Anti-CD93 antibodies and uses thereof

Abstract

The present disclosure provides an antibody or an antigen-binding fragment thereof, which specifically binds to CD93. The present invention aims to screen and obtain anti-CD93 antibody molecules with stronger blocking activity for the interaction between CD93 and IGFBP7 and / or MMRN2 through antibody discovery technology, thereby obtaining an anti-tumor drug with better tumor inhibition effect by promoting normalization of tumor tissue blood vessels, inhibiting vascular leakage, and increasing immune cell infiltration.

Description

[0001] Reference to a sequence listing

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of antibodies, and more specifically to an antibody against CD93 and applications thereof. Background Art

[0004] CD93, or Cluster of Differentiation 93, is encoded by the CD93 gene. Human CD93 protein consists of 629 amino acids, and the sequence is shown in SEQ ID NO:1 (Uniprot Q9NPY3). CD93 belongs to the type I transmembrane glycoprotein of the XIV family protein, which contains a C-class lectin-like domain, an EGF-like repeat sequence, a highly glycosylated mucin domain, a transmembrane domain, and an intracellular domain. CD93 is mainly expressed on endothelial cells, platelets, immature B cells, and monocytes. CD93 is considered to be a downstream signal regulator of VEGF and plays an important role in regulating vascular normalization. The ligands that bind to CD93 are mainly IGFBP7 and MMRN-2 proteins. IGFBP7 is a secreted protein of the IGFBP family, consisting of an N-terminal IGF binding domain (IB), a Kazal-like serine protease inhibitor domain, and a C-terminal Ig-like C2-like domain (IgC2). IGFBP7 is hardly expressed in normal human tissue vessels, but its expression is upregulated in tumor tissue vasculature, such as head and neck cancer tumor tissue. The hypoxic environment caused by the upregulation of VEGF signaling pathway induces the expression of IGFBP7. MMRN-2 protein can bind to a variety of group 14 C-type lectin proteins, while IGFBP7 only binds to CD93. IGFBP7 does not affect the expression of CD93, and MMRN-2 stabilizes CD93 protein by inhibiting the degradation of CD93. IGFBP7 and MMRN-2 do not compete with each other when binding to CD93. The interaction between IGFBP7 and CD93 is mediated by the IB domain of IGFBP7 and the epidermal growth factor (EGF)-like domain of CD93, respectively, while MMRN-2 interacts with the C-type lectin-like domain (CTLD) of CD93. Studies on tumor-bearing mouse animal models have found that by blocking the interaction pathway between CD93 and IGFBP7, the tumor infiltration of immune cells can be increased, the normalization of blood vessels in tumor tissue can be promoted, and the tumor growth can be inhibited. At the same time, CD93 pathway blockade combined with anti-tumor small molecule drugs or immune checkpoint drugs can achieve the effect of enhancing tumor growth inhibition.

[0005] VEGF pathway targeted therapy plays a role in inhibiting tumor growth by inhibiting tumor angiogenesis. As a downstream signaling pathway molecule of VEGF, the CD93 signaling pathway plays an important role in the normalization of tumor cell blood vessels. Therefore, when used in combination with chemotherapy drugs, it can increase the anti-tumor value of the combination by promoting the delivery of small molecule drugs in solid tumor blood vessels. The blocking effect of the CD93 pathway is an extension of VEGF's regulation of tumor cell blood vessels.

[0006] At present, DyanmiCure Biotechnology LLC has reported the discovery of anti-human CD93 antibodies that block the interaction between CD93 and IGFBP7 or CD93 and MMRN2. Although these antibodies show blocking activity at high concentrations, the tumor inhibitory effect on tumor-bearing mice is not significant (WO2022067262A1). This technology aims to screen and obtain anti-CD93 antibody molecules with stronger blocking activity for the interaction between CD93 and IGFBP7 and / or MMRN2 through antibody discovery technology, thereby obtaining an anti-tumor drug with better tumor inhibition effect by promoting normalization of tumor tissue blood vessels, inhibiting vascular leakage, and increasing immune cell infiltration. Summary of the invention

[0007] In view of the above problems, the present disclosure provides antibodies, preparation methods and compositions thereof, etc. The benefits provided by the present disclosure are widely applicable to the fields of antibody therapy and diagnosis, and can be used in combination with antibodies reactive with various targets.

[0008] The present invention discloses an isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to CD93 and comprises a heavy chain variable region (VH) and a light chain variable region (VL),

[0009] The heavy chain variable region comprises:

[0010] (i) HCDR1 comprising a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 12, 15, 18, 20, and 23 or consisting of SEQ ID NOs: 12, 15, 18, 20, and 23;

[0011] (ii) HCDR2 comprising or consisting of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 13, 16, 19, 21, and 24; and

[0012] (iii) HCDR3 comprising or consisting of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 14, 17, 22, and 25;

[0013] The light chain variable region comprises:

[0014] (i) LCDR1, comprising or consisting of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 26, 29, 32, and 35; and

[0015] (ii) LCDR2, comprising or consisting of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 27, 30, 33, and 36; and

[0016] (iii) LCDR3, comprising or consisting of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to one of SEQ ID NOs: 28, 31, 34, and 37.

[0017] In some embodiments of the present invention, the antibody or fragment comprises the following combination:

[0018] (i) HCDR1 shown in SEQ ID NO:12, HCDR2 shown in SEQ ID NO:13, and HCDR3 shown in SEQ ID NO:14, LCDR1 shown in SEQ ID NO:26, LCDR2 shown in SEQ ID NO:27, and LCDR3 shown in SEQ ID NO:28; or

[0019] (ii) HCDR1 set forth in SEQ ID NO:15, HCDR2 set forth in SEQ ID NO:16, and HCDR3 set forth in SEQ ID NO:17, LCDR1 set forth in SEQ ID NO:29, LCDR2 set forth in SEQ ID NO:30, and LCDR3 set forth in SEQ ID NO:31; or

[0020] (iii) HCDR1 set forth in SEQ ID NO:18, HCDR2 set forth in SEQ ID NO:19, and HCDR3 set forth in SEQ ID NO:17, LCDR1 set forth in SEQ ID NO:29, LCDR2 set forth in SEQ ID NO:30, and LCDR3 set forth in SEQ ID NO:31; or

[0021] (iv) HCDR1 of SEQ ID NO:20, HCDR2 of SEQ ID NO:21, and HCDR3 of SEQ ID NO:22, LCDR1 of SEQ ID NO:32, LCDR2 of SEQ ID NO:33, and LCDR3 of SEQ ID NO:34; or

[0022] (v) HCDR1 shown in SEQ ID NO:23, HCDR2 shown in SEQ ID NO:24, and HCDR3 shown in SEQ ID NO:25, LCDR1 shown in SEQ ID NO:35, LCDR2 shown in SEQ ID NO:36, and LCDR3 shown in SEQ ID NO:37.

[0023] In some embodiments of the invention, the heavy chain variable region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 2, 4, 6, 8 and 10;

[0024] wherein the light chain variable region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 3, 5, 7, 9 and 11, or consists of one of SEQ ID NOs: 3, 5, 7, 9 and 11.

[0025] In some embodiments of the present invention, the antibody or fragment comprises one of the following combinations:

[0026] (i) a heavy chain variable region comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2;

[0027] a light chain variable region comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3;

[0028] (ii) a heavy chain variable region comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4;

[0029] a light chain variable region comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5;

[0030] (iii) a heavy chain variable region comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:6;

[0031] a light chain variable region comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7;

[0032] (iv) a heavy chain variable region comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8;

[0033] a light chain variable region comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9;

[0034] (v) a heavy chain variable region comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10;

[0035] A light chain variable region comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 11.

[0036] In some embodiments of the present invention, the antibody or fragment further comprises a heavy chain constant region and a light chain constant region, wherein the antibody heavy chain constant region is selected from the IgG series antibodies, and the light chain constant region is selected from κ or λ chain.

[0037] In some embodiments of the present invention, the antibody or fragment, wherein the antibody heavy chain constant region is preferably selected from one of IgG1, IgG2c, and IgG2b, and the light chain constant region is preferably a κ chain.

[0038] In some embodiments of the present invention, the antibody or fragment, wherein the antibody is selected from the group consisting of a whole antibody, a bispecific antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody;

[0039] The fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab)2 fragment, a Fv fragment and a ScFv.

[0040] On the other hand, the present invention also provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or fragment.

[0041] On the other hand, the present invention also provides a vector comprising the nucleic acid molecule.

[0042] On the other hand, the present invention also provides a host cell, comprising the nucleic acid molecule or the vector.

[0043] In another aspect, the present invention also provides a conjugate comprising the antibody or fragment coupled to at least one detectable label.

[0044] In another aspect, the present invention also provides an antibody drug conjugate comprising an antibody, wherein the antibody comprises one or more drug moieties, wherein the drug moieties are covalently linked to the antibody or fragment directly or via a linker.

[0045] On the other hand, the present invention also provides a multispecific molecule, which comprises the antibody or antigen-binding fragment; preferably, the multispecific molecule specifically binds to CD93, and additionally specifically binds to one or more other targets; further preferably, the multispecific molecule also comprises at least one molecule having a second binding specificity for a second target.

[0046] On the other hand, the present invention also provides a pharmaceutical composition or kit, comprising the antibody or fragment, or the nucleic acid molecule, or the vector, or the host cell, or the conjugate, or the antibody-drug conjugate, or the multispecific molecule, and a pharmaceutically acceptable carrier.

[0047] On the other hand, the present invention also provides use of the antibody or fragment, or the nucleic acid molecule, or the vector, or the host cell, or the conjugate, or the antibody-drug conjugate, or the multispecific molecule, or the pharmaceutical composition or kit in the preparation of a kit for diagnosing, detecting or monitoring diseases related to CD93 expression.

[0048] On the other hand, the present invention also provides use of the antibody or fragment, or the nucleic acid molecule, or the vector, or the host cell, or the conjugate, or the antibody-drug conjugate, or the multispecific molecule, or the pharmaceutical composition or kit in the preparation of a drug for treating a disease associated with CD93 expression or determining its prognosis.

[0049] On the other hand, the present invention also provides the use, wherein the disease associated with CD93 expression is cancer, and the cancer is selected from the following group: renal cancer, pancreatic cancer, breast cancer, gastric cancer, glioma and other cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The accompanying drawings are used to provide further understanding of the present disclosure and constitute a part of the specification. They are used to explain the present disclosure together with the embodiments of the present disclosure and do not constitute a limitation of the present disclosure.

[0051] Figure 1 is a binding activity curve of anti-human CD39 antibody and human CD93 overexpressing cells detected by flow cytometry, wherein the abscissa mAb conc.Log is the concentration of the antibody of the present invention, in nM, and the ordinate MFI-PE is the mean fluorescence intensity;

[0052] Figure 2 is a binding activity curve of anti-human CD39 antibody and cynomolgus monkey CD93 overexpressing cells detected by flow cytometry, wherein the abscissa mAb conc.Log is the concentration of the antibody of the present invention, in nM, and the ordinate MFI-PE is the mean fluorescence intensity;

[0053] Figure 3 The binding activity of anti-human CD39 antibody to mouse CD93 overexpressing cells is detected by flow cytometry, wherein the abscissa is the antibody number of the present invention, and the ordinate MFI-PE is the mean fluorescence intensity;

[0054] Figure 4 is the result of flow cytometry detection of the binding specificity of anti-human CD39 antibody to CHOZN-K1, wherein the abscissa is the antibody number of the present invention, and the ordinate MFI-PE is the mean fluorescence intensity;

[0055] Figure 5 is a flow cytometry-based curve for detecting the blocking activity of anti-human CD39 antibody against human CD93 / human IGFBP7 binding, wherein the abscissa mAb conc.Log is the concentration of the antibody of the present invention, in nM, and the ordinate MFI-PE is the mean fluorescence intensity;

[0056] Figure 6 The figure is a flow cytometry detection curve of anti-human CD39 antibody blocking human CD93 / human MMRN-2 binding activity, wherein the abscissa mAb conc.Log is the antibody concentration of the present invention, the unit is nM, and the ordinate MFI-PE is the mean fluorescence intensity. DETAILED DESCRIPTION

[0057] In order to make those skilled in the art better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only illustrative embodiments of a part of the present invention, rather than all embodiments. Therefore, the present invention is not limited to the specific embodiments illustrated. In addition, any section titles used herein are not to be construed as limiting the subject matter described.

[0058] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, terms in the singular shall include plural forms, and terms in the plural shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural indicators. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include endpoints and all values ​​between the endpoints.

[0059] definition

[0060] In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0061] The term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of antibodies can be divided into κ or λ light chains. The heavy chains can be divided into μ, δ, γ, α or ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA or IgE, respectively. In the light and heavy chains, the variable region is connected to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementary determining regions, or CDRs) separated by relatively conserved regions (called framework regions, or FRs). Each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from N-terminus to C-terminus. The CDRs on VH are HCDR1, HCDR2, and HCDR3; the CDRs on VL are LCDR1, LCDR2, and LCDR3. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites / parts, respectively. The distribution of amino acids in various regions or domains follows the numbering definitions in common systems such as Kabat, IMGT, or Chothia. In the specific embodiments of the present disclosure, the CDR sequences are determined using the numbering definitions in the Kabat system.

[0062] Antibodies in the present disclosure also include antigen binding portions (interchangeably used with the term "antigen binding fragment"). Antigen binding portions refer to polypeptides comprising fragments of intact antibodies that retain the ability to specifically bind to an antigen that specifically binds to a full-length or intact antibody, and / or that compete with a full-length antibody for binding to the same antigen. Under some conditions, antigen binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and antibodies that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to a polypeptide. The antigen binding portion of an antibody can be obtained from a given antibody by conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and can be screened for specificity in the same manner as intact antibodies.

[0063] The term "isotype" refers to the antibody class (eg, IgM or IgG1) encoded by the heavy chain constant region genes.

[0064] The term "monoclonal antibody" or "mAb" refers to an antibody molecule / preparation of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. The antibodies of the present invention may be derived from different species, including but not limited to mouse, rat, rabbit, guinea pig and human.

[0065] The term "epitope" refers to an antigenic determinant in a molecule, which refers to a portion of a molecule that is recognized by the immune system (e.g., by an antibody), such as a discontinuous three-dimensional site on an antigen recognized by the immune system. In the present invention, the epitope is, for example, the CD93 protein.

[0066] The term "chimeric antibody" as used herein refers to an antibody whose variable region sequence is from one species and the constant region sequence is from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0067] The term "humanized antibody" is intended to refer to antibodies in which CDR sequences / antigen binding portions or sites derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In addition, additional framework region modifications may also be made within the human framework sequences.

[0068] The term "KD value" is the equilibrium dissociation constant between an antibody and its antigen, i.e., the ratio of koff / kon or kd / ka (determined by SPR technology). Therefore, the lower the KD value (the lower the concentration), the higher the affinity of the antibody. Therefore, the "KD value" can be used to measure the binding affinity between an antibody and its antigen.

[0069] The terms "CD93" and "CD93 antigen" are used interchangeably herein and include any variants, isoforms, and species homologs of human CD93 that are naturally expressed by cells or expressed on cells transfected with the CD93 gene. In some embodiments, binding of the antibodies of the present disclosure to the CD93 antigen mediates killing of cells expressing CD93 (e.g., tumor cells) by inactivating CD93. Killing of cells expressing CD93 may occur by one or more of the following mechanisms: cell death / apoptosis induction, ADCC, and CDC.

[0070] The term "anti-CD93 antibody" or "CD93 antibody" refers to an antibody capable of binding to the CD93 antigen or to a cell expressing CD93 as defined herein.

[0071] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.

[0072] The term "isolated" refers to a state obtained from a natural state by artificial means. If a certain "isolated" substance or component exists in nature, it may be because its natural environment has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide exists naturally in a living organism, and the same polynucleotide or polypeptide with high purity separated from this natural state is called an isolated polynucleotide or polypeptide. The term "isolated" neither excludes mixed artificial or synthetic substances, nor excludes other impure substances that do not affect the activity of the isolated substance. For example, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0073] The term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by a polynucleotide inserted therein, the vector is called an expression vector. The vector can be transformed, transduced or transfected into a host cell to express the genetic material elements carried in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). The vector may contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may contain an origin of replication. As for the vector for expressing the antibody, a vector type in which the antibody heavy chain and light chain exist in different vectors or a vector type in which the heavy chain and light chain exist in the same vector can be used.

[0074] The term "host cell" refers to a cell system that can be engineered to produce a target protein, protein fragment or peptide. Host cells include, but are not limited to, cultured cells, such as cultured mammalian cells derived from rodents (rat, mouse, guinea pig or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells and insect cells, as well as cells contained in transgenic animals or cultured tissues. The term covers not only specific test cells, but also the offspring of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such offspring may be different from the parental cells, but are still included in the scope of the term "host cell".

[0075] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules (or protein molecules) or two or more nucleic acid molecules determined by alignment and comparison of the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecules being compared. For these calculations, gaps in the alignment, if any, are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAM J. Applied Math. 48:1073.

[0076] The term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It not only refers to the property of antigens to stimulate the activation, proliferation and differentiation of specific immune cells to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also refers to the specific immune response of antibodies or sensitized T lymphocytes that can be formed in the immune system of an organism after stimulating the organism with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host and the means of immunity.

[0077] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13:197.

[0078] The term "hybridoma" and the term "hybridoma cell line" are used interchangeably. When referring to the term "hybridoma" and the term "hybridoma cell line", they also include subclones and progeny cells of the hybridoma.

[0079] The term "immune effector function" includes any function mediated by components of the immune system that results in inhibition of tumor growth and / or inhibition of tumorigenesis, including inhibition of tumor dissemination and metastasis. Preferably, the immune effector function results in killing tumor cells. Preferably, the immune effector function in the present invention is an antibody-mediated effector function. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), induction of apoptosis in cells carrying tumor-associated antigens (e.g., by binding of antibodies to surface antigens) and / or inhibition of proliferation of cells carrying tumor-associated antigens, preferably ADCC and / or CDC. Antibodies can also simply act by binding to tumor-associated antigens on the surface of tumor cells. For example, antibodies can block the function of tumor-associated antigens or induce apoptosis simply by binding to tumor-associated antigens on the surface of tumor cells.

[0080] The term "cancer" refers to any tumor or malignant cell growth, proliferation or metastasis-mediated solid tumors and non-solid tumors such as leukemia that cause a medical condition. For example, cancers associated with CD93 expression or caused by abnormal expression include, but are not limited to: B-cell lymphomas, including NHL, pre-B-cell lymphocytic leukemia / lymphomas and mature B-cell tumors, such as B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT type, nodal and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasma cell neoplasm, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia and anaplastic large cell lymphoma (ALCL)

[0081] The term "pharmaceutically acceptable" means that the carrier, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0082] The term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19 th Ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to pH adjusters, surfactants, adjuvants and ionic strength enhancers. For example, pH adjusters include but are not limited to phosphate buffer; surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.

[0083] The term "adjuvant" refers to a non-specific immunopotentiator, which can enhance the immune response to the antigen in the organism or change the type of immune response when it is delivered to the organism together with the antigen or delivered to the organism in advance. There are multiple adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium brevis, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in current animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0084] Anti-CD93 antibody

[0085] In some aspects, the invention includes isolated antibodies or antigen-binding fragments thereof.

[0086] In the context of the present application, "antibodies" may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intracellular antibodies, bifunctional antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including mutant proteins and variants thereof, improved antibodies; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecules, as long as they show preferential association or binding with CD93 protein. In addition, unless the context otherwise dictates, the term also includes antibodies of all classes (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a chimeric monoclonal antibody or a humanized monoclonal antibody or an improved chimeric monoclonal antibody.

[0087] The variable regions and CDRs in an antibody sequence can be identified according to the general rules (such as Kabat) numbering system developed in the art or by aligning the sequence with a database of known variable regions.

[0088] Regardless of how the antibody is produced, methods for testing the ability of an antibody to bind to an antigen (e.g., CD93) are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266 and the above section regarding competition assays).

[0089] According to the present invention, in a standard assay (such as the assay described in the present invention), if the antibody has a significant affinity to the predetermined target (such as CD93 protein or cells expressing CD93), the antibody is capable of binding to the predetermined target. In order to test the binding of the monoclonal antibody to living cells expressing CD93, flow cytometry (FCM) can be used. Preferably, in a flow cytometric fluorescence sorting technique (FACS) analysis, the antibody is measured for binding to a target expressed on the cell surface. If the antibody can detectably bind to the target (CD93 protein or cells expressing CD93), the antibody is capable of binding to the target and has "affinity".

[0090] The CD93 specificity described in the present invention refers to the ability to bind to one or more CD93 epitopes, especially CD93 epitopes in natural conformation, in particular human CD93 specificity.

[0091] In the art, various methods are used to modify the desired properties of antibodies without changing them, such as the method used in the present disclosure of recombining the light chain and heavy chain of the antibody, replacing amino acids, etc. For example, the sequences of the present invention, including chimeric antibody sequences or humanized antibody sequences, can be subjected to conservative amino acid substitutions.

[0092] Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementary determining regions (CDRs). For this reason, the amino acid sequences of CDRs are more diverse between antibodies than other sequences. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody by constructing an expression vector comprising CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from different antibodies with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germ lines are sequences that are different from mature antibody gene sequences because they do not contain fully assembled variable genes, which are formed by V(D)J joining during B cell maturation. Germline gene sequences will also have sequences that are different from the high-affinity second antibody library (secondary repertoireantibody) at individual locations evenly across the variable region.

[0093] Mouse antibodies are highly immunogenic in humans, resulting in reduced therapeutic efficacy when used repeatedly, with the main immunogenicity mediated by the heavy chain constant region. If the individual antibodies are chimerized or humanized, the immunogenicity of mouse antibodies in humans can be reduced or completely avoided.

[0094] Chimeric antibodies refer to antibodies in which different parts are from different animal species, for example, antibodies with variable regions from mouse antibodies and human immunoglobulin constant regions. The variable regions of mouse antibody heavy and light chains are linked together with the constant regions of human heavy and light chains to obtain the chimeric nature of the antibody (for example, as described in Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are produced by connecting human kappa light chain constant regions to mouse light chain variable regions. In another preferred embodiment, chimeric antibodies can be produced by connecting human lambda light chain constant regions to mouse light chain variable regions.

[0095] Humanized antibodies are antibodies in which CDR sequences / antigen-binding portions or sites derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0096] In order to reduce the immunogenicity of the antibody to humans, the sequence of the CD93 antibody of the present disclosure is used to produce a humanized anti-CD93 antibody, and the CDR region of the anti-CD93 antibody of mouse origin is combined with the framework region of human origin (e.g., human immunoglobulin) to form the humanized anti-CD93 antibody of the present disclosure. The humanized antibody is expected to retain the function of binding to human CD93 as well as the function of binding to monkey CD93.

[0097] Preparation or generation of antibodies

[0098] The antibodies of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975), and other techniques for producing monoclonal antibodies, such as virus or oncogene transformation of B lymphocytes or phage display technology using antibody gene libraries, somatic cell hybridization methods, and for example, obtained by genetic engineering recombination technology. For example, DNA molecules encoding the heavy chain and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification, the obtained DNA molecules are inserted into expression vectors, and then transfected into host cells, and then, the transfected host cells are cultured under specific conditions, and the antibodies of the present invention are expressed.

[0099] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are rat and rabbit systems (described, for example, in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92: 9348 (1995), see also Rossie et al.. Am. J. Clin. Pathol. 124: 295 (2005)). Hybridoma production in mice is a very well established method. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion methods are also known.

[0100] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals or some combination thereof. For example, hybridomas and biochemical and genetic engineering techniques recognized in the art can be used to produce monoclonal antibodies, as described in detail in An, Zhiqiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1st ed.2009; Shire et al. (eds.) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science + Business Media LLC, 1st ed.2010; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.1988; Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety.

[0101] It should be understood that the selected binding sequence can be further altered, for example, to increase affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies comprising altered target binding sequences are also antibodies of the present invention.

[0102] In some embodiments, the method of producing the antibodies or fragments described herein comprises the following steps:

[0103] (i) expressing the antibody or fragment in a host cell; and optionally

[0104] (ii) isolating the antibody or antigen-binding fragment thereof from the host cell.

[0105] In a preferred embodiment, anti-CD93 monoclonal antibodies are prepared by using hybridomas.

[0106] In order to obtain hybridomas that produce antibodies of the invention, such as human monoclonal antibodies of the invention, spleen cells and / or lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas are screened for the production of antigen-specific antibodies. The production of hybridomas is well known in the art. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0107] The antibodies of the present invention can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229: 1202). In some embodiments, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors so that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is linked to the vector so that the transcriptional and translational control sequences within the vector perform their intended functions of regulating transcription and translation of the antibody gene.

[0108] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors.In some embodiments, the variable region is used to produce the full-length antibody gene of any antibody isotype by inserting it into the expression vector of the heavy chain constant region and the light chain constant region encoding the desired isotype, so that the VH segment is operably connected to the CH segment and the VL segment in the carrier are operably connected to the CL segment in the carrier.In addition or alternatively, the recombinant expression vector can encode the signal peptide that promotes the secretion of the antibody chain from the host cell.The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the amino terminal of the antibody chain gene.The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0109] To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. The antibodies of the present invention can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells, which can assemble and secrete appropriately folded and immunologically active antibodies.

[0110] Mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary cells (CHO cells) (including dhfr CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220) used with a DHFR selection marker (e.g., as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159: 601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036 and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell or secreted into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0111] In another preferred embodiment, transgenic or transchromosomal mice carrying parts of the human immune system (rather than the mouse system) can be used to generate human monoclonal antibodies against CD93.

[0112] Another strategy for generating monoclonal antibodies is to directly isolate the gene encoding the antibody from the lymphocytes producing antibodies of a defined strategy, see for example Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined strategy. For details of recombinant antibody engineering, see also Welschof and Krau, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny KC Lo Antibody Engineering ISBN 1-58829-092-1.

[0113] To prepare chimeric antibodies, murine immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably linking the nucleic acid encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat et al. (1991), Sequences Of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but more preferably an IgG1 or IgG4 constant region. The isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat et al., supra), and DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region. Once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding VL or VH is operably connected to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably connected" as used herein is intended to indicate that two DNA fragments are connected so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0114] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter, U.S. Pat. No. 5,225,539; Queen et al., U.S. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,762; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can be used that are capable of not producing endogenous immunoglobulins after immunization and are capable of producing a complete human antibody repertoire. For example, it has been reported that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice can completely inhibit endogenous antibody production, and then transfer of the human germ-line immunoglobulin gene array into the germ-line mutant mice will result in the mice producing human antibodies when challenged with antigen (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258). Non-limiting examples of the above transgenic animals include, HuMAb mice (Medarex, Inc.), which contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, plus targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g., Lonberg et al. (1994) Nature 368 (6474): 856-859); or "KM mice TM" carrying human heavy chain transgenes and human light chain transchromosomes (see patent application WO02 / 43478). Other methods for antibody humanization include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227: 381; Marks et al., J. Mol. Biol. 1991, 222: 581-597; Vaughan et al., 1996, Nature Biotech 14: 309).

[0115] Nucleic acid molecules encoding the antibodies of the present invention

[0116] In some aspects, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the above-mentioned isolated antibody or fragment thereof as disclosed herein.

[0117] Nucleic acids of the present invention can be obtained using standard molecular biology techniques. For hybridoma-expressed antibodies (e.g., hybridomas prepared by transgenic mice carrying human immunoglobulin genes as further described below), cDNA encoding the light and heavy chains of antibodies prepared by hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from gene libraries.

[0118] In order to prepare chimeric antibodies, methods known in the art can be used to connect the mouse immunoglobulin variable region to the human immunoglobulin constant region (see, for example, U.S. Pat. No. 4,816,567 to Cabilly et al.). The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably connecting the nucleic acid encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3), and DNA fragments containing these regions can be obtained by standard PCR amplification. The isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operably connecting the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene or a scFv gene. In these operations, the DNA fragment encoding VL or VH is operably connected to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.

[0119] Conjugate

[0120] In one aspect, the present disclosure provides a conjugate comprising an antibody or fragment thereof as described above coupled to at least one detectable marker. Detectable markers include, but are not limited to: (i) providing a detectable signal; (ii) interacting with a second marker to modify the detectable signal provided by the first or second marker, such as FRET (Fluorescence Resonance Energy Transfer); (iii) affecting mobility (e.g., electrophoretic mobility) by charge, hydrophobicity, shape or other physical parameters, or (iv) providing a capture moiety, such as affinity, antibody / antigen or ion complexation.

[0121] Suitable structures as labels include fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotope labels, preferably stable isotope labels, isobaric labels, enzyme labels (such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), particle labels (especially metal particle labels, magnetic particle labels, polymer particle labels), organic small molecules (such as biotin, receptor ligands or binding molecules (such as cell adhesion proteins or phosphatidylcholine), which can be used to bind The marker sequence containing nucleic acid and / or amino acid residues can be detected by the marker. The marker includes, but is not limited to, barium sulfate, iodine, iopanoic acid, amiopromide calcium, diatrizoate sodium, diatrizoate meglumine, meglumine, sodium tyropanate, and radiodiagnostic agents (including positron emitters, (e.g., fluorine-18 and carbon-11), gamma emitters (e.g., iodine-123, iodine-125, technetium-99m, iodine-131 and indium-111), nuclear magnetic resonance nuclides (e.g., fluorine and gadolinium)), luminescent substances (e.g., isoluminol and acridinium esters), fluorescent substances (e.g., fluorescein and rhodamine), and colored substances (e.g., latex particles and colloidal gold).

[0122] Detectable labels as described above can be detected by methods known in the art. For example, fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, detectable labels as described above can be connected to antibodies or antigen-binding fragments thereof of the present invention by linkers of varying lengths to reduce potential steric hindrance.

[0123] Antibody Drug Conjugates / Immunoconjugates

[0124] On the one hand, the present disclosure provides an antibody-drug conjugate comprising an antibody, which includes one or more drug moieties / therapeutic agents, wherein the drug moieties are directly or via a linker (e.g., covalently linked) to an antibody or fragment thereof as described above. In the antibody-drug conjugate of the present application, the linker structure for conjugating the anti-CD93 antibody to the drug is not particularly limited, as long as the resulting antibody-drug conjugate can be used.

[0125] Since antibody-drug conjugates have the ability to selectively deliver one or more drugs to target tissues (e.g., tumor-associated antigens, such as tumors expressing CD93), antibody-drug conjugates can improve the therapeutic efficacy of the antibodies or antigen-binding fragments thereof of the present invention in treating diseases (e.g., cancer).

[0126] Multispecific molecules

[0127] The antibodies or antigen-binding fragments thereof of the present invention can be used to form multispecific molecules (e.g., bispecific molecules). The antibodies or antigen-binding fragments thereof of the present invention can be part of a multispecific molecule (e.g., a bispecific molecule), wherein the bispecific or multispecific molecule comprises a second functional module (e.g., a second antibody) or a third functional module (e.g., a third antibody) having a binding specificity different from that of the antibodies or antigen-binding fragments thereof of the present invention, thereby being able to bind to at least two different binding sites and / or target molecules. For example, the antibodies or antigen-binding fragments thereof of the present invention can be connected to a second antibody or antigen-binding fragment thereof that can specifically bind to any protein that can be used as a potential target for combined therapy. To produce the bispecific or multispecific molecules, the antibodies or antigen-binding fragments thereof of the present invention can be connected (e.g., by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimetics).

[0128] Thus, in some aspects, the invention provides a multispecific molecule comprising an antibody or antigen-binding fragment thereof of the invention.

[0129] In certain preferred embodiments, the multispecific molecule specifically binds CD93 (eg, human CD93 or monkey CD93) and specifically binds one or more additional targets.

[0130] In certain preferred embodiments, the multispecific molecule further comprises at least one molecule having a second binding specificity for a second target (eg, a second antibody).

[0131] In certain preferred embodiments, the multispecific molecule is a bispecific antibody.

[0132] Pharmaceutical composition

[0133] In some aspects, the present invention relates to a pharmaceutical composition. The present disclosure provides a pharmaceutical composition or kit comprising the antibody or fragment as described above, the nucleic acid molecule as described above, the vector as described above, the host cell as described above, the conjugate as described above, the antibody-drug conjugate as described above, the multispecific molecule as described above; and a pharmaceutically acceptable carrier.

[0134] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent or vaccine, so that the anti-CD93 antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, combinations of various components known in the art or more.

[0135] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene and / or propyl arsenate. As disclosed in the present invention, in a solvent containing an antibody or its antigen-binding fragment of the composition disclosed in the present invention containing one or more antioxidants such as methionine that reduce the antibody or its antigen-binding fragment, it may be oxidized. Redox can prevent or reduce the reduction of binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, the present invention provides a composition comprising one or more antibodies or their antigen-binding fragments and one or more antioxidants such as methionine. The present invention further provides methods wherein the antibodies or antigen-binding fragments thereof are mixed with one or more antioxidants such as methionine, whereby the antibodies or antigen-binding fragments thereof can be protected from oxidation to extend their shelf life and / or increase their activity.

[0136] To further illustrate, pharmaceutically acceptable carriers can include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN-80), partitioning agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multiple dose containers comprising phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

[0137] Administration, formulation and dosage

[0138] The pharmaceutical compositions of the present invention can be administered to a subject in need thereof in vivo by various routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The compositions of the present invention can be formulated into solid, semisolid, liquid or gaseous preparations; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. Suitable preparations and routes of administration can be selected according to the intended application and treatment regimen.

[0139] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups or inhalants and controlled release forms thereof.

[0140] Preparations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickeners, and solutes that make the preparation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohol, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such preparations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosage regimens (including dosage, time, and repetition) will depend on specific individuals and individual medical history as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0141] The requirements for effective pharmaceutical carriers for injectable formulations / compositions are well known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, eds. Banker and Chalmers, pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pp. 622-630 (1986)).

[0142] The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferating or tumorigenic cells, maintaining the reduction of such tumor cells, reducing the proliferation of tumor cells or delaying the development of metastasis. In some embodiments, the dose administered can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a sustained continuous release formulation of the therapeutic composition of the present invention may be suitable.

[0143] Those skilled in the art will appreciate that the appropriate dosage may vary from patient to patient. Determining the optimal dosage generally involves a balance between the level of therapeutic benefit and any risk or adverse side effects. The selected dosage level will depend on a variety of factors, including but not limited to the activity of the specific compound, administration, administration time, compound clearance rate, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and species, sex, age, weight, condition, general health and previous medical history of the patient. However, the dosage is generally selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or adverse side effects.

[0144] Generally, the antibodies or antigen-binding fragments thereof of the present invention can be administered in various ranges.

[0145] In certain preferred embodiments, the course of treatment involving the antibodies or antigen-binding fragments thereof of the present invention will include multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibodies or antigen-binding fragments thereof of the present invention may be administered every day, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two months, every ten weeks or every three months. In this regard, it is understood that the dosage may be changed or the interval may be adjusted based on patient response and clinical practice.

[0146] Compatible formulations for parenteral administration (eg, intravenous injection) will contain an antibody or antigen-binding fragment thereof as disclosed herein at a concentration of about 5 μg / mL to about 100 mg / mL.

[0147] The antibodies of the invention can be co-administered with one or more other therapeutic agents (e.g., cytotoxic agents, radiotoxic agents, anti-tumor agents, anti-angiogenic agents or and immunosuppressive agents) to reduce the induction of an immune response against the antibodies of the invention. The antibody can be linked to the therapeutic agent (as an immune complex) or can be administered separately from the therapeutic agent.

[0148] In the context of administering a treatment, the term "combination" or "co-administration" as used herein refers to the use of more than one treatment or therapeutic agent. The use of the term "combination" does not limit the order in which the treatment or therapeutic agent is administered to a subject. The treatment or therapeutic agent may be administered before, simultaneously, or after the second treatment or therapeutic agent is administered to the patient. Preferably, the treatment or therapeutic agent is administered to the subject in a certain order, amount, and / or within a certain time interval so that the treatment or therapeutic agent can work together. In a specific embodiment, the treatment or therapeutic agent is administered to the subject in a certain order, amount, and / or within a certain time interval so that they provide an increased benefit than if they were administered in other ways (particularly independently of each other). Preferably, the increased benefit is a synergistic effect.

[0149] Medical Uses

[0150] The antibodies, antibody compositions and methods of the invention have many in vitro and in vivo uses, including, for example, detection of CD93 or enhancement of immune responses. For example, these molecules can be administered to cultured cells in vitro or ex vivo, or, for example, administered to human subjects in vivo.

[0151] Preferred subjects include mammals, such as humans / patients. Mammals in the context of the present invention are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cows, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and captive animals, such as zoo animals.

[0152] Treating conditions associated with CD93 expression

[0153] In some aspects, the invention provides methods of treating a disorder in a mammal comprising administering to a subject (eg, a human) in need of treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[0154] As described herein, the antibodies disclosed herein have one or more activities that can be therapeutically applied to kill cells and / or inhibit cells. In particular, killing cells, inhibiting the proliferation of cells and / or inhibiting the colony formation of cells can be used to treat or prevent cancer (including cancer metastasis). Inhibiting the proliferation, colony formation and / or metastasis of cells can be applied, especially for treating or preventing cancer metastasis and metastatic spread of cancer cells.

[0155] In some aspects, the present disclosure provides a method for treating a disease associated with CD93 expression or determining the prognosis thereof in a subject, comprising administering an effective dose of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit to a subject in need thereof.

[0156] In some aspects, the present disclosure provides the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method for treating a disease associated with CD93 expression or determining the prognosis thereof in a subject.

[0157] In some aspects, the present disclosure provides use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of an agent (or drug) for treating a disease associated with CD93 expression or determining the prognosis thereof.

[0158] In one embodiment, the disease associated with CD93 expression includes a tumor disease, such as cancer.

[0159] The antibodies or antigen-binding fragments thereof can be used alone as a monotherapy or can be used in combination with chemotherapy or radiation therapy.

[0160] The antibodies or antigen-binding fragments thereof can be used in combination with anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0161] The term "anticancer agent" or "antiproliferative agent" means any agent that can be used to treat a cell proliferative disorder such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, anti-metastatic agents, and immunotherapeutic agents. It should be understood that in selected embodiments as described above, such anticancer agents may comprise a conjugate and may be combined with the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, the selected anticancer agent is linked to an unpaired cysteine ​​of an engineered antibody to provide an engineered conjugate as described herein. Therefore, such an engineered conjugate is explicitly contemplated within the scope of the present invention. In other embodiments, the disclosed anticancer agent will be administered in combination with a site-specific conjugate comprising a different therapeutic agent as described above.

[0162] diagnosis

[0163] The present invention provides in vitro and in vivo methods for detecting, diagnosing or monitoring proliferative disorders and methods for screening cells from patients to identify tumor cells including tumorigenic cells. Such methods include identifying individuals with cancer for treatment or monitoring the progression of cancer, including contacting the patient or a sample obtained from the patient (in vivo or in vitro) with an antibody as described herein, and detecting the presence or absence or binding level of the antibody bound to a bound or free target molecule in the sample. In some embodiments, the antibody will comprise a detectable label or reporter molecule as described herein.

[0164] In some aspects, the present disclosure provides a method for diagnosing, detecting or monitoring a disease associated with CD93 expression, comprising administering an effective dose of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit to a subject in need thereof.

[0165] In some aspects, the present disclosure provides the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method for diagnosing, detecting or monitoring a disease associated with CD93 expression in a subject.

[0166] In yet another aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of an agent (or drug) for diagnosing, detecting or monitoring a disease associated with CD93 expression.

[0167] Samples can be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblotting assays, Western blot analysis, and flow cytometry assays. Compatible in vivo diagnostics or diagnostic assays can include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scans), positron emission tomography (e.g., PET scans), radiography, ultrasound, etc., known to those skilled in the art.

[0168] The methods described in the present invention for detecting or monitoring CD93 expression or the level of cells expressing CD93 in vitro can also be used for non-diagnostic purposes.

[0169] Preferred subjects include mammals, such as humans / patients in need thereof.

[0170] The sample from the subject is blood, excreta (urine or feces), oral or nasal secretions, or alveolar lavage fluid, tissue fluid, sweat, or extracts thereof from the subject.

[0171] Pharmaceutical packaging and kits

[0172] Also provided are pharmaceutical packaging and kits containing one or more containers of one or more doses of an antibody or an antigen-binding fragment thereof. In certain embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or an antigen-binding fragment thereof, with or without one or more other agents. For other embodiments, such a unit dose is supplied in a disposable prefilled syringe for injection. In other embodiments, the composition contained in the unit dose may contain saline, sucrose or the like; a buffer such as phosphate, etc.; and / or be formulated in a stable and effective pH range. Alternatively, in certain embodiments, the conjugate composition may be provided as a lyophilized powder, which may be reconstituted after adding a suitable liquid (e.g., sterile water or saline). In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the encapsulated conjugate composition is used to treat a selected tumor disease condition.

[0173] Such kits typically include a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally include one or more anticancer agents or other agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnosis or combined therapy.

[0174] More specifically, the kit can have a single container containing an antibody or antigen-binding fragment thereof of the present disclosure, with or without additional components, or they can have different containers for each required reagent. In the case of providing a combination therapeutic agent for conjugation, a single solution can be premixed in a molar equivalent combination or one component more than another. Alternatively, the conjugate of the kit and any optional anticancer agent can be stored separately in different containers before being administered to a patient. The kit can also include a second / third container device for containing a sterile pharmaceutically acceptable buffer or other diluent such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution, and glucose solution.

[0175] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or a saline solution. However, the components of the kit may be provided as dry powders. When the reagents or components are provided in dry powder form, the powder may be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in another container.

[0176] Example

[0177] The invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. In addition, the experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples are commercially available products unless otherwise specified.

[0178] Example 1 Generation of mouse anti-human CD93 antibodies

[0179] Five SJL mice (provided by Ruizhi Chemical) and five Balb / c mice (provided by Ruizhi Chemical) were intraperitoneally injected with 50-25 μg of human CD93 extracellular region fusion protein carrying human Fc tag (provided by Ruizhi Chemical), and Mn 2+ Five SJL mice and five Balb / c mice were immunized once a week. 4 μg of pCP-hCD93FL encoding full-length human CD93 plasmid (provided by Ruizhi Chemical) was used for gene gun immunization, supplemented with GM-CSF (R&D, Cat.215-GM-050 / CF) and Mn 2+ Immunize once every week or two weeks. After two immunizations, collect mouse serum for serum titer detection. When the serum titer detection reaches the fusion requirement, take the mouse lymph node, spleen or bone marrow for cell fusion.

[0180] The collected spleen cells were mixed with mouse myeloma cells SP2 / 0 (provided by Ruizhi Chemical) and cell fusion was performed using high-efficiency electrofusion technology. The fused cells were diluted into DMEM medium containing HT, plated into 96-well plates in proportion, and the 96-well plates were cultured overnight in a 5% CO2, 37°C incubator. After 24 hours, DMEM medium containing 2xHAT was added.

[0181] After 10-14 days, the supernatant of hybridoma cells in a 96-well plate was taken for enzyme-linked immunosorbent assay (ELISA). The experimental process is briefly described as follows: dilute the human CD93 recombinant protein to 1 μg / ml with CB buffer (Sigma, Cat.C3041), mix well, add 100 μl / well to the 96-well plate, and leave to coat overnight at 2-8°C. Wash the plate 3 times with 1xPBS containing 0.05% Tween-20, i.e., 1xPBST, and add 1% BSA in 1xPBS blocking solution at 300 μl / well, and incubate at 37°C for 2 hours. After washing the plate 3 times with 1xPBST, add the hybridoma cell supernatant, 100 μl / well, and incubate at 37°C for 1 hour. After washing 3 times with 1xPBST, add HRP-labeled goat anti-mouse IgG (Invitrogen, Cat.G-21040) diluted with 1xPBS at 100 μl / well and incubate at 37°C for 1 hour. After washing 3 times with 1xPBST, add TMB colorimetric solution (Biyuntian, Cat.P0209) at 100 μl / well, incubate at 37°C for 10 minutes, and then add ELISA stop solution (Solebo, Cat.C1058) at 100 μl / well. Place the 96-well plate in an ELISA reader for OD450nm reading. The positive clones obtained in the initial screening were expanded and cultured to 24-well plates for human CD93 binding rescreening. In this experiment, the positive clones will be preferentially used for subcloning in order to obtain stable monoclonal hybridoma cell lines, and the subclone plates will be screened and rescreened again.

[0182] The selected hybridoma cell lines were further expanded to 100 ml culture bottles for production, and the supernatant was collected and purified using a protein A affinity column. The antibody concentration was detected by A280, and the purity of the antibody was detected by polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions and size exclusion chromatography (SEC-HPLC). The purity analysis of anti-human CD93 monoclonal antibodies using SEC-HPLC is shown in Table 1.

[0183] Table 1. Purity analysis of anti-human CD93 monoclonal antibodies using SEC-HPLC

[0184]

[0185]

[0186] Enzyme-linked immunosorbent assay was used to identify the subtypes of anti-human CD93 monoclonal antibodies. The experimental process is briefly described as follows. Add anti-mouse IgG capture antibody (Invitrogen, Cat. A16080) diluted to 1 μg / ml with 1xPBS to a 96-well plate at 50 μl / well and incubate at 2-8 ° C overnight. After washing once with 1xPBST, add 1xPBS blocking solution containing 1% BSA at 250 μl / well and incubate at room temperature for 2 hours. After diluting 3 times with 1xPBST, add hybridoma cell culture supernatant at 50 μl / well and incubate at 37 ° C for 1 hour. After washing 3 times with 1xPBST, add SBAclonotyping System-HRP (SouthernBiotech, Cat. 5300-05) diluted with 1xPBS containing 1% BSA at 50 μl / well and incubate at 37 ° C for 30 minutes. After washing three times with 1xPBST, TMB colorimetric solution (Biyuntian, Cat. P0209) was added at 100 μl / well, incubated at 37°C for 10-15 minutes, and ELISA stop solution (Solebol, Cat. C1058) was added at 100 μl / well. The subtypes of anti-human CD93 monoclonal antibodies are shown in Table 2.

[0187] Table 2. Identification of subtypes of anti-human CD93 monoclonal antibodies using ELISA

[0188] Clone number Subtype 11A11B6 mIgG1, κ 20F1B9 mIgG2c, κ 32D10G7 mIgG2b, κ 61C6A1 mIgG1, κ 86B2E10 mIgG1, κ

[0189] Example 2 Cloning and sequencing analysis of CD93 monoclonal antibody

[0190] Total RNA samples were extracted as follows: 1x10 7For hybridoma cells, remove the cell culture medium and add at least 1 ml of NucleoZOL cell lysis buffer (MACHEREY-NAGEL, Cat. 740406.200) to the culture dish to lyse the cells. Repeated pipetting ensures that the cells are completely lysed. In the lysis buffer, add 200 μl of RNase-free water to 500 μl of NucleoZOL. Shake the sample vigorously for 15 seconds, incubate at room temperature for 5 minutes, centrifuge at 12,000xg for 15 minutes at room temperature, and transfer 500 μl of supernatant to a new 1.5 ml centrifuge tube. Leave a layer of supernatant on the DNA / protein precipitate. Add 500 μl of isopropanol to each 500 μl of supernatant to precipitate the RNA. After incubating the sample at room temperature for 10 minutes, centrifuge the sample at 12,000xg for 10 minutes. Remove and discard the supernatant. Add 500 μl of 75% ethanol to precipitate and centrifuge at 8,000xg for 3 minutes. The ethanol in the precipitate was discarded, and the RNA precipitate was washed once with 75% ethanol, and then dissolved in RNase-free water to obtain about 1 μg / μl RNA.

[0191] Synthesis and amplification of heavy and light chain variable regions: RACE 5' / 3' Kit Instructions. Random primers were used for the synthesis of the first cDNA strand. RACE was used to amplify the heavy and light chain variable regions, i.e., cDNA was used as a template and Mouse Ig-Primer Set primers were used as gene-specific primers (GSP) to perform PCR amplification of the heavy and light chain variable regions. The PCR products were stained and analyzed by electrophoresis in a 1.2% agarose gel. The desired DNA fragments were collected for purification.

[0192] TA cloning of VH and VL fragments: Transfer the target gene into the pMD 18-T construction vector and transform the ligation product into DH5α competent cells. Thaw a tube of DH5αTM cells on ice, gently mix the cells, transfer 50μl of cells to a 1.5ml centrifuge tube, add 1-5μl (1-10ng) of DNA, and mix gently. Incubate the test tube on ice for 30 minutes. Heat shock the cells in a 42℃ water bath for 20 seconds. Place the centrifuge tube on ice for 2 minutes. Add 950μl of preheated LB medium to each centrifuge tube. Incubate at 37℃, 225rpm for 1 hour. Take 20-200μl and spread it on a preheated LB plate containing 100μg / ml ampicillin. Incubate overnight at 37℃.

[0193] Cloning and sequencing: 15 transformant colonies were selected from each plate, and the sequences of the inserts were analyzed by DNA sequencing. The sequences were analyzed for the heavy chain and light chain CDR regions. Table 3 shows the heavy chain variable region and light chain variable region sequences of the anti-CD93 monoclonal antibodies. Tables 4 and 5 show the CDR sequences of the heavy chain variable region and the light chain variable region CDR sequences of the anti-CD93 monoclonal antibodies, respectively.

[0194] Table 3. Sequences of heavy chain variable region and light chain variable region of anti-human CD93 monoclonal antibody

[0195] Clone number Heavy chain variable region Light chain variable region 11A11B6 SEQ ID NO: 2 SEQ ID NO: 3 20F1B9 SEQ ID NO: 4 SEQ ID NO: 5 32D10G7 SEQ ID NO: 6 SEQ ID NO: 7 61C6A1 SEQ ID NO: 8 SEQ ID NO: 9 86B2E10 SEQ ID NO: 10 SEQ ID NO: 11

[0196] Table 4. Heavy chain variable region VH CDRs of anti-human CD93 monoclonal antibodies

[0197]

[0198]

[0199] Table 5. Light chain variable region VL CDRs of anti-human CD93 antibodies

[0200]

[0201] Example 3 Detection of the binding activity of anti-human CD93 monoclonal antibody to CD93 by flow cytometry

[0202] The binding activity of anti-human CD93 monoclonal antibody to CD93 overexpressing cell lines was detected by flow cytometry. Briefly, human CD93, cynomolgus monkey CD93 and mouse CD93 overexpressing CHO-K1 cell lines, namely CHO-K1-CD93 (Jiman Biotechnology, Cat.GM-C22126), CHO-K1-cynoCD93 (Jiman Biotechnology, Cat.GM-C22128) and CHO-K1-mCD93 (Jiman Biotechnology, Cat.GM-C22125) were grown to the logarithmic growth phase, and the cells were digested with Accutase solution (Invitrogen Cat.00-4555-56), neutralized with complete medium, washed, centrifuged, and resuspended with FASC buffer, and the cells were cultured at 2x10 5 / well added to a 96-well plate. Prepare the antibody with FACS buffer, dilute it 3-fold, mix well, take 100μl / well and add it to the corresponding CHO-K1 overexpression cell line, mix well, and incubate at 2-8℃ for 30 minutes. Centrifuge at 300xg for 5 minutes, and remove the supernatant. Wash the cells with FACS buffer. Add 100μl / well of PE-labeled goat anti-mouse IgG fluorescent secondary antibody (Biolegend, Cat.405305) diluted with FACS buffer, mix well, and incubate at 2-8℃ for 30 minutes. Centrifuge at 300xg for 5 minutes, and remove the supernatant. Wash the cells with FACS buffer. Resuspend the cells with 1xPBS and detect using a BD lyric flow cytometer. The results are as follows. Figure 1 , Figure 2 and Figure 3 The binding affinity is shown in Table 6. The experimental results show that among the five anti-human CD93 antibodies, 20F1B9, 32D10G7 and 61C6A1 can bind to human CD93 and cynomolgus CD93 overexpressing cell lines with high affinity, and 11A11B6 and 86B2E10 have low binding affinity to human CD93 and cynomolgus CD93 overexpressing cell lines. All five anti-human CD93 antibodies do not bind to mouse CD93 overexpressing cell lines.

[0203] Table 6. Binding affinity of anti-human CD93 antibodies to human CD93 and cynomolgus monkey CD93 overexpressing cell lines

[0204]

[0205] Example 4 Flow cytometry detection of binding specificity of anti-human CD93 antibodies

[0206] The specificity of anti-human CD93 antibody binding to CHO cells is briefly described as follows. When CHOZN-K1 cells (Sigma, Cat.) grow to the logarithmic growth phase, take an appropriate amount of cells into a 15 ml centrifuge tube, centrifuge at 200 x g for 7 minutes, resuspend the cells in FASC buffer, and press 2x10 5 / well added to a 96-well plate. Prepare the antibody with FACS buffer, mix well, take 100μl / well and add it to the CHOZN-K1 cell line, mix well, and incubate at 2-8℃ for 30 minutes. Centrifuge at 300xg for 5 minutes, and remove the supernatant. Wash the cells with FACS buffer. Add 100μl / well of PE-labeled goat anti-mouse IgG fluorescent secondary antibody (Biolegend, Cat.405305) diluted with FACS buffer, mix well, and incubate at 2-8℃ for 30 minutes. Centrifuge at 300xg for 5 minutes, and remove the supernatant. Wash the cells with FACS buffer. Resuspend the cells with 1xPBS and detect using a BD lyric flow cytometer. The results are as follows. Figure 4The experimental results showed that none of the five anti-human CD93 antibodies non-specifically bound to CHOZN-K1 cells.

[0207] Example 5 Detection of the binding affinity of anti-human CD93 antibody to CD93 by Biacore

[0208] Biacore was used to detect the affinity of anti-human CD93 antibodies, and the experimental process is briefly described as follows. An-mFc was coupled using a mouse anti-capture kit (Cytiva, Cat.BR100838) and an amino coupling kit (Cytiva, Cat.BR100633), that is, 50mM NHS and 200mM EDC were obtained by mixing in a ratio of 1:1 to activate the 1-8 channel surface of the CM5 chip (Cytiva, Cat.BR100530), with a flow rate of 10μl / min and a time of 420 seconds. Then, the anti-mFc antibody diluted with pH 5.0 sodium acetate solution was injected at a flow rate of 10μl / min, with a concentration of 30μg / ml, for 420 seconds. 1M ethanolamine was injected at 10μl / min for 420 seconds to block the excess active carboxyl groups on the chip. In the affinity KD test, the running buffer was 1xHBS-EP+ (pH7.4) solution (Cytiva, Cat.BR100669). Anti-human CD93 antibody was diluted to a concentration of 2 μg / ml using running buffer, and 2 μg / ml anti-human CD93 antibody was injected into the detection channel flow cell at a flow rate of 10 μl / min for 60 seconds for capture. Human CD93 extracellular region fusion protein carrying hFc tag was diluted with running buffer to a concentration of 400, 200, 100 or 25 nM, and double dilution was performed. The sample human CD93 ECD-hFc to be tested was injected into the detection channel at a flow rate of 30 μl / min at each concentration. The binding and dissociation times of the antigen and antibody were set to 180 seconds and 400 seconds, respectively. Finally, 10 mM glycine pH 1.5 was injected into the detection channel at a flow rate of 30 μl / min for 30 seconds. Data analysis was performed using Biacore Insight Evaluation Software (Version 2.0.15.12933). The 1:1 binding model was selected for curve fitting and calculation of kinetic parameters. As shown in Table 7, the experimental results showed that the binding affinity of the five anti-human CD93 monoclonal antibodies to human CD93 was 1.0E-09 to 1.0E-10.

[0209] Table 7. Anti-human CD93 monoclonal antibody binding kinetics results

[0210] Clone number Ka(1 / Ms) Kd(1 / s) KD(M) 11A11B6 1.26E+06 6.21E-04 4.94E-10 20F1B9 4.90E+04 1.20E-04 2.44E-09 32D10G7 3.56E+04 8.02E-06 2.26E-10 61C6A1 1.92E+05 1.18E-04 6.14E-10 86B2E10 3.20E+05 7.29E-04 2.27E-09

[0211] Example 6 Flow cytometry detection of anti-human CD93 antibody-mediated human CD93 / human IGFBP7 binding blocking activity

[0212] The anti-human CD93 antibody-mediated human CD93 / human IGFBP7 binding blocking activity, the experimental process is briefly described as follows. After labeling the hFc-tagged recombinant human IGFBP7 protein (provided by Ruizhi Chemical) with EZLink Sulfo-NHS-Biotin (Thermo scientific, Cat.21217), the Zeba TM Desalting columns (Thermo Scientific, Cat. 89882) were used to remove uncoupled biotin. When the human CD93 overexpressing cell line CHO-K1-hCD93 grew to the logarithmic growth phase, the cells were digested with Accutase solution (Invitrogen Cat. 00-4555-56), neutralized with complete medium, washed, centrifuged, and resuspended with FASC buffer. 5 / well is added to a 96-well plate. Biotin-labeled human IGFBP7 recombinant protein is diluted with FACS buffer to a concentration of 40nM, added to the cells at 50μl / well, and incubated at 2-8℃ for 30 minutes. Anti-human CD93 monoclonal antibody is diluted with FACS buffer, diluted 3 times, mixed, added to the cells at 50μl / well, and incubated at 2-8℃ for 30 minutes. After centrifugation at 300xg for 5 minutes, the supernatant is removed. Wash the cells with FACS buffer. Streptavidin-PE (Jackson ImmunoResearch, Cat.016-110-084) diluted in FACS buffer is added at 100μl / well, mixed, and incubated at 2-8℃ for 30 minutes. After centrifugation at 300xg for 5 minutes, the supernatant is removed. Wash the cells with FACS buffer. Resuspend the cells with 1xPBS and detect using a BDlyric flow cytometer. The results are as follows Figure 5 The blocking activity is shown in Table 8. The experimental results showed that among the five anti-human CD93 antibodies, 20F1B9, 32D10G7 and 61C6A1 could block the binding of human CD93 to human IGFBP7.

[0213] Example 7 Flow cytometry detection of anti-human CD93 antibody-mediated human CD93 / human MMRN-2 binding blocking activity

[0214] The anti-human CD93 antibody-mediated human CD93 / human MMRN-2 binding blocking activity was briefly described as follows. After labeling the hFc-tagged recombinant human MMRN-2 protein (provided by Ruizhi Chemical) with EZLink Sulfo-NHS-Biotin (Thermo scientific, Cat. 21217), the hFc-tagged recombinant human MMRN-2 protein (provided by Ruizhi Chemical) was labeled with Zeba TM Desalting columns (Thermo Scientific, Cat. 89882) were used to remove uncoupled biotin. When the human CD93 overexpressing cell line CHO-K1-hCD93 grew to the logarithmic growth phase, the cells were digested with Accutase solution (Invitrogen Cat. 00-4555-56), neutralized with complete medium, washed, centrifuged, and resuspended with FASC buffer. 5 / well is added to a 96-well plate. The biotin-labeled human MMRN-2 recombinant protein is diluted with FACS buffer to a concentration of 70nM, and added to the cells at 50μl / well, and incubated at 2-8℃ for 30 minutes. The anti-human CD93 monoclonal antibody is diluted with FACS buffer, diluted 3 times, mixed, and added to the cells at 50μl / well, and incubated at 2-8℃ for 30 minutes. After centrifugation at 300xg for 5 minutes, the supernatant is removed. The cells are washed with FACS buffer. Streptavidin-PE (Jackson ImmunoResearch, Cat.016-110-084) diluted in FACS buffer is added at 100μl / well, mixed, and incubated at 2-8℃ for 30 minutes. After centrifugation at 300xg for 5 minutes, the supernatant is removed. The cells are washed with FACS buffer. Resuspend the cells with 1xPBS and detect using a BDlyric flow cytometer. The results are as follows Figure 6 The blocking activity is shown in Table 8. The experimental results showed that among the five anti-human CD93 antibodies, 11A11B6 and 86B2E10 could block the binding of human CD93 to human MMRN-2.

[0215] Table 8. Blocking activity of anti-human CD93 monoclonal antibodies

[0216]

[0217]

[0218] Example 8 Epitope grouping experiment at protein level

[0219] The epitope grouping of anti-human CD93 antibodies was analyzed at the protein level using enzyme-linked immunosorbent assay. The experimental process is briefly described as follows: dilute the anti-human CD93 monoclonal antibody to a concentration of 1 μg / ml with 1xPBS, divide it into 96-well plates at 100 μl / well, and incubate overnight at 2-8°C. After washing 3 times with 1xPBS, add 1xPBS blocking solution containing 1% BSA at 300 μl / well and block at 37°C for 2 hours. Add human CD93 recombinant protein diluted with 1xPBS (provided by Ruizhi Chemical) with a starting concentration of 10 μg / ml, 5-fold gradient dilution, mix well, add at 100 μl / well, and incubate at 37°C for 1 hour. After washing 3 times with 1xPBST, add HRP-labeled goat anti-human IgG (Sigma, Cat.A0170) diluted with 1xPBS at 100 μl / well and incubate at 37°C for 1 hour. After washing 3 times with 1xPBST, TMB colorimetric solution (Biyuntian, Cat.P0209) was added at 100 μl / well, and after incubation at 37°C for 10 minutes, ELISA stop solution (Solebo, Cat.C1058) was added at 100 μl / well. The 96-well plate was placed in an ELISA reader for OD450nm reading. GraphPad Prism was used for four-parameter fitting to calculate the EC80 value.

[0220] Use 1xPBS to dilute the anti-human CD93 monoclonal antibody to a concentration of 1μg / ml, divide it into 96-well plates at 100μl / well, and incubate overnight at 2-8°C. After washing 3 times with 1xPBS, add 1% BSA in 1xPBS blocking solution at 300μl / well and block at 37°C for 2 hours. Add 50μl 60μg / ml second anti-human CD93 monoclonal antibody (provided by Ruizhi Chemical) and 50μl hFc-tagged human CD93 recombinant protein (concentration of 2xEC80), a total of 100μl / well, and incubate at 37°C for 1 hour. After washing 3 times with 1xPBST, add HRP-labeled goat anti-human IgG (Sigma, Cat.A0170) diluted with 1xPBS at 100μl / well and incubate at 37°C for 1 hour. After washing 3 times with 1xPBST, TMB colorimetric solution (Biyuntian, Cat.P0209) was added at 100 μl / well, and after incubation at 37°C for 10 minutes, ELISA stop solution (Solebo, Cat.C1058) was added at 100 μl / well. The 96-well plate was placed in an ELISA reader for OD450nm reading, the inhibition rate was calculated, and the epitope grouping of anti-human CD93 monoclonal antibodies was performed according to the results. The experimental results are shown in Table 9. The results show that 11A11B6 has the same epitope as 86B2E10, 20F1B9 has the same recognition epitope as 32D10G7, and 61C6A1 has different recognition epitopes from 11A11B6 and 32D10G7.

[0221] Table 9. Epitope binning analysis of anti-human CD93 monoclonal antibodies

[0222] Inhibition rate, % 11A11B6 20F1B9 32D10G7 61C6A1 86B2E10 11A11B6 87.49 1.81 0.27 15.11 71.24 20F1B9 -2.08 89.01 82.40 <![CDATA[ND * ]]> <![CDATA[ND * ]]> 32D10G7 -6.60 92.71 84.76 5.46 -3.42 61C6A1 -3.25 <![CDATA[ND * ]]> 8.07 93.43 <![CDATA[ND * ]]> 86B2E10 79.36 <![CDATA[ND * ]]> 26.78 <![CDATA[ND * ]]> 78.38

[0223] ND * : Indicates that no detection was performed.

[0224] Incorporated by Reference

[0225] Each patent document and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0226] Equivalence

[0227] The present invention may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be regarded as illustrative in all cases, rather than limiting the invention described herein. Therefore, the scope of the present invention is indicated by the appended claims rather than by the above description, and it is intended that all changes within the equivalent meaning and scope of the claims are included therein.

Claims

1. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to CD93 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), The heavy chain variable region and the light chain variable region comprise: HCDR1 shown in SEQ ID NO:12, HCDR2 shown in SEQ ID NO:13, and HCDR3 shown in SEQ ID NO:14, LCDR1 shown in SEQ ID NO:26, LCDR2 shown in SEQ ID NO:27, and LCDR3 shown in SEQ ID NO:

28.

2. The antibody or fragment according to claim 1, comprising: a heavy chain variable region as shown in SEQ ID NO: 2; The light chain variable region is shown in SEQ ID NO:

3.

3. The antibody or fragment according to claim 2, further comprising a heavy chain constant region and a light chain constant region, wherein: The antibody heavy chain constant region is selected from the IgG series of antibodies, and the light chain constant region is selected from κ or λ chain.

4. The antibody or fragment according to claim 3, wherein the antibody heavy chain constant region is selected from one of IgG1, IgG2c, and IgG2b, and the light chain constant region is a κ chain.

5. The antibody or fragment of any of the above claims, wherein the antibody is selected from the group consisting of a whole antibody, a bispecific antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody; The fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab)2 fragment, a Fv fragment and a ScFv.

6. An isolated nucleic acid molecule encoding the antibody or fragment as claimed in any one of claims 1 to 5. A vector comprising the nucleic acid molecule according to claim 6 .

8. A host cell comprising the nucleic acid molecule according to claim 6 or the vector according to claim 7.

9. A conjugate comprising the antibody or fragment of any one of claims 1 to 5 coupled to at least one detectable label.

10. An antibody drug conjugate comprising an antibody, wherein one or more drug moieties are covalently linked directly or via a linker to the antibody or fragment according to any one of claims 1 to 5.

11. A multispecific molecule comprising the antibody or antigen-binding fragment of any one of claims 1-5.

12. The multispecific molecule of claim 11, which specifically binds CD93 and additionally specifically binds one or more other targets.

13. The multispecific molecule of claim 12, further comprising at least one molecule having a second binding specificity for a second target.

14. A pharmaceutical composition or kit comprising the antibody or fragment of any one of claims 1 to 5, or the nucleic acid molecule of claim 6, or the vector of claim 7, or the host cell of claim 8, or the conjugate of claim 9, or the antibody-drug conjugate of claim 10, or the multispecific molecule of claims 11 to 13, and a pharmaceutically acceptable carrier.

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