GPC4) typically binds to the target with at least twice the affinity of the non-target compound. Specificity can be determined using standard methods, such as solid-phase ELISA immunoassays (for a description of the forms and conditions of immunoassays that can be used to determine specific immunoreactivity, see, for example, Harlow & Lane). Using Antibodies, A Laboratory Manual (1998)).

[0091] The term "binding" for antibody targets (e.g., antigens, analytes, immune complexes) generally indicates that an antibody binds to the majority of the antibody target in a pure population (assuming a suitable molar ratio). For example, an antibody binding to a given antibody target typically binds to at least two-thirds of the antibody target in solution (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). Those skilled in the art will recognize that some variability will arise depending on the method used to determine the binding and / or the threshold.

[0092] A "control" sample or value refers to a sample used as a reference, typically a known reference, for comparison with the test sample. For example, a test sample may be taken from test conditions (e.g., in the presence of the test compound) and compared to a sample from known conditions, such as in the absence of the test compound (negative control) or in the presence of the known compound (positive control). A control may also represent an average or range obtained from a large number of tests or results. Those skilled in the art will recognize that controls can be designed to evaluate any number of parameters. For example, controls can be designed to compare therapeutic benefits based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of benefits and / or side effects). Controls can be designed for in vitro applications. Those skilled in the art will understand which controls are valuable in a given context and are able to analyze data based on comparisons with control values. Controls are also valuable for determining the significance of data. For example, if the value of a given parameter varies widely in controls, the variation in the test sample will not be considered significant.

[0093] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "percentage of identity" refers to two or more sequences or subsequences being identical, or having a specified percentage of identical amino acid residues or nucleotides (i.e., approximately 60% identity in a specified region when performing maximum correspondence comparisons and alignments within a comparison window or specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher), as measured using the BLAST 2.0 sequence comparison algorithm and the default parameters described below, or as measured by manual alignment and visual inspection (see, for example, the NCBI website ncbi.nlm.nih.gov / BLAST / etc.). Such sequences are referred to as "substantially identical." As described herein, preferred algorithms may take into account vacancies, etc. Preferably, the identity exists in a region of at least about 25 amino acids or nucleotides in length, or more preferably, in a region of 50-100 or more amino acids or nucleotides in length.

[0094] For sequence comparisons, typically one sequence serves as a reference sequence, which is compared to a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer (with subsequence coordinates specified if necessary), and the sequence algorithm program parameters are specified. Preferably, default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the program parameters.

[0095] As used herein, a “comparison window” refers to a segment selected from any number of consecutive positions between 20 and 600 (typically about 50 to about 200, more typically about 100 to about 150), whereby, after optimal alignment of two sequences, a sequence can be compared with a reference sequence of the same number of consecutive positions. The sequence alignment methods used for comparison are well known in the art.

[0096] The algorithms suitable for determining sequence identity percentage and sequence similarity are BLAST and BLAST 2.0, which are based on Altschul. et al., Nuc.Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol.Biol As described in .215:403-410 (1990). BLAST and BLAST 2.0, along with the parameters described herein, are used to determine the percentage of sequence identity between nucleic acids and proteins of this disclosure. The software used for performing the BLAST analysis is publicly available from the National Center for Biotechnology Information ( / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by recognizing short words of length W in the query sequence. These short words match or satisfy a positive threshold score T when compared to words of the same length in the database sequence. T is called the nearest neighbor score threshold (Altschul). et al.(Same as above). These initial neighbor word hits act as seeds to initiate the search for longer HSPs containing them. Word hits extend in both directions along each sequence until the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Word hits stop extending in each direction when: the cumulative alignment score decreases by an amount X from its maximum value; the cumulative score becomes zero or lower due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expected value I of 10, M=5, N=-4, and compares two strands. For amino acid sequences, the BLASTP program uses a word length of 3, an expected value I of 10, and a BLOSUM62 scoring matrix by default (see Henikoff & Henikoff). Proc.Natl.Acad.Sci.USA 89:10915 (1989)) The alignment (B) is 50, the expected value I is 10, M=5, N=-4, and the two chains are compared.

[0097] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, their polymers, and their complements. This term includes nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0098] Unless otherwise stated, a specific nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer). et al ., Nucleic Acid Res .19:5081 (1991); Ohtsuka et al ., J. Biol.Chem. 260:2605-2608 (1985); Rossolini et al ., Mol.Cell.Probes 8:91-98 (1994)).

[0099] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms encompass amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0100] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bonded to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

[0101] Amino acids can be referred to in this article using their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides can be referred to using their commonly accepted single-letter codes.

[0102] The term "competition" used herein to refer to the competition for binding between a first antibody or its antigen-binding portion and a second antibody or its antigen-binding portion, wherein the binding of the first antibody to its corresponding epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody to its corresponding epitope in the absence of the second antibody. Alternatively, the binding of the second antibody to its epitope may also be detectably reduced in the presence of the first antibody, but this is not necessary. That is, the first antibody may inhibit the binding of the second antibody to its epitope, while the second antibody does not inhibit the binding of the first antibody to its corresponding epitope. However, when each antibody detectably inhibits the binding of other antibodies to their respective epitopes or ligands, whether to the same, greater, or lesser extent, these antibodies are considered to "cross-compete" for binding to their respective epitopes. Both competitive and cross-competitive antibodies are covered by this disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or its portion), those skilled in the art will understand, based on the teachings provided herein, that such competitive and / or cross-competitive antibodies are covered and can be used in the methods disclosed herein.

[0103] Many types of competitive binding assays are known, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (see Stahli). et al. , Methods in Enzymology 9:242-253 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland) et al. , J. Immunol. 137:3614-3619 (1986)); direct labeling determination of solid phases, direct labeling sandwich determination of solid phases (see Harlow and Lane, Antibodies, A Laboratory Manual Cold Spring Harbor Press (1988); solid-phase direct labeling of RIA using I-125 markers (see Morel). et al. , Molec.Immunol .25(1):7-15 (1988)); Solid-phase direct biotin-avidin EIA (Cheung et al. , Virology 176:546-552 (1990)); and directly labeled RIA (Moldenhauer) et al. , Scand.J. Immunol. 32:77-82 (1990)). Typically, this assay involves using a purified antigen or cells carrying said purified antigen bound to a solid surface, an unlabeled test immunoglobulin, and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of the label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, an excess of the test immunoglobulin is present. Antibodies identified by the competitive assay (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to adjacent epitopes, which are sufficiently close to the epitope bound by the reference antibody to create steric hindrance. Typically, when an excess of the competitive antibody is present, it inhibits the specific binding of the reference antibody to the common antigen by at least 50% or 75%.

[0104] As used herein, the term "GPC4" refers to human phosphatidylinositol proteoglycan 4 (GPC4) protein, its isotypes, or variants, including naturally occurring variants of human GPC4, such as splice variants or allelic variants. An exemplary amino acid sequence of human GPC4 is shown in SEQ ID NO: 1 (Uniprot ID: O75487). In some embodiments, human GPC4 may refer to variants, such as allelic variants or splice variants, that show at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 1. In some embodiments, it should be understood that the provided antibody or antigen-binding fragment may show cross-reactive binding to another mammalian GPC4 protein, such as mouse GPC4 or primate GPC4.

[0105] The amino acid sequence of GPC4 is as follows: .

[0106] "Solid support" refers to a non-aqueous matrix to which antibodies can adhere or attach, according to the provided disclosure. Examples of solid supports include, but are not limited to, microtiter plates, membranes (e.g., nitrocellulose), beads, dipsticks, thin-layer chromatography plates, or other solid media.

[0107] As used herein, "individual" or "subject" refers to a mammal. "Mammalian" used for therapeutic purposes includes humans, livestock and farm animals, as well as zoo animals, sporting animals, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some implementations, the individual or subject is a human.

[0108] II. Antibodies that bind to GPC4 This article provides antibodies that specifically bind to GPC4, including their antigen-binding fragments. The provided antibodies include monoclonal antibodies that bind to GPC4 and their antigen-binding fragments, and offer superior target specificity, signal-to-noise ratio, etc., compared to other reported antibodies. This article also provides methods for generating anti-GPC4 antibodies, as well as methods for detecting and using said antibodies.

[0109] Any antibody or antigen-binding fragment thereof provided herein may bind to all or part of GPC4. Optionally, any antibody or antigen-binding fragment thereof provided herein may bind to all or part of the extracellular domains of GPC4.

[0110] Any antibody or antigen-binding fragment thereof is a GPC4 antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment is isolated (e.g., separated from components of its natural environment (e.g., animal, biological sample)). In some embodiments, the anti-antibody is a humanized antibody or its antigen-binding fragment. In some embodiments, the antibody is a derivative of a bound humanized antibody. In some embodiments, the antibody binds under laboratory conditions (e.g., in vitro, in flow cytometry assays, in ELISA). In some embodiments, the antibody binds under physiological conditions (e.g., in the subject's cells).

[0111] Typically, the antibodies provided herein comprise at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain. In some embodiments, the antibodies described herein comprise two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains. Typically, each immunoglobulin heavy chain variable domain of the antibody comprises a first, second, and third heavy chain complementarity-determining region (CDR; CDRH1, CDRH2, and CDRH3), and each immunoglobulin light chain variable domain of the antibody comprises a first, second, and third light chain CDR (CDRL1, CDRL2, and CDRL3).

[0112] Antibodies can be antigen-binding fragments, such as Fab, F(ab')2, Fv, or scFv. Antigen-binding fragments can be generated using any method known in the art, including chemical digestion (e.g., papain or pepsin) and recombinant methods. Methods for isolating and preparing recombinant nucleic acids are known to those skilled in the art (see Sambrook et al., Molecular Cloning.A Laboratory Manual (2d ed. 1989); Ausubel et al., Current Protocols in Molecular Biology (1995)). Antibodies can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells, such as COS, CHO, and HeLa cell lines and myeloma cell lines.

[0113] This document provides antibodies or antigen-binding fragments thereof, including antibodies or antigen-binding fragments thereof that specifically bind to GPC4 (e.g., human GPC4), wherein the antibody or antigen-binding fragment contains specific complementarity-determining regions (CDRs), including heavy chain CDRs (i.e., CDRH1, CDRH2, and / or CDRH3) and light chain CDRs (i.e., CDRL1, CDRL2, and / or CDRL3), such as any CDRs described herein. In some embodiments, the antibody or antigen-binding fragment thereof includes a heavy chain variable domain and a light chain variable domain, such as any variable domain described herein.

[0114] This document provides antibodies or antigen-binding fragments thereof that bind to GPC4 or a portion thereof, the antibodies or antigen-binding fragments thereof comprising a) an immunoglobulin heavy chain variable domain comprising: (i) a heavy chain complementarity-determining region 1 (CDRH1) comprising SEQ ID NO:2; (ii) a heavy chain complementarity-determining region 2 (CDRH2) comprising SEQ ID NO:3; (iii) a heavy chain complementarity-determining region 3 (CDRH3) comprising SEQ ID NO:4; and b) an immunoglobulin light chain variable domain comprising: (i) a light chain complementarity-determining region 1 (CDRL1) comprising SEQ ID NO:5; (ii) a light chain complementarity-determining region 2 (CDRL2) comprising SEQ ID NO:6; and (iii) a light chain complementarity-determining region 3 (CDRL3) comprising SEQ ID NO:7.

[0115] Optionally, the immunoglobulin heavy chain variable domain comprises: CDRH1, wherein CDRH1 comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:2; CDRH2, wherein CDRH2 comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:3; and CDRH3, wherein CDRH3 comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:2; and CDRH3, wherein CDRH3 comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or more sequence identity with SEQ ID NO:2; and CDRH3, wherein CDRH3 comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, NO:4 shows an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0116] Optionally, the variable domain of the immunoglobulin heavy chain comprises: CDRH1, which contains the amino acid sequence shown in SEQ ID NO:2; CDRH2, which contains the amino acid sequence shown in SEQ ID NO:3; and CDRH3, which contains the amino acid sequence shown in SEQ ID NO:4.

[0117] Optionally, the immunoglobulin light chain variable domain comprises: CDRL1, wherein CDRL1 comprises the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:5; CDRL2, wherein CDRL2 comprises the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:6; and CDRL3, wherein CDRL3 comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:5; and CDRL3, wherein CDRL3 comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 99% or more sequence identity with SEQ ID NO:5; and CDRL3, wherein CDRL3 comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, NO:7 shows an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0118] Optionally, the immunoglobulin light chain variable domain comprises: CDRL1, which contains the amino acid sequence shown in SEQ ID NO:5; CDRL2, which contains the amino acid sequence shown in SEQ ID NO:6; and CDRL3, which contains the amino acid sequence shown in SEQ ID NO:7.

[0119] Optionally, CDRH1 contains the amino acid sequence shown in SEQ ID NO:2; CDRH2 contains the amino acid sequence shown in SEQ ID NO:3; CDRH3 contains the amino acid sequence shown in SEQ ID NO:4; CDRL1 contains the amino acid sequence shown in SEQ ID NO:5; CDRL2 contains the amino acid sequence shown in SEQ ID NO:6; and CDRL3 contains the amino acid sequence shown in SEQ ID NO:7.

[0120] Optionally, CDRH1 comprises the sequence shown in SEQ ID NO:2 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:2; CDRH2 comprises the sequence shown in SEQ ID NO:3 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:2; CDRH3 comprises the sequence shown in SEQ ID NO:4 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:2; NO:4 shows an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity; CDRL1 contains the sequence shown in SEQ ID NO:5 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:5; CDRL2 contains the sequence shown in SEQ ID NO:6 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:5; NO:6 shows an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identical; and CDRL3 contains the sequence shown in SEQ ID NO:7 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identical to SEQ ID NO:7.

[0121] Optionally, the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO:15 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:15.

[0122] Optionally, the immunoglobulin heavy chain comprises the amino acid sequence shown in any of SEQ ID NO:15.

[0123] Optionally, the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:17 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:17.

[0124] Optionally, the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:17.

[0125] Optionally, the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO:15 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:15; and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:17 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:17.

[0126] Optionally, the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO:15; and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:17.

[0127] III. Antibodies that competitively bind to anti-GPC4 antibodies This document also provides antibodies (e.g., competitive agents) that competitively bind to or are capable of competitively binding to one or more GPC4 antibodies described herein. In some cases, an antibody (e.g., a competitive agent antibody) is considered to competitively bind to GPC4 when the competitive agent binds to the overall same GPC4 region as the antibody described herein. In some cases, an antibody (e.g., a competitive agent antibody) is considered to competitively bind to GPC4 when the competitive agent binds to the exact same GPC4 region (e.g., the exact same peptide (linear epitope) or the exact same surface amino acid (conformational epitope)) as the antibody described herein. In some cases, an antibody (e.g., a competitive agent antibody) is considered capable of competitively binding to GPC4 when the competitive agent binds to the overall same GPC4 region (e.g., an extracellular region or a leucine-rich binding domain) as the antibody described herein under suitable assay conditions. In some cases, an antibody (e.g., a competitive agent) is considered capable of competitively binding to GPC4 when the competitive agent binds to the exact same GPC4 region (e.g., the exact same peptide (linear epitope) or the exact same surface amino acid (conformational epitope)) as the antibody described herein under suitable assay conditions.

[0128] In certain circumstances, when a competitive agent, for example under suitable assay conditions, blocks the binding of one or more antibodies described herein to GPC4, the antibody (e.g., the competitive agent antibody) can be considered to competitively bind to GPC4. Suitable competition or blocking assays (such as, for example, the blocking assays described herein) can be used to determine whether the competitive agent blocks the binding of one or more antibodies described herein to GPC4. The competitive agent antibody may block the binding of one or more antibodies described herein to GPC4 by 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%) in a competition or blocking assay, and conversely, one or more antibodies described herein may block the binding of the competitive agent antibody to GPC4 by about 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%) in a competition or blocking assay.

[0129] In certain circumstances, an antibody (e.g., a competing antibody) is considered to competitively bind to GPC4 when a competing agent binds to GPC4 with an affinity similar to that of one or more antibodies described herein, for example, under suitable assay conditions. In some embodiments, an antibody (i.e., a competing antibody) is considered to competitively bind to GPC4 when a competing agent binds to GPC4 with an affinity of at least about 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the affinity of one or more antibodies described herein.

[0130] This document also provides antibodies that bind to or are capable of binding to the same epitopes as one or more antibodies described herein. In particular, this document provides antibodies that competitively bind to the same epitopes on GPC4 (e.g., the same peptide (linear epitope) or the same surface amino acid (conformational epitope)) as one or more antibodies described herein. Such antibodies that bind to the same epitopes may be referred to as epitope competitors.

[0131] IV. Polyclonal and Monoclonal Antibodies Polyclonal antibodies can be generated in animals (vertebrates or invertebrates, including mammals, birds, and fish, including cartilaginous fish) by repeated subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Conjugation of the relevant antigen to a protein or other carrier (e.g., keyhole hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor) that is immunogenic in the species to be immunized can be useful, using bifunctional or derivatizing agents (e.g., maleimide benzoyl sulfosuccinimide ester (via cysteine ​​residue conjugation), N-hydroxysuccinimide (via lysine residue conjugation), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR, where R and R1 are different alkyl groups). Non-protein carriers (e.g., colloidal gold) can also be used for antibody production.

[0132] Animals can be immunized against antigens, immunogenic conjugates, or derivatives by combining, for example, 100 µg or 5 µg of protein or conjugate (for rabbits or mice, respectively) with three times the volume of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, animals are boosted with one-fifth to one-tenth of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to fourteen days later, blood is drawn from the animals, and the antibody titer in the serum is measured. Animals are boosted until the titer reaches a stable level. Typically, animals are boosted with conjugates of the same antigen but with different proteins and / or conjugated via different cross-linking agents. Conjugates can also be prepared as protein fusions in recombinant cell cultures. Furthermore, aggregation agents such as alum are suitable for enhancing the immune response.

[0133] Monoclonal antibodies can be prepared using hybridomas (e.g., the hybridoma method first described in Kohler et al., Nature, 256:495 (1975)) or by other methods such as recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). In the hybridoma method, mice or other suitable host animals (such as hamsters or macaques) are immunized to induce lymphocytes to produce or be able to produce antibodies that specifically bind to proteins used for immunization. Optionally, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent (such as polyethylene glycol) to form hybridoma cells (see, for example, Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0134] Hybridoma cells thus prepared are seeded in a suitable culture medium and allowed to grow, the medium containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium used for hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibit the growth of HGPRT-deficient cells. Preferred myeloma cells are those that fuse efficiently, support stable high levels of antibody production, and are sensitive to culture media such as HAT medium. Preferred myeloma cell lines are mouse myeloma lines, such as SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection (Rockville, Md. USA). Human myeloma and mouse-human heterologous myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0135] The culture medium in which hybridoma cells are grown is analyzed to determine the production of monoclonal antibodies against antigens. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation, by in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)), or by flow cytometry analysis of cells expressing membrane antigens. The binding affinity of monoclonal antibodies can be determined, for example, by the Skachard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0136] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the clone can be subcloned using limiting dilution methods and grown using standard methods (see, for example, Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 media. Alternatively, hybridoma cells can be grown in vivo as ascites tumors in animals. The monoclonal antibodies secreted by the subclones can be appropriately separated from the culture medium, ascites, or serum using conventional immunoglobulin purification methods (e.g., protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography).

[0137] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional methods, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of monoclonal antibodies. Alternatively, cDNA can be prepared from mRNA and then sequenced. Hybridoma cells are a preferred source of such genomic DNA or RNA for cDNA preparation. After isolation, the DNA can be placed in expression vectors well known in the art and then transfected into host cells that do not normally produce immunoglobulins (such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells), thereby achieving monoclonal antibody synthesis in recombinant host cells.

[0138] V. Humanization and Amino Acid Variants General methods for antibody humanization are described, for example, in U.S. Patent Nos. 5,861,155, 6,479,284, 6,407,213, 6,639,055, 6,500,931, 5,530,101, 5,585,089, 5,693,761, 5,693,762, 6,180,370, 5,714,350, 6,350,861, 5,777,085, 5,834,597, 5,882,644, 5,932,448, 6,013,256, 6,129,914, 6,210,671, 6,329,511, 5,225,539, 6,548,640, and 5,624,821. In some implementations, it may be desirable to generate amino acid sequence variants of these humanized antibodies, particularly when these variants enhance the binding affinity or other biological properties of the antibody (e.g., half-life).

[0139] In some implementations, the antibody is a humanized antibody, i.e., an antibody that retains the reactivity of a non-human antibody while exhibiting low immunogenicity in humans. This can be achieved, for example, by retaining the non-human CDR region and replacing the remainder of the antibody with its human counterpart. See, for example, Morrison et al., PNAS USA , 81:6851-6855 (1984); Morrison andOi, Adv. Immunol. , 44:65-92 (1988); Verhoeyen et al., Science , 239:1534-1536(1988);Padlan, Molec.Immun. , 28:489-498 (1991); Padlan, Molec.Immun ., 31(3):169-217 (1994). The technology of humanized antibodies is well known in the art and is used in, for example, U.S. Patent Nos. 4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and 6,329,511; WO 87 / 02671; EP Patent Application 0173494; Jones et al. (1986). Nature 321:522; and Verhoyen et al. (1988) Science Described in 239:1534. Humanized antibodies are, for example, described in Winter and Milstein (1991). NatureAs further described in 349:293. For example, a set of polynucleotides comprising a first sequence encoding a humanized immunoglobulin framework region and a second sequence encoding a desired immunoglobulin complementarity-determining region can be produced by synthesis or by combining a suitable cDNA with a genomic DNA segment. Human constant region DNA sequences can be isolated from a variety of human cells using well-known methods. The CDR used to produce the immunoglobulins of this disclosure can similarly be derived from a monoclonal antibody capable of specifically binding to GPC4.

[0140] Amino acid sequence variants of antibodies can be prepared by introducing appropriate nucleotide alterations into antibody DNA or through peptide synthesis. Such variants include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antibodies exemplified herein. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics. Amino acid alterations can also modify the post-translational processes of humanized or variant antibodies, such as changing the number or location of glycosylation sites.

[0141] A method for identifying specific residues or regions of an antibody as preferred sites for mutagenesis is called "alanine scanning mutagenesis," as exemplified by Cunningham and Wells. Science As described in 244:1081-1085 (1989). In this paper, groups of residues or target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and substituted with neutral or negatively charged amino acids (preferably Ala or poly-Ala) to affect the interaction of the amino acid with the antigen. Then, the positions of amino acids that exhibit functional sensitivity to substitution are refined by introducing further or other variants at or against the substitution site. Therefore, although the sites where amino acid sequence variations are introduced are predetermined, the nature of the mutation itself does not need to be predetermined. For example, to analyze the performance of mutations at a given site, alanine scans or random mutagenesis are performed at the target codon or region, and the desired activity of expressed antibody variants is screened. Amino acid sequence inserts include N-terminal and / or C-terminal fusions of polypeptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence inserts of single or multiple amino acid residues. Examples of terminal inserts include N-terminal methionine residues or antibodies fused with epitope tags. Other insertion variants include the fusion of enzymes or peptides that increase the serum half-life of the antibody with the N-terminus or C-terminus of the antibody.

[0142] Another type of variant is the amino acid substitution variant. These variants remove at least one amino acid residue from the antibody molecule and insert a different residue at its position. The most interesting sites for substitution mutagenesis include hypervariable regions, but FR alterations are also considered. Conservative substitutions are preferred, but more substantial changes can be introduced, and products can be screened. Examples of substitutions are listed below: Ala (A): Val; Leu; Ile; Val Arg €: Lys; Gln; Asn; Lys Asn (N): Gln; His; Asp, Lys; Gln; Arg Asp (D): Glu; Asn Cys €:Ser;Ala Gln (Q): Asn; Glu Glu €:Asp;Gln Gly (G): Ala His (H): Asn; Gln; Lys; Arg Ile (I): Leu; Val; Met; Ala; Leu; Phe; Leucine Leu (L): Leucine; Ile; Val; Ile; Met; Ala; Phe Lys (K): Arg; Gln; Asn Met (M): Leu; Phe; Ile Phe (F): Leu; Val; Ile; Ala; Tyr Pro (P): Ala Ser (S): Thr Thr (T): Ser Trp (W): Tyr; Phe Tyr (Y): Trp; Phe; Thr; Ser Val (V): Ile; Leu; Met; Phe; Ala; Leucine Substantial modifications to antibody biological properties are achieved by selecting substitutions that significantly differ in maintaining the following: (a) the polypeptide backbone structure (e.g., folded or helical conformation) of the substituted region, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Naturally occurring residues are grouped according to their common side chain properties as follows: (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr; (3) Acidity: Asp, Glu; (4) Alkaline: Asn, Gln, His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatic compounds: Trp, Tyr, Phe Non-conservative replacement requires replacing a member of one of the above categories with another category.

[0143] Any cysteine ​​residue that does not participate in maintaining the correct conformation of the antibody can be substituted to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds can be added to the antibody to improve its stability (especially when the antibody is an antigen-binding fragment such as an Fv fragment).

[0144] One type of substitution variant involves replacing one or more hypervariable residues of the parent antibody. Typically, variants selected for further development have improved biological properties relative to the parent antibody from which they originate. A convenient method for generating such substitution variants is phage display affinity maturation. Briefly, several hypervariable sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated can be displayed in monovalent form from filamentous phage particles as fusions with the M13 gene III product packaged within each particle. The phage-displayed variants are then screened as disclosed herein to determine their biological activity (e.g., binding affinity).

[0145] To identify candidate hypervariable sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable residues that significantly contribute to antigen binding. Optionally or additionally, analyzing the crystal structure of the antigen-antibody complex to identify the contact sites between the antibody and antigen can be beneficial. According to the techniques detailed herein, such contact residues and adjacent residues are candidates for substitution. After generating such variants, the group of variants is screened as described herein, and antibodies exhibiting superior properties in one or more relevant assays can be selected for further development.

[0146] Another type of amino acid variant of an antibody alters its original glycosylation pattern. This alteration refers to the deletion of one or more carbohydrate moieties present in the antibody, and / or the addition of one or more glycosylation sites not present in the antibody. Antibody glycosylation is typically N-linked and / or O-linked. N-linking refers to the attachment of a carbohydrate moieties to a side chain of asparagine residues. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are the most common recognition sequences for the enzymatic attachment of carbohydrate moieties to asparagine side chains. Therefore, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. Adding glycosylation sites to an antibody can be achieved by altering the amino acid sequence to include one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by adding one or more serine or threonine residues to the original antibody sequence or by replacing them with one or more serine or threonine residues (for O-linked glycosylation sites).

[0147] VI. Other Modifications Other modifications to antibodies have been considered. For example, the techniques described herein also relate to immunoconjugates comprising the antibodies described herein, said antibodies conjugated to cytotoxic agents such as toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof) or radioisotopes (e.g., radioconjugates) or cytotoxic drugs. Such conjugates are sometimes referred to as “antibody-drug conjugates” or “ADCs”. A variety of bifunctional protein coupling agents can be used to prepare conjugates, such as N-succinimide-3-(2-pyridyldithiool)propionate (SPDP), iminothiacyclopentane (IT), bifunctional derivatives of imine esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimide octanoate), aldehydes (e.g., glutaraldehyde), diazid compounds (e.g., bis-(p-azidobenzoyl)hexamethylenediamine), diazido compound derivatives (e.g., bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bifunctional fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0148] In some embodiments, any antibody or antigen-binding fragment thereof disclosed herein can be formulated as immunoliposomes. Antibody-containing liposomes are prepared by methods known in the art, for example, those described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980) and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with extended cycle times are disclosed in U.S. Patent No. 5,013,556. For example, liposomes can be generated by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of defined pore size to produce liposomes with the desired diameter. The Fab' fragment of the antibody presented herein can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem. 257:286-288 (1982). The liposomes optionally contain another active ingredient.

[0149] Enzymes or other peptides can be covalently bound to antibodies using techniques well known in the art, such as the heterobifunctional crosslinking reagents discussed above. In some embodiments, fusion proteins can be constructed using recombinant DNA techniques well known in the art, the fusion proteins comprising at least the antigen-binding region of the antibody provided herein, the antigen-binding region being linked to at least one functionally active portion of the enzyme (see, for example, Neuberger et al., Nature 312:604-608 (1984)).

[0150] In some implementations, using an antigen-binding fragment instead of the intact antibody may be desirable to, for example, increase penetration into target tissues and cells. In this case, modifying the antigen-binding fragment to increase its serum half-life may be desirable. This can be achieved, for example, by incorporating a salvage receptor-binding epitope into the antigen-binding fragment (e.g., by mutation of an appropriate region in the antigen-binding fragment, or by incorporating the epitope into a peptide tag that is then fused to the end or middle of the antigen-binding fragment, for example, through DNA or peptide synthesis; see, for example, WO96 / 32478, published October 17, 1996).

[0151] In some embodiments, any antibody or antigen fragment disclosed herein is conjugated or hybridized with an oligonucleotide. In some embodiments, the oligonucleotide includes a sample barcode sequence, a primer binding site, and an anchor. In some embodiments, the oligonucleotide may be conjugated or hybridized with any detectable biomarker or label disclosed herein. In some embodiments, the oligonucleotide is a polymer sequence. In some embodiments, the terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to a single-stranded nucleotide multimer of about 2 to about 500 nucleotides in length. In some embodiments, any oligonucleotide described herein may be synthetic, enzymatically prepared (e.g., via polymerization), or prepared using a "split-pool" method. In some embodiments, any oligonucleotide described herein may comprise ribonucleotide monomers (i.e., may be oligoribonucleotides) and / or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides). In some embodiments, any oligonucleotide described herein may comprise a combination of deoxyribonucleotide monomers and ribonucleotide monomers (e.g., a random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers). In some embodiments, the length of the oligonucleotide can be 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, or 400 to 500 nucleotides. In some embodiments, any oligonucleotide described herein may include one or more functional portions attached to another structure (e.g., covalently or non-covalently). In some embodiments, any oligonucleotide described herein may include one or more detectable markers (e.g., radioisotopes or fluorophores). In some embodiments, the anchor is a defined polymer, such as a polynucleotide or oligonucleotide sequence, designed to hybridize with a complementary oligonucleotide sequence. In some embodiments, the anchor is designed for the purpose of generating a double-stranded construct oligonucleotide sequence. In some embodiments, the anchor is located at the 3' end of the construct oligonucleotide sequence. In other embodiments, the anchor is located at the 5' end of the construct oligonucleotide sequence. Each anchor is specific to its intended complementary sequence.

[0152] In some embodiments, the sample barcode sequence is a polymer (e.g., a polynucleotide) that, when it is a functional element, is specific to a single ligand. In some embodiments, the sample barcode sequence can be used to identify specific cells or substrates, such as drop-seq microbeads. In some embodiments, the sample barcode sequence may be formed from a defined sequence of DNA, RNA, modified bases, or combinations of these bases, and any other polymers as defined above. In some embodiments, the sample barcode sequence is about 2 to 4 monomeric components, such as nucleotide bases, in length. In other embodiments, the barcode is at least about 1 to 100 monomeric components (e.g., nucleotides) in length. Therefore, in various implementations, the barcode is composed of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 The sequence (e.g., nucleic acid) of 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomeric components is formed. In some embodiments, the sample barcode sequence is a specific barcode that can be unique relative to other barcodes.

[0153] In some implementations, the sample barcode sequence can take many different forms. For example, the sample barcode sequence may include polynucleotide barcodes, random nucleic acid and / or amino acid sequences, and synthetic nucleic acid and / or amino acid sequences. The sample barcode sequence can be attached to an analyte or another part or structure in a reversible or irreversible manner. The sample barcode sequence can be added to fragments of, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) samples before or during sample sequencing. The sample barcode sequence can allow for the identification and / or quantification of individual sequencing reads (e.g., the barcode can be or may include a unique molecular identifier or "UMI").

[0154] Sample barcode sequences can, for example, spatially resolve molecular components present in a biological sample at single-cell resolution (e.g., the barcode may be or may include a "spatial barcode"). In some embodiments, the barcode includes UMI and spatial barcodes. In some embodiments, the barcode includes two or more sub-barcodes that function together as a single barcode. For example, a polynucleotide barcode may include two or more polynucleotide sequences separated by one or more non-barcode sequences (e.g., sub-barcodes).

[0155] In some embodiments, the primer binding site is a functional component of an oligonucleotide, which is itself an oligonucleotide or polynucleotide sequence that provides an annealing site for the amplification of the oligonucleotide. The primer binding site can be formed from DNA, RNA, PNA, modified bases, or combinations of these bases, polymers, or polyamides. In some embodiments, the primer binding site is about 10 such monomeric components (e.g., nucleotide bases). In other embodiments, the primer binding site is at least about 5 to 100 monomeric components (e.g., nucleotides). Therefore, in multiple embodiments, the primer binding sites are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5 The sequence (e.g., nucleic acid) of 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99, or up to 100 monomeric components is formed. In some embodiments, the primer binding site can be a universal sequence suitable as an annealing site for various amplification techniques. Amplification techniques include, but are not limited to, DNA polymerase-based amplification systems, such as polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP, MALBAC), strand displacement amplification (SDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and polymerization by any number of DNA polymerases (e.g., T4 DNA polymerase, Sulfulobus DNA polymerase, Klenow DNA polymerase, Bst polymerase, Phi29 polymerase), as well as RNA polymerase-based amplification systems (e.g., T7-, T3-, and SP6-RNA polymerase amplification), nucleic acid sequence-based amplification (NASBA), self-sustaining sequence replication (3SR), rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification (I), branching amplification, and RNA-seq. The methods for conjugating or hybridizing oligonucleotides can be carried out in the manner described in WO / 2018 / 144813, WO / 2016 / 018960, WO / 2018 / 089438, WO / 2014 / 182528, WO / 2018 / 026873, and WO / 2021 / 188838.

[0156] In some embodiments, modifications may optionally be introduced into the antibody (e.g., within the polypeptide chain or at the N-terminus or C-terminus) to, for example, prolong the in vivo half-life; said modifications include, for example, PEGylation or incorporation into a long-chain polyethylene glycol (PEG) polymer. The introduction of PEG or a long-chain PEG polymer increases the effective molecular weight of the polypeptide, for example, to prevent rapid filtration into urine. In some embodiments, lysine residues in the sequence are conjugated to PEG directly or via a linker. The linker may be, for example, a Glu residue or an acyl residue containing a thiol functional group for attachment to a suitably modified PEG chain. An alternative method for introducing the PEG chain is to first introduce a Cys residue, for example, replacing an Arg or Lys residue, at the C-terminus or a residue exposed to a solvent. This Cys residue site is then specifically attached to a PEG chain containing, for example, a maleimide functional group. Methods of incorporating PEG or a long-chain PEG polymer are known in the art (e.g., in Veronese, FM, et al., Drug Disc.Today 10: 1451-8(2005); Greenwald, RB, et al., Adv.Drug Deliv.Rev. 55: 217-50 (2003); Roberts, MJ, et al., Adv.Drug Deliv.Rev. (as described in , 54: 459-76 (2002), the contents of which are incorporated herein by reference).

[0157] Covalent modification of antibodies is also included within the scope of this technology. For example, modification can be achieved through chemical synthesis or enzymatic or chemical cleavage of the antibody. Other types of covalent modification of antibodies are introduced into the molecule by reacting the target amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chains or N-terminal or C-terminal residues. Examples of peptide covalent modification are described in U.S. Patent No. 5,534,615, which is specifically incorporated herein by reference. Preferred types of antibody covalent modification include linking the antibody to one of a variety of non-protein polymers (e.g., polyethylene glycol, polypropylene glycol, or polyoxyethylene) in a manner described, for example, in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0158] VII. Nucleic Acids, Vectors, Host Cells, and Recombinant Methods This disclosure also provides isolated nucleic acids encoding antibodies, vectors containing the nucleic acids, and host cells, as well as recombinant technologies for antibody production. The nucleic acids described herein may include one or more subsequences, each subsequence being referred to as a polynucleotide. Nucleic acids comprising SEQ ID NOs: 8-14 and 16 are provided herein.

[0159] This document provides nucleic acids (e.g., isolated nucleic acids) comprising a nucleotide sequence encoding an antibody or a fragment thereof. In some embodiments, the nucleic acid encodes an immunoglobulin heavy chain variable domain of the antibody provided herein. In some embodiments, the nucleic acid encodes an immunoglobulin light chain variable domain of the antibody provided herein. In some embodiments, the nucleic acid encodes both the immunoglobulin heavy chain variable domain and the immunoglobulin light chain variable domain of the antibody provided herein. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of any one of SEQ ID Nos: 2-7, 15, or 17.

[0160] This article provides nucleotide sequences that encode the immunoglobulin heavy chain variable domain and immunoglobulin light chain variable domain of any antibody or antigen-binding fragment provided herein.

[0161] To generate antibodies through recombination, the nucleic acid encoding the antibody can be isolated and inserted into a reproducible vector for further cloning (DNA amplification) or expression. In some cases, antibodies can be generated through homologous recombination. The DNA encoding the antibody can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence and origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence.

[0162] Suitable host cells for cloning or expressing DNA in vectors can be prokaryotes, yeast, or higher eukaryotic cells. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, and species like Escherichia coli. Escherichia For example, Escherichia coli, Enterobacter spp. Enterobacter Erwinia ( ) Erwinia Klebsiella spp. Klebsiella ), Proteus spp. Proteus Salmonella ( Salmonella ), such as Salmonella typhimurium ( Salmonella typhimurium ), Serratia, such as Serratia marcescens ( Serratia marcescans ) and Shigella, and Bacilli, such as Bacillus subtilis ( B. subtilis ) and Bacillus licheniformis ( B. licheniformis ), Pseudomonas, such as Pseudomonas aeruginosa ( P. aeruginosaThe strains are *Streptomyces* and *Escherichia coli*. A preferred host for *E. coli* cloning is *E. coli* 294 (ATCC 31,446), although other strains such as *E. coli* B, *E. coli* X1776 (ATCC 31,537), and *E. coli* W3110 (ATCC 27,325) may also be suitable. These examples are illustrative and not limiting.

[0163] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for vectors encoding antibodies. (Saccharomyces cerevisiae) Saccharomyces cerevisiae *Saccharomyces cerevisiae*, or common baker's yeast, is the most commonly used of the microorganisms in lower eukaryotic hosts. Many other genera, species, and strains are also commonly available and useful in this paper, such as *Schizosaccharomyces cerevisiae*. Schizosaccharomyces pombe ); Kluyveromyces ( Kluyveromyces ) hosts, such as, for example, Kluyveromyces lactis ( K. lactis Kluyveromyces brittle-walled K. fragilis (ATCC 12,424), Kluyveromyces bulgaricus ( K. bulgaricus (ATCC 16,045), Kluyveromyces hominis ( K. wickeramii (ATCC 24,178) K. waltii (ATCC 56,500), Kluyveromyces davidianus ( K. drosophilarum (ATCC 36,906), heat-resistant Kluyveromyces ( K. thermotolerans ) and Max Kluyveromyces ( K. marxianus ); Yarrowia (EP 402,226); Pichia pastoris ( Pichia pastoris (EP 183,070); Candida; Trichoderma reesei Trichoderma reesia (EP 244,234); Neurospora crassa ( Neurospora crassa ); *Schönleinstrogenus* ( Schwanniomyces ), such as Western Schwann yeast ( Schwanniomyces occidentalis ); and filamentous fungi, such as, for example, Neurospora ( Neurospora ), Penicillium genus ( Penicillium ), genus *Cyclophorus* Tolypocladium ) and Aspergillus ( Aspergillus Hosts such as Aspergillus nidus ( A. nidulans ) and Aspergillus niger ( A. niger ).

[0164] Suitable host cells for antibody expression can also be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Various baculovirus strains and variants have been identified, as well as those derived from hosts such as the fall armyworm (Pterocarya stenoptera). Spodoptera frugiperda (caterpillars), Aedes aegypti mosquitoes ( Aedes aegypti (mosquitoes), Aedes albopictus ( Aedes albopictus (mosquitoes), fruit flies (black-bellied flies) Drosophila melanogaster (fruit fly) and silkworm ( Bombyx mori (Silkworm moth) corresponding permissible insect host cells. Various viral strains used for transfection are publicly available, such as those from the alfalfa silver-striped moth (…). Autographa californica The L-1 variant of NPV and the Bm-5 strain of silkworm NPV are used, and these viruses can be used as viruses in this paper according to the technique, especially for transfection of fall armyworm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0165] Suitable host cells for antibody expression may also include vertebrate cells (e.g., mammalian cells). Vertebrate cells can be propagated in cultures (tissue cultures). Examples of useful mammalian host cell lines include: SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or subclones used for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); juvenile hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad.Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2).

[0166] Host cells can be transformed using the expression or cloning vectors described above for antibody production and cultured in standard nutrient media, which can be modified as needed to induce promoters, screen transformants, or amplify genes encoding desired sequences. Host cells used to produce the antibodies presented herein can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco Modified Eagle Medium (DMEM, Sigma), are suitable for culturing host cells. In addition, any culture medium described in Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Reissue Patent No. 30,985 may be used as a culture medium for host cells. Any of these culture media may be supplemented as needed with the following substances: hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN™), trace elements (defined as inorganic compounds typically present in micromolar final concentrations), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. The culture conditions (e.g., temperature, pH, etc.) are those previously used to express the selected host cells and are obvious to those skilled in the art.

[0167] When using recombinant technology, antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If antibodies are produced intracellularly, the first step is to remove particulate debris (host cell or lysed fragments), which is done, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a method for isolating antibodies secreted into the periplasmic space of *E. coli*. Briefly, the cell paste is thawed for approximately 30 min in the presence of sodium acetate (pH 3.5), EDTA, and benzyl sulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. When antibodies are secreted into the culture medium, the supernatant of such expression systems is typically concentrated first using a commercially available protein concentrater (e.g., an Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors (e.g., PMSF) may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of incidental contaminants.

[0168] Antibody compositions prepared from cells can be purified using methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domains present in the antibody. Protein A can be used to purify human heavy chain-based antibodies (Lindmark et al., J. Immunol.Meth .62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix for attachment of affinity ligands is most commonly agarose, but other matrices are also available. Mechanically stable matrices such as controlled-porosity glass or poly(divinyl styrene)benzene allow for faster flow rates and shorter processing times compared to the flow rates and processing times achieved with agarose. When the antibody contains a CH3 domain, Bakerbond ABX.TM. resin (JT Baker, Phillipsburg, NJ) can be used for purification. Depending on the antibody to be recovered, other protein purification techniques, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, heparin SEPHAROSE™ chromatography, anion exchange resin or cation exchange resin (such as polyaspartic acid column) chromatography, chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation, are also available.

[0169] After any initial purification steps, the mixture containing the antibody of interest and contaminants can be subjected to low-pH hydrophobic interaction chromatography using an elution buffer with a pH of, for example, about 2.5-4.5, and can be performed at low salt concentrations (e.g., about 0-0.2 5 M salt).

[0170] VIII. Drug Formulation, Dosage, and Route of Administration This disclosure provides antibodies and related compositions that can be used, for example, to eliminate pathogens expressing GPC4 from the body, and for, for example, to identify and quantify the amount of pathogens expressing GPC4 in biological samples.

[0171] In some embodiments, any antibody or its antigen-binding fragment can be formulated into a pharmaceutical composition that can be used for a variety of purposes, including treating diseases or conditions. A pharmaceutical device can be used to administer the pharmaceutical composition containing one or more antibodies to a patient in need, and according to one embodiment of this technology, a kit including such a device is provided. Such devices and kits can be designed for routine administration (including self-administration) of the pharmaceutical compositions described herein.

[0172] This can be achieved by mixing an agent or antibody with the desired purity with an optional physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16). th The therapeutic formulation of antibodies is prepared for storage in the form of lyophilized or aqueous solutions, according to edition, Osol, A. Ed. (1980). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as blood... Albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEENTM, PLURONICS™, or polyethylene glycol (PEG).

[0173] In some embodiments, the disease or condition is associated with GPC4 expression. In some embodiments, the disease or condition is associated with abnormal GPC4 expression. In some embodiments, the disease or condition is associated with natural killer (NK) cells, αβT cells, γδT cells, CD8+ T cells, monocytes, or dendritic cells. In some embodiments, the disease or condition is associated with natural killer (NK) cells. In some embodiments, the disease or condition is associated with αβT cells. In some embodiments, the disease or condition is associated with γδT cells. In some embodiments, the disease or condition is associated with CD8+ T cells. In some embodiments, the disease or condition is associated with monocytes. In some embodiments, the disease or condition is associated with dendritic cells.

[0174] In some implementations, the disease or ailment is cancer, an infectious disease, or an autoimmune disease.

[0175] In some implementations, the disease or condition is cancer. In some implementations, cancer is, for example, metastatic melanoma, solid tumor, bladder cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, liver metastases originating from the colon, papillary thyroid carcinoma, acute myeloid leukemia, or asymptomatic myeloma.

[0176] In some implementations, the disease or condition is an infectious disease. In some implementations, the infectious disease is, for example, human immunodeficiency virus (HIV), chronic hepatitis C, cytomegalovirus, or hantavirus.

[0177] In some implementations, the disease or condition is an autoimmune disease. In some implementations, an autoimmune disease is, for example, Crohn's disease, multiple sclerosis, systemic sclerosis, ocular myasthenia gravis, psoriasis, or rheumatoid arthritis.

[0178] In some implementations, any antibody or antigen-binding fragment thereof described herein may be used to reduce the production of androgens in prostate cancer cells.

[0179] In some embodiments, any antibody or antigen-binding fragment thereof described herein may be used to inhibit or reduce the cleavage of the coronavirus spike glycoprotein. In some embodiments, any antibody or antigen-binding fragment thereof described herein may be used to inhibit or reduce the uptake of the virus by host cells.

[0180] The formulations described herein may also contain more than one active compound, preferably those with complementary activities that do not adversely affect each other, for the specific indication to be treated. Such molecules are appropriately combined in amounts effective for the intended purpose.

[0181] Formulations intended for internal administration are typically sterile. This can be achieved, for example, by filtration through a sterile filter membrane.

[0182] Sustained-release articles can be prepared. Suitable examples of sustained-release articles include semi-permeable matrices of solid hydrophobic polymers containing agents / antibodies, which are in the form of shaped articles, such as membranes or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactide (US Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, and degradable lactic-glycolic acid copolymers such as Lupron. Depot® (injectable microspheres containing lactic-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic-glycolic acid can release molecules for more than 100 days, while some hydrogels release proteins for shorter periods. When encapsulated agents / antibodies are retained in vivo for extended periods, they may denature or aggregate due to exposure to moisture at 37°C, leading to loss of biological activity and possible changes in immunogenicity. Depending on the mechanism involved, appropriate stabilization strategies can be designed. For example, if the aggregation mechanism is found to be the formation of intermolecular SS bonds through thiol-disulfide exchange, stabilization can be achieved by modifying thiol residues, lyophilizing from acidic solutions, controlling moisture content, using suitable additives, and developing specific polymer matrix compositions.

[0183] For therapeutic applications, the antibodies described herein are administered to mammals (e.g., humans) in pharmaceutically acceptable dosage forms (e.g., those discussed above). These dosage forms include those that can be administered to humans intravenously as a bolus or via continuous infusion over a period of time, or via intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, local, or inhalation routes. For the prevention or treatment of disease, the appropriate dosage of the agent or antibody depends on the type of disease to be treated (as defined above), the severity and duration of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, prior treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody may be administered to the patient once or as part of a series of treatments.

[0184] Depending on the type and severity of the disease, antibodies from approximately 1 µg / kg to approximately 50 mg / kg (e.g., 0.1–20 mg / kg) can be administered to the patient as an initial candidate dose, whether, for example, by a single or multiple administrations alone or by continuous infusion. Depending on the factors described above, typical daily or weekly doses can range from approximately 1 µg / kg to approximately 20 mg / kg or higher. For repeated administration over several days or longer, treatment should be repeated as needed until the desired suppression of disease symptoms is achieved. However, other dosing regimens may be useful. Progression of the therapy can be readily monitored using routine techniques and assays, including, for example, radiographic imaging. Detection methods for determining GPC4 levels in body fluids or tissues using antibodies can be used to optimize patient exposure to the therapeutic antibody.

[0185] In some embodiments, the composition containing the antibody described herein may be administered as a monotherapy; and in some embodiments, the antibody-containing composition may be administered as part of a combination therapy. In some cases, the effectiveness of the antibody in preventing or treating a disease may be enhanced by administering the antibody sequentially or in combination with another drug effective for the aforementioned purpose (e.g., a chemotherapy drug for treating cancer or microbial infections). In other cases, the antibody may be used to enhance the effects of chemotherapy or to sensitize cells to chemotherapy, thereby allowing efficacy to be achieved at lower doses and with lower toxicity. Some combination therapies, in addition to administering the antibody-containing composition (which reduces the number of expressing cells), include the delivery of a second treatment regimen selected from the group consisting of: chemotherapy agents, radiotherapy, surgery, and combinations of any of the foregoing regimens. These other agents may be present in the administered composition or may be administered alone. Furthermore, the antibody may be administered sequentially or in combination with other agents or modalities (e.g., chemotherapy drugs or radiotherapy for treating cancer, infections, etc., or immunosuppressive drugs).

[0186] IX. Research and Diagnosis This document also provides diagnostic reagents comprising the antibodies described herein. For example, the antibodies provided herein can be used to detect and / or purify GPC4 from body fluids or tissues. This document also provides methods for detecting GPC4. For example, methods may include contacting a sample (e.g., a biological sample known or suspected of containing GPC4) with the antibodies provided herein, and detecting the GPC4:antibody complex if the sample contains GPC4. This document also provides reagents comprising the antibodies described herein and detection methods for research purposes.

[0187] Any antibody or antigen-binding fragment disclosed herein can be used for diagnostic assays to detect its presence in specific cells, tissues, or body fluids. Such diagnostic methods can be used to diagnose, for example, hyperplastic diseases or conditions. Therefore, this document includes both clinical diagnostic and research uses. In some embodiments, the antibody contains a detectable biomarker or marker. In some embodiments, the antibody is conjugated to a detectable biomarker or marker. For example, for research and diagnostic applications, the antibody may be labeled with a detectable portion. Many biomarkers are available, and these biomarkers are generally grouped into the following categories: (a) Radioactive isotopes, such as 35S, 14C, 125I, 3H, and 131I. Antibodies can be labeled with radioactive isotopes using techniques described, for example, Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, NY, Pubs. (1991), and radioactivity can be measured using scintillation counting.

[0188] (b) Fluorescent labels, such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin, Texas red, and Brilliant Violet™, are available. Fluorescent labels can be conjugated with antibodies using techniques disclosed, for example, in Current Protocols in Immunology described above. Fluorescence can be quantified using flow cytometry, imaging microscopy, or a fluorometer.

[0189] A variety of enzyme substrate labels are available. Enzymes typically catalyze chemical changes in chromogenic substrates, and these changes can be measured using various techniques. For example, enzymes can catalyze color changes in substrates, which can be measured spectrophotometrically. Optionally, enzymes can alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying fluorescence changes are described above. Chemiluminescent substrates are excited by electrons through a chemical reaction and can then emit measurable light (e.g., using a chemiluminometer) or provide energy to a fluorescent acceptor. Examples of enzyme-labeled markers include luciferases (e.g., firefly luciferase and bacterial luciferase), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. The technique of conjugating enzymes with antibodies is described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for Use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York, 73:147-166 (1981).

[0190] Examples of enzyme-substrate combinations include, for example: (i) Horseradish peroxidase (HRP) with catalase as a substrate, wherein catalase oxidizes dye precursors (e.g., o-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB)). (ii) an alkaline phosphatase (AP) with p-nitrophenyl phosphate as a chromogenic substrate; and (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or a fluorescent substrate 4-methylumbelliferyl-β-D-galactosidase.

[0191] In some cases, the marker binds indirectly to an agent or antibody. Various techniques for achieving this are known to those skilled in the art. For example, an antibody can be conjugated to biotin, and any of the three classes of markers mentioned above can be conjugated to avidin, and vice versa. Biotin binds selectively to avidin, and therefore, the marker can be conjugated to an antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the marker to an antibody, the antibody is conjugated to a small hapten (e.g., digoxigenin), and one of the different types of markers mentioned above is conjugated to an anti-hapten antibody (e.g., an anti-digoxigenin antibody). Thus, indirect binding of the marker to an antibody can be achieved.

[0192] In some implementations, the antibody or its antigen-binding fragment does not need to be labeled, and its presence can be detected, for example, using a labeled antibody that binds to the antibody.

[0193] In some embodiments, the antibodies described herein are immobilized on a solid support or substrate. In some embodiments, the antibodies described herein are non-diffusely immobilized on the solid support (e.g., the antibodies do not detach from the solid support). The solid support or substrate can be any physically separable solid to which the antibody can be directly or indirectly attached, including but not limited to surfaces provided by microarrays and pores, and particles such as beads (e.g., paramagnetic beads, magnetic beads, microbeads, nanobeads), micron particles, and nanoparticles. Solid supports may also include, for example, chips, columns, optical fibers, wipes, filters (e.g., planar filters), one or more capillaries, glass and modified or functionalized glass (e.g., controlled porosity glass (CPG)), quartz, mica, diazotized membranes (paper or nylon), polyoxymethylene, cellulose, cellulose acetate, paper, ceramics, metals, quasi-metals, semiconductor materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylic resins, polystyrene, copolymers of styrene or other materials, polybutene, polyurethane, TEFLON™, polyethylene, polypropylene, polyamide, polyester, etc.). The solid support or substrate may be made of a variety of materials, including polyvinylidene fluoride (PVDF), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials, including silicon, silica gel and modified silicon, Sephadex®, Sepharose®, carbon, metals (e.g., steel, gold, silver, aluminum, silicon and copper), inorganic glasses, conductive polymers (including polymers such as polypyrrole and polyindole); micron or nanostructured surfaces, such as nucleic acid tiled arrays, nanotubes, nanowires or nanoparticle-modified surfaces; or porous surfaces or gels, such as methacrylates, acrylamide, sugar polymers, cellulose, silicates or other fibrous or chain polymers. In some embodiments, the solid support or substrate may be coated with a variety of materials (including polymers such as dextran, acrylamide, gelatin or agarose) using passive or chemically derived coatings. Beads and / or particles may be non-linked (free) or linked to each other (e.g., sintered). In some embodiments, the solid support or substrate may be an aggregate of particles. In some embodiments, the particles may include silica, and the silica may include silica dioxide. In some embodiments, the silica-based material may be porous, and in other embodiments, it may be non-porous. In some embodiments, the particles also include an agent that imparts paramagnetism to the particles. In some embodiments, the agent comprises a metal, and in other embodiments, the agent is a metal oxide (e.g., iron or iron oxide, wherein the iron oxide comprises a mixture of Fe2+ and Fe3+). Antibodies can be attached to a solid support via covalent bonds or non-covalent interactions, and can be attached to the solid support directly or indirectly (e.g., via an intermediate agent, such as a spacer molecule or biotin).

[0194] The antibodies and their antigen-binding fragments described herein can be used with any known assay, such as flow cytometry, immunohistochemistry, immunofluorescence, mass cytometry (e.g., Cytof instruments), competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. (Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987)). Flow cytometry and mass cytometry assays typically involve the use of a single primary antibody to specifically identify the presence of a target molecule expressed on the surface of individual cells in a dispersed suspension. Dispersed cells are typically obtained from biological fluid samples (e.g., blood), but can also be obtained from single-cell dispersions prepared from solid tissue samples (e.g., spleen or tumor biopsies). The primary antibody can be directly conjugated to a detectable moiety, such as a fluorophore like phycoerythrin for flow cytometry or a heavy metal chelate for mass cytometry. Optionally, the primary antibody may be unlabeled or labeled with an undetectable tag such as biotin, and the primary antibody is then detected by a detectably labeled secondary antibody that specifically recognizes the primary antibody itself or the tag on the primary antibody. The labeled cells are then analyzed in an instrument capable of single-cell detection (e.g., flow cytometry, mass spectrometry, fluorescence microscopy, or bright-field microscopy) to identify those individual cells in a dispersed population or tissue sample expressing the target recognized by the primary antibody. A detailed description of the technical basis and practical applications of flow cytometry can be found, for example, in Shapiro, Practical Flow Cytometry, 4. th Edition, Wiley, 2003.

[0195] Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic moiety or epitope of the protein being detected. In a sandwich assay, the test sample analyte is bound by a first antibody immobilized on a solid support, and then a second antibody binds to the analyte, forming an insoluble three-part complex. See, for example, U.S. Patent No. 4,376,110. The second antibody itself can be labeled with a detectable moiety (direct sandwich assay), or it can be measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which the detectable moiety is an enzyme. In a cell ELISA, a target cell population is attached to a solid support using antibodies that first attach to a support and recognize different cell surface proteins. These first antibodies capture the cells to the support. GPC4 on the cell surface can then be detected by adding any of the anti-GPC4 antibodies described herein or their antigen-binding fragments to the captured cells and detecting the amount of anti-GPC4 antibody or its antigen-binding fragment attached to the cells. In some cases, fixed and permeabilized cells can be used, and in such cases, surface GPC4 and intracellular GPC4 can be detected.

[0196] In some embodiments, any antibody or antigen-binding fragment thereof provided herein may be formulated for immunohistochemical analysis. In some embodiments, the immunohistochemical analysis includes the use of a sample. In some embodiments, the immunohistochemical analysis includes the use of blood and / or tissue samples. In some embodiments, the sample may be fresh or frozen, or may be embedded in paraffin and fixed with a preservative such as formalin. In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) sample. In some embodiments, the FFPE sample is saturated with formalin (i.e., formaldehyde) and then embedded in a paraffin block. In some embodiments, the FFPE sample is stable at room temperature. In some embodiments, all structures in the FFPE sample are preserved. In some embodiments, intracellular and surface proteins in the FFPE sample are preserved. In some embodiments, mRNA in the FFPE sample is preserved. In some embodiments, mRNA, intracellular, and surface proteins in the FFPE sample are preserved. In some embodiments, surface proteins in the FFPE sample are denatured.

[0197] In some embodiments, any anti-GPC4 antibody or antigen-binding fragment thereof provided herein can detect GPC4 in formalin-fixed paraffin-embedded samples. In some embodiments, any anti-GPC4 antibody or antigen-binding fragment thereof provided herein can detect GPC4 on the cell surface in formalin-fixed paraffin-embedded samples. In some embodiments, any anti-GPC4 antibody or antigen-binding fragment thereof provided herein can detect intracellular GPC4 in formalin-fixed paraffin-embedded samples. In some embodiments, any anti-GPC4 antibody or antigen-binding fragment thereof provided herein can detect both intracellular and surface GPC4 in formalin-fixed paraffin-embedded samples.

[0198] In some embodiments, the sample is a fresh sample that has already been frozen. In some embodiments, the sample is a fresh sample that has already been cryogenically frozen. In some embodiments, the sample is flash-frozen. In some embodiments, the sample is flash-frozen and stored at 80°C. In some embodiments, all structures in the flash-frozen sample are preserved. In some embodiments, intracellular and surface proteins in the flash-frozen sample are preserved. In some embodiments, mRNA in the flash-frozen sample is preserved. In some embodiments, mRNA, intracellular, and surface proteins in the flash-frozen sample are preserved. In some embodiments, surface proteins in the flash-frozen sample are denatured.

[0199] In some embodiments, any anti-GPC4 antibody or antigen-binding fragment thereof provided herein can detect GPC4 in frozen samples. In some embodiments, any anti-GPC4 antibody or antigen-binding fragment thereof provided herein can detect GPC4 on the surface of frozen samples. In some embodiments, any anti-GPC4 antibody or antigen-binding fragment thereof provided herein can detect intracellular GPC4 in frozen samples. In some embodiments, any anti-GPC4 antibody or antigen-binding fragment thereof provided herein can detect both intracellular and surface GPC4 in frozen samples.

[0200] The antibodies described in this article can also be used for in vivo diagnostic assays. Typically, antibodies are labeled with radionuclides (e.g., 111In, 99Tc, 14C, 131I, 125I, 3H, 32P, or 35S) so that the bound target molecule can be localized using immunoscintillation imaging. This article also provides reagents for non-diagnostic use, which contain the anti-GPC4 agents / antibodies described herein. This article also provides reagents for non-therapeutic use, which contain the anti-GPC4 agents / antibodies described herein. This article also provides reagents for non-diagnostic and non-therapeutic use, which contain the anti-GPC4 agents / antibodies described herein. For example, this article provides reagents for research applications, which contain the anti-GPC4 agents / antibodies described herein. Research applications may include studying GPC4 and its role in one or more of the following: cell division, growth regulation, cell signaling (e.g., Wnt, BMP, FGF signaling), cancer (e.g., pancreatic cancer, colorectal cancer), etc. This article also provides methods for detecting GPC4 and / or measuring GPC4 levels in non-biological samples. For example, the method may include contacting a non-biological sample (e.g., a laboratory research sample known to contain or suspected of containing GPC4) with an antibody provided herein, and detecting a GPC4:antibody complex if the sample contains GPC4. Laboratory research samples may include non-human animal models, samples derived from non-human animal models, cell lines, products generated from cell lines, etc.

[0201] X. GPC4 detection This document provides antibodies and methods for detecting GPC4. In some embodiments, antibodies and methods for detecting GPC4 in biological samples are provided. In some embodiments, the biological sample is a solid tissue, fluid, or cells. In some embodiments, GPC4 is detected on the cell surface. In some embodiments, GPC4 is detected intracellularly. In some embodiments, the detection of GPC4 is in vitro. In some embodiments, the detection of GPC4 is in vivo.

[0202] Solid tissues may include one or more of the following: adipose tissue, bladder, bone, brain, mammary gland, cervix, endothelium, gallbladder, kidney, liver, lung, lymph, ovary, prostate, salivary gland, stomach, testis, thyroid gland, urethra, uterus, vagina, and vulva. In some embodiments, fluids include one or more of the following: amniotic fluid, bile, blood, breast milk, mammary fluid, cerebrospinal fluid, lavage fluid, lymph, mucus, plasma, saliva, semen, serum, cerebrospinal fluid, sputum, tears, umbilical cord blood, urine, and vaginal fluid.

[0203] In some embodiments, the sample includes immune cells. In some embodiments, the sample includes a heterogeneous population of immune cells. In some embodiments, the immune cells are selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and peripheral blood mononuclear cells (PBMCs).

[0204] In some of the embodiments described herein, any antibody or antigen-binding fragment thereof provided herein may be used to characterize single cells by measuring gene expression levels and cellular proteins. Among such known single-cell sequencing platforms suitable for integration with the antibodies or antigen-binding fragments thereof described herein, Drop-seq methods exist, including but not limited to microfluidic, plate- or microwell-based, Seq-Well™ methods and adaptations of the basic protocol, and InDrop™ methods. In another embodiment, a single-cell sequencing platform suitable for integration with the antibodies or antigen-binding fragments thereof described herein is the l0x genomics single-cell 3' protocol or the single-cell V(D)J protocol, which runs on a Chromium controller or a dedicated Chromium single-cell controller. Other suitable sequencing methods include the Wafergen iCell8™ method, the Microwell-seq method, the Fluidigm CI™ method, and equivalent single-cell products. In addition, other known sequencing protocols that can be used with the antibodies or antigen-binding fragments thereof described herein include the BD Resolve™ single-cell analysis platform and ddSeq (from Illumina® Bio-Rad® SureCell™ WTA 3' Library Prep Kit for the ddSEQ™ System, 2017, Pub.No.1070-2016-014-B, Illumina Inc., Bio-Rad Laboratories, Inc.). In yet other embodiments, the antibodies or antigen-binding fragments thereof described herein can be used with combined indexing methods (sci-RNA-seq™ or SPLiT-seq™ methods) and spatial transcriptomics or similar spatially resolved sequencing methods. The methods and compositions described herein can also be used as an additional layer of information for standard index sorting (FACS) and mRNA-based sequencing methods.

[0205] In some of the embodiments described herein, any antibody or antigen-binding fragment thereof may be used to detect the presence, absence, or quantity of the various nucleic acids, proteins, targets, oligonucleotides, amplification products, and barcodes described herein.

[0206] In some implementations, the biological sample is obtained from a healthy subject. In some implementations, the sample is obtained from a subject suffering from a disease or condition. In some implementations, the detection of GPC4 indicates the presence or absence of a disease or condition. In some implementations, the disease or condition is cancer, an autoimmune disease, an inflammatory disease, a neurological disease, or an infection. In some implementations, the cancer is acute myeloid leukemia, acute lymphoblastic leukemia, colorectal cancer, ovarian cancer, gynecological cancer, liver cancer, glioblastoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, esophageal cancer, gastric cancer, pancreatic cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, and melanoma.

[0207] In some embodiments, the disease or condition is associated with GPC4 expression; in other embodiments, the disease or condition is associated with abnormal GPC4 expression. In some embodiments, the disease or condition is associated with natural killer (NK) cells, αβT cells, γδT cells, CD8+ T cells, monocytes, or dendritic cells. In some embodiments, the disease or condition is associated with natural killer (NK) cells. In some embodiments, the disease or condition is associated with αβT cells. In some embodiments, the disease or condition is associated with γδT cells. In some embodiments, the disease or condition is associated with CD8+ T cells. In some embodiments, the disease or condition is associated with monocytes. In some embodiments, the disease or condition is associated with dendritic cells. In some of the implementation schemes, the disease or condition is selected from nonviral cancer, virus-associated cancer, cancer associated with HBV infection, cancer associated with EBV infection, cancer associated with polyomavirus infection, erythema nodosum (ENL), autoimmune diseases, autoimmune inflammation, autoimmune thyroid diseases, B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's disease, acute myeloid leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, B-cell leukemia, myelodysplastic syndrome, solid tumors, herpes virus infection, and / or rejection of transplanted tissues or organs.

[0208] In some implementations, the disease or ailment is cancer, an infectious disease, or an autoimmune disease.

[0209] In some implementations, the disease or condition is cancer. In some implementations, cancer is metastatic melanoma, solid tumor, bladder cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, liver metastases originating from the colon, papillary thyroid carcinoma, acute myeloid leukemia, or asymptomatic myeloma.

[0210] In some implementations, the disease or symptom is an infectious disease. In some implementations, the infectious disease is human immunodeficiency virus (HIV), chronic hepatitis C, cytomegalovirus, or hantavirus.

[0211] In some implementations, the disease or condition is an autoimmune disease. In some implementations, the autoimmune disease is Crohn's disease, multiple sclerosis, systemic sclerosis, ocular myasthenia gravis, psoriasis, or rheumatoid arthritis.

[0212] In some embodiments, any antibody or antigen-binding fragment thereof may be used to generate a nucleic acid molecule comprising all or part of the sequence of an oligonucleotide or its complement. In some of any embodiments, the antibody or antigen-binding fragment thereof may be used in methods for correlating the presence or abundance of GPC4 with a location of interest in a tissue sample.

[0213] In some embodiments, any antibody or its antigen-binding fragment may be used for the construction of the protein library. In some embodiments, the construction of the protein library includes sequencing. In some embodiments, the construction of the protein library includes the use of flow cytometry.

[0214] In some of the various implementations, this paper provides methods for detecting GPC4, including: a) Contacting the sample with the antibody or antigen-binding fragment thereof under conditions that bind any antibody or antigen-binding fragment thereof to the GPC4 receptor on the sample, wherein the binding results in the formation of a receptor / antibody or antigen-binding fragment complex; b) Detecting the presence of the receptor / antibody or antigen-binding fragment complex thereof; c) Detecting the presence or absence of the GPC4 receptor on the sample.

[0215] In some of the embodiments, this document provides a method for treating or preventing a disease or condition related to GPC4 in a subject, comprising: a) contacting a sample known to contain or suspected to contain GPC4 with any antibody or an antibody or an antigen-binding fragment thereof; b) detecting the presence of a complex comprising GPC4 and an antibody or an antigen-binding fragment thereof; wherein the presence of the complex indicates the presence of a disease or condition; and c) administering any antibody or an antibody or an antigen-binding fragment thereof to the subject.

[0216] In some of the embodiments, this document provides a method for diagnosing a disease or condition, comprising: a) isolating a sample from a subject; b) incubating the sample with any antibody or an antigen-binding fragment thereof for a period of time sufficient to generate a GPC4:anti-GPC4 complex; c) detecting the presence or absence of a GPC4:anti-GPC4 complex from the isolated tissue; and d) correlating the presence or abundance of GPC4 with the location of interest in the tissue sample.

[0217] In some of the implementation schemes, an increase in GPC4 at the site of interest in the tissue sample exceeding the control level indicates a disease or condition in the subject.

[0218] In some embodiments, the detection includes hybridization of the detectable portion with an antibody or an antigen-binding fragment thereof. In some embodiments, the sample is contacted with a second antibody. In some embodiments, the second antibody is an antibody containing the detectable portion. In some embodiments, the detectable portion contains an oligonucleotide. In some embodiments, the detectable portion contains a fluorescent label. In some embodiments, the measurement includes sequencing. In some embodiments, the detectable portion includes immunofluorescence. In some embodiments, the sample is a formalin-fixed paraffin-embedded sample. In some embodiments, the sample includes cells. In some embodiments, the sample includes a tissue sample.

[0219] XI. Kits containing anti-antibodies The antibodies described herein may be provided in the form of a kit (e.g., a package combining pre-quantitative reagents with instructions for use (e.g., instructions for use in diagnostic assays; instructions for use in laboratory assays)). In some embodiments, the kit is a diagnostic kit configured to detect GPC4 in a sample (e.g., a biological sample). When the antibody is labeled with a fluorophore, the kit may include the same isotype negative control antibody as a control for nonspecific binding of the antibody. When the antibody is labeled with an enzyme, the kit may include the substrate and cofactor required for the enzyme (e.g., a substrate precursor providing a detectable chromophore or fluorophore). Additional additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), etc. The relative amounts of various reagents can vary widely to provide reagent concentrations in solutions that significantly optimize assay sensitivity. In some cases, the reagents may be provided as a dry powder (e.g., a lyophilized powder) containing an excipient that, upon dissolution, provides a reagent solution with an appropriate concentration.

[0220] XI. Some implementation methods The following are some non-limiting examples of implementations of this technology.

[0221] A1. An antibody or antigen-binding fragment thereof that binds to GPC4 or a portion thereof, comprising: a) An immunoglobulin heavy chain variable domain, wherein the immunoglobulin heavy chain variable domain comprises: (i) Contains the heavy chain complementarity determination region 1 (CDRH1) of SEQ ID NO:2; (ii) Contains the heavy chain complementarity determination region 2 (CDRH2) of SEQ ID NO:3; (iii) Heavy chain complementarity determination region 3 (CDRH3) containing SEQ ID NO:4; and b) An immunoglobulin light chain variable domain, wherein the immunoglobulin light chain variable domain comprises: (i) Contains the light chain complementarity determination region 1 (CDRL1) of SEQ ID NO:5; (ii) Containing the light chain complementarity determination region 2 (CDRL3) of SEQ ID NO:6; and (iii) Light chain complementarity determination region 3 (CDRL3) containing SEQ ID NO:7.

[0222] A2. The antibody or antigen-binding fragment thereof according to embodiment A1, wherein the immunoglobulin heavy chain variable domain comprises: CDRH1, wherein CDRH1 comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:2; CDRH2, wherein CDRH2 comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:3; and CDRH3, wherein CDRH3 comprises the amino acid sequence shown in SEQ ID NO:4 ... NO:4 shows an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0223] A3. The antibody or antigen-binding fragment thereof according to embodiment A1 or A2, wherein the immunoglobulin heavy chain variable domain comprises: CDRH1, wherein CDRH1 comprises the amino acid sequence shown in SEQ ID NO:2; CDRH2, wherein CDRH2 comprises the amino acid sequence shown in SEQ ID NO:3; and CDRH3, wherein CDRH3 comprises the amino acid sequence shown in SEQ ID NO:4.

[0224] A4. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A3, wherein the immunoglobulin light chain variable domain comprises: CDRL1, wherein CDRL1 comprises the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:5; CDRL2, wherein CDRL2 comprises the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:6; and CDRL3, wherein CDRL3 comprises the amino acid sequence shown in SEQ ID NO:7 ... NO:7 shows an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0225] A5. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A4, wherein the immunoglobulin light chain variable domain comprises: CDRL1, wherein CDRL1 comprises the amino acid sequence shown in SEQ ID NO:5; CDRL2, wherein CDRL2 comprises the amino acid sequence shown in SEQ ID NO:6; and CDRL3, wherein CDRL3 comprises the amino acid sequence shown in SEQ ID NO:7.

[0226] A6. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A5, wherein CDRH1 comprises the amino acid sequence shown in SEQ ID NO:2; CDRH2 comprises the amino acid sequence shown in SEQ ID NO:3; CDRH3 comprises the amino acid sequence shown in SEQ ID NO:4; CDRL1 comprises the amino acid sequence shown in SEQ ID NO:5; CDRL2 comprises the amino acid sequence shown in SEQ ID NO:6; and CDRL3 comprises the amino acid sequence shown in SEQ ID NO:7.

[0227] A7. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A5, wherein CDRH1 comprises the sequence shown in SEQ ID NO:2 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:2; wherein CDRH2 comprises the sequence shown in SEQ ID NO:3 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:3; wherein CDRH3 comprises the sequence shown in SEQ ID NO:4 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:3; and wherein CDRH3 comprises the sequence shown in SEQ ID NO:4 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or more sequence identity with SEQ ID NO:2. NO:4 shows an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity; CDRL1 contains the sequence shown in SEQ ID NO:5 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:5; CDRL2 contains the sequence shown in SEQ ID NO:6 or an amino acid sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:5; NO:6 shows an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity; and the CDRL3 contains the sequence shown in SEQ ID NO:7 or an amino acid sequence that shows at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:7.

[0228] A8. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A7, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO:15 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:15.

[0229] A9. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A8, wherein the immunoglobulin heavy chain comprises an amino acid sequence shown in any one of SEQ ID NO:15.

[0230] A10. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A9, wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:17 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:17.

[0231] A11. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A10, wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:17.

[0232] A12. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A11. The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO:15 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:15; and The immunoglobulin light chain therein comprises the amino acid sequence shown in SEQ ID NO:17 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:17.

[0233] A13. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A12. The immunoglobulin heavy chain contains the amino acid sequence shown in SEQ ID NO:15; and The immunoglobulin light chain contains the amino acid sequence shown in SEQ ID NO:17.

[0234] A14. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A13, comprising an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain.

[0235] A15. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A13, comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.

[0236] A16. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A15, wherein the antibody or antigen-binding fragment thereof is isolated.

[0237] A17. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A16, wherein the antibody or antigen-binding fragment thereof is humanized.

[0238] A18. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A17, wherein the antibody or antigen-binding fragment thereof is conjugated.

[0239] A19. The antibody or antigen-binding fragment thereof according to any one of embodiments A1-A18 further comprises oligonucleotides.

[0240] A20. The antibody or antigen-binding fragment thereof according to embodiment A19, wherein the oligonucleotide comprises a sample barcode sequence.

[0241] A21. An antibody or antigen-binding fragment thereof according to any one of embodiments A19 or A20, wherein the oligonucleotide comprises a primer binding site and an anchor.

[0242] A22. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A21, wherein the antibody or antigen-binding fragment thereof is conjugated to a detectable biomarker or label.

[0243] A23. The antibody or antigen-binding fragment thereof according to embodiment A22, wherein the detectable marker or label is directly conjugated to the antigen or antigen-binding fragment thereof.

[0244] A24. The antibody or antigen-binding fragment thereof according to embodiment A22, wherein the detectable marker or label is conjugated to an oligonucleotide.

[0245] A25. The antibody or antigen-binding fragment thereof according to embodiment A22, wherein the detectable biomarker or marker comprises a detectable portion.

[0246] A26. The antibody or antigen-binding fragment thereof according to embodiment A22, wherein the detectable portion is a radioactive isotope, a fluorescent label, or an enzyme substrate label.

[0247] A27. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A26, wherein the antibody or antigen-binding fragment thereof is non-diffusely immobilized on a solid support.

[0248] A28. The antibody or antigen-binding fragment thereof according to any one of embodiments A1-A27 is a single-chain fragment.

[0249] A29. The antibody or antigen-binding fragment thereof according to embodiment A28, wherein the single-chain fragment is a single-chain variable fragment (scFv).

[0250] A30. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A29, used for detecting GPC4 in a sample.

[0251] A31. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A29, wherein the antibody or antigen-binding fragment thereof binds to cells expressing GPC4 in a sample.

[0252] A32. An antibody or antigen-binding fragment according to embodiment A30 or A31, wherein the sample comprises immune cells.

[0253] A33. An antibody or antigen-binding fragment according to embodiment A30 or A31, wherein the sample comprises a heterogeneous population of immune cells.

[0254] A34. The antibody or antigen-binding fragment according to embodiment A32 or A33, wherein the immune cells are selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and peripheral blood mononuclear cells (PBMCs).

[0255] A35. An antibody or antigen-binding fragment thereof according to embodiment A30 or A31, wherein the sample comprises cells having a disease or condition.

[0256] A36. The antibody or antigen-binding fragment thereof as described in embodiment A35, wherein the disease or condition is cancer, an autoimmune disease, an inflammatory disease, a neurological disease, or an infection.

[0257] A37. The antibody or antigen-binding fragment thereof according to embodiment A36, wherein the cancer is acute myeloid leukemia, acute lymphoblastic leukemia, colorectal cancer, ovarian cancer, breast cancer, gynecological cancer, liver cancer, glioblastoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, esophageal cancer, gastric cancer, pancreatic cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, and melanoma.

[0258] A38. An antibody or antigen-binding fragment thereof according to any one of embodiments A30-A37, wherein the detection comprises using a single antibody or antigen-binding fragment thereof to bind a portion of GPC4.

[0259] A39. An antibody or antigen-binding fragment thereof according to any one of embodiments A30-A37, wherein the detection comprises using two antibodies or antigen-binding fragments thereof, each antibody or antigen-binding fragment thereof being capable of binding to different portions of GPC4.

[0260] A40. An antibody or antigen-binding fragment thereof according to any one of embodiments A30-A37, wherein the detection of GPC4 is performed on the cell surface.

[0261] A41. An antibody or antigen-binding fragment thereof according to any one of embodiments A30-A37, wherein the detection of GPC4 is performed intracellularly.

[0262] A42. An antibody or antigen-binding fragment thereof according to any one of embodiments A30-A37, wherein detection of GPC4 indicates the presence or absence of a disease or condition.

[0263] A43. An antibody or antigen-binding fragment thereof according to any one of embodiments A30-A42, wherein the detection is performed in vitro.

[0264] A44. An antibody or antigen-binding fragment thereof according to any one of embodiments A30-A42, wherein the detection is performed in vivo.

[0265] A45. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A44, wherein the antibody or antigen-binding fragment thereof binds to cells expressing GPC4.

[0266] A46. A diagnostic antibody or an antigen-binding fragment thereof comprising an antibody or an antigen-binding fragment thereof according to any one of embodiments A1-A45.

[0267] A47. An isolated antibody or antigen-binding fragment thereof according to any one of embodiments A1-A44, for non-diagnostic and / or non-therapeutic purposes.

[0268] B1. A kit comprising an antibody or antigen-binding fragment thereof according to any one of embodiments A1-A45 or a diagnostic antibody or antigen-binding fragment thereof according to embodiment A46.

[0269] B2. The kit according to embodiment B1, comprising a diagnostic kit configured to detect GPC4 in a biological sample.

[0270] C1. A composition comprising an antibody or an antigen-binding fragment thereof according to any one of embodiments A1-A45 and a pharmaceutically acceptable excipient.

[0271] C2. The composition according to embodiment C1, wherein the antibody or its antigen-binding fragment is used as an adjuvant or in combination with an adjuvant.

[0272] D1. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain encoding an antibody or an antigen-binding fragment thereof according to any one of embodiments A1-A45.

[0273] D2. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin light chain variable domain encoding an antibody or an antigen-binding fragment thereof according to any one of embodiments A1-A45.

[0274] D3. A recombinant expression vector comprising isolated nucleic acids according to embodiment D1 or D2.

[0275] D4. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid molecule containing a nucleotide sequence encoding an immunoglobulin heavy chain variable domain according to any one of embodiments A1-A45, and the second expression cassette comprises a nucleic acid molecule containing a nucleotide sequence encoding an immunoglobulin light chain variable domain of an antibody or an antigen-binding fragment thereof according to any one of embodiments A1-A45.

[0276] D5. The recombinant expression vector according to embodiment D4, wherein the first expression cassette and the second expression cassette comprise promoters.

[0277] D6. A host cell transfected with the recombinant expression vector according to embodiments D3-D5.

[0278] E1. A drug-pharmaceutical conjugate comprising an antibody or an antigen-binding fragment thereof according to any one of embodiments A1-A45.

[0279] E2. A composition comprising an antibody-drug conjugate according to embodiment E1 and a pharmaceutically acceptable carrier.

[0280] F1. A method for detecting GPC4, comprising: a) Under conditions that allow the antibody or its antigen-binding fragment according to any one of embodiments A1-A45 to bind to the GPC4 receptor on the sample, the sample is contacted with the antibody or its antigen-binding fragment, wherein the binding results in the formation of a receptor / antibody or its antigen-binding fragment complex. b) Detect the presence of a complex of the receptor / antibody or its antigen-binding fragment; c) The detection includes the presence or absence of the GPC4 receptor on the sample.

[0281] F2. A method for treating or preventing a disease or condition related to GPC4 in a subject, comprising: a) Contact a sample known to contain or suspected to contain GPC4 with an antibody or antigen-binding fragment thereof according to any one of embodiments A1-A45; b) Detect the presence of a complex containing GPC4 and an antibody or its antigen-binding fragment; wherein the presence of the complex indicates the presence of a disease or condition; and c) Administer to the subject an antibody or antigen-binding fragment thereof according to any one of embodiments A1-A45.

[0282] F3. A method for diagnosing a disease or condition, comprising: a) Separating samples from the subject; b) Incubate the sample with the antibody or its antigen-binding fragment according to any one of embodiments A1-A45 for a period of time sufficient to generate the GPC4:anti-GPC4 complex; c) Detect the presence or absence of GPC4:anti-GPC4 complex from isolated tissues, and d) Correlate the presence or abundance of GPC4 with the location of interest in the tissue sample.

[0283] F4. The method according to implementation scheme F3, wherein an increase in GPC4 at a location of interest in a tissue sample exceeding the control level indicates a disease or condition in the subject.

[0284] F5. The method according to implementation scheme F2 or F3, wherein the method is performed in vitro.

[0285] F6. The method according to implementation scheme F2 or F3, wherein the method is performed in vivo.

[0286] F7. The method according to any one of embodiments F1-F6, wherein the detection includes intracellular detection.

[0287] F8. The method according to any one of embodiments F1-F6, wherein the detection includes detection of cell surface.

[0288] F9. The method according to any one of embodiments F1-F6, wherein the detection comprises hybridization of the detectable portion with an antibody or an antigen-binding fragment thereof.

[0289] F10. The method according to any one of embodiments F1-F9, wherein the sample is contacted with the second antibody.

[0290] F11. The method according to embodiment F10, wherein the second antibody is an antibody containing a detectable portion.

[0291] F12. The method according to embodiment F11, wherein the detectable portion comprises an oligonucleotide.

[0292] F13. The method according to embodiment F11, wherein the detectable portion comprises a fluorescent marker.

[0293] F14. The method according to any one of embodiments F1-F9, wherein the detection includes sequencing.

[0294] F15. The method according to embodiment F11, wherein the detectable portion comprises immunofluorescence.

[0295] F16. The method according to any one of embodiments F1-F13 and F15, wherein the sample is a formalin-fixed paraffin-embedded sample.

[0296] F17. The method according to any one of embodiments F1-F16, wherein the sample comprises cells.

[0297] F18. The method according to any one of embodiments F1-F16, wherein the sample comprises a tissue sample.

[0298] F19. The method according to any one of embodiments F1-F16, wherein the sample comprises immune cells.

[0299] F20. The method according to embodiment F19, wherein the immune cells are selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and peripheral blood mononuclear cells (PBMCs).

[0300] F21. The method according to any one of embodiments F1-F16, wherein the sample comprises tissue or cells related to a disease or condition.

[0301] F22. The method according to implementation scheme F21, wherein the disease or condition is cancer, an autoimmune disease, an inflammatory disease, or an infection.

[0302] F23. The method according to implementation plan F21, wherein the disease or condition is selected from non-viral cancer, virus-associated cancer, cancer associated with HBV infection, cancer associated with EBV infection, cancer associated with polyomavirus infection, erythema nodosum (ENL), autoimmune diseases, autoimmune inflammation, autoimmune thyroid diseases, B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's disease, acute myeloid leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, B-cell leukemia, myelodysplastic syndrome, solid tumors, herpes virus infection and / or rejection of transplanted tissues or organs.

[0303] G1. An antibody or antigen-binding fragment thereof according to any one of embodiments A1-A45, used in a method for associating the presence or abundance of GPC4 with a location of interest in a tissue sample.

[0304] G2. A method for detecting GPC4 in a tissue sample using an antibody or antigen-binding fragment thereof according to any one of embodiments A1-A45.

[0305] G3. An antibody or antigen-binding fragment thereof according to embodiment G1 or G2, wherein the method includes generating a nucleic acid molecule comprising all or part of the sequence of an oligonucleotide or its complement.

[0306] G4. An antibody or antigen-binding fragment thereof, according to any one of embodiments A1-A45, is used to construct a protein library.

[0307] G5. An antibody or its antigen-binding fragment as described in implementation scheme G4, wherein the construction of the protein library includes sequencing.

[0308] G6. The antibody or its antigen-binding fragment as described in implementation scheme G4, wherein the construction of the protein library includes the use of flow cytometry.

[0309] Example The embodiments described below illustrate certain implementations and do not limit the technology.

[0310] Example 1: Generation and characterization of GPC4-resistant hybridomas The hybridoma is prepared as described in this embodiment, wherein the hybridoma secretes a monoclonal antibody that reacts with GPC4 expressed in vivo.

[0311] Immunization of laboratory mice (e.g., BALB / c or C57 / Bl6) or rats (e.g., Sprague Dawley) with the GPC4 immunogen was performed. Following successful immunization, myeloma cells were fused with cells collected from the spleen and draining lymph nodes of the animals to form hybridomas using a standard protocol. Successful fusions were selected in HAT medium and cloned into approximately one cell / well in a microtiter plate. The culture supernatant was then tested by flow cytometry for GPC4-expressing cell transfectants (e.g., HEK 293 or RBL). Wells with successful staining characteristics were passaged into larger containers until sufficient cells were present to allow for subcloning. Cells transfected with GPC4 were further characterized by flow cytometry for hybridoma subclonal candidates. Clones selected as optimal candidates were further screened by flow cytometry for human blood cells divided into different subsets (e.g., lymphocytes, monocytes, etc.) and for one or more cell lines derived from diseased and / or infected human cells. The percentage of positive cells in each blood cell subset was quantified compared to the isotype control.

[0312] Exemplary clones were selected based on their strong reactivity to GPC4-expressing immune cells (e.g., lymphocytes) but not to other blood cell populations, the detection of GPC4 on the surface of immune cells (e.g., lymphocytes), and / or the intracellular detection of GPC4.

[0313] Example 2: Sequencing of the variable region of anti-GPC4 antibody Cells from well-performing anti-GPC4 hybridoma cell lines (as described in Example 1 above) were grown in standard mammalian tissue culture medium. Total RNA was isolated from hybridoma cells from multiple clones expressing anti-GPC4 monoclonal antibodies using a procedure based on the RNeasy mini kit (Qiagen). The RNA was used to generate first-strand cDNA. The light and heavy chain variable domain cDNAs were amplified by 5'-RACE technology. Positive clones were prepared by PCR, and multiple clones were then sequenced.

[0314] The individual variable domains (CDRs and framework regions) of the AB2 heavy and light chains, including the amino acid sequences of CDR1, CDR2, and CDR3 regions, are shown in Table 1 below. The full-length sequence of AB2 is shown below Table 1.

[0315] Table 1. Amino acid and nucleic acid sequences of CDR. Heavy chain: DNA sequence (411 bp) (SEQ ID NO:14) Signal sequence - FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 Heavy chain: amino acid sequence (137 aa) (SEQ ID NO:15) Light chain: DNA sequence (384 bp) (SEQ ID NO:16) Signal sequence - FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 Light chain: amino acid sequence (128 aa) (SEQ ID NO:17) Example 3: Detection of GPC4-expressing cells This embodiment describes the ability of an exemplary generated anti-GPC4 antibody AB2 to detect GPC4-expressing cells by flow cytometry.

[0316] In the first experiment, the exemplary anti-GPC4 antibody AB2 was evaluated on cells from a human cell line that exhibited a phenotype similar to directed CNS neuronal precursor cells (NTERA-2; ATCC® CRL-1973). TM ).

[0317] ANTERA-2 cells were grown in T75 flasks in DMEM medium supplemented with 10% FBS until approximately 80% confluence was reached. An exemplary PE-conjugated anti-GPC4 antibody was added at a concentration of 1 µg / 1 million cells and incubated for 15 minutes. A commercially available antibody (CAb) was used as a control. Cells were washed with FACS buffer and analyzed on a BD LSRII flow cytometer. Figure 1 As shown, AB2 was positive for staining NTERA-2 cells.

[0318] In similar experiments, targeting cell lines that do not express GPC4 (U-937; ATCC® CRL-1539.2) TM AB2 was further evaluated. (For example...) Figure 2 As shown, AB2 showed negative staining in U-937 cells.

[0319] In another experiment, using cell lines known to express their respective GPCs, including A431 cells (GPC1), Molt4 cells (GPC2), and HepG2 cells (GPC6), the cross-reactivity of anti-GPC4 antibody AB2 or commercially available antibodies with GPC homologues GPC1 (45% homology), GPC2 (42% homology), and GPC6 (65% homology) was tested. Figures 3A-3C As shown, anti-GPC4 antibody AB2 did not show any interaction with GPC1 ( Figure 3A ), GPC2 ( Figure 3B ) or GPC6 ( Figure 3C Cross-reactivity of ).

[0320] These results demonstrate the ability of the exemplary anti-GPC4 antibody to specifically recognize cells expressing the corresponding receptor GPC4, with minimal to no nonspecific binding.

[0321] Example 4: Evaluation of GPC4-mediated Wnt3a signaling This embodiment describes a functional determination of the ability of an exemplary anti-GPC4 antibody AB2 to block GPC4-mediated enhancement of Wnt3a signaling.

[0322] In another experiment, the ability of AB2 to block Wnt3a signaling in NTERA-2 cells was evaluated. Briefly, NTERA-2 cells were cultured in black 96-well plates with glass bottoms to approximately 80% confluence, followed by the addition of 5 µg / mL of the exemplary antibody AB2, a commercially available antibody (CAb), or an allotype control to the culture medium for 15 minutes. After antibody incubation, cells were stimulated with 200 ng / mL recombinant human Wnt3a (R&D Systems) for 3 hours or without stimulation. Cells were fixed for 30 minutes with 1x working solution of True Nuclear Transcription Factor Fix (BioLegend True Nuclear Buffer Set, catalog number 424401) and washed twice with True Nuclear Perm Buffer (BioLegend True Nuclear Buffer Set, catalog number 424401). Cells were then stained with anti-human β-catenin antibody (BioLegendclone 12F7, catalog number 844601) followed by staining with Alexa Fluor® 594 anti-mouse IgG. Cells were imaged using an inverted epifluorescence microscope (Olympus IX83). Fluorescence intensity in the cytoplasm and nucleus was quantified using ImageJ, and the nucleus / cytoplasm fluorescence ratio was calculated. Untreated cells and cells stimulated with Wnt3a only served as controls. Figure 4 As shown, AB2 can reduce Wnt3a-induced translocation of β-catenin to the nucleus. Figure 5 As shown, the exemplary antibody AB2 reduces Wnt3a-induced translocation of β-catenin to the cell nucleus, while commercially available antibodies do not show this effect. Figure 6 As shown, the exemplary antibody AB2 was able to reduce Wnt3a-induced translocation of β-catenin to the cell nucleus at all tested concentrations, while commercially available antibodies did not show this effect.

Claims

1. An antibody or antigen-binding fragment thereof that binds to GPC4 or a portion thereof, comprising: a) An immunoglobulin heavy chain variable domain, wherein the immunoglobulin heavy chain variable domain comprises: i) Heavy chain complementarity-determining region 1 (CDRH1), which contains an amino acid sequence that shows at least 80% sequence identity with SEQ ID NO:2; ii) Heavy chain complementarity-determining region 2 (CDRH2), which contains an amino acid sequence that shows at least 80% sequence identity with SEQ ID NO:3; iii) Heavy chain complementarity-determining region 3 (CDRH3), which contains an amino acid sequence that shows at least 80% sequence identity with SEQ ID NO:4; and / or b) An immunoglobulin light chain variable domain, wherein the immunoglobulin light chain variable domain comprises: i) Light chain complementarity-determining region 1 (CDRL1), which contains an amino acid sequence that shows at least 80% sequence identity with SEQ ID NO:5; ii) Light chain complementarity-determining region 2 (CDRL2), which contains an amino acid sequence exhibiting at least 80% sequence identity with SEQ ID NO:6; and iii) Light chain complementarity determination region 3 (CDRL3), which contains an amino acid sequence that shows at least 80% sequence identity with SEQ ID NO:

7.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain variable domain comprises: CDRH1, wherein CDRH1 comprises the amino acid sequence shown in SEQ ID NO:2; CDRH2, wherein CDRH2 comprises the amino acid sequence shown in SEQ ID NO:3; and CDRH3, wherein CDRH3 comprises the amino acid sequence shown in SEQ ID NO:

4.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, wherein the immunoglobulin light chain variable domain comprises: CDRL1, wherein CDRL1 comprises the amino acid sequence shown in SEQ ID NO:5; CDRL2, wherein CDRL2 comprises the amino acid sequence shown in SEQ ID NO:6; and CDRL3, wherein CDRL3 comprises the amino acid sequence shown in SEQ ID NO:

7.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO:15 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

15.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:17 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

17.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, used for detecting GPC4 in a sample.

7. A diagnostic antibody or an antigen-binding fragment thereof, comprising the antibody or an antigen-binding fragment thereof according to any one of claims 1-6.

8. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-6.

9. A composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-6 and a pharmaceutically acceptable excipient.

10. The composition of claim 9, wherein the antibody or its antigen-binding fragment is used as an adjuvant or in combination with an adjuvant.

11. An isolated nucleic acid comprising a nucleotide sequence encoding a variable domain of an immunoglobulin heavy chain that encodes an antibody or an antigen-binding fragment thereof according to any one of claims 1-6.

12. A recombinant expression vector comprising the isolated nucleic acid according to claim 11.

13. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid molecule containing a nucleotide sequence encoding an immunoglobulin heavy chain variable domain according to any one of claims 1-6, and the second expression cassette comprises a nucleic acid molecule containing a nucleotide sequence encoding an immunoglobulin light chain variable domain of an antibody or antigen-binding fragment thereof according to any one of claims 1-6.

14. A host cell comprising the recombinant expression vector according to claim 12 or claim 13.

15. A method for detecting the presence or absence of GPC4 in an in vitro sample, the method comprising: a) Under conditions in which the antibody or antigen-binding fragment thereof according to any one of claims 1-6 is bound to GPC4 in an in vitro sample, the sample is contacted with the antibody or antigen-binding fragment thereof, wherein the binding results in the generation of at least one complex comprising GPC4 and the antibody or antigen-binding fragment thereof. and b) Detect the presence or absence of at least one complex containing GPC4 and the antibody or its antigen-binding fragment to detect the presence or absence of GPC4 in the in vitro sample.

16. A method for treating or preventing a GPC4-related disease or condition in a subject, the method comprising: a) Contact a sample known to contain or suspected to contain GPC4 with the antibody or its antigen-binding fragment according to any one of claims 1-6; b) Detecting the presence of a complex comprising GPC4 and the antibody or an antigen-binding fragment thereof; wherein the presence of the complex indicates the presence of a disease or condition; and c) Administer the antibody or its antigen-binding fragment according to any one of claims 1-6 to the subject.

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